JP2002516067A - Novel FDRG proteins and nucleic acid molecules and uses therefor - Google Patents

Abstract

  (57) [Summary] Disclosed are novel FDRG polypeptides, proteins, and nucleic acid molecules. In addition to an isolated full-length FDRG protein, the present invention further provides an isolated FDRG fusion protein, an antigenic peptide and an anti-FDRG antibody. The present invention also provides FDRG nucleic acid molecules, recombinant expression vectors containing the nucleic acid molecules of the present invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals into which the FDRG gene has been introduced or disrupted. provide. Diagnostic, screening and therapeutic methods utilizing the compositions of the invention are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

GOVERNMENT FUNDING This invention relates, in part, to the National Institutes of Health.
This funding was funded by the government under USPHS grant DK31405 awarded by e. of Health. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION [0002] The formation of blood vessels occurs by two processes: angiogenesis and angiogenesis.
Angiogenesis requires the establishment of a primitive vascular network during embryogenesis. afterwards,
Angiogenesis rebuilds this network to form the mature cardiovascular system. In addition, angiogenesis occurs in adult tissues during tissue repair, reshaping of female reproductive tissues and tumor growth and metastasis (Maisonpiee).
rre) et al. (1997) Science 277: 55-60).

[0003] Endothelial cells have been found to be centrally involved in both angiogenesis and angiogenesis. These cells migrate, proliferate, and congregate to form tubes with tight cell-cell connections. Peri-endothelial feeder cells are then recruited to completely enclose the endothelial tubules, thereby maintaining and modulating the function of the blood vessels.
(Hanahan (1997) Science)
e 277: 48-50).

[0004] The process of vascular establishment and reconstruction is controlled by the interaction of several protein ligands and tyrosine kinase-like receptors expressed on endothelial cells. Such receptors include, for example, the recently discovered TIE receptor family consisting of TIE1 and TIE2, which has been found to be critically involved in the formation of the vasculature. Dumont et al. (1994) Genes.
Dev. 8: 1897-1909; Sato et al. (1995) Natu.
re 376: 70-74. In addition, ligands have been discovered that bind to the TIE2 receptor and belong to the protein family that includes fibrinogen and ficolin. These ligands are angiopoietin-1 that binds TIE2 and induces its phosphorylation (Davis et al. (1996) Ce
11 87: 1161-1169), and Angiopoietin-1 and TI
Angiopoietin-2 (Maisonpierre (1997) Science 277: 55) acting as a natural antagonist of E2
-60). Both of these ligands, Angiopoietin-1 and Angiopoietin-2, are involved in vascular formation and reconstruction.

[0005] Recently, an interesting connection has been made between the different fields of angiogenesis and obesity. In particular, leptin, a protein secreted by adipocytes, has been demonstrated to function as both a metabolic hormone and a genuine angiogenic factor (
(1998) Sc. Sierra-Honigmann et al.
issue 281: 1683-1686). The finding that factors secreted by adipocytes, which have a known role in metabolism and obesity, also play a role in angiogenesis, establishes a novel association between adipocytes and the vasculature. The complex functionality of leptin raises the possibility that there are other molecules that may play a dual role in both angiogenesis and obesity. Thus, there is a need to identify and characterize such molecules and to understand the mechanisms by which organisms utilize one factor in controlling multiple cellular functions. Further, there is a need to identify molecules with which such dual regulators interact, as well as to identify modulators of such dual regulators.

SUMMARY OF THE INVENTION [0006] The present invention is directed, at least in part, to a novel homology to angiopoietin that is thought to modulate angiogenesis and mediate adipocyte contribution to diverse metabolic processes. Based on the discovery of secretory molecules. These new molecules are
Herein fibrinogen domain-related (F ibrinogen D omai
n R elated) (herein referred to as "FDRG" or referred to as TANGO 115) nucleic acid and protein molecules, and regulation of various cellular processes (e.g., angiogenesis-related and metabolic-related processes) ( regulating)
(modulating agent). Thus, in one aspect, the present invention relates to F
Provided are isolated nucleic acid molecules encoding a DRG protein or a biologically active portion thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of nucleic acids encoding FDRG.

In one embodiment, the FDRG nucleic acid molecule is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4.
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, 60% homologous to the nucleotide sequence of the DNA insert of the plasmid deposited with the ATCC under accession number 98632, or their complement. In another embodiment, the isolated FDRG nucleic acid molecule encodes the amino acid sequence of human FDRG set forth in SEQ ID NO: 2. In another embodiment, the isolated FDRG nucleic acid molecule encodes the amino acid sequence of mouse FDRG shown in SEQ ID NO: 12.

[0008] In one preferred embodiment, the isolated FDRG nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1 or its complement. In another embodiment, the FDRG nucleic acid molecule further comprises nucleotides 1-165 of SEQ ID NO: 1. In another embodiment, the FDRG nucleic acid molecule further comprises nucleotides 1384-1894 of SEQ ID NO: 1. In yet another embodiment, the FDRG nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3.

[0009] In another preferred embodiment, the isolated FDRG nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 11, or its complement. In another embodiment, the FDRG nucleic acid molecule further comprises nucleotides 1-148 of SEQ ID NO: 11. In yet another embodiment, the FDRG nucleic acid molecule is nucleotides 1379-18 of SEQ ID NO: 11.
93 further. In yet another embodiment, the FDRG nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 13.

[0010] In another preferred embodiment of the invention, the isolated FDRG nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, or a complement thereof. In yet another embodiment, the FDRG nucleic acid molecule comprises nucleotides 1-556 or SEQ ID NO: 13 of SEQ ID NO: 3, or nucleotides 1-481 or SEQ ID NO: 14 of SEQ ID NO: 4.

[0011] In yet another preferred embodiment, the isolated FDRG nucleic acid molecule has accession number 9
No. 8632 has the nucleotide sequence of the DNA insert of the plasmid deposited at the ATCC or its complement.

In another embodiment, the FDRG nucleic acid molecule comprises a nucleotide sequence that encodes a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. In yet another aspect,
An FDRG nucleic acid molecule includes a nucleotide sequence that encodes a protein having an amino acid sequence at least 60% homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. In one preferred embodiment, the FDRG nucleic acid molecule comprises a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15.

In another aspect, the isolated nucleic acid molecules of the invention encode a secreted FDRG protein that includes a signal sequence, a unique domain at the N-terminus, a fibrinogen-like domain at the C-terminus. In another embodiment, the FDRG nucleic acid molecule is FD
RG protein is a naturally occurring nucleotide sequence. In yet another embodiment, the isolated nucleic acid molecule of the present invention encodes an FDRG protein and has SEQ ID NO: 1 under stringent hybridization conditions.
, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 or a nucleotide sequence that hybridizes to a nucleic acid molecule having the nucleotide sequence of the DNA insert of the plasmid deposited with the ATCC as accession number 98632. Consists of

[0014] Another aspect of the present invention relates to a nucleic acid molecule encoding a non-FDRG protein.
It features an FDRG nucleic acid molecule that specifically detects a DRG nucleic acid molecule. For example, in one embodiment, the FDRG nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising nucleotides 1-721 or SEQ ID NO: 11 of SEQ ID NO: 1. In another exemplary embodiment, the FDRG nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising nucleotides 1-556 of SEQ ID NO: 3 or SEQ ID NO: 13. In another embodiment, the FDRG nucleic acid molecule is
Hybridizes under stringent conditions to a nucleic acid molecule comprising nucleotides 1-481 of SEQ ID NO: 4 or SEQ ID NO: 14. In another embodiment, the FD
The RG nucleic acid molecule is at least 500 to 550 nucleotides in length, 600 to 650 nucleotides in length, 700 to 750 nucleotides in length, 800 to 850 nucleotides in length, and has SEQ ID NO: 1 under stringent conditions.
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, the nucleotide sequence of the DNA insert of the plasmid deposited at the ATCC as Accession No.98632, or the complement thereof. Hybridize to the nucleic acid molecule comprising.

[0015] Another aspect of the invention provides an isolated nucleic acid molecule that is antisense to the coding strand of an FDRG nucleic acid.

[0016] Another aspect of the present invention provides a vector comprising the FDRG nucleic acid molecule. In some embodiments, the vector is a recombinant expression vector. In another aspect, the invention provides a host cell containing a vector of the invention. The present invention also provides FDR
Also provided is a method for producing an FDRG protein by culturing a host cell of the invention containing a recombinant expression vector in a suitable medium so that the G protein is produced.

[0017] Another aspect of the invention features isolated or recombinant FDRG proteins and polypeptides. In one aspect, the isolated FDRG protein comprises:
It has a signal sequence, a unique domain at the N-terminus, a fibrinogen-like domain at the C-terminus, and is secreted. In another embodiment, the isolated FDRG protein is N
It has a unique terminal domain and a C-terminal fibrinogen-like domain.

In another embodiment, the isolated FDRG protein has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. In one preferred embodiment, the FDRG protein is SEQ ID NO: 2,
At least about 60% in the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15
It has a homologous amino acid sequence. In another embodiment, the FDRG protein has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15.

Another aspect of the present invention relates to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11,
SEQ ID NO: 13, nucleotide sequence of SEQ ID NO: 14, or accession number 98632
As an isolated FDRG protein encoded by a nucleic acid molecule having a nucleotide sequence at least about 60% homologous to the DNA insert of the plasmid deposited with the ATCC, or their complement. The present invention also provides
SEQ ID NO: 1, SEQ ID NO: 3, under stringent hybridization conditions
SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, nucleotide sequence of SEQ ID NO: 14,
An isolated FDR encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes to a nucleic acid molecule that comprises a nucleotide sequence of a DNA insert of the plasmid deposited at the ATCC under accession number 98632, or their complement.
It also features a G protein. In another embodiment, the polypeptide is SEQ ID NO: 2.
A fragment of the FDRG polypeptide comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or the amino acid sequence encoded by the DNA insert of the plasmid deposited at the ATCC as Accession No. 98632;
Here, the fragment is the D of the plasmid deposited at the ATCC as SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or Accession No. 98632.
It comprises at least 15 contiguous amino acids of the amino acid sequence encoded by the NA insert.

The FDRG protein of the present invention or a biologically active part thereof is
The non-FDRG polypeptide can be operatively linked to form a fusion protein. The invention further features an antibody that specifically binds an FDRG protein, such as a monoclonal or polyclonal antibody. In addition, the FDR
The G protein or biologically active portion thereof can be incorporated into a pharmaceutical composition, optionally including a pharmaceutically acceptable carrier.

[0021] In another aspect, the present invention provides a method for producing a biological sample comprising the steps of: Methods are provided for detecting FDRG expression in a biological sample by contacting the polypeptide with an agent capable of detecting the polypeptide.

In another aspect, the present invention relates to a method for detecting the presence of FDRG activity in a biological sample, wherein the biological sample comprises an agent capable of detecting an indicator of FDRG activity. Provided is a method of detecting the presence of FDRG activity in said biological sample by contacting.

In another aspect, the invention comprises contacting a cell with an agent that modulates FDRG activity such that FDRG activity in the cell is modulated.
Methods for modulating RG activity are provided. In one aspect, the agent inhibits FDRG activity. In another embodiment, the agent stimulates FDRG activity. In one aspect, the agent is an antibody that specifically binds to the FDRG protein. In another embodiment, the agent is transcription of the FDRG gene or mRN of FDRG.
FDRG expression is regulated by regulating the translation of A. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the FDRG mRNA or coding strand of the FDRG gene.

In one embodiment, the method of the invention comprises treating an agent that is an FDRG modulator by administering the agent to a subject having a disorder characterized by abnormal FDRG protein or nucleic acid expression or activity. Used for In one aspect, the FDRG modulator is an FDRG protein. In another aspect, the FDRG modulator is an FDRG nucleic acid molecule. In yet another embodiment, the FDRG modulator is a peptide, peptidomimetic or other small molecule. In a preferred embodiment, the disorder characterized by abnormal FDRG protein or nucleic acid expression is a hematopoietic disorder, a differentiation or developmental disorder, a proliferative disorder or a cell mobilization disorder.

The present invention also relates to (i) abnormal modifications or mutations of the gene encoding the FDRG protein; (ii) mis-regulation of said gene;
iii) providing a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of aberrant post-translational modifications of the FDRG protein, wherein the wild-type form of the gene has FDRG activity. Encodes a protein that has

[0026] In another aspect, the invention provides an indicator composition comprising an FDRG protein having FDRG activity, contacting the indicator composition with a test compound, and Determining the effect of a test compound on FDRG activity to identify compounds that modulate the activity of the FDRG protein;
Methods for identifying compounds that bind to RG protein or modulate its activity are provided.

In another aspect, the invention provides a method of treating obesity or diabetes in a subject, the method comprising administering to the subject an agent (eg, a small molecule modulator of FDRG, FD
RG nucleic acid molecule or FDRG antibody). In yet another aspect, the present invention provides a method for diagnosing obesity or diabetes in a subject using an FDRG nucleic acid molecule. In still another aspect, the present invention provides a method for identifying a compound that regulates metabolic abnormality, using a cell that expresses an FDRG receptor. In yet another aspect, the invention provides a method of modulating angiogenesis in a subject.

[0028] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel homology to a family of proteins, including fibrinogen, angiopoietin (s) and ficolin, referred to herein as FDRG proteins and nucleic acid molecules. Based on the discovery of molecules, they comprise a family of molecules with certain conserved structural and functional characteristics. Nucleotide sequence of human FDRG nucleic acid molecule and putative human FDR
The amino acid sequence of the G protein molecule is depicted in FIG. The nucleotide sequence of the mouse FDRG nucleic acid molecule and the predicted amino acid sequence of the mouse FDRG protein molecule are depicted in FIG.

The term “family” when referring to the proteins and nucleic acid molecules of the present invention refers to those that share a common structural domain and have sufficient amino acid or nucleotide sequence homology as defined herein. It is intended to mean species or more protein or nucleic acid molecules. The members of such a family can be naturally occurring and from either the same or different species. For example, a family may contain a first protein of human origin and a homolog of that protein of mouse origin, and a second distinct protein of human origin and a mouse homolog of that protein. Members of a family may also have common functional characteristics. An exemplary family of the invention is the fibrinogen-like protein family, members of which include at least human angiopoietin 1, human angiopoietin 2, ficorin and fibrinogen.
Another exemplary family of the invention is the FDRG protein family.

In one embodiment, members of the FDRG family are identified based on the presence of a “C-terminal fibrinogen-like domain” in the protein or corresponding nucleic acid molecule. As used herein, the term "C-terminal fibrinogen-like domain" is at least about 200-225 amino acid residues in length and at least about 38-41% of the β or γ chain of human fibrinogen. Includes protein domains with homology. In another embodiment, the "C-terminal fibrinogen-like domain" is at least about 100-300 amino acid residues in length, and preferably at least about 150-250 amino acid residues in length, and the human fibrinogen β
Alternatively, it has at least about 25-60%, preferably at least about 30-50%, and more preferably at least about 35-45% homology with the gamma chain. In yet another aspect, "
A "C-terminal fibrinogen-like domain" is at least about 100-300 amino acid residues in length, preferably at least about 150-250 amino acid residues in length, and more preferably at least about 200 amino acid residues in length. And amino acid residue 1 of SEQ ID NO: 2
84-400 at least about 25-60%, preferably at least about 30-50%, more preferably at least about 35-45%, and even more preferably at least about 38-4.
Refers to a protein domain with 1% homology. In one preferred embodiment, C
The terminal fibrinogen domain is involved in protein-protein interactions.

“C-terminal fibrinogen-like domain” with members of the FDRG family
To identify the presence of the protein family member, the amino acid sequence of the protein family member can be determined using, for example, the HMM database (eg, Pfam) using default parameters.
Database, Release 3.3). For example, the search is hm
It can be performed using the msf program (family specific) and a threshold score of 15 to determine hits. hmmsf is available as part of the HMMER package of search programs (HMMER 2.1.1, December 1998), which is freely distributed by the University of Washington School of Medicine. In one aspect, at least 150-200, preferably at least 200-250, more preferably at least 250
A hit to fibrinogen_C (fibrinogen β and γ chains, C-terminal globular domain) HMM with a score of ~ 300, and more preferably a minimum of 300-350 or higher, is a C-terminal fibrinogen-like in the query protein. Determine the existence of a domain. A search using the amino acid sequence of SEQ ID NO: 2 was performed against the HMM database, resulting in the identification of the C-terminal fibrinogen-like domain in the amino acid sequence of SEQ ID NO: 2. Thus, in one aspect of the invention, the FDRG protein has a C-substituted at about amino acids 184-400 of SEQ ID NO: 2.
It has a terminal fibrinogen-like domain. (193 score for fibrinogen_C domain profile HMM accession number PF00147). In another embodiment, the FDRG protein has a C-terminal fibrinogen-like domain at about amino acids 188-404 of SEQ ID NO: 2 (xxx.x score). The C-terminal fibrinogen-like domain of human and mouse FDRG, and those of human angiopoietin, mouse angiopoietin-2 and ficolin are shown in bold italics in FIG.

The C-terminal fibrinogen-like domain has at least about 2, more preferably at least 3,
And even more preferably, it may further contain at least four cysteine residues, which are between the FDRG molecule and members of the fibrinogen-like protein family (eg, human angiopoietin 2, mouse angiopoietin 2, human angiopoietin 1 and ficolin). Has been saved. Preferably, the C-terminal fibrinogen-like domain of FDRG has a cysteine residue located at the same or similar position as a cysteine residue in a member of the fibrinogen-like protein family. For example, if an FDRG protein of the invention is aligned with a member of the FDRG family or a member of the fibrinogen-like protein family for comparative purposes (see, eg, FIG. 3), a preferred cysteine residue of the invention will have The cysteine residue in the amino acid sequence of FDRG is
It is located at the same or similar position as the cysteine residue in the family or members of the fibrinogen-like family. As one specific example, FIG. 3 shows that cysteine residues located at the same or similar positions of human FDRG protein (corresponding to SEQ ID NO: 2) and human angiopoietin-2 are changed to the following positions: Amino acid number 188 of human FDRG and amino acid number 284 of human angiopoietin 2 protein; amino acid number 2 of human FDRG
Amino acid No. 313 of human 16 and human angiopoietin 2 protein; human FD
Amino acid number 341 of RG and amino acid number 437 of human angiopoietin 2 protein; and amino acid number 354 of human FDRG and amino acid number 450 of human angiopoietin 2 protein are shown. Figure 3 shows the cysteines that are conserved.
It is indicated by an asterisk in it.

Another aspect of the invention features an FDRG molecule containing a unique domain at the N-terminus. As used herein, the term “N-terminal unique domain” refers to a member of the FDRG protein family that includes the N-terminal amino acid residues of the N-terminal to C-terminal fibrinogen-like domain of the amino acid sequence of the FDRG protein. One protein domain of the member, for example, a protein domain that includes the amino acid residues from the N-terminal amino acid residue of the amino acid sequence of the protein to the N-terminal of the C-terminal fibrinogen-like domain. As used herein, an "N-terminal unique domain" is at least about 170-190 amino acid residues in length and at least about 75-85 amino acid sequences of a second FDRG family member.
% Protein domains with homology. In another embodiment, the unique domain at the N-terminus is at least about 150-210 amino acid residues in length, preferably at least about 160-210 amino acid residues in length, and the amino acid sequence of a second FDRG family member And at least about 65-95%, preferably at least about 70-90%. As further defined herein, the N-terminal unique domain of a member of the FDRG protein family, however, is not sufficiently homologous to the amino acid sequence of another protein family member, such as the angiopoietin protein family. For example, the N-terminal unique domain of human FDRG (containing about amino acids 1-183 of SEQ ID NO: 2) is the lowest relative to the N-terminal unique domain of mouse FDRG (amino acid residues 1-187 of SEQ ID NO: 12). About 8
Has 0% homology, but has no significant homology to the amino acid sequence of human angiopoietin 2.

As further defined herein, the N-terminal unique domain may further contain an “N-terminal signal sequence”. As used herein, "signal sequence" refers to a peptide containing about 25 amino acids, which is located at the extreme N-terminal end of secreted and integral membrane proteins and has numerous hydrophobic Contains amino acid residues. For example, the signal sequence contains at least about 15-35 amino acid residues, preferably about 18-32 amino acid residues, more preferably about 20-30 amino acid residues, and more preferably about 22-28 amino acid residues. And has a minimum of about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (eg, alanine, valine, leucine, isoleucine, phenylalanine or proline). Such "signal sequences", also referred to in the art as "signal peptides", serve to direct proteins containing such sequences to the lipid bilayer.

In one preferred embodiment, the FDRG protein contains both a unique N-terminal domain and a C-terminal fibrinogen-like domain. In another preferred embodiment, the N-terminal unique domain of FDRG further contains a signal sequence.
In one exemplary embodiment, the FDRG protein is amino acids 1-25 of SEQ ID NO: 2.
Contains a unique domain at the N-terminus comprising amino acids 1-183 of SEQ ID NO: 2 further containing a signal sequence. In yet another exemplary embodiment, the FDRG protein contains a unique N-terminal domain comprising amino acids 1-158 of SEQ ID NO: 5. In another exemplary embodiment, the FDRG protein comprises SEQ ID NO: 1
It contains a unique domain at the N-terminus comprising amino acids 1-187 of SEQ ID NO: 12 further containing a signal sequence at amino acids 1-23 of two. In yet another exemplary embodiment, the FDRG protein contains a unique domain at the N-terminus comprising amino acids 1-162 of SEQ ID NO: 15.

[0037] In yet another embodiment, the FDRG protein encodes a mature FDRG protein. The term "mature FDRG protein" as used herein refers to an FDRG protein from which the signal peptide has been cleaved. In one exemplary embodiment, the mature FDRG protein comprises amino acid residues 1- of SEQ ID NO: 5.
381. In another exemplary embodiment, the mature FDRG protein contains amino acid residues 1-387 of SEQ ID NO: 15. In one preferred embodiment, the FDR
G protein is a mature FDR comprising a C-terminal fibrinogen-like domain
G protein.

Preferred FDRG molecules of the present invention have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. As used herein, the term "sufficiently homologous" refers to a first or second amino acid or nucleotide sequence, such that they share a common structural domain and / or a common functional activity. Refers to a first amino acid or nucleotide sequence that contains a sufficient or minimal number of amino acid residues or nucleotides that are identical or equivalent (eg, amino acid residues having similar side chains) to two amino acid or nucleotide sequences. For example, at least about 60-65% homology over the amino acid sequence of the domain, preferably 70-75% homology, more preferably 80%
Share a common structural domain with ８５85%, and more preferably 90-95% homology and at least 1, preferably 2, and more preferably 3 or 4
Amino acid or nucleotide sequences containing two structural domains are defined herein as sufficiently homologous. Furthermore, at least 60-65%, preferably 70-75
%, More preferably 80-85%, and even more preferably 90-95%, amino acid or nucleotide sequences that share a common functional activity are defined herein as sufficiently homologous. .

“FDRG activity”, “FDRG biological activity” or “FDRG functional activity” as used interchangeably herein, may be measured in vivo or in vitro according to standard techniques. In addition, it refers to the activity exerted by FDRG proteins, polypeptides or nucleic acid molecules on FDRG-responsive cells. In one embodiment, the FDRG activity is a direct activity, such as association with a cell surface protein (eg, an FDRG receptor or FDRG binding protein). In another embodiment, the FDRG activity is an indirect activity, such as modulation of the activity of a second protein (eg, a cell signaling molecule) mediated by the interaction of the FDRG protein with a cell surface protein. In a preferred embodiment, the FDRG activity is the following activity: (i) FRD in the extracellular environment with a non-FDRG protein molecule (eg, FDRG receptor) on the surface of the same cell that secreted the FDRG protein molecule.
DRG protein interaction; (ii) FDRG protein interaction in the extracellular environment with non-FDRG protein molecules (eg, FDRG receptor) on the surface of cells different from those that secreted FDRG protein molecules: (iii) FDRG protein (Iv) complex formation between FDRG protein and non-FDRG receptor; (v) modulation of signaling through cell surface proteins (eg, FDRG receptor); (vi) fat (Vii) modulation of PPARγ function or modulation by PPARγ function; (viii) modulation of cellular response to PPARγ ligands (eg, thiazolidinedione) and (ix) a second protein in the extracellular milieu (eg, FDRG binding protein) and F Interaction RG protein is a minimum one or more.

In yet another preferred embodiment, the FDRG activity is the following activity: (i
) Activation of FDRG-dependent signaling pathways; (ii) regulation of angiogenesis; (ii)
(iv) regulation of hematopoiesis; (iv) regulation of proliferation, development or differentiation of FDRG-expressing cells or FDRG receptor-expressing cells (eg, mediating the growth and / or differentiation of adipocytes, such as white or brown adipocytes); v) regulation of proliferation, development or differentiation of non-FDRG expressing cells or FDRG expressing cells; (vi) F
(Vii) regulation of insulin sensitivity and / or insulin responsiveness; (viii) regulation of insulin secretion; (ix) regulation of cell recruitment; (x) regulation of homeostasis of non-FDRG-expressing cells. And (xi) regulation of glucose metabolism.

The present invention relates to a novel secreted protein (amino acid sequence of SEQ ID NO: 2) isolated at least in part from an aortic endothelial cDNA library, highly expressed in placenta and fat, and having homology to angiopoietin. Human FD having
RG protein). The invention further relates, in part, to the amino acid sequence of SEQ ID NO: 12, which was isolated in a screen for the transcription target of the nuclear hormone receptor PPARγ, which can act as a potent regulator of adipogenic transcription factors and systemic insulin action. Based on the discovery of the mouse FDRG protein with

PPARγ exists in two isoforms (γ1 and γ2) formed by alternative splicing (Zhu et al. (1995) PNAS 92:
7921-7925) and acts both as a direct regulator of many fat-specific genes and also as a "master" regulator that can trigger an entire program of adipogenesis (Spiegelman and Flier).
(1996) Cell 87: 377-389). PPARγ forms a heterodimer with RXRα and adipocyte P
Fully characterized fat-specific enhancer from 2 (aP2: Tontonose (
(Tontonoz) (1994) Genes Dev. 8: 1224-1234
) And the phosphoenolpyruvate carboxykinase (PEPCK) gene (
Tontonoz (1994) Mol. Cell. Biol. 1
5: 351-357). The identification of FDRG as a target of PPARγ places it in an important biological context, especially in light of the prominent role played by the latter molecule in adipocyte differentiation and insulin signaling.

Mouse FDRG is highly expressed in brown and white fat and placenta, and expression is induced acutely in cells cultured with PPARγ ligands such as thiazolidinedione (TZD). Mouse FDRG expression is also up-regulated in rodent tissues chronically treated with TZD. Furthermore, FDRG mRNA levels are elevated in obese mouse models, including ob / ob and db / db mice, and are regulated acutely by nutritional intake.

Thus, in another preferred embodiment, the FDRG activity comprises the following activities: (1) maintaining energy homeostasis (eg, regulating the balance and / or imbalance between energy storage and energy consumption, eg, Increase / decrease in energy consumption)
(2) adaptive thermogenetic regulation (eg, regulation of mitochondrial biogenesis;
(3) regulation of mitochondrial enzyme expression, uncoupling protein expression); (3) regulation of obesity; (4) regulation of energy storage efficiency; (5) regulation of appetite; (6) angiogenesis (ie vascular (7) regulation of tumor angiogenesis; (8) regulation of wound healing; (9) tumor mass growth / contraction; and (10) fat mass growth /
Shrinkage, at least one or more.

Further, in another aspect of the invention, the FDRG molecule or preferably FDRG
Alternatively, modulators of the FDRG receptor may include the following diseases or disorders: (1) obesity (eg, excessive storage of energy as fat or a chronic imbalance between energy intake and consumption); (2) obesity (3) diabetes (eg, non-insulin dependent diabetes); (4) impaired energy homeostasis; (5) metabolic abnormalities typical of obesity (eg, high insulin) (6) insulin resistance; (7) disorders associated with lipid metabolism (eg, cachexia); and (8) disorders involving abnormal angiogenesis (eg, epithelial cancer (eg, pancreas, Stomach, liver, secretory glands (eg, adenocarcinoma), bladder, lung, breast, skin (eg, fibromatosis or malignant melanoma), prostate, ovary, carcinoma of the reproductive organs including the cervix and body) Hematopoietic and immune systems in cancer (e.g., leukemias and lymphomas)
Cancers of the central nervous system, brain system and eyes (eg glioma, neuroblastoma and retinoblastoma)
Modulating at least one or more of cancers of the connective tissue, bone, muscle and vasculature, including but not limited to hemangiomas and sarcomas;
Useful for prevention and / or treatment. In another embodiment, the FDRG molecule or preferably a modulator of the FDRG or FDRG receptor is an antidiabetic or an insulin sensitizer. In still another embodiment, the FDRG molecule or preferably a modulator of FDRG or FDRG receptor is an angiogenic factor.

Thus, another aspect of the invention features isolated FDRG proteins and polypeptides that have FDRG activity. Preferred FDRG proteins have a C-terminal fibrinogen-like domain and FDRG activity. In another embodiment, the FDRG protein has a unique domain at the N-terminus, a fibrinogen-like domain at the C-terminus, and FDRG activity. In another preferred embodiment, FDRG
The protein has at least a fibrinogen-like domain at the C-terminus, a unique domain at the N-terminus, FDRG activity, and an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. .

In one particularly preferred embodiment, the FDRG proteins and nucleic acid molecules of the invention are human FDRG molecules. The human FDRG cDNA molecule was obtained from a human aortic endothelial cDNA library as described in Example 1. Isolated human F
The nucleotide sequence of the DRG cDNA and the predicted amino acid sequence of the human FDRG protein are shown in FIG. 1 and in SEQ ID NOs: 1 and 2, respectively. In addition, the nucleotide sequence corresponding to the coding region of the human FDRG cDNA is represented as SEQ ID NO: 3.

The cDNA of human FDRG I shown in SEQ ID NO: 1 has a length of about 1894.
Encodes a protein (SEQ ID NO: 2) that is nucleotides and is approximately 406 amino acid residues in length. The human FDRG protein contains a signal sequence as defined herein, a unique domain at the N-terminus and a fibrinogen-like domain at the C-terminus. The unique domain at the N-terminus of FDRG is at least
For example, it can be found from about amino acids 1-183 of SEQ ID NO: 2 and, for example, about amino acids 1-158 of SEQ ID NO: 5. The C-terminal fibrinogen-like domain of FDRG may be found at least, for example, from about amino acids 184-400 of SEQ ID NO: 2 and, for example, from about amino acids 159-374 of SEQ ID NO: 5. The signal sequence can be found at least, for example, from about amino acids 1-25 of SEQ ID NO: 2. The prediction of such a signal peptide is performed, for example, by using a computer algorithm S
IGNALP (Henrik et al. (1997) Protein E
ngineering 10: 1-6).

[0049] In another particularly preferred embodiment, the FDRG proteins and nucleic acid molecules of the invention are mouse FDRG molecules. The mouse FDRG cDNA molecule was prepared in Example 4
, Obtained from a screen designed to detect nucleic acids regulated by the fat-specific transcription factor PPARγ. The nucleotide sequence of the isolated mouse FDRG cDNA and the predicted amino acid sequence of the mouse FDRG protein are shown in FIG. 4 and in SEQ ID NOs: 11 and 12, respectively. In addition, the nucleotide sequence corresponding to the coding region of the mouse FDRG cDNA is represented as SEQ ID NO: 13.

The cDNA of mouse FDRG1 shown in SEQ ID NO: 11 has a length of about 189
Encodes a protein that is 3 nucleotides and is approximately 410 amino acid residues in length (SEQ ID NO: 12). Murine FDRG protein contains a signal sequence as defined herein, a unique domain at the N-terminus, and a fibrinogen-like domain at the C-terminus. A unique domain at the N-terminus of FDRG can be found at least, for example, from about amino acids 1-187 of SEQ ID NO: 12, and, for example, from about amino acids 1-162 of SEQ ID NO: 15. The fibrinogen-like domain at the C-terminus of FDRG is at least about amino acid 188 of SEQ ID NO: 12, for example.
-404 and, for example, from about amino acids 163-379 of SEQ ID NO: 15. The signal sequence is at least about amino acids 1-
23. Prediction of such signal peptides is performed, for example, by the computer algorithm SIGNALP (Henrik et al. (1997) P
This can be accomplished using protein engineering 10: 1-6).

The expression of FDRG mRNA was particularly high in placenta and fat as determined by Northern blot analysis of various human and mouse tissues (see Examples 2 and 8).

Human FDRG, mouse FDRG, human angiopoietin 2 (Genbank [G
enbank] ™ accession number AF004327), mouse angiopoietin 2 (Genbank ™ accession number AF004326) and ficolin (Genbank accession number L12345).
3) shows the alignment of the amino acid sequences. (This alignment is based on the megualine [MegA
light] ™ sequence alignment software. The first paired alignment step is one K-tuple, three gap penalties (
GAP penalty, window of 5, and diagonals saved set to 5 were performed using Lipman Pearson algorithm. Multiple alignment steps were performed using a PAM250 residue weight table (residue).
weight Table), a cluster with a gap penalty of 10 and a gap length penalty of 10 (GAP length penalty).
Clustal) algorithm).

Various aspects of the invention are described in further detail in the following subsections. That is, I. Isolated nucleic acid molecules One aspect of the invention is an isolated nucleic acid molecule that encodes a FDRG protein or a biologically active portion thereof, as well as nucleic acids that encode FDRG (eg, FDRG mRN
A) nucleic acid fragments sufficient for use as hybridization probes to identify A) and fragments for use as PCR primers for amplification or mutation of FDRG nucleic acid molecules. As used herein, the term "nucleic acid molecule" includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and homologs of DNA or RNA produced using nucleotide homologs. Shall be considered. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid molecule does not include the sequence that naturally flanks the nucleic acid (ie, the sequences located at the 5 ′ and 3 ′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid molecule is derived. . For example, in various embodiments, an isolated FDRG nucleic acid molecule can comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb that naturally sandwiches the nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived. it can. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or culture media when produced by recombinant methods, or other chemicals when synthesized or chemically synthesized. Not included.

A nucleic acid molecule of the invention, for example, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1
1, a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 14, the nucleotide sequence of the DNA insert of the plasmid deposited at the ATCC under accession number 98632, or portions thereof, can be prepared by standard molecular biology techniques and methods described herein. Can be isolated using the sequence information provided in the textbook. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 or all nucleotide sequences of the DNA insert of the plasmid deposited at the ATCC under accession number 98632 as hybridization probes. Alternatively, or in part, FDRG nucleic acid molecules can be synthesized using standard hybridization and cloning techniques (eg, Sambrook, J., French, EF).
., and Maniatis, T., Molecular Cloning: Laboratory Manual (
Molecular Cloning: A Laboratory Manual ), 2nd edition, as described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). .

Further, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13,
A nucleic acid molecule encompassing all or part of the nucleotide sequence of SEQ ID NO: 14, or the DNA insert of the plasmid deposited at the ATCC under accession number 98632, has the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or the ATCC. It can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the nucleotide sequence of the DNA insert of the plasmid deposited under accession number 98632.

The nucleic acids of the invention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification methods. The nucleic acid so amplified is cloned into an appropriate vector, and the DNA
It can be characterized by sequence analysis. In addition, oligonucleotides corresponding to the FDRG nucleotide sequences can be prepared by standard synthetic methods, eg, using an automated DNA synthesizer.

In a preferred embodiment, the isolated nucleic acid analysis of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ ID NO: 1 corresponds to human FDRG cDNA. This c
The DNA comprises the human FDRG protein (ie, the "coding region" from nucleotides 166 to 1386), as well as 5 'untranslated sequences (nucleotides 1-165) and 3' untranslated sequences (
Nucleotides 1387-1894). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO: 1 (nucleotides 166 to 1386).

In another preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 3. The sequence of SEQ ID NO: 3 corresponds to human FDRG cDNA. This cDNA comprises the sequence encoding the human FDRG protein (ie, the "coding sequence" from nucleotides 1-1218 of SEQ ID NO: 3).

[0060] In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 4. The sequence of SEQ ID NO: 4 corresponds to human FDRG cDNA. This cDNA comprises the sequence encoding the mature FDRG protein (ie,
From nucleotides 241-1386 of SEQ ID NO: 1 after the signal sequence has been cleaved).

[0061] In yet another preferred embodiment, the isolated nucleic acid molecule of the present invention is deposited with the ATCC under accession number 98.
632, comprising the nucleotide sequence of the DNA insert of the plasmid deposited.
A plasmid containing the full-length nucleotide sequence encoding FDRG was produced on January 14, 1977.
American type culture collection in Rockville, Maryland (
ATCC) as deposit number 98632.

In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 13. The sequence of SEQ ID NO: 13 is a mouse FDRG cDNA
Corresponding to This cDNA is a mouse FDRG protein (ie, nucleotides 149-137).
8 "coding region"), as well as 5 'untranslated sequences (nucleotides 1-148) and 3' untranslated sequences (nucleotides 1379-1893). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO: 13 (eg, nucleotides 149-1378).

In another preferred embodiment, the isolated nucleic acid molecule of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 13. The sequence of SEQ ID NO: 3 corresponds to mouse FDRG cDNA. This cDNA comprises the sequence encoding the mouse FDRG protein (ie,
"Coding region" from nucleotides 1 to 1230 of SEQ ID NO: 13).

In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 14. The sequence of SEQ ID NO: 14 corresponds to mouse FDRG cDNA. This cDNA comprises the sequence encoding the mature FDRG protein (i.e., from nucleotides 710 to 1380 of SEQ ID NO: 11 after cleavage of the signal sequence).

In another preferred aspect, the isolated nucleic acid molecule of the invention comprises SEQ ID NO: 1, SEQ ID NO: 3
SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, the nucleotide sequence of the DNA insert of the plasmid deposited at the ATCC under accession number 98632, or any portion of such a nucleotide sequence. Comprising a nucleic acid molecule that is the complement. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or complementary to the nucleotide sequence of the DNA insert of the plasmid deposited at the ATCC under accession number 98632. Certain nucleic acid molecules have the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or the nucleotides of the DNA insert of the plasmid deposited with the ATCC under accession number 98632. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1 so that they can hybridize to the sequence and thereby form a stable duplex.
1, which is sufficiently complementary to the nucleotide sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, or the DNA insert of the plasmid deposited with the ATCC under accession number 98632.

In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, ATCC The nucleotide sequence of the DNA insert of the plasmid deposited under accession number 98632, or a portion of any such nucleotide sequence, for at least about 60-65.
%, Preferably at least about 70-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95% or more nucleotide sequence homology. In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, accession number to the ATCC. Nucleotide sequence of the DNA insert of the plasmid deposited at 98632, or a portion of any such nucleotide sequence, at least about 61%, 62%
, 63%, 64%, 65%, 66%, 67%, 68% or 69% homologous, preferably at least about 71
%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% homologous, more preferably at least about 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88% or 89% homologous, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%
, 98% or 99% or more nucleotide sequence homology.

Further, the nucleic acid molecule of the present invention may be a nucleic acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or accession number 9863 at the ATCC.
Only part of the nucleotide sequence of the DNA insert of the plasmid deposited in 2, e.g. FD
It can comprise a fragment that can be used as a probe or primer or fragment that encodes a biologically active portion of RG. Nucleotide sequences determined from the cloning of the human and mouse FDRG genes include, for example, FDRG homologues in other cell types from other tissues, as well as FDRG from other mammals.
It allows the generation of probes and primers designed for use in identifying and / or cloning homologs. The probe / primer typically comprises a substantially pure oligonucleotide. The oligonucleotide is typically a polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or the plasmid deposited at the ATCC under accession number 98632 under stringent conditions. DNA insert sense or SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or accession number 9863 to the ATCC.
Antisense sequence of the DNA insert of the plasmid deposited in step 2, or SEQ ID NO: 1
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or at least about 12 naturally occurring mutants of the DNA insert of the plasmid deposited at the ATCC under accession number 98632, Preferably, it comprises a region of nucleotides that hybridizes to about 25, more preferably about 40, 50 or 75 contiguous nucleotides.

[0068] Probes based on the human FDRG nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In a preferred embodiment, the probes further comprise a label group attached thereto,
For example, the labeling group can be a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. Such probes can be used to detect FD in a sample of cells from an individual.
Misexpressing FDRG protein, such as by measuring the level of nucleic acid encoding RG, for example, by detecting FDRG mRNA levels or determining whether the genomic FDRG gene is mutated or deleted It can be used as part of a diagnostic test kit for identifying cells or tissues.

The nucleic acid fragment encoding the “biologically active part of FDRG” is SEQ ID NO: 1.
FDRG biological activity of the DNA insert of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or the plasmid deposited at the ATCC under accession number 98632 (FD
The biological activity of the RG protein has been described previously), isolating the portion encoding the polypeptide, expressing the encoded portion of the FDRG protein (eg, by recombinant expression in vitro), and removing the portion of the encoded FDRG. It can be prepared by evaluating the activity.

Further, the present invention relates to nucleotides that differ from the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 due to the degeneracy of the gene codons, and the DNA insert of the plasmid deposited with the ATCC under accession number 98632 SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 or encoded by the nucleotide sequence of the DNA insert of the plasmid deposited at the ATCC under accession number 98632 Includes nucleic acid sequences encoding the same FDRG protein. In another aspect, the isolated nucleic acid molecule of the invention encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15.

In addition to the human FDRG nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, respectively, those skilled in the art will appreciate that polymorphisms in the DNA sequence that lead to changes in the amino acid sequence of FDRG are present in the population (eg, the human population). ) May be present. Such genetic polymorphisms in the FDRG gene may be present in individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene”
Refers to a nucleic acid molecule comprising an open reading frame encoding a FDRG protein, preferably a mammalian FDRG protein. Such natural allelic variations can typically result in 1-5% variation in the nucleotide sequence of the FDRG gene. Any that is the result of a natural allelic variation and does not alter the functional activity of FDRG,
And all such nucleotide variations and amino acid polymorphisms that occur in the FDRG are intended to be within the scope of the present invention.

A nucleic acid molecule encoding a FDRG protein from yet another species, and thus the human sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or Plasmid DNA deposited with ATCC under accession number 98632
Those having a nucleotide sequence that differs from the nucleotide sequence of the insert are intended to be within the scope of the present invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the FDRG cDNA of the present invention can be prepared using human cDNA or a portion thereof as a hybridization probe according to standard hybridization methods under stringent hybridization conditions. And can be isolated based on their homology to human FDRG cDNA nucleic acids as disclosed herein. For example, viral FDRG cDNA can be isolated based on its homology to human FDRG.

Thus, in another embodiment, an isolated nucleic acid molecule of the present invention is at least 15 nucleotides in length and under stringent conditions SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, the nucleotide sequence of SEQ ID NO: 14, or AT
Hybridizes to a nucleic acid molecule comprising the nucleotide sequence of the DNA insert of the plasmid deposited at the CC under accession number 98613. In another embodiment, the nucleic acid is at least 30, 50,
It is 100, 250 or 500 nucleotides long. As used herein, the term "hybridizes under stringent conditions" refers to hybridization and washing conditions in which nucleotide sequences that are at least 60% homologous to each other typically remain hybridized to each other. It is intended to explain. Preferably, the conditions are such that sequences that are at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% homologous to each other typically remain hybridized to each other. It is. Such stringent conditions are known to those skilled in the art, and are described in Current Protocols in Molecular Biology.
Molecular Biology ), John Wiley & Sons, NY (
1989), 6.3.1-6.3.6. Suitable non-limiting examples of stringent hybridization conditions include hybridization in 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 0.2 × SS at 50-65 ° C.
C, one or more washes in 0.1% SDS. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule having a naturally occurring nucleotide sequence (encoding a naturally occurring protein).

In addition to the naturally occurring allelic variants of the FDRG sequence that may be present in the population, those skilled in the art may further deposit the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or plasmid deposited at the ATCC under accession number 98632 Mutations to the nucleotide sequence of the DNA insert will introduce changes that could lead to changes in the amino acid sequence of the encoded FDRG protein without altering the functional capacity of the FDRG protein. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues are:
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or the sequence of the DNA insert of the plasmid deposited at the ATCC under accession number 98632. “Non-essential” amino acid residues are those that do not alter the biological activity of the wild-type sequence of FDRG (eg, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or 1).
5), while “essential” amino acid residues are required for biological activity. For example, amino acid residues conserved between the FDRG proteins of the present invention are not expected to be particularly easily modified. In addition, conserved amino acid residues between the FDRG protein and other proteins with a fibrinogen-like domain do not appear to be amenable to modification.

Thus, another aspect of the invention pertains to nucleic acid molecules encoding FDRG proteins that contain changes in amino acid residues that are not essential for activity. Such FDRG proteins differ from the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 13 or SEQ ID NO: 15, but retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, wherein the protein comprises an amino acid sequence that is at least about 60-65% homologous to the amino acid sequence of SEQ ID NO: 2. Preferably, the protein encoded by the nucleic acid molecule is SEQ ID NO: 2,
At least about 70-75% homologous to SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15, more preferably at least about 80-85% homologous to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15 And even more preferably is at least about 90-95% homologous to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15.

In another aspect, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, wherein the protein has at least about 61 nucleotides in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, or SEQ ID NO: 15. %, 62%, 63%, 64%, 65%, 66%, 67%, 68%
Or 69% homologous, preferably at least about 71%, 72%, 73%, 74%, 75%, 76%
, 77%, 78% or 79% homologous, more preferably at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88% or 89% homologous, and even more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% % Or 99% or more homologous.

An isolated nucleic acid molecule encoding a FDRG protein that is homologous to the protein of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15 encodes one or more amino acid substitutions, additions or deletions A plasmid deposited with one or more nucleotide substitutions, additions or deletions in the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, or with the ATCC under accession number 98632, for introduction into the inserted protein. By introducing the DNA into the nucleotide sequence of the DNA insert. Mutations were made by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 1.
1, SEQ ID NO: 13, SEQ ID NO: 14 or the DNA insert of the plasmid deposited at the ATCC under accession number 98632. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Such families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine,
Tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg,
For example, amino acids having tyrosine, phenylalanine, tryptophan, histidine) are included. Thus, a predicted nonessential amino acid residue in a FDRG is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutation is FDR, such as in a saturation mutagenesis method.
Mutants can be introduced randomly along all or part of the G coding sequence, and the resulting mutants can be screened for FDRG biological activity to identify those that retain activity. Following mutagenesis of the DNA insert of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 or the plasmid deposited at the ATCC under accession number 98632, The protein can be expressed recombinantly, and the activity of the protein can be measured.

In a preferred embodiment, the mutant FDRG protein comprises (1) activation of the FDRG-dependent signaling circuit; (2) regulation of secretion of non-IL-17 cytokines; (4) regulation of surface expression of cell adhesion molecules; (5) regulation of proinflammatory cytokines; (6) regulation of hematopoietic cytokines; (7) regulation of development or differentiation of FDRG-expressing cells; (8) non-regulation. -Regulation of the development or differentiation of FDRG-expressing cells; (9) regulation of homeostasis of FDRG-expressing cells; (10) regulation of homeostasis of non-FDRG-expressing cells; (11) regulation of adipocyte development and physiology; (12) It can be assayed for modulation of insulin signaling.

In addition to the nucleic acid molecules encoding the FDRG proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. An “antisense” nucleic acid includes a nucleotide sequence that is complementary to a “sense” nucleic acid that encodes a protein, for example, a sequence that is complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Become. Thus, an antisense nucleic acid can hydrogen bond with a sense nucleic acid. The antisense nucleic acid can be complementary to the entire FDRG coding strand or only a portion thereof. In one embodiment, the antisense nucleic acid molecule is FD
Antisense to the "coding region" of the coding strand of the nucleotide sequence encoding RG. The term "coding region" refers to a region of a nucleotide sequence comprising codons translated into amino acid residues (eg, the coding region of human FDRG corresponding to SEQ ID NO: 3). In another embodiment, the antisense nucleic acid molecule is antisense to a "non-coding region" of the coding strand of a nucleotide sequence encoding FDRG. The term "non-coding region" refers to the 5 'and 3' flanking a coding region that is not translated into amino acids.
The sequences are referred to (ie, also referred to as the 5 'and 3' untranslated regions).

In view of the coding strand sequence encoding FDRG disclosed herein (eg, SEQ ID NO: 3 or SEQ ID NO: 13), the antisense nucleic acids of the invention should be designed according to the Watson and Crick base pairing rules. Can be. The antisense nucleic acid molecule can be complementary to the entire coding region of the FDRG mRNA, but more preferably the FDR
Oligonucleotides that are antisense to only a portion of the coding or non-coding region of G mRNA. For example, antisense oligonucleotides are FDRG mRNA
Can be complementary to the region surrounding the translation initiation site of Antisense oligonucleotides are, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. The antisense nucleic acids of the invention can be constructed using chemical synthesis or enzymatic ligation using procedures known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) are naturally-occurring nucleotides or double-stranded physical forms formed between antisense and sense nucleic acids to increase the biological stability of the molecule. Can be chemically synthesized using a variety of modified nucleotides designed to increase stability, for example, phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides that can be used to generate antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxy (Hydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylcuosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1- Methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino Methyl-2-thiouracil, beta-D-mannosylcuosin, 5'-methoxycal Carboxymethyl uracil, 5-methoxy uracil, 2-methylthio -N6- isopentenyladenine, uracil-5-oxyacetic acid (v),
Witoxosin, pseudouracil, cueosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-
Includes thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, antisense nucleic acids can be produced biologically using expression vectors in which the nucleic acid has been subcloned in the antisense direction (ie, as described in further detail in the subsections below).
RNA transcribed from the inserted nucleic acid is in the antisense orientation with respect to the target nucleic acid of interest).

The antisense nucleic acid molecules of the invention hybridize or bind to cellular mRNA and / or genomic DNA encoding the FDRG protein, thereby repressing protein expression, eg, repressing transcription and / or translation. And is typically administered to an individual or produced in situ. Hybridization can form a stable duplex by conventional nucleotide complementarity, or, for example, in the case of an antisense nucleic acid molecule that binds to a DNA duplex,
This can be through the specific interaction of the major groove of the double-stranded helix. Examples of routes of administration of the antisense nucleic acid molecules of the invention include direct injection into a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules
For example, an antisense nucleic acid molecule can be modified to specifically bind to a receptor or antigen expressed on a selected cell surface by linking it to a peptide or antibody that binds to a cell surface receptor or antigen. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve a sufficient intracellular concentration of the antisense molecule, a vector construct in which the antisense nucleic acid molecule is placed under the control of a strong polII or polIII promoter is preferred.

[0082] In yet another aspect, the antisense nucleic acid molecules of the invention are α-anomeric nucleic acid molecules. α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA, where the strands run parallel to each other (Gaultier, in contrast to the usual β-units).
et al. (1987) Nucleic Acids Res. 15: 6625-6641). Also, antisense nucleic acid molecules
2'-o-methyl ribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15: 613
1-6148) or a chimeric RNA-DNA homolog (Inoue et al., (1987) FEBS Lett. 215: 32) .
7-330).

[0083] In yet another aspect, the antisense nucleic acids of the invention are ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave single-stranded nucleic acids such as mRNA into those with complementary regions. Thus, ribozymes (for example, hammerhead ribozymes (Haselhof and Gerlach (1988) Nature 344:
585) as described in 585-591) can be used to catalytically cleave FDRG mRNA transcripts and thereby suppress translation of FDRG mRNA. A ribozyme having specificity for a nucleic acid encoding FDRG is the nucleotide sequence of the FDRG cDNA disclosed herein (ie, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11).
, SEQ ID NO: 13, SEQ ID NO: 14, or the DNA insert of the plasmid deposited at the ATCC under accession number 98632). For example, Tetrahymena L
Derivatives of -19IVS RNA can be constructed such that the nucleotide sequence of the active site is complementary to the nucleotide sequence that is cleaved in the mRNA encoding FDRG. See, for example, Cech et al., US Pat. No. 4,987,071; and Cechet al., US Pat.
See, for example, U.S. Pat. Alternatively, FDRG mRNA can be used to select catalytic RNA with specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel, D. and Szostak, JW (1993) Science 261: 1411-1418.

Alternatively, FDRG gene expression is suppressed by a target nucleotide sequence complementary to a regulatory region of FDRG (eg, FDRG5 promoter and / or enhancer)
It can form a triple helix structure that prevents transcription of the FDRG gene in target cells. Helence, C. (1991) Anticancer Drug Des. 6 (6): 569-84; Helence, C
et al., (1992) Ann, NY Acad . Sci. 660: 27-36; and Maher. LJ (1992) Bioass.
See ay 14 (12): 807-15.

In a preferred embodiment, the nucleic acid of the FDRG can be modified at the base moiety, sugar moiety or phosphate backbone to improve, for example, the stability or solubility of the molecule, hybridization. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (Hyrup B. et al., (1996) Bioorganic & Medical Chemistr ).
y 4 (1): 5-23). As used herein, the term "peptide nucleic acid" or "PN
"A" refers to a nucleic acid mimic, such as a DNA mimic, where the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four nucleobases are retained. The neutral backbone of the PNA is designed for DNA and
It was shown that it can specifically hybridize to RNA. The synthesis of PNA oligomers
Hyrup B. et al., (1996) supra ; Perry-O'Keefe et al., PNAS 93: 14670-675.

The FDRG PNA can be used for therapeutic and diagnostic applications. For example, PNA
Antisense or antigenic agents for sequence-specific regulation of gene expression, for example by introducing transcription or translational blockade or by inhibiting replication (
antigene agent). PNAs of FDRG include, for example, PNA-specific P
For analysis of single base pair mutations in genes by CR clamping; as an artificial restriction enzyme when used in combination with other enzymes such as S1 nuclease (
Hyrup B. (1996) supra); or can be used as a probe or primer for DNA sequence and hybridization (Hyrup B. (1996) supra;
y-O'Keefe Same as above).

In another embodiment, the PNAs of FDRG can be modified by attaching a lipophilic or other helper group to the PNA, eg, to enhance their stability or cellular uptake.
It can be modified by forming NA chimeras or by the use of liposomes or drug delivery techniques known in the art. For example, a PNA-DNA chimera of FDRG that can combine the advantageous properties of PNA and DNA can be generated.
Such chimeras are those in which DNA recognition enzymes (e.g., RNAseH and DNA polymerase)
While allowing to interact with the moiety, the PNA moiety will provide high binding affinity and specificity. PNA-DNA chimeras can be ligated using appropriate length linkers selected for base stacking, number of bonds between nucleotides and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras is described in Hyrup B. (1996).
) Ditto and as described in Finn PJ et al., (1996) Nucleic Acids Research 24 (17): 3357-63. For example, a DNA strand can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and is a modified nucleoside homolog, such as 5 ′-(4-methoxytrityl) amino-5′- Deoxy-thymidine phosphoramidite can be used between the PNA and the 5 'end of the DNA (Mag, M. et al., (1989) Nucleic Acids Res. 17: 5973-88). The PNA monomers are then coupled in a stepwise fashion to generate a chimeric molecule with a 5 'DNA segment and a 3' PNA segment (Finn PJ et al., (1996) ibid). Alternatively, chimeric molecules can be synthesized using 5 'PNA and 3' DNA segments (Pe
terser. KH et al., (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

In another embodiment, the oligonucleotides may have other associated groups, such as peptides (eg, to target host cell receptors in vivo), agents that can be transported across the cell membrane (eg, For example, Letsinger et al., 1989, Proc. Natl.
Acad.Sci.USA.86: 6553-6556; Lemaitre et al., Proc.Natl.Acad.Sci.84: 648-652
See WO 88/09810 published December 15, 1988)
Or a blood-brain barrier (eg, WO 89/1013 published April 25, 1988)
No. 4). In addition, oligonucleotides can be used with cleavage agents that are triggered by hybridization (eg, Krol et al., BioT
echniques 6: 958-976) or intercalating agents (e.g., Zon, 1
988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, for example, a peptide, a hybridization-triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like. II. Isolated FDRG proteins and anti-FDRG antibodies One aspect of the present invention relates to isolated FDRG proteins and biologically active portions thereof, and use as immunogens to generate anti-FDRG antibodies Polypeptide fragments suitable for In one embodiment, the native FDRG protein can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification methods. In another embodiment, the FDRG protein is produced by a recombinant DNA method. Instead of recombinant expression, the FDRG protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the FDRG protein is derived. Or is substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of FDRG proteins in which the protein has been isolated or the protein has been separated from the cellular components of a recombinantly produced cell. In one embodiment, the term "substantially free of cellular material" refers to less than about 30% (by dry weight) of non-FDRG protein (also referred to herein as "contaminating protein").
, More preferably less than about 20% non-FDRG protein, even more preferably about 10%
% Of non-FDRG proteins, and most preferably less than about 5% of non-FDRG proteins. When the FDRG proteins or biologically active portions thereof are produced recombinantly, they are preferably free of culture medium, ie, the culture medium is less than about 20% of the protein preparation, more preferably less than about 10%, and Most preferably, it represents less than about 5% capacity.

The term “substantially free of chemical precursors or other chemicals” refers to the preparation of FDRG proteins in which the protein is separated from the chemical precursors or other chemicals involved in the synthesis of the protein. including. In one embodiment, the term "substantially free of chemical precursors or other chemicals" refers to less than about 30% (by dry weight) of chemical precursors or non-FDRG chemicals, more preferably Less than about 20% chemical precursor or non-FDRG chemical, even more preferably less than about 10% chemical precursor or non-FDRG chemical, and most preferably less than about 5% chemical precursor or Non-FD
Includes preparation of FDRG protein with RG chemicals.

The biologically active portion of the FDRG protein is sufficiently homologous to or derived from the amino acid sequence of the FDRG protein, eg, SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 12 or SEQ ID NO: 15. Wherein the peptide comprises fewer amino acids than the full length FDRG protein and exhibits at least one activity of the FDRG protein. Typically, the biologically active portion comprises at least one active domain or motif of the FDRG protein. Biologically active portions of the FDRG protein include, for example,
The polypeptide can be 10, 25, 50, 100 or more amino acids in length.

[0092] In one embodiment, the biologically active portion of the FDRG protein comprises at least one C-terminal fibrinogen-like domain. In yet another aspect, the FDRG
The biologically active portion of the protein comprises at least one signal sequence. In another embodiment, the biologically active portion of the FDRG protein is an FDRG lacking a signal sequence.
Comprising the RG amino acid sequence.

It is understood that suitable biologically active portions of the FDRG proteins of the present invention can include at least one of the above-identified structural domains. More preferred biologically active portions of a FDRG protein can include at least two of the above-identified structural domains. An even more preferred biologically active portion of a FDRG protein can comprise at least three or more of the above-identified structural domains.

Still other biologically active portions (where other regions of the protein have been deleted) can be prepared by recombinant methods and the functional activity of one or more native FDRG proteins Can be evaluated.

[0095] In a preferred embodiment, the FDRG protein has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. In another embodiment, the FDRG protein is substantially homologous to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15, and of the protein of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15. It retains functional activity, but differs in amino acid sequence due to natural allelic variation or mutation, as described in detail in Section I above. Thus, in another embodiment, the FDRG protein is at least about 60% homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15, and is SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 12. No. 15 retains the functional activity of the FDRG protein. Preferably, the protein is SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12
Or at least about 70% homologous to SEQ ID NO: 15, and more preferably SEQ ID NO: 2,
At least about 80% homologous to SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15, even more preferably at least about 90% homologous to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15, and most preferably Are SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 1
2 or SEQ ID NO: 15 is at least about 95% homologous.

To determine the percent homology of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (eg, for optimal alignment with a second amino acid or nucleic acid sequence, Gaps can be introduced in the sequence of the first amino acid or nucleic acid sequence, and non-homologous sequences can be ignored for comparative purposes). In a preferred embodiment, the length of the reference sequence aligned for comparative purposes is at least
30%, preferably at least 40%, more preferably at least 50%. Even more preferably, is at least 60%, and even more preferably at least 70%, 80% or 90% of the reference sequence (eg, the second sequence is a FDRG amino acid sequence of SEQ ID NO: 2 having 86 amino acid residues). Align at least 66, preferably at least 46, more preferably at least 26 amino acid residues). Next, amino acid residues or nucleotides corresponding to the amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are homologous at that position (ie, as used herein, the amino acid Or "homology" of a nucleic acid is equivalent to "identity" of an amino acid or nucleic acid). The percent homology between two sequences is a function of the number of identical positions shared by the sequences (ie,% homology = # of identical positions / total # of positions x 100).

The comparison of sequences and the percent homology between two sequences can be performed using mathematical methods. Non-limiting examples of suitable calculations used for sequence comparisons are described in Karlin and Altschul (1993), Karlin and Altschul (1990), modified by Proc. Natl. Acad. Sci. USA 90.5873-77, Proc.Natl.Acad.Sci.USA 87.2264-68. Such a calculation method is described in Altschul et al., (1990) J. Mol. Biol., 215: 4.
03-10 are included in the NBLAST and XBLAST programs (version 2.0). BLA
ST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the FDRG nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the TAP-1 protein molecule of the present invention. Gapped to obtain gapped sequences for comparison
BLAST was performed using Altschul et al., (1997) Nucleic Acids Research 25 (17), 3389-3402.
Can be used as described in When using BLAST and Gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used. http: //www.ncbi.nlm.nih.go
See v. A non-limiting example of another suitable mathematical calculation utilized for sequence comparison is the formula of Myers and Miller, CABIOS (1989). Such calculations are included in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Or PAM250 residue weight table, 10 GAP
A penalty, and a GAP length penalty of 10 can be used. Another suitable, non-limiting example of a mathematical calculation utilized for sequence comparison is the Wilbur-Lipman calculation, which is part of the MegAlign ™ sequence alignment software. Wilbur-Li
When using the pman algorithm, a K-tuple of 1, a GAP penalty of 3, a window of 5, and a term set to 5 can be used. Many alignments can be performed using the Clustal algorithm.

[0098] The present invention also provides FDRG chimeric or fusion proteins. As used herein, FDRG "chimeric protein" or "fusion protein" refers to non-FDRG
Comprising an FDRG polypeptide operably linked to the polypeptide. `` FDRG polypeptide '' refers to a polypeptide having an amino acid sequence corresponding to FDRG, while `` non-FDRG polypeptide '' is a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to FDRG protein, For example, a protein different from the FDRG protein and derived from the same or a different organism. Within the scope of the FDRG fusion protein, the FDRG polypeptide can correspond to all or a portion of the FDRG protein. In a preferred embodiment, the FDRG fusion protein comprises at least one biologically active portion of a FDRG protein. In another preferred embodiment, the FDRG fusion protein comprises at least two biologically active portions of the FDRG protein. In another preferred embodiment, the FDRG fusion protein comprises at least three biologically active portions of the FDRG protein. Within the scope of the fusion protein,
The term "operably linked" is intended to indicate that the FDRG polypeptide and the non-FDRG polypeptide are fused together in frame. Non-FDRG polypeptides can be fused to the N-terminus or C-terminus of the FDRG polypeptide.

For example, in one embodiment, the fusion protein is a GST-FDRG fusion protein, wherein the FDRG sequence is fused to the C-terminus of the GST sequence. Such a fusion protein can facilitate the purification of the recombinant FDRG.

In another embodiment, the fusion protein is a FDRG protein that contains a heterologous signal sequence at its N-terminus. For example, the native FDRG signal sequence (ie, SEQ ID NO: 2)
About amino acids 1-25) can be removed and replaced with a signal sequence from another protein. In certain host cells (eg, mammalian host cells), FD
RG expression and / or secretion can be increased through the use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a FDRG-immunoglobulin fusion protein, wherein the FDRG sequence comprising predominantly the FDRG-extracellular domain is a sequence derived from a member of the immunoglobulin protein family. Will be fused. Soluble derivatives have also been made from cell surface glycoproteins in the immunoglobulin gene superfamily consisting of the extracellular domain of cell surface glycoproteins fused to immunoglobulin constant (Fc) regions (see, eg, Capon, DJ, et al. , (1989) Nature 337 : 525-53
1 and Capon U.S. Patent Nos. 5,116,964 and 5,428,130 [CD4-IgG1
Constructs]; Linsley, PSet al., (1991) J. Exp . Med . 173 , 721-730 [CD28-IgG1 and B7-1-IgG1 constructs]; and Linsley, PSet al., (1991) J. Exp. .Med . 174 :
561-569 and US Pat. No. 5,434,131 [CTLA4-IgG1]).
Such fusion proteins have been shown to be useful for modulating receptor-ligand interactions. Soluble derivatives of the cell surface proteins of the tumor necrosis factor receptor (TNFR) superfamily proteins have been made from the extracellular domain of the cell surface receptor fused to the immunoglobulin constant (Fc) region (see, eg, Moreland et al., (1997) N. Engl. J. Med 337 (3): 141-147; van der Po
ll et al., (1997) Blood 89 (10); 3727-3734; and Ammann et al., (1997) J. Cl.
in. Invest. 99 (7) 1699-1703). In addition, fusion proteins were made using the CH2 and CH3 domains of IgG fused downstream of the mouse IL-17 leader sequence and upstream of the mouse CTLA-8 sequence and upstream of the HVS13 sequence (eg, Y
ao et al., (1995) Immunity 8; 811-821).

The FDRG-immunoglobulin fusion protein of the present invention is included in a pharmaceutical composition and is administered to an individual to inhibit the interaction between the FDRG protein and the FDRG receptor on the cell surface, thereby administering to the individual Mediated cell function in vivo. An FDRG-immunoglobulin fusion protein can be used to affect the bioavailability of the FDRG protein. Inhibition of FDRG protein / FDRG receptor interaction can be, for example, regulation of cellular immune response, regulation of inflammation, regulation of hematopoiesis,
It may be therapeutically useful in regulating adipogenesis, suppressing angiogenesis. Further according to the invention
FDRG-immunoglobulin fusion proteins are used as immunogens to generate anti-FDRG antibodies in individuals, to purify FDRG receptors, and to combine FDRG proteins with FDRG
It can be used in screening assays to identify molecules that inhibit interaction with the receptor.

[0103] Preferably, the FDRG chimeric or fusion protein of the present invention comprises standard recombinant DNA.
Generated by the method. For example, DNA fragments encoding various polypeptide sequences can be blunted or cohesive terminated, for example, for ligation, restriction enzyme digestion to provide suitable termini, filling-in of suitable cohesive ends. in)
Ligated together in a frame according to customary techniques by using alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including an automated DNA synthesizer. Alternatively, PCR amplification of a gene fragment can be performed using an anchor primer that creates a complementary overhang between two consecutive gene fragments, which is subsequently annealed and re-amplified to yield a chimeric gene sequence. (Eg, the latest protocol in molecular biology ( Current Prot
ocols in Molecular Biology ), edited by Ausubel et al., John Willy and
Sands: 1992). In addition, many expression vectors are commercially available that already encode a fusion moiety (eg, a GST polypeptide). Fusion part is FDRG
The nucleic acid encoding FDRG can be cloned into such an expression vector so as to link in frame with the protein.

The present invention also relates to variants of the FDRG protein that function as either FDRG agonists (mimics) or FDRG antagonists. Mutants of the FDRG protein can be obtained by mutagenesis, for example, discrete point mutations of the FDRG protein.
mutation) or truncation.
An agonist of the FDRG protein can retain substantially the same or a subset of the biological activities of the naturally occurring form of the FDRG protein. An antagonist of the FDRG protein can inhibit one or more activities of a naturally occurring form of the FDRG protein, for example, by competitively binding to the FDRG receptor of the FDRG-binding protein. By treating with the mutant having such a limited function, a special biological effect can be exhibited. In one embodiment, treatment of an individual with a variant having a subset of the biological activity of the naturally occurring protein form has fewer side effects in the individual as compared to treatment with the naturally occurring FDRG protein form.

In one embodiment, a variant of the FDRG protein that functions as either a FDRG agonist (mock) or an FDRG antagonist is a FDRG protein antagonist or mutant of the FDRG protein with respect to antagonist activity (eg, a truncation mutant) Can be identified by screening a combination library. In one embodiment, the library that gave the FDRG variant alterations is a combination mutagenesis method at the nucleic acid level.
s) and encoded by a modified gene library. Libraries that have given FDRG variant changes may have the degenerate set of possible FDRG sequences as individual polypeptides or as a larger set of fusion proteins containing the set of FDRG sequences therein (eg, for protein display phage). To be expressed)
For example, it can be made by enzymatically linking a mixture of synthetic oligonucleotides to a gene sequence. Various methods can be used to generate a library of possible FDRG variants from the degenerate oligonucleotide sequence. Chemical synthesis of the degenerate gene sequence is performed on a fully automatic DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector. The use of a degenerate set of genes makes it possible to prepare in a mixture all sequences encoding the desired set of possible FDRG sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (eg,
Narang, SA (1983) Tetrahedron 39: 3; Itakura et al., (1984) Annu . Rev. Bioch
em .53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al., (1983) Nuc
See leic Acids Res 11.477.

[0106] In addition, libraries of fragments of the FDRG protein coding sequence can be used to generate altered populations of FDRG fragments for screening and subsequent selection of FDRG protein variants. In one embodiment, the library of coding sequence fragments is prepared by treating a double-stranded PCR fragment of the FDRG coding sequence with a nuclease under conditions that result in only about one nick per molecule. Denature, regenerate DNA and sense from various nick products
/ Forming a double-stranded DNA capable of containing an antisense pair, removing a single-stranded portion from the double-stranded DNA re-formed by treatment with S1 nuclease, and adding the resulting fragment library to an expression vector. It can be generated by linking. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the FDRG protein.

The gene products of the combinatorial library created by point mutation or truncation are screened and the gene products having the selected properties are screened.
Several techniques are known in the art for screening cDNA libraries. Such techniques can be applied to rapid screening of gene libraries created by combinatorial mutagenesis of FDRG proteins. For screening large gene libraries, the most widely used techniques that can accommodate high-throughput analysis are typically cloning of gene libraries into replicable expression vectors, library of generated vectors And the expression of the combined gene under conditions in which the desired activity is detected which facilitates isolation of the vector encoding the gene for which the product was detected. Recrusive ensemble mutation, a new technology that enhances the frequency of functional mutants in libraries
agenesis: REM) can be used in combination with screening assays to identify FDRG variants (Arkin and Yourvan (1992) PNAS 89: 7811-7815; Delgr
ave et al., (1993) Protein Engineering 6 (3) 327-331).

In one embodiment, a cell-based assay can be utilized to analyze the altered FDRG library. For example, a library of expression vectors can be transfected into a cell line that normally secretes FDRG protein. The supernatant from the transfected cells is then contacted with FDRG-reacting cells and FD
The effect of the mutant in RG can be detected, for example, by measuring the number of responses of FDRG-reactive cells. The plasmid DNA is then used for suppression or F
Mutant FDRG-secreting cells scored for enhanced DRG-dependent response can be recovered and individual clones can be further characterized.

The isolated FDRG proteins, or portions or fragments thereof, can be purified using standard techniques for preparing polyclonal and monoclonal antibodies.
Can be used as an immunogen to generate antibodies that bind to. The full length FDRG protein can be used, or the invention can be used for FDRG for use as an immunogen.
The present invention provides an antigenic peptide fragment of The antigenic peptide of FDRG was identified as SEQ ID NO: 2 such that antibodies raised against the peptide formed specific immune complexes with FDRG.
, Comprising at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15, and encompasses the epitope of FDRG. Preferably, the antigenic peptide contains at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Consists of

[0110] FDRG immunogens are typically used to prepare antibodies by immunizing a suitable individual (eg, a rabbit, goat, mouse or other mammal) with the immunogen. Suitable immunogenic preparations can include, for example, recombinantly expressed FDRG proteins or chemically synthesized FDRG polypeptides. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant or a similar immunostimulant. Immunization of a suitable individual with an immunogenic FDRG preparation induces a polyclonal anti-FDRG antibody response.

Accordingly, another aspect of the present invention relates to anti-FDRG antibodies. As used herein, the term “antibody” refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, ie, an antigen that specifically binds (immunoreactive) to an antigen such as FDRG. Refers to a molecule that contains a binding site. Examples of immunologically active portions of an immunoglobulin molecule include F (ab) and F (ab ') ₂ fragments that can be generated by treating an antibody with an enzyme such as pepsin. The present invention provides polyclonal and monoclonal antibodies that bind to FDRG. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to FD
Refers to a population of antibody molecules that contains only one of the antigen binding sites capable of immunoreacting with a particular epitope of RG. A monoclonal antibody composition thus typically exhibits one binding affinity for a particular FDRG protein with which it immunoreacts.

[0112] Polyclonal anti-FDRG antibodies can be prepared by immunizing a suitable individual with an FDRG immunogen as described above. Anti-FDRG antibody titers in immunized individuals can be monitored over time by standard techniques, such as using an enzyme-linked immunosorbent assay (ELISA) using immobilized FDRG. FDRG, if desired
Antibody molecules directed to are isolated from mammals (eg, from blood) and can be further purified by well-known techniques, such as protein A chromatography to obtain an IgG fraction. At an appropriate time after immunization (eg, when anti-FDRG antibody titers are at their highest), antibody-producing cells are obtained from the individual, and first obtained by Kohler and Milstei.
n (1975) Nature 256: 495-497 (also Brown et al., (1981) J.Immumol.127 : 539-4
6; Brown et al., (1980) J. Biol . Chem. 255: 4980-83; Yeh et al., (1976) PNAS
76: 2927-31; and also Yeh et al., (1982) Int . J. Cancer 29: 269-75), and more recently the human B cell hybridoma method (Kozbor et al. ., (1983) Immumol Today 4:72), EBV-hybridoma method (
Cole et al., (1985) Monoclonal Antibodi
es and Cancer Threapy ), Alan R. Liss, Inc., pp. 77-96), or can be used to prepare monoclonal antibodies by standard techniques such as the trioma method. Techniques for generating monoclonal antibody hybridomas are well known (in general, RHKenneth, Monoclonal Antibodies : A New Dimension In Biolog.
ical Analysis .) Plenum Publishing Corp., New York, New York (1980); EALerner (1981) Yale J. Biol. Med. , 54: 387-402; MLGeft.
er et al. (1977) Somatic Cell Genet 3: 231-36). Briefly, an immortalized cell line (typically myeloma) is fused with lymphocytes (typically spleen cells) from a mammal immunized with an FDRG immunogen as described above, and The resulting culture supernatant of the hybridoma cells is screened to identify a monoclonal antibody produced by the hybridoma that binds to FDRG.

[0113] Any of the many well-known protocols used to fuse lymphocytes with immortalized cell lines can be applied for the purpose of generating anti-FDRG monoclonal antibodies (eg, G. Galfre et al. , (1977) Nature 266: 55052; Gafter et al., Somat
ic Cell Genet . id .; Lerner, Yale J. Biol. Med. , supra; Kenneth, Monoclonal Antibodies , supra.). More ordinary technicians will envision many modifications of such methods that will be useful. Typically,
Immortalized cell lines (eg, myeloma cell lines) are derived from the same mammalian species as lymphocytes. For example, mouse hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the invention with an immortalized mouse cell line. Suitable immortalized cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any number of myeloma cell lines can be used as fusion partners according to standard techniques, e.g., P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653
Or Sp2 / O-Ag14 myeloma. These myelomas are available from the ATCC. Typically, HAT-sensitive mouse myeloma cells are fused with mouse spleen cells using polyethylene glycol ("PEG"). The hybridoma cells generated from the fusion then kill the unfused and non-productively fused myeloma cells (the unfused spleen cells die after a few days because they are not transformed).
Select using HAT medium. Hybridoma cells producing the monoclonal antibodies of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind to FDRG, eg, using a standard ELISA assay.

Instead of preparing a hybridoma that secretes a monoclonal antibody, one can identify a monoclonal anti-FDRG antibody and screen a recombinant combinatorial immunoglobulin library (eg, an antibody protein display phage library) using FDRG. Isolation, whereby members of the immunoglobulin library that bind to FDRG can be isolated. Kits for generating and screening protein display phage libraries are commercially available (eg, the Pharmacia Recombinant Phage Antib
ody System ), Catalog No. 27-9400-1; and Stratagene Sur
fZAP ™ protein display kit ( Phage Display Kit ), catalog No. 240612).
Examples of methods and reagents that can be particularly used to generate and screen antibody protein display phage libraries are also described, for example, in Lander et al., US Pat. No. 5,223,409; Kang et al., International Publication No. No. 18619; Dowe
r et al., WO 91/17271; Winter et al., WO 92/20791.
Markland et al., WO 92/15679; Breitling et al.
WO 93/01288; McCafferty et al., WO 92/01047; Garrard et al., WO 92/09690; Ladner et al., WO 92. / 02809; Fuchs et al., (1991) Bio / Technology 9: 1370-1372; H
ay et al., (1992) Hum . Antibod . Hybridomas 3: 81-85; Huse et al., (1989) Sc
ience 246: 1275-1281; Griffiths et al., (1993) EMBO J 12: 725-734; Hawkins
et al., (1992) J. Mol. Biol. 226: 889-896; Clarkson et al., (1991) Nature 35.
2: 624-628; Gram et al., (1992) PNAS 89: 3576-3580; Garrad et al., (1991) Bi
o / Technology 9: 1373-1377; Hoogenboom et al., (1991) Nuc. Acid Res. 19: 4133
-4137; Barbas et al., (1991) PNA S 88: 7978-7982; and McCafferty et al., N
ature (1990) 348: 552-554.

Recombinant anti-FDRG antibodies, such as chimeric and humanized monoclonal antibodies, further comprising both human and non-human portions, and which can be made by standard recombinant DNA techniques, are within the scope of the present invention. It is. Such chimeric and humanized monoclonal antibodies can be obtained using recombinant DNA techniques known in the art, e.g., Robinson et al.,
International Application No. PCT / US86 / 02269 No .; Akira et al., European Patent Application Publication No. 18
4,187; Taniguchi, M., EP 171,496; Morrison
et al., EP 173,494; Neuberger et al., International Publication No.
86/01533; Cabilly et al., US Pat. No. 4,816,567;
t al., EP 125,023; Better et al., (1988) Science
240: 1041-1043; Liu et al., (1987) PNAS 84: 3439-3443; Liu et al., (1987)
J.Immumol 139:. 3521-3526; Sun et al, (1987) PNAS 84:. 214-218; Nishimura
et al., (1987) Canc. Res. 47: 999-1005; Wood et al., (1985) Nature 314: 446-.
449; and Shaw et al., (1988) J. Natl . Cancer Inst. 80: 1533-1559); Morriso.
n, SL (1985) Science 229: 1202-1207; Oi et al., (1986) BioTechniques 4:21.
4; Winter, U.S. Patent No. 5,225,539; Jones et al., (1986) Nature 321:
552-525; Verhoeyan et al., (1988) Science 239: 1534; and Beidler et al.
, (1998) J. Immunol. 141 : 4053-4060.

An anti-FDRG antibody (eg, a monoclonal antibody) can be used to isolate FDRG by standard techniques, such as affinity chromatography or immunoprecipitation. Anti-FDRG antibodies facilitate the purification of native FDRG from cells and of recombinantly produced FDRG expressed in host cells. Furthermore, anti-FDRG antibody
It can be used to detect FDRG protein (eg, in cell lysates or cell supernatants) to assess the abundance and pattern of DRG protein expression. Anti-FDR
G antibodies can be used diagnostically to monitor protein levels in tissues as part of a clinical test, for example, to determine the efficacy of a given treatment dose. Detection can be accomplished by coupling (ie, physically linking) the antibody to a detectable substrate. Examples of detectable substrates include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-glucosidase or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin / biotin and avidin / biotin; suitable fluorescence Examples of materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase; It includes luciferin and aequorin, and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H. III. Recombinant Expression Vectors and Host Cells Another aspect of the present invention relates to vectors, preferably expression vectors containing nucleic acids encoding FDRG (or portions thereof). As used herein,
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which is a circular, double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors may control the expression of an operably linked gene. Such vectors are referred to herein as "expression vectors." Generally, expression vectors suitable for practical use in the recombinant DNA method are often in the form of plasmids. In this specification, "
“Plasmid” and “vector” can be used interchangeably, and plasmids are the most frequently used form of vector. However, this specification is intended to include other forms of expression vectors, such as viral vectors that provide equivalent functions (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

A recombinant expression vector of the invention comprises a nucleic acid of the invention in a state suitable for expression of the nucleic acid in a host cell, which is selected based on the host cell in which the recombinant expression vector is used for expression. Means including one or more regulatory sequences operably linked to the nucleic acid to be expressed. "Operably linked" in the context of a recombinant expression vector means that the nucleic acid sequence of interest is ligated to one or more regulatory sequences in a manner that allows expression of the nucleotide sequence. (Eg, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Methods: ( Gene Express
ion Technology ): Methods in Enzymology 185, Academic Publishing, San Diego, CA (1990). Regulatory sequences include those that govern constitutive expression of the nucleotide sequence in many types of host cells and those that govern expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). Including. Those skilled in the art can design an expression vector by selecting a host cell to be transformed,
It will be appreciated that it may depend on factors such as the level of expression of the desired protein.
The expression vector of the invention is introduced into a host cell, whereby a protein or peptide comprising a fusion protein or peptide encoded by a nucleic acid as described herein (eg, a FDRG protein, a mutant form of FDRG, a fusion protein, etc.) )
Can be produced.

The recombinant expression vector of the present invention can design the expression of FDRG in prokaryotic or eukaryotic cells. For example, FDRG is used for bacterial cells such as Escherichia coli ( E. coli ) and insect cells (
(Using a baculovirus expression vector), yeast cells or mammalian cells. Suitable host cells are further described in Goeddle, Gene Expression Methods: ( Ge
ne Expression Technology ): Methods in Enzymology 185, Academic Publishing,
Considered in San Diego, California (1990). Alternatively, a recombinant expression vector can be transcribed and translated in vitro using, for example, a T7 promoter regulatory sequence and T7 polymerase.

[0119] Expression of proteins in prokaryotes is most often performed in E. coli containing a constitutive or inducible promoter that governs the expression of either fused or unfused proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) increase the expression of the recombinant protein; 2) increase the solubility of the recombinant protein; and 3) act as a ligand in affinity purification. Purification of recombinant protein by Often in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and allows for separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin and enterokinase. A typical fusion expression vector is pGEX (Pharmacia Biotech: Pharmacia Biotech).
Inc .: Smith, DB and Johnson, KS, (1988) Gene 67: 31-40), including pMAL (New England Biolabs: New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) These are glutathione S-transferase (GST) and maltose E, respectively.
The binding protein, or protein A, is fused to the targeted recombinant protein.

[0120] The purified fusion protein was tested for FDRG ligand binding (
Direct assays or competition assays, eg, as described in detail below), for example, to generate antibodies specific for the FDRG protein.
In a preferred embodiment, FDRG fusions expressed with a retroviral expression vector of the invention can be used to infect bone marrow cells that are subsequently transplanted into a radiation-treated receptor. The condition of the individual receptor is then investigated after a sufficient time has elapsed (eg 6 weeks).

Examples of suitable inducible non-fused E. coli expression vectors include pTrc (Amann e
t al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technology : Methods in Enzymology 185, Academic Publishing, San Diego, CA (1990 (60-89 The expression of the gene of interest from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.The expression of the gene of interest from the pET 11d vector is T7 gn10 mediated by the coexpressed viral RNA polymerase. -by transcription from a lac fusion promoter (T7 gn1) This viral polymerase is transformed from a resident λ phage with the T7 gn1 gene under the transcriptional control of the lacUV5 promoter into the host strain BL.
Supplied by 21 (DE3) or HMS174 (DE3).

One method for maximizing recombinant protein expression in E. coli is to transform the protein in a host bacterium with the added ability to proteolytically cleave the recombinant protein. Expression (Gottesman, S., Gene Expression Method ( G
ene Expression Technology) : Methods in Enzymology 185, Academic Publishing,
San Diego, California (1990) 119-128). Another method is to modify the nucleic acid sequence of the nucleic acid inserted into the expression vector such that the individual codons for each amino acid are preferentially used in E. coli ( E. coli ). Wada et al.,
(1992) Nucleic Acids Res . 20: 2111-2118). Such modifications of the nucleic acid sequences of the present invention can be made by standard DNA synthesis methods.

[0123] In another embodiment, the FDRG expression vector is a yeast expression vector. S. a yeast. Examples of vectors for expression in S. cerivisae include pYepSec1 (B
aldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1
982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123),
pYES2 (Invitrogen Corporation, San Diego, Calif.) and picZ (Invitrogen, Carlsbad, Calif.).

Alternatively, FDRG can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors that can be used for protein expression in cultured insect cells (eg, Sf9 cells) include the pAc series (Smith et al., (1983) M
ol. Cell. Biol. 3: 2156-2165) and pVL series (Lucklow and Summers (1989) V
irology 170: 31-39).

In yet another aspect, the nucleic acids of the invention are expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed. B., (1987)
) Nature 329: 840) and pMT2PC (Kaufman et al., (1987) EMBO J. 6: 187-195).
)including. When used in mammalian cells, the control functions on the expression vectors are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Sambro.
ok, J., French, EF and Maniatis. T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, NY, 1989 See Chapters 16 and 17 of the book.

In another embodiment, the recombinant mammalian expression vector can preferentially control the expression of the nucleic acid in certain types of cells (eg, where tissue-specific regulatory elements are used to express the nucleic acid). used). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific: Pinkert et al., (1987) Gene Dev . 1: 268-277), the lymphoid system-
Specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), especially promoters for T cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-7)
33) and immunoglobulins (Banerji et al., (1983) Cell 33: 729-740; Queen
and Baltimore (1983) Cell 33: 741-748), a neuron-specific promoter (eg, a neurofilament promoter: Byrne and Ruddle (1989) PNAS 86: 547).
3-5477), pancreas-specific promoter (Edlund et al., (1985) Science 230: 912-9
16) and mammary gland-specific promoters (eg, whey promoter; US Pat.
873,316 and EP-A-264,166). Developmental-regulated promoters, such as the mouse hox promoter (Kessel and Gruss (1990) Scie
nce 249: 374-379) and the α-fetoprotein promoter (Campes and Tilgh)
am (1989) Gene Dev. 3: 537-546).

The present invention further provides a recombinant expression vector comprising a DNA molecule of the present invention cloned in an antisense orientation into the expression vector. That is, the DNA molecule
Expression of RNA molecules that are antisense to FDRG mRNA (by transcription of DNA molecules)
Operably linked to a regulatory sequence in a manner that allows Selecting regulatory sequences, such as viral promoters and / or enhancers, operably linked to the nucleic acid cloned in the antisense orientation that governs the continuous expression of the antisense RNA molecule in various types of cells may be selected. Alternatively, constitutive, tissue-specific or cell-type-specific regulatory sequences that govern antisense RNA expression can be selected. An antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which the antisense nucleic acid is produced under the control of a highly efficient regulatory region, the activity of which is determined by the cell type into which the vector has been introduced. Is done. For a discussion on regulating gene expression using antisense genes, see Weintr.
aub.H. et al., Antisense RNA as a molecular tool for gene analysis (Antis
ence RNA as a molecular tool for genetic analysis), Reviews-Trends in Ge
See netics . Vol. 1 (1), 1986.

[0128] Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms are intended to refer not only to the cells of a particular individual, but also to the progeny or possible progeny of such cells. Since certain modifications may occur in later generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but nevertheless the scope of terms used herein Included in

[0129] The host cell can be any prokaryotic or eukaryotic cell. For example, the FDRG protein can be expressed in bacterial cells, such as E. coli , insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host bacteria are known to those skilled in the art.

[0130] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection methods. As used herein, the terms “transformation” and “transfection” refer to foreign nucleic acids (eg, DNA)
Are intended to refer to various art-recognized techniques for introducing DNA into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE
-Including dextran-mediated transfection, lipofectin or electroporation. Suitable methods for transforming or transfecting host cells are described in Sambrook.
et al., (Molecular Cloning: Laboratory Manual, 2nd edition,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Publications, Cold Spring Harbor, NY, 1989) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection method used, only a small fraction of the cells can integrate foreign DNA into their genome. I have. To identify and select for such integrations, a gene encoding a selectable marker (eg, resistance to an antibiotic) is generally introduced into the host cell along with the gene of interest. Suitable selectable markers include those that confer resistance to drugs such as, for example, G418, hygromycin and methotrexate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as the vector encoding FDRG, or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acids can be identified by drug selection (eg, cells that contain a selectable marker gene will survive,
Other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ie, express) the FDRG protein. Accordingly, the present invention further provides a method for producing a FDRG protein using the host cell of the present invention. In one embodiment, the method comprises culturing the host cell of the present invention, into which a recombinant expression vector encoding FDRG has been introduced, in a suitable medium to produce FDRG protein. Consists of In another embodiment, the method further comprises FDRG
From the medium or the host cell.

[0133] The host cells of the present invention can also be used to create non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or embryonic stem cell into which the FDRG-coding sequence has been introduced. Such host cells are then used to create non-human transgenic animals in which exogenous FDRG sequences have been introduced into their genome, or homologous recombinant animals in which the endogenous FDRG sequences have been modified. be able to. Such animals are useful for studying FDRG function and / or activity, and for identifying and / or evaluating modulators of FDRG activity. As used herein, “transgenic animal”
Is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, wherein one or more cells of the animal contain the transgene. Examples of other transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA that is integrated into the genome of a cell from which the transgenic animal has developed and remains in the genome of the mature animal, thereby being encoded in one or more cell types or tissues in the transgenic animal. Controls the expression of the gene product. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, and more preferably a mouse, wherein the endogenous FDRG gene is an animal that has an endogenous gene and an animal. Before animal cells (
(Eg, embryonic cells of animals) by homologous recombination with exogenous DNA molecules introduced into the cells.

The transgenic animals of the present invention are those wherein the nucleic acid encoding FDRG is introduced into the male pronucleus of fertilized oocytes, for example, by microinjection, retroviral infection, and the oocytes are expressed in pseudopregnant females. It is produced by growing foster animals. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13
The FDRG cDNA sequence of SEQ ID NO: 14, or the DNA insert of the plasmid deposited with the ATCC under accession number 98632, can be introduced as a transgene into the genome of a non-human animal. An intron sequence and a polyadenylation signal can also be included in the transgene to increase transgene expression efficiency. Tissue-specific regulatory sequence (s) are operably linked to the FDRG transgene and
Can control the expression of FDRG protein. Methods for producing transgenic animals, particularly animals such as mice, via embryo manipulation and microinjection are routine in the art and, for example, both are described by Leder et al.
U.S. Patent Nos. 4,736,866 and 4,870,009 to Wanger et al.
U.S. Patent No. 4,873,191 and Hogan. B., Manipulation of mouse embryos ( Manip.
ulating the Mouse Embryo ), (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Similar methods are used to generate other transgenic animals. Transgenic founders can be identified based on the presence of the FDRG transgene in its genome and / or expression of FDRG mRNA in animal tissues or cells. The transgenic founder can then be used to breed additional animals with the transgene. In addition, transgenic animals having a transgene encoding FDRG can be bred to other transgenic animals having other animal genes.

To create a homologous recombinant animal, a vector containing at least one portion of the FDRG gene is prepared and FDRG is introduced by deleting, adding or substituting therein.
Modifying (eg, functionally disrupting) a gene. The FDRG gene can be a human gene (eg, the cDNA of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4).
), More preferably a non-human homolog of the human FDRG gene. For example, mouse FDRG
The gene (eg, the cDNA of SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14) can be used to construct a homologous recombination vector suitable for modifying the endogenous FDRG gene in the mouse genome. In a preferred embodiment, the vector is designed such that the homologous recombination functionally disrupts the endogenous FDRG gene (ie, no longer encodes a functional protein: also referred to as a "knockout" vector). Alternatively, the vector is designed such that the endogenous FDRG gene is mutated or otherwise modified by homologous recombination, but the functional protein is still encoded (eg, the upstream regulatory region has been modified, thereby Can alter the expression of the endogenous FDRG protein). In a homologous recombination vector, the modified part of the FDRG gene is
It is flanked at its 5 'and 3' ends by additional nucleic acids of the FDRG gene to allow homologous recombination between the exogenous and endogenous FDRG genes carried by the vector in embryonic stem cells. The flanking FDRG nucleic acid to be added is of sufficient length for successful homologous recombination using an endogenous gene. Typically, several kilobases of flanking DNA (both at the 5 'and 3' ends) are included in the vector (eg, see Thomas, KRand Capecchi, MR (198 for the description of homologous recombination vectors).
7) See Cell 51: 503). The vector is introduced into an embryonic stem cell line (eg, by electroporation), and selects cells in which the introduced FDRG gene has been homologously recombined with the endogenous FDRG gene (eg, Li, E. et al. , (1992) Cell 69: 915)
. The selected cells are then injected into a blastocyst of an animal (eg, a mouse) and the assembled chimera (
(eg, Bradley, A., Teratocarcinoma and Embryonic Stem Cells: A Practical Study)
Approach ), edited by EJ Robertson (IRL, Oxford, 1987), 113th to 15th
See page 2). The chimeric embryo is then implanted into a suitable pseudopregnant female foster animal and the embryo is brought to term. Homologously recombined DNA can be used to breed offspring that have their germ cells in animals, where all cells of the animal are homologously recombined by germline inheritance of the transgene. Containing DNA. The method of constructing a homologous recombination vector or generating a homologous recombination animal is further described in Bradley, A. (1991) Current Opinion in Biotechnology 2: 823-829.
And WO 90/11354 by Smith and Le Mouellec et al .: Smithies et al.
WO 91/01140 by Z.I .; WO 92/096 by Zijlstra et al.
No. 8; and WO 93/04169 by Berns et al.

In another aspect, transgenic non-human animals can be created that contain a selected system that can regulate the expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, for example, Lakso et al., (1992) PNAS 89: 6232-6236. Another example of a recombinase system is Saccharomyces cerevisiae .
omyces cerevisiae ) (O'Gorman et al., (1991) S
cience 251: 1351-1355). If the cre / loxP recombinase system is used to regulate transgene expression, an animal is needed that contains the transgene encoding both the Cre recombinase and the selected protein. Such animals can be "double" transgenic, for example, by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. It can be provided through the construction of transgenic animals.

The clones of the non-human transgenic animals described herein were obtained from Wilmut, Ie.
t al., (1997) Nature 385: 810-813 and WO 97/07668 and 97/0.
It can also be prepared according to the method described in the 7669 specification. Briefly, cells, eg, stem cells from a transgenic animal, can be isolated and induced to exit the growth cycle and enter the _Go phase. The quiescent cells are then fused, eg, via the use of electric pulses, with enucleated oocytes from the same animal as the animal from which the quiescent cells were isolated. The reconstituted oocyte is then cultured so that it develops into a morula or blastocyst and then transferred to a pseudopregnant female foster animal. The progeny of this female foster parent is an animal clone from which the cells, eg, stem cells, have been isolated. IV. Pharmaceutical Compositions FDRG nucleic acid molecules, FDRG proteins and anti-FDRG antibodies of the invention (herein "
Active compounds) can be included in pharmaceutical compositions suitable for administration.
Such a composition typically comprises the nucleic acid molecule, protein or antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to any solvent compatible with drug administration,
It is intended to include dispersing media, coatings, antibacterial and antifungal agents, agents that are isotonic and delay absorption. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except where any customary medium or agent is incompatible with the active compound, their use in the compositions is intended. Supplementary active compounds can also be included in the composition.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (eg,
Topical), transmucosal and rectal administration. Solvents or suspensions used for parenteral, intradermal or subcutaneous administration may contain the following ingredients: water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents. Sterilizing diluents such as; antimicrobial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; acetates, citrates and phosphates Buffer and tonicity adjusting agent such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the ready-to-use preparation of injectable solutions or dispersions. For intravenous administration, suitable carriers are saline, bacteriostatic water, Cremophor EL ™ (BASF
Or Parsippany, NJ) or phosphate buffered salt solution (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and transport and must be preserved against the contaminating action of microorganisms such as bacteria and mold. The carrier is, for example, water, ethanol, polyol (
For example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof can be solvents or dispersion media. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, tonicity agents, for example, sugars, polyalcohols such as mannitol, sorbitol,
Preferably, the composition contains sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

A sterile injectable solution may contain an active compound (eg, FDRG protein or anti-FDR
G antibody) in the required amount in a suitable solvent containing a combination of one or more of the above-listed components, if necessary, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, in which the active ingredient powder is further added with the desired ingredients from a previously sterile filtered solution. This produces a powder.

[0141] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is administered orally and and swished in the mouth; Exhale or swallow. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose; Disintegrants such as alginic acid, Primogel ™ or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; peppermint; Flavors such as methyl salicylate or orange flavors.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser or nebulizer which contains a suitable propellant, such as carbon dioxide.

[0143] Systemic administration can also be through the mucosa or by transdermal means. For transmucosal or transdermal administration, penetrants appropriate to cross the barrier (pene
trants) are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Administration through the mucosa can be through nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is a carrier that protects the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. It is prepared using Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. The preparation of such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals Inc. Liposomal suspensions (including liposomes targeting infected cells using monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically divided units suitable as unit doses for the individual to be treated; each unit being a predetermined amount calculated to produce the desired therapeutic effect. Of the active compound together with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by and unique to the unique properties of the active compounds and the particular therapeutic effect to be achieved, as well as the limitations of the pharmaceutical field such as the active compound for treatment of an individual. Dependent.

The toxic and therapeutic effects of such compounds can be determined, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). , Cell cultures or standard pharmacological techniques for experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but target such compounds to the affected tissue site to minimize the potential for damage to uninfected cells and thereby reduce side effects. Care should be taken in the design of delivery systems that do this.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within a range depending on the dosage form employed and the route of administration utilized. For compounds used in the methods of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. The dose is formulated in animal models to determine the IC50 as determined in cell culture (ie, the concentration of test compound that achieves half-maximal suppression of symptoms)
To achieve a circulating plasma concentration range that includes Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

The nucleic acid molecules of the invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be administered to an individual, for example, by intravenous injection, topical administration (see US Pat. No. 5,328,470) or stereotactic injection (eg, Che
n et al., (1994) PNAS 91: 3054-3057). Pharmaceutical preparations of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where a complete gene delivery vector can be produced seamlessly from recombinant cells (eg, a retroviral vector), the pharmaceutical composition can include one or more cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologs and antibodies described herein can be obtained by the following methods: a) screening assays; b) detection assays (eg, chromosome mapping, tissue typing, forensic Biology); c) predictive medicine (eg, diagnostic assays, prognostic assays, tracking of clinical trials; and d) methods of treatment (eg,
Therapeutic and prophylactic methods, and such methods associated with genetic pharmacology). The F of the present invention, as described herein,
DRG proteins have the following activities: (i) activation of FDRG-dependent signaling pathways; (ii) regulation of angiogenesis; (iii) regulation of hematopoiesis; (iv) regulation of development or differentiation of FDRG-expressing cells. (E.g., mediating proliferation and / or differentiation of adipocytes, e.g., white or brown adipocytes); (v) regulating the development or differentiation of non-FDRG expressing cells; (vi) regulating homeostasis of FDRG expressing cells; vii) modulation of insulin sensitivity and / or insulin responsiveness;
viii) regulating insulin secretion; (ix) regulating cell recruitment; and (x) regulating homeostasis of non-FDRG expressing cells. Thus, FDRG proteins, FDRG modulators or FDRG receptors
Modulators include, for example, (1) activation of the FDRG-dependent signaling pathway;
(2) regulation of angiogenesis; (3) regulation of hematopoiesis; (4) regulation of development or differentiation of FDRG-expressing cells (eg, of proliferation and / or differentiation of adipocytes, such as white or brown adipocytes). (5) regulation of the development or differentiation of non-FDRG-expressing cells; (6) regulation of homeostasis of FDRG-expressing cells; (7) regulation of insulin sensitivity and / or insulin responsiveness; (8) regulation of insulin secretion; (9) regulating cell recruitment; and (10) regulating homeostasis of non-FDRG expressing cells; (11) maintaining energy homeostasis (eg, regulating the balance and / or imbalance between energy storage and energy consumption, eg, energy consumption) (12) regulation of adaptive thermogenesis (eg, regulation of mitochondrial biosynthesis, mi Regulation of the expression of Kondoria enzymes, regulation of the expression of uncoupling protein); regulatory efficiency (14) energy storage; (13) the regulation of adiposity (1
5) regulating appetite; and (16) regulating angiogenesis.

The isolated nucleic acid molecules of the present invention may be, for example, F, as described further below.
To express DRG proteins (eg, via recombinant expression vectors in host cells in gene therapy applications), to detect FDRG mRNA (eg, in biological samples) or genetic mutations in the FDRG gene, and to modulate FDRG activity Can be used. In addition, FDRG proteins may be used to screen for drugs or compounds that modulate FDRG activity, as well as to under- or over-produce FDRG proteins, or FDRG
Use for treating a disorder characterized by the production of an FDRG protein type having reduced or abnormal activity compared to a wild-type protein (eg, a differentiation or developmental disorder, a proliferative disorder, or a cell replacement disorder). Can be. In addition, soluble forms of the FDRG protein bind to membrane-bound FDRG receptors and can be used to affect the bioavailability of such receptor cognate ligands. Moreover, the anti-FDRG antibodies of the present invention can be used to detect and isolate FDRG proteins and to modulate FDRG activity.

A. Screening Assays : The present invention relates to modulators that bind to the FDRG protein or have, for example, a promoting or inhibiting effect on FDRG expression or FDRG activity, ie, candidate or test compounds or agents (eg, peptides, peptidomimetics,
Methods for identifying small molecules or other drugs (also referred to herein as "screening assays") are provided.

In one embodiment, the invention provides an assay for screening candidate or test compounds that bind to or modulate the activity of an FDRG protein or polypeptide or a biologically active portion thereof. I do. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of FDRG receptor.

The test compounds of the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, provided that these libraries are biological libraries; Can be approached (adre
ssable) parallel solid or solution phase library; synthetic library method requiring deconvolution; "one bead one compound" library method; and synthetic live using affinity chromatography sorting Including the rally method. Biological library approaches are limited to peptide libraries, while the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam, KS (1997)). Anticance
r Drug Des , 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt e.
tal, (1993) Proc. Natl. Acad. Sci. U. S. A.
90: 6909; Erb et al. (1994) Proc. Natl. Ac
ad. Sci. U. S. A. 91: 11422; Zuckermann et.
al. (1994) J. Am. Med. Chem . 37: 2678; Cho et a
l. (1993) Science 261: 1303; Carrell et.
al. (1994) Angew. Chem. Int. Ed. Engl . 33: 2
059; Carrell et al. (1994) Angew. Chem. I
nt. Ed. Engl . 33: 2061; and Gallop et al. (
1994) J. Med. Chem . 37: 1233.

A library of compounds is available in solution (eg, Houghten (1992) Bi ).
otechniques 13: 412-421) or beads (L
am (1991) Nature 354: 82-84), chip (Fodor (
1993) Nature 364: 555-556), bacteria (Ladne).
US Patent No. 5,223,409), spores (Ladner US Patent '409).
No.), a plasmid (Cul et al (1992) Proc. Natl. A ) .
cad. Sci. U. S. A. 89: 1865-1869) or phage (
(Scott and Smith (1990) Science 249: 38.
6-390); (Devlin (1990) Science 249: 404-
(Cwirla et al. (1990) Proc. Natl . A.)
cad. Sci. U. S. A. 87: 6378-6382): (Felici (
1991). Mol. Biol. 222: 301-310); (Ladner
I) above).

[0158] In one embodiment, the assay is a cell-based assay, wherein:
A cell expressing FDRG is contacted with a test compound and the test compound F
The ability to regulate DRG expression and / or activity is determined. Determining the ability of a compound to regulate FDRG expression can be accomplished, for example, by detecting the presence, absence, or amount of FDRG transcripts of the protein (eg, using a probe based on the nucleotide sequence of the present invention or an anti-FDRG antibody). Can be. FDRG of compound
The determination of the activity regulating ability can be performed, for example, by detecting FDRG activity in the cell supernatant (for example, by contacting a second cell with the supernatant).

In another embodiment, the assay is a cell-based assay, wherein cells expressing the FDRG receptor on the cell surface are contacted with a test compound and binding of the test compound to the FDRG receptor. Noh is determined. The cells may be, for example, of mammalian origin or yeast cells. Determination of the ability of the test compound to bind to the FDRG receptor can be determined, for example, by coupling the test compound to a radioisotope or enzyme label, and detecting the binding of the test compound to the FDRG receptor by detecting the labeled compound in the complex. Can be accomplished in this manner. For example, either directly or indirectly, a test compound may be labeled with ¹²⁵ I, ³⁵ S, ¹⁴ C or ³ H, and the radioactive isotope detected by direct measurement of radioemission or by scintillation counting. Otherwise, the test compound may be enzymatically labeled, for example, with horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label can be detected by determining the conversion of the appropriate substrate to product.

It is also within the scope of the present invention to determine the ability of a test compound to interact with the FDRG receptor without labeling any of the interactants. For example, a microphysiometer can be used to detect the interaction of a test compound with the FDRG receptor without any labeling of the test compound or receptor. McConnell, H .; M. et al. (
1992) Science 257: 1906-1912. As used herein, a “microphysiometer” (eg, Cytosensor ^™ ) is a light-addressable potentiometric sensor (light-addressable).
An analytical instrument that measures the rate at which cells acidify their environment using an epotentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between ligand and receptor.
In a preferred embodiment, the assay comprises contacting cells expressing the FDRG receptor on the cell surface with the FDRG receptor or a biologically active portion thereof to form an assay mixture, and testing the assay mixture. Contacting with a compound and determining the ability of the test compound to interact with FDRG, wherein
Determining the ability of a test compound to interact with FDRG comprises determining the ability of the test compound to preferentially bind to its receptor as compared to the ability of FDRG or a biologically active portion thereof to bind to the FDRG receptor. Comprising. Alternatively, the assay may comprise determining the ability of the test compound to modulate the cellular activity of FDRG and / or FDRG receptor.

Determining the ability of a FDRG protein to bind or interact with an FDRG target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the FDRG protein to bind or interact with the FDRG target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule may detect the induction of a target cellular second messenger (ie, intracellular Ca ²⁺ , diacylglycerol, IP _3, etc.)
Whether to detect the catalytic / enzymatic activity of the target on a suitable substrate or to detect the induction of a reporter gene (eg, an FDRG responsive regulatory element operably linked to a detectable marker, eg, a nucleic acid encoding luciferase) Alternatively, it can be determined by detecting a cellular response, eg, expression of a differentiated phenotype.

In still other embodiments, the assays of the invention are cell-free assays, wherein an FDRG protein or a biologically active portion thereof is contacted with a test compound and the FDRG of the test compound is The ability to bind to the protein or a biologically active portion thereof is determined. Binding of a test compound to the FDRG protein can be determined, directly or indirectly, as described above. In a preferred embodiment, the assay comprises contacting the FDRG protein or a biologically active portion thereof with a known compound that binds to FDRG to form an assay mixture, contacting the assay mixture with a test compound, and Determining the ability of the test compound to interact with the FDRG protein, wherein determining the ability of the test compound to interact with the FDRG protein comprises determining the ability of the test compound to interact with the FDRG protein relative to a known compound. Determining the ability to preferentially bind to the more active moiety.

In another embodiment, is a cell-free assay, wherein the FDRG protein or a biologically active portion thereof is contacted with a test compound and the test compound is a FDRG protein or a biologically active portion thereof. The ability to modulate the activity of the active moiety is determined. The ability of a test compound to modulate FDRG activity can be accomplished, for example, by determining the ability of the FDRG protein to bind to a target molecule (eg, an FDRG binding protein) by one of the methods described above for determining direct binding.
In addition, the determination of the binding ability of the FDRG protein to the FDRG target molecule is performed by real-time analysis of biomolecular interaction (Biomolecular Interaction).
n analysis (BIA). Sjol
ander, S.M. and Urbaniczky, C.I. (1991) Anal.
Chem . 63: 2338-2345 and Szabo et al. (199
5) Curr. Opin. Struct. Biol . 5: 699-705. As used herein, "BIA" is a technique for studying biospecific interactions in real time without labeling all interactants (eg, BIA).
IAcore ^™ ). Optical phenomenon surface plus resonance (surfac
Changes in eplasmon response (SPR) can be used as an indicator of real-time reactions between biological molecules.

In another embodiment, determining the ability of a test compound to modulate FDRG activity comprises measuring F
This can be accomplished by determining the ability of the DRG protein to further regulate the FDRG target molecule. For example, the activity of the target molecule on the receptor can be determined as described above.

In still other embodiments, the cell-free assay comprises contacting an FDRG protein or a biologically active portion thereof with a known compound that binds to FDRG to form an assay mixture, and testing the assay mixture. Contacting with the compound and determining the ability of the test compound to interact with the FDRG protein, wherein determining the ability of the test compound to interact with the FDRG protein comprises:
Determining the ability of the protein to bind preferentially to the FDRG target molecule or to modulate activity.

The cell-free assays of the present invention rely on the use of both soluble and / or membrane-bound forms of the isolated protein (eg, the FDRG protein or a biologically active portion thereof or the FDRG target molecule). In the case of cell-free assays in which a membrane-bound form of the isolated protein is used (eg, FDRG target molecule or receptor), a solubilizing agent is utilized so that the membrane-bound form of the isolated protein is maintained in solution. Would be desirable. Examples of such solubilizers are nonionic surfactants such as n-octyl glucoside, n
- dodecyl glucoside, n- dodecyl maltoside, octanoyl -N- methyl glucammonium amide, decanoyl -N- methyl glucammonium amide, Triton ^R X-100
^{, Triton R X-114, Thesit} R, Isotorideshipori (ethylene glycol ether) _n, 3 - [(3- chloro amidopropyl) dimethylammonio Mini O (amminio)] - 1- propane sulfonate (CHAPS), 3 - [( 3
-Chloroamidopropyl) dimethylamminio] -2-hydroxy-1-propanesulfonate (CHAPSO) or N-dodecyl = N, N-dimethyl-3
-Ammonio-1-propanesulfonate.

In one or more embodiments of the foregoing assay methods of the invention, to facilitate the separation of the complex form from the uncomplexed form of one or both of the proteins, and to accommodate the automation of the assay, It may be desirable to immobilize any FDRG or its target molecule. Binding of the test compound to the FDRG, or interaction of the FDRG with the target molecule in the presence and absence of the candidate compound, can be accomplished in any container suitable for containing the reactants. Examples of such containers include plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein may be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase / FDRG fusion protein or glutathione-S-transferase / target fusion protein can be obtained by using glutathione sepharose beads (Sigma).
a Chemicals, St. (Louis, MO) or glutathione-derivatized microtiter plates, which are then used to bind the test compound, or test compound, and any unadsorbed target protein or FDR.
The G protein is combined and the mixture is incubated under conditions that favor complex formation (eg, under physiological conditions with respect to salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, and in the case of beads, the immobilized matrix or complex, directly or indirectly, as described above. Is determined as was done. Alternatively, the complex may be dissociated from the matrix, and the level of FDRG binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, FDRG or any of its target molecules can be immobilized utilizing the conjugation of biotin and streptavidin. Biotinylated FDRG or target molecule can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemica).
ls, Rockford, IL), and 96 coated with streptavidin.
Immobilized in the wells of a well plate (Pierce Chemical). Alternatively, an antibody that is reactive to the FDRG or target molecule, but does not interfere with the binding of the FDRG protein to its target molecule, is derivatized to the wells of the plate, and the unbound target or FDRG is bound by antibody conjugation. Captured in the well. Methods for detecting such a complex include, in addition to those described above for the GST-immobilized complex, immunodetection of the complex using antibodies reactive with FDRG or the target molecule, as well as FDRG or target molecule related Includes an enzyme ligation assay that relies on detecting enzyme activity.

In another embodiment, modulators of FDRG expression are identified in a method in which a cell is contacted with a candidate compound and the expression of FDRG mRNA or protein in the cell is determined. FDRG mRNA in the presence of candidate compounds
Alternatively, the expression level of the protein is determined in the absence of the candidate compound by FDRG mRNA.
Alternatively, it is compared to the expression level of the protein. Candidate compounds can then be identified as modulators of FDRG expression based on this comparison. For example, if the expression of FDRG mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as FD
Identified as a stimulator of RG mRNA or protein expression. Alternatively, a candidate compound is identified as an inhibitor of FDRG mRNA or protein expression if expression of the FDRG mRNA or protein is less in the presence of the candidate compound than in its absence (statistically significantly less). The level of FDRG mRNA or protein expression in cells is determined by FDRG
It may be determined by the methods described herein for detecting mRNA or protein.

In still other aspects of the invention, the FDRG protein is a two-hybrid assay or a three-hybrid assay (see, eg, US Pat. No. 5,283,283).
No. 317; Zerovos et al. (1993) Cell 72: 223-.
232; Madura et al. (1993) J. Bil. Chem . 26
8: 12046-12054; Bartel et al. (1993) Bio
techniques 14: 920-924; Iwabuchi et al.
. (1993) Oncogene 8: 1693-1696; and Brent
WO94 / 10300, see as “bait protein” and used as FDRG (“FDRG binding protein” or “FDRG-
bp ") and other proteins that modulate FDRG activity can be identified. Such FDRG binding proteins also appear to be involved in signal transmission by the FDRG protein, for example, as a downstream element of the FDRG-mediated signaling pathway. Alternatively, such an FDR
G-binding proteins are considered to be cell surface molecules associated with non-FDRG expressing cells, in which case such FDRG-binding proteins are required for second cytokine production.

The two-hybrid system is based on the modularity of most transcription factors,
It consists of separable DNA binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, FDRG
Is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In other constructs, the DN encoding an unidentified protein ("prey" or "sample")
DNA sequences from a library of A sequences are fused to a gene encoding the activation domain of a known transcription factor. If the "bait" and "prey" proteins can interact in vivo to form an FDRG-dependent complex, the DNA binding and activation domains of the transcription factor are brought into close proximity. This access allows transcription of a receptor gene (eg, LacZ) operably linked to a transcription regulatory site responsive to the transcription factor. Expression of the receptor gene can be detected, and cell colonies containing the functional transcription factor are isolated and used to obtain a cloned gene encoding a protein that interacts with FDRG be able to.

The present invention further relates to novel agents identified by the above screening assays. Thus, it is within the scope of the present invention to use an agent identified as described herein in a suitable animal model. For example, an agent identified as described herein (eg, FD
RG modulator, antisense FDRG nucleic acid molecule, FDRG specific antibody or F
DRG binding partners) can be used in animal models to determine the efficacy, toxicity or side effects of treatment with such agents. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Further, the present invention relates to the use of the novel agents identified by the above screening assays for the treatment as described herein.

B. Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in various ways as polynucleotide reagents. For example, these sequences may (i) map their respective genes on the chromosome, thereby locating gene regions associated with genetic diseases; (ii) identify individuals from small biological samples (tissue typing) );
And (iii) aid in the legal identification of biological samples. These applications are described in the following subsections.

[0174] 1. If the sequence (or a portion of the sequence) of a chromosome mapping gene has been isolated, this sequence can be used to map the location of the gene on the chromosome. This process is called chromosome mapping. Accordingly, a portion or fragment of the FDRG sequence described herein may comprise a FDRG sequence on a chromosome.
It can be used to map the locus of each gene. FDR for chromosome
Mapping of G sequences is an important first step in correlating these sequences with genes associated with disease.

[0175] Briefly, the FDRG gene can be mapped to a chromosome by making PCR primers (preferably 15-25 bp in length) from the FDRG sequence. FDRG, a computer analysis of sequences, can be used to rapidly select primers that do not span more than one exon in genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. FDR
Only those hybrids containing the human gene corresponding to the G sequence can produce the amplified fragment.

[0176] Somatic cell hybrids are made by fusing somatic cells (eg, human and mouse cells) from different mammals. As hybrids of human and mouse cells proliferate and divide, they gradually lose human chromosomes randomly, but retain mouse chromosomes. Mouse cells cannot grow because they lack certain enzymes, but human cells can sustain one human chromosome containing the gene encoding the required enzyme by using a medium that can grow. . By using various media, a panel of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio
P. et al. (1983) Science 220: 919-924).
Somatic cell hybrids containing only fragments of the human chromosome can be made by using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid operation to assign a particular sequence to a particular chromosome. More than two sequences can be specified in one day by using one thermal cycle. Sublocalization (subloc) using the FDRG sequence to design oligonucleotide primers
is achieved by a panel of fragments from a particular chromosome. Another mapping strategy that can also be used to map the 9o, 1p or 1v sequence for that chromosome is in situ hybridization (Fan, Y. et al. (1990) PNAS , 87: 6223-2).
7), pre-screening with labeled and flow-sorted chromosomes and pre-selection by hybridizing a specific cDNA library to the chromosomes.

Further, fluorescence in situ hybridization (FISH) of DNA sequences to metaphase chromosomal spread (spread) can be used to provide a precise chromosomal locus in one step. Chromosome spreads can be made using cells in which cell division has been arrested in metaphase by a chemical agent-like colcemide that disrupts the mitotic spindle. Chromosomes can be easily treated with trypsin and then stained with Giemsa. A bright and dark band pattern develops on each chromosome so that the chromosomes can be individually identified. FISH technology can be used with DNA sequences as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher probability of binding to a unique chromosomal locus with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will be sufficient to obtain good results in a reasonable amount of time. For a review of this technology, see Verma et al. , Human Chrom
osomes: A Manual of Basic Technologies (
Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used separately to mark a single chromosome or a single site on that chromosome, and a panel of reagents can be used for multiple sites and / or multiple sites. Can be used to mark the chromosome of
Reagents corresponding to non-coding regions of the gene are in fact suitable for mapping purposes. The coding sequence is more likely to be conserved within the gene family, thus increasing the chance of cross-hybridization during chromosome mapping.

If the sequence has been mapped to a precise chromosomal locus, the physical location of the sequence on the chromosome can be corrected with the genetic map data. (Such data is available, for example, from Johns Hopkins University Welch.
V. available online through the Medical Library. McKu
sick, found in Mendelian Inheritance in Man ). The relationship between a disease and a gene mapped to the same chromosomal region is
Next, for example, Egelland, J. et al. et al. (1987) Nature ,
325: 783-787, and can be identified through linkage analysis (joint inheritance of physically adjacent genes).

In addition, differences in DNA sequence between individuals with and without a disease associated with the FDRG gene can be determined. If the mutation is observed in some or all affected individuals but not in all unaffected individuals, the mutation may be a causative agent of a particular disease . In general, the comparison of affected and unaffected individuals involves first looking for structural changes in the chromosome, such as deletions or translocations that are visualized from chromosomal spreads or detected using PCR based on their DNA sequence. Need. Finally, complete sequencing of the gene from several individuals can be performed to confirm the presence of the mutation and to discriminate between the mutation and the polymorphism.

[0182] 2. Tissue Typing The FDRG sequences of the present invention can also be used to identify individuals from small biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphisms (RFLPs) for the identification of its members. In this technique, an individual's genome D
NA is digested with one or more restriction enzymes and probed on a Southern blot to obtain a unique band for identification. This method does not suffer from the current limitations of "dog tags" that can be lost, switched, or stolen, creating positive identification difficulties. The sequences of the present invention are RF
Useful as additional DNA markers for LPs (U.S. Pat.
057).

Still further, the sequences of the present invention can be used to provide an alternative technique for determining the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the FDRG sequence described herein has two PCs from the 5 'and 3' ends of the sequence.
It can be used to prepare R primers. These primers
The individual's DNA can then be amplified and subsequently used for sequencing.

Since each individual will have a unique set of such DNA sequences due to allelic differences, a panel of corresponding DNA sequences from individuals prepared in this manner will require unique individual identification. Can be provided. The sequences of the present invention can be used to obtain such identified sequences from individuals and tissues. FDRG of the present invention
The sequence uniquely represents a portion of the human genome. Allelic variation occurs to some extent in the coding regions of these sequences and to a greater extent in the non-coding regions. Allelic variation between individual humans occurs with a frequency of about once for each 500 bases. Each of the sequences described herein can be used to some extent as a standard against which DNA from an individual can be compared for identification purposes. In non-coding regions, fewer sequences are needed to distinguish individuals, as more polymorphisms occur. SEQ ID NO:
One non-coding sequence can easily provide positive individual identification with a panel of possibly 10-1,000 primers, where each primer yields a non-coding amplified sequence of 100 bases. Predicted coding sequence, eg, SEQ ID NO:
If the sequence in 3 is used, a more suitable number of primers for positive individual identification is 500-2,000.

If a panel of reagents from the FDRG sequences described herein is used to create a unique identification database for an individual, those same reagents will then be used from that individual. Can be used to identify the tissue.

[0186] 3. Use of Partial FDRG Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific discipline that uses the genetic typing of biological evidence found at crime scenes, for example, as a means to positively identify perpetrators of crime. To perform such identification, PCR techniques are used to obtain very small biological samples, such as tissues, such as hair or skin, or body fluids, such as blood, saliva, or semen, found at the scene of a crime. The obtained DNA sequence can be amplified. The amplified sequence is then compared to a standard, thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to provide a polynucleotide reagent, eg, a PCR primer, that targets a particular locus in the human genome, for example, by using other “identifying markers” (ie, , Or other DNA sequences that are unique to a particular individual) can increase the reliability of DNA-based legal identification. As described above, actual nucleotide sequence information can be used for identification as an accurate alternative to the pattern formed by fragments generated with restriction enzymes. Sequences targeting the non-coding region of SEQ ID NO: 1 are particularly suitable for this use, as more polymorphisms occur in the non-coding region, and the use of this technique to distinguish individuals To make it easier. Examples of polynucleotide reagents include FDRG sequences or portions thereof, eg, at least 20
Includes fragments derived from the non-coding region of SEQ ID NO: 1 having a length of bases, preferably at least 30 bases.

In addition, the FDRG sequences described herein provide polynucleotide reagents, eg, labeled or labelable probes, that can be used in in situ hybridization techniques, eg, to identify a particular tissue, eg, brain tissue. Can be used to This is very useful if forensic pathologists are presented with tissues of unknown origin. Such a panel of FDRG probes can be used to identify tissue by species and / or organ type.

Similarly, these reagents, eg, FDRG primers or probes,
It can be used to screen tissue cultures for contamination (ie, to screen for the presence of a mixture of various types of cells in the culture).

C. Predictive Medicine : The present invention also relates to the field of predictive medicine, wherein diagnostic assays, prognostic assays and monitoring of clinical trials are used for prognostic (predictive) purposes, thereby treating the individual prophylactically. Accordingly, one embodiment of the present invention is directed to measuring FDRG protein and / or nucleic acid expression and FDRG activity in relation to a biological sample (eg, blood, serum, cells, tissues) to thereby render an individual abnormal. Diagnostic assays for determining whether a subject is afflicted with, or at risk of developing, a disease or disorder associated with FDRG expression or activity. Also,
The invention also provides prognostic (or predictive) assays for determining whether an individual is at risk of developing a disease associated with FDRG protein, nucleic acid expression or activity. For example, mutations in the FDRG gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purposes, thereby treating an individual prophylactically prior to the onset of a disease characterized by or associated with FDRG protein, nucleic acid expression or activity.

Another aspect of the invention relates to monitoring the effect of an agent (eg, drug, compound) on FDRG expression or activity in a clinical trial.

[0192] These and other agents are described in further detail in the following sections.

[0193] 1. Diagnostic assaysA typical method for detecting the presence or absence of FDRG in a biological sample is to obtain a biological sample from the test subject, and to detect the presence of the FDRG protein in the biological sample. Or contacting a biological sample with a compound or agent capable of detecting a nucleic acid (eg, mRNA, genomic DNA) encoding a FDRG protein. A preferred agent for detecting FDRG mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to FDRG mRNA or genomic DNA. The nucleic acid probe may be, for example, a DNA insert of the plasmid deposited at the ATCC as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, Accession No. 98632. Full-length FDRG nucleic acid, such as the nucleic acid of the invention, or at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize to FDRG mRNA or genomic DNA under stringent conditions. They may be part of them, such as oligonucleotides. Other suitable probes for use in the diagnostic assays of the invention are described herein.

Preferred agents for detecting FDRG protein are antibodies capable of binding to FDRG protein, preferably those with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody or a fragment thereof (eg, Fab or F (ab ′) ₂ ) can be used. The term "labeled" with respect to a probe or antibody refers to direct labeling of the probe or antibody by coupling (ie, physically linking) the detectable substance to the probe or antibody, as well as directly labeled It is intended to include indirect labeling of the probe or antibody by reactivity with another reagent. Examples of indirect labeling include detection of the primary antibody using a fluorescently labeled secondary antibody and DNA with biotin such that it can be detected with fluorescently labeled streptavidin
Includes probe end labels. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject as well as tissues, cells and fluids present in a subject. That is, the detection method of the present invention can be used to detect FDRG mRNA, protein or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detecting FDRG mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting FDRG protein include enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of FDRG genomic DNA include Southern hybridizations. In addition, in vivo techniques for the detection of FDRG protein include introducing a labeled anti-FDRG antibody into a subject. For example, the antibody can be labeled with a radioactive marker, and its presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules from a test subject. Alternatively, the biological sample can contain mRNA molecules from a test subject or genomic DNA molecules from a test subject. Preferred biological samples are serum samples routinely isolated from a subject.

In another embodiment, the method further comprises obtaining a control biological sample from the control subject, such that the presence of the FDRG protein, mRNA or genomic DNA is detected in the biological sample. Contacting a control sample with a compound or agent capable of detecting genomic DNA and comparing the presence of FDRG protein, mRNA or genomic DNA in the test sample with the presence of FDRG protein, mRNA or genomic DNA in the control sample Including.

[0197] The present invention also encompasses kits for detecting the presence of FDRG in a biological sample. For example, a kit may include a FDRG protein or mR in a biological sample.
A labeled compound or agent capable of detecting NA; FDRG in the sample
Means for measuring the amount of FDRG; and means for comparing the amount of FDRG in the sample to a reference. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit for detecting a FDRG protein or nucleic acid.

[0198] 2. Prognostic Assays The diagnostic methods described herein can further be utilized to identify subjects at risk of developing or at risk for developing a disease or disorder associated with abnormal FDRG expression or activity. For example, to identify a subject having or at risk of developing a disease associated with FDRG protein, nucleic acid expression or activity, such as a proliferative disease, a differentiation or developmental disease, a cell migration disease, or a hematopoietic disease. The assays described herein, such as the diagnostic assays described above or the assays below, can be utilized. Accordingly, the present invention provides a method for obtaining a test sample from a subject and detecting a FDRG protein or nucleic acid (eg, mRNA, genomic DNA) for identifying a disease or disorder associated with abnormal FDRG expression or activity. However, in that case, the presence of the FDRG protein or nucleic acid is useful in diagnosing a subject having or at risk of developing a disease or disorder associated with abnormal FDRG expression or activity. As used herein, "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (eg, serum), a cell sample or tissue.

In addition, agents (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecules or other drug candidates) can be used to treat diseases or disorders associated with abnormal FDRG expression or activity. The prognostic assays described herein can be used to determine if they can be administered to a subject. For example, using such methods to determine whether a subject can be effectively treated with an agent for a disease such as a proliferative disease, a differentiation or developmental disease, a cell migration or recruitment disease, or a hematopoietic disease. Can be. Thus, the present invention provides an unusual method for obtaining test samples and detecting FDRG proteins or nucleic acids.
Provided are methods for determining whether a subject can be effectively treated with an agent for a disease associated with FDRG expression or activity (eg, where the presence of FDRG protein or nucleic acid indicates abnormal FDRG expression or activity). Or for the diagnosis of a subject who can be administered an agent to treat a disease associated with activity).

In addition, a gene alteration in the FDRG gene is detected, whereby a subject having the altered gene is characterized by abnormal differentiation, abnormal angiogenesis, abnormal proliferative response or abnormal hematopoietic response. The method of the present invention can also be used to determine whether a person is at risk. In a preferred embodiment, the method comprises detecting, in a sample of cells from a subject, the presence or absence of a genetic alteration characterized by at least one of the following: Detecting. For example, 1) deletion of one or more nucleotides from the FDRG gene, 2) addition of one or more nucleotides to the FDRG gene; 3) substitution of one or more nucleotides of the FDRG gene; 4) Chromosomal rearrangement of FDRG gene; 5) Changes in messenger RNA transcript level of FDRG gene; 6) Abnormal modification of FDRG gene such as methylation pattern of genomic DNA; 7) Messenger RNA transcription of FDRG gene. By ascertaining the presence of at least one of the following: a non-wild-type splicing pattern of the product, 8) non-wild-type levels of the FDRG protein, 9) allelic loss of the FDRG gene, and 10) inappropriate post-translational modification of the FDRG protein. Such a genetic change can be detected. As described herein, there are a number of assay techniques known in the art that can be used to detect changes in the FDRG gene.
Preferred biological samples are serum samples that have been routinely isolated from a subject.

In some embodiments, the detection of the alteration is accomplished by polymerase chain reaction (PCR) such as anchor PCR or RACE PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,202), or alternatively, by ligation chain reaction (LCR) ( For example, Landegran et al. (1988) Scie
241: 1077-1080; and Nakazawa et al. (1994) PNAS 91: 360-364), the latter of which are particularly useful for detecting point mutations in the FDRG gene. May be useful (Abravaya et al. (1995) Nucleic
Acids Res. 23: 675-682). This method involves collecting a sample of cells from a patient,
Isolating nucleic acids (eg, genomic, mRNA, or both) from the cells of the sample;
Contacting the nucleic acid sample with one or more primers that specifically hybridize to the FDRG gene under conditions such that hybridization and amplification of the FDRG gene occurs, if any, and whether to detect the presence or absence of the amplification product Or detecting the size of the amplification product and comparing its length to a control sample. It is anticipated that PCR and / or LCR may be desirable to use as a preliminary amplification step with any of the techniques used to detect mutations described herein.

Another amplification method is: self-sustained sequence replication (Guatelli, JC et al., 1990, Proc
Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (Kwoh, DY et al., 1989, Proc. Natl. A.
cad.Sci.USA 86: 1173-1177), Q-β replicase (Lizardi, PM et al., 1988, Bio / Te
chnology 6: 1197) or any other nucleic acid amplification method followed by detection of the amplified molecule using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when they are present in very small numbers.

[0203] In another embodiment, mutations in the FDRG gene from a sample cell can be identified by an altered restriction enzyme cleavage pattern. For example, sample and control DNA is isolated, (optionally) amplified, digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length size between the sample and the control DNA indicate a mutation in the sample DNA. In addition, the use of sequence-specific ribozymes (see, eg, US Pat. No. 5,498,531) can be used to score for the presence of a particular mutation due to the occurrence or loss of a ribozyme cleavage site.

In another embodiment, identifying genetic mutations in FDRG by hybridizing sample and control nucleic acids, such as DNA or RNA, to a high density array containing hundreds or thousands of oligonucleotide probes. it can(
Cronin, MT et al. (1996) Human Mutation 7: 244-255; Kozal, MJ et al. (1996) Nature Med
icine 2: 753-759). Light generation as described above, e.g., Cronin, MT
Gene mutations in FDRG can be identified in two-dimensional arrays containing (light-generated) DNA probes. Briefly, a primary hybridization array of probes can be used to scan long distances of DNA in samples and controls and identify base changes between sequences by creating a linear array of consecutive overlapping probes. it can. This step allows point mutations to be identified. This step is followed by a secondary hybridization array that can characterize a particular mutation by using a smaller, specialized probe array that is complementary to all the mutants or mutations detected. Each mutant array is composed of a corresponding set of probes, one complementary to the wild-type gene and one complementary to the mutant gene.

In yet another embodiment, the FDRG gene is sequenced directly and various sequences known in the art for detecting mutations by comparing the sequence of the sample FDRG to the corresponding wild-type (control) sequence. Any of the Sing reactions can be used. Examples of sequencing reactions are described in Maxim and Gilbert ((1977) PNAS
74: 560) or those based on techniques developed by Sanger (Naeves et al. (1977) PNAS 74: 5463). When performing a diagnostic assay ((1995) Biotechniques 19: 448), sequencing by mass spectrometry (for example, PCT International Publication No. WO 94/16101)
No .; Cohen et al. (1996) Adv.Chromatogr. 36: 127-162; and Griffin et al. (1993) Appl.
m. Biotechnol. 38: 147-159), and any of a variety of automated sequencing methods could be used.

[0206] Another method for detecting mutations in the FDRG gene is RNA / RNA or RNA / DNA.
Includes methods using protection from cleavage agents to detect mismatched bases in heteroduplexes (Myers et al. (1985) Science 230: 1242). In general, the art of "mismatch cleavage" consists in hybridizing (labeled) RNA or DNA containing the wild-type FDRG sequence to potentially mutant RNA or DNA obtained from a tissue sample. Begin by giving the heteroduplex that is formed. Treat the double stranded duplex with an agent that cleaves the single stranded region of the duplex as present due to base pair mismatch between the control and sample strands. For example, RNA / DNA duplexes can be treated with RNase to enzymatically digest mismatched regions, and DNA / DNA hybrids can be treated with S1 nuclease. In another embodiment, either the DNA / DNA or RNA / DNA duplex can be treated with hydroxylamine or osmium tetroxide and with piperidine to digest the mismatched region. After digestion of the mismatched regions, the resulting material is then separated by size on a denaturing polyacrylamide gel to determine the site of mutation. For example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85: 4397; Saleeba et al. (1992) Methods En.
zymol.217: 286-295. In a preferred embodiment, control DNA or RNA can be labeled for detection.

In yet another aspect, the mismatch cleavage reaction was obtained from a sample of cells.
Use one or more proteins that recognize mismatched base pairs in double-stranded DNA in certain systems (the so-called "DNA mismatch repair" enzymes) to detect and map point mutations in FDRG cDNA . For example, the Escherichia coli mutY enzyme cleaves A at a G / A mismatch, and thymidine DNA glycosylase from HeLa cells cleaves T at a G / T mismatch (Hsu et al. (1994) Carcinogene).
sis 15: 1657-1662). According to an exemplary embodiment, a probe based on a FDRG sequence, eg, a wild-type FDRG sequence, is used to convert cDNA or other DN from a test cell (one or more).
Hybridize to A product. This duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols and the like. See, for example, U.S. Patent No. 5,459,039.

In another embodiment, alterations in electrophoretic mobility are used to identify mutations in the FDRG gene. For example, single-stranded structural polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989
) Proc Natl. Acad. Sci USA: 86: 2766, and Cotton (1993) Mutat Res 285: 125-144;
And Hayashi (1992) Genet Anal Tech Appl 9: 73-79). The single-stranded DNA fragment of the sample and control FDRG nucleic acids is denatured and regenerated. The secondary structure of single-stranded nucleic acids varies with sequence, and changes occurring in electrophoretic mobility can detect even single base changes. The DNA fragment can be labeled or detected with a labeled probe. The use of RNA (rather than DNA) can increase the sensitivity of the assay, in which case the secondary structure is more sensitive to sequence changes. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7 :Five).

In yet another embodiment, the migration of mutant or wild-type fragments in polyacrylamide gels containing denaturing agent gradients is determined by denaturing gradient gel electrophoresis (DGGE).
(Myers et al. (1985) Nature 313: 495). When DGGE is used as a method of analysis, it is modified to ensure that the DNA is not completely denatured, for example by adding a GC clamp of approximately 40 bp of high melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used instead of a denaturing gradient to identify differences in mobility between control and sample DNA (Rosenbaum and Rei
ssner (1987) Biophys Chem 265: 12753).

[0210] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification or selective primer extension. For example, an oligonucleotide primer can be prepared in which the known mutation is centered, and then hybridized to the target DNA under conditions that allow hybridization only when a perfect match is found (Sa
(1986) Nature 324: 163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86: 6230)
. When such allele-specific oligonucleotides bind to the hybridizing membrane and hybridize to the labeled target DNA, they hybridize to the PCR-amplified target DNA or a number of different mutations. .

Alternatively, allele-specific amplification techniques that rely on selective PCR amplification can be used with the present invention. Oligonucleotides used as primers for specific amplification are located at the center of the molecule (Gibbs et al. (1989) Nucleic Acids Res. 17: 2437-244, so that amplification depends on differential hybridization).
8) or at the most 3 'end of one primer where mismatches can prevent or reduce polymerase extension (Prossner (1993) Tibtec
h 11: 238) Can carry the desired mutation. In addition, it may be desirable to introduce a new restriction site in the region of the mutation to provide cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6: 1). In one embodiment, it is expected that amplification may be performed using Taq ligase for amplification (Barany (
1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation will occur only if there is a perfect match at the 3 'end of the 5' sequence, and the presence of a known mutation can be detected at a particular site by checking for amplification. .

For example, by utilizing a prepackaged diagnostic kit comprising at least one probe nucleic acid or antibody reagent described herein, the methods described herein can be performed, For example, they can be conveniently used in a clinical setting to diagnose patients exhibiting symptoms or a family history of a disease or disorder involving the FDRG gene.

Further, any cell type or tissue in which FDRG is expressed can be utilized in the prognostic assays described in the present invention. 3. Monitoring effects during clinical trials Effects of agents (eg, drugs, compounds) on FDRG expression or activity (eg, activation of FDRG-dependent signaling pathways; regulation of angiogenesis or cell proliferation; cells Monitoring of migration or recruitment; or modulation of hematopoietic response) can be applied not only in basic drug screening, but also in clinical trials. For example, as described herein, increasing FDRG gene expression, protein levels or up-regulating FDRG activity in a subject in a clinical trial showing reduced FDRG gene expression, protein levels or down-regulated FDRG activity. The effectiveness of the agent as determined by such screening assays can be monitored. Alternatively, increased FDRG gene expression,
Monitor efficacy of agents determined by screening assays that reduce FDRG gene expression, protein levels, or down-regulate FDRG activity in clinical trials of subjects with protein levels or up-regulated FDRG activity can do. In such clinical trials, the expression or activity of FDRG and, preferably, for example, other genes involved in proliferative disorders, can be used as a "indicator" or marker of the proliferation of particular cells.

For example, but not by way of limitation, cells may be treated by treatment with an agent (eg, a compound, drug or small molecule) that modulates FDRG activity (eg, identified in a screening assay as described herein). FDRG and other genes that are regulated in can be identified. Thus, for example, in a clinical trial to study the effect of an agent on a proliferative, developmental or hematopoietic disorder,
Cells can be isolated, RNA prepared and analyzed for levels of expression of FDRG and other genes involved in proliferative, developmental or hematopoietic disorders, respectively. As described herein, by measuring the amount of protein produced, either by Northern blot analysis or RT-PCR, or also by any of the methods as described herein, or by FDRG or other By measuring the level of gene activity, the level of gene expression (ie, gene expression pattern)
Can be determined. Thus, the gene expression pattern can be used as a marker that indicates the physiological response of the cell to the agent. Thus, this response state can be determined before and at various points during the treatment of the individual with the agent.

In a preferred embodiment, the present invention provides (i) obtaining a pre-dose sample from a subject prior to administration of the agent; (ii) detecting the level of expression of FDRG protein, mRNA or genomic DNA in the pre-dose sample. (Iii) obtaining one or more post-dose samples from the subject; (iv) detecting the level of expression or activity of FDRG protein, mRNA or genomic DNA in the post-dose sample; (v) the pre-dose sample Comparing the level of expression or activity of the FDRG protein, mRNA or genomic DNA in the sample or the plurality of samples after administration with the FDRG protein, mRNA or genomic DNA; and (vi) correspondingly administering the agent to the subject. Agents (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, subunits) comprising altering the administration. Or the efficacy of the treatment of the subject with the other drug candidate) identified by a screening assay described herein provides a method of monitoring. For example, increased administration of an agent may be desirable to increase the expression or activity of FDRG to higher levels than detected, ie, to increase the effectiveness of the agent. Alternatively, reduced administration of an agent may be desirable to reduce the expression or activity of FDRG to lower levels than that detected, ie, to reduce the effectiveness of the agent. With such an aspect,
Even in the absence of a discernable phenotypic response, FDRG expression or activity can be used as an indicator of agent efficacy.

C. Methods of Treatment : The present invention provides both prophylactic and therapeutic methods of treating a subject at risk for (or predisposed to) or having a disease associated with abnormal FDRG expression or activity. I will provide a. For both prophylactic and therapeutic methods of treatment, such treatments can be specifically tailored or modified based on the knowledge gained from the field of pharmacogenomics. As used herein, "pharmacogenomics" refers to the application of genomics techniques such as gene sequencing, statistical genetics and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers to a study of how a patient's gene determines his or her response to a drug (eg, a patient's "drug response phenotype" or "drug response genotype"). Point out. Accordingly, another aspect of the present invention provides a method for tailoring prophylactic or therapeutic treatment of an individual using either a FDRG molecule or a FDRG modulator of the present invention according to the drug response genotype of the individual. Pharmacogenomics allows clinicians or physicians to direct prophylactic or therapeutic treatment to patients who will most benefit from treatment and avoid treatment of patients who will experience toxic side effects associated with the drug can do.

[0217] 1. Prophylactic Methods In one embodiment, the present invention provides that an agent that modulates FDRG expression or at least one FDRG activity is administered to the subject to cause abnormal FDRG in the subject.
Methods are provided for preventing a disease or disorder associated with expression or activity. Subjects at risk for a disease caused or caused by abnormal FDRG expression or activity can be identified, for example, by any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the onset of symptoms characterized by FDRG abnormalities, so as to prevent or slow the progression of the disease or disorder. Depending on the type of FDRG abnormality, for example, a FDRG agonist or FDRG antagonist agent can be used to treat the subject. Suitable agents can be determined based on the screening assays described herein.

[0219] 2. Therapeutic Methods Another aspect of the present invention relates to methods of modulating FDRG expression or activity for therapeutic purposes. The modulation methods of the invention include contacting a cell with an agent that modulates one or more FDRG protein activities associated with the cell. The agent that modulates FDRG protein activity can be an agent as described herein, such as a nucleic acid or a reservoir, a naturally occurring target molecule of the FDRG protein, a peptide, a FDRG peptidomimetic or other small molecule. It may be. In one embodiment,
An agent stimulates one or more FDRG protein activities. Examples of such stimulants include active FDRG proteins and nucleic acid molecules encoding FDRG that have been introduced into cells. In another embodiment, the agent comprises one or more FDRGs
Inhibits activity. Examples of such inhibitors include antisense FDRG nucleic acid molecules and anti-F
Contains DRG antibodies. These modulating methods can be performed in vitro (eg, by culturing cells with the agent) or alternatively in vivo (eg, by administering the agent to a subject). As such, the invention provides a method of treating an individual afflicted with a disease or disorder characterized by abnormal expression or activity of a FDRG protein or nucleic acid molecule. In one embodiment, the method comprises an agent (eg, an agent identified by a screening assay described herein) or an agent that modulates (eg, up-regulates or down-regulates) FDRG expression or activity. Administering a combination of substances. In another embodiment, the method comprises administering a FDRG protein or nucleic acid molecule as a treatment to compensate for reduced or abnormal FDRG expression or activity.

A preferred embodiment of the present invention includes a method of treating a disease or disorder associated with a FDRG protein comprising administering to a subject a therapeutically effective amount of an antibody against the FDRG protein. As defined herein, a therapeutically effective amount of an antibody (ie, an effective dose) is from about 0.001 to 30 mg / kg body weight, preferably from about 0.01 to 25 mg / kg body weight,
More preferably about 0.1-20 mg / kg body weight, and even more preferably about 1-10 mg / kg
, 2-9 mg / kg, 3-8 mg / kg, 4-7 mg / kg or 5-6 mg / kg body weight. One of skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatment, the general health and / or age of the subject, and other diseases present, may effectively affect the subject. Recognize that it may affect the dosage required to treat. Further, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments. As a preferred example, an antibody in the range between about 0.1-20 mg / kg body weight, once a week, about 1
The subject is treated for 〜10 weeks, preferably for 2-8 weeks, more preferably for about 3-7 weeks, and even more preferably for about 4,5 or 6 weeks. It is also understood that the effective dosage of the antibody used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may be due to the results of a diagnostic assay as described herein.

In situations where FDRG is abnormally down-regulated and / or increased FDRG activity appears to have a beneficial effect, stimulation of FDRG activity is desirable. One example of such a situation is when the subject has a disease characterized by an abnormal proliferative disease (eg, cancer). Yet another example of such a situation is where the subject has a disease characterized by an abnormal hematopoietic response. Yet another example of such a situation is where the subject has a disease characterized by abnormal differentiation or development or abnormal cell migration.

[0221] 3. Pharmacogenomics To treat (prophylactically or therapeutically) a disease associated with abnormal FDRG activity (eg, a proliferative or developmental disease), an FDRG molecule of the invention as well as an agent or described herein. FDRG activity as identified by screening assays (
For example, a modulator having a stimulatory or inhibitory effect on FDRG gene expression) can be administered to an individual. With such treatment, pharmacogenomics (ie, a study of the relationship between an individual's genotype and the individual's response to a foreign compound or agent) can be considered. Differences in therapeutic drug metabolism can lead to severe toxicity or treatment failure by altering the relationship between pharmacologically active drug dose and blood concentration. Accordingly, a physician or clinician may determine whether to administer a FDRG molecule or FDRG modulator and customize the dosage and / or treatment regimen for treatment with the FDRG molecule or FDRG modulator.
One can consider applying the knowledge gained from the relevant pharmacogenomic studies.

[0222] Pharmacogenomics deals with clinically significant genetic changes in the response to drugs due to altered drug predisposition and abnormal effects in patients. For example, Eichelbaum, M
., Clin Exp Pharmacol Physiol, 1996, 23 (10-11): 983-985 and Linder, MW, Clin
Chem, 1997, 43 (2): 254-266. In general, two types of pharmacogenomic diseases can be distinguished. A genetic disease transmitted as a single factor that alters the way drugs act on the body (altered drug action) or multiple singles that alter the way the body acts on drugs Genetic disorders transmitted as factors (unusual drug metabolism). These pharmacogenomic diseases can occur either as rare genetic defects or as naturally occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary enzyme deficiency,
In that case, the main clinical complications are haemolysis after ingestion of oxidizing agents (antimalarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomic method for identifying genes that predict drug response, known as “genome-wide association”, is primarily based on:
A high-resolution map of the human genome consisting of already known gene-related markers (eg,
"Biallelic" genetic marker map consisting of 60,000 to 100,000 polymorphisms or variable sites on the human genome, each of which has two variants. Markers associated with specific observed drug responses or side effects by comparing such a high resolution genetic map to a genomic map of each of a statistically significant number of patients participating in phase II / III drug trials Can be identified. Alternatively, such high resolution maps can be generated from combinations of about 10 million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, "SNP" is a common change that occurs at a single nucleotide base in a stretch of DNA. For example, a SNP can occur once for every 1000 bases of DNA. SNPs may be involved in the disease process, however, the majority may not be associated with the disease. Given a genetic map based on the occurrence of such SNPs, individuals can be categorized into gene categories according to the particular pattern of SNPs in their individual genomes. In that way, a treatment plan can be tailored for a group of genetically similar individuals, taking into account the traits that may be common between such genetically similar individuals.

Alternatively, a method called “candidate gene method” can be used to identify a gene that predicts a drug response. By this method, if the gene encoding the drug target is known (eg, the FDRG protein or FDRG receptor of the invention), all common variants of that gene can be identified quite easily in the population, One can then determine whether having one type of the gene for another is associated with a particular drug response.

In an exemplary embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of gene polymorphisms for drug metabolizing enzymes (eg, N-acetyltransferase 2 (NAT2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) may indicate the expected drug effect after taking a standard and safe dose of drug. An explanation is given as to why unacceptable or excessive drug reactions and severe toxicity may occur. These polymorphisms have two phenotypes in the population, the extensive metabolites (extensi
ve metabolizer (EM) and poor metabolizer (PM). PM
Prevalence varies among distinct populations. For example, the gene encoding CYP2D6 is very polymorphic, and several mutations have been identified in PM, all of which result in a lack of functional CYP2D6. Minor metabolites of CYP2D6 and CYP2C19 very frequently experience excessive drug response and side effects when given standard doses. If the metabolite is an effective therapeutic component, PM does not show a therapeutic response, as shown for the analgesic effect of codeine provided by its metabolite morphine formed by CYP2D6. The other extreme is the so-called ultra-rapid metabolite, which does not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified as being due to CYP2D6 gene amplification.

Alternatively, a method called “gene expression profiling” can be used to identify genes that predict drug response. For example, gene expression in an animal that has been administered an agent (eg, an FDRG molecule or FDRG modulator of the present invention) can provide an indication of whether a gene pathway associated with toxicity is operating.

[0227] Information from one or more of the above pharmacogenomic methods can be used to determine the appropriate dosage and treatment regimen for prophylactic or therapeutic treatment of the individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failures, and is thus identified by FDRG molecules or any of the exemplary screening assays described herein. Treatment of a subject with a FDRG modulator, such as a modulator, can increase therapeutic or prophylactic efficacy.

The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

[0229] EXAMPLES Example 1: Isolation and characterization of cDNAs for the human FDRG This example describes the isolation and characterization of the gene encoding the human FDRG (also referred to as "FDRG").

Isolation of cDNA for human FDRG Human aortic endothelial cDNA library (human aortic endo)
human FDRG cDNA was isolated from the same cDNA library. To construct the library, three micrograms of polyA + RNA were isolated from human aortic endothelial cells, reverse transcribed, and Superscript cDNA Synthesis Kit ^™ (Gib
co BRL; Gaithersburg, MD) was used to synthesize a cDNA library. The complementary DNA is SalI in the polylinker.
And directionally cloned into the expression plasmid pMET7 using the NotI site to construct a plasmid library. Transformants are removed (pic
ked), and single-pass sequence
amplifying). In addition, human aortic endothelial cDNA was
Λ linked to the SalI / NotI site of OX ^TM vector (Gibco BRL)
A phage cDNA library was constructed.

A partial cDNA clone encoding FDRG was identified from the above cDNA library using the following method. First, each sequence was checked to determine if it was a bacterial, ribosome, or mitochondrial contaminant. Such sequences were excluded from further analysis. Second, sequence artifacts, such as vectors and repetitive elements, were masked and / or removed from each sequence. Third, the remaining sequences are stored in the BLASTN ^™ program (BLASTN1
. 3MP: Altschul et al. , J. et al. Mol. Bio. 215: 4
03, 1990) against a copy of the GenBank nucleotide database. Fourth, the sequence translates the nucleic acid sequence of all six frames and obtains it from the available protein databases (BLASTX1.3MP).
: Altschul et al. , Supra) against a non-redundant protein database by the BLASTX ^™ program. This protein database is Swiss-
Combination of Prot, PIR and NCBI GenPept protein databases.

The original first pass sequence of the partial cDNA clone (original first)
pass sequence) showed homology to angiopoietin 2 using the BLASTX ^™ program. Additional screening of the phage library allows for a full length clone of human FDRG (full length cl).
one) was isolated. The nucleotide sequence and predicted amino acid sequence are shown in FIG. 1 (corresponding to SEQ ID NO: 1 and SEQ ID NO: 2, respectively). The FDRG protein (corresponding to amino acids 1-406 of the predicted amino acid sequence, SEQ ID NO: 2) has a 31.0% identity to human ficolin
ty) and 29.0% identity to human angiopoietin 2 (see FIG. 3). Percent identity is determined by the Needleman-Wunsch alignment algorithm.
um), Needlman and Wunsch (1970) J. Am. Mol. B
iol. 48: 444-453, which incorporates GCG (Wisconsin).
Calculated using a gap weight of 12 and a length weight of 4 using the Gap program from the Genetics Computer Group.

This human FDRG protein has a C-terminal fibrinogen-like domain (corresponding to amino acids 186-399 of the predicted amino acid sequence, SEQ ID NO: 2 and amino acids 161-376 of SEQ ID NO: 5) and a signal sequence (expected). (Corresponding to amino acids 1-25 of the amino acid sequence, SEQ ID NO: 2), which is cleaved to form the mature FDRG protein (corresponding to amino acids 1-381 of SEQ ID NO: 5).

Example 2 Distribution of FDRG mRNA in Human Tissue Northern Blot Analysis Northern blot hybridization was used to analyze FDRG expression. For analysis of human FDRG, the entire FDRG insert was converted to SalI / N
Following otI digestion, it was isolated from the plasmid and used as a probe. The probe DNA was purchased from Prime-It kit ^™ (Strata) according to the supplier's instructions.
tagene, La Jolla, CA) were radioactively labeled with ³² P-dCTP using. Filters containing human mRNA (Clontech, Palo A
lto, CA; MTNI and MTNII) from an ExpressHyb ^™ hybridization solution (Clontech).
obed and high stringency according to the manufacturer's recommendations
Gency).

[0235] Approximately 2.0 kb FDRG transcript is the only exception for peripheral blood leukocytes with all examined tissues (spleen, thymus, prostate, testis, ovary, small intestine, colon, heart, brain, placenta, Lung, liver, skeletal muscle, kidney and pancreas) at various levels. The highest levels of FDRG expression were observed in the placenta.

Example 3 Screening of Mouse Tissue for FDRG Binding Site This example demonstrates mouse embryos for the T115 binding site.
2 illustrates the use of cell supernatants containing AP-hFDRG for screening tissue sections of yos) and whole mice.

Screening of tissue sections with supernatants containing alkaline phosphatase (AP) fusion protein was performed as described in Cheng and Flagan (1994) Cell.
79: 157-168, as described. In short, mouse embryos (
14.5 sunrise prenatal development, E14.5) and whole mice
Fresh frozen sections (8 μm) of (1.5 days after birth, P1.5) were made and HB
HA (Hank's balanced salt supplemented with 20 mM Hepes, pH 7, 0.05% BSA and 0.1% sodium azide)
solution)). Tissue sections were then incubated for 1 hour at room temperature with supernatant containing AP-hFDRG (alkaline phosphatase fused to the N-terminus of human FDRG) or AP at a concentration of 5 nM. After incubation, the tissue sections were washed six times in HBHA, 60% acetone, 3%
Fix in a solution containing formaldehyde and 20 mM Hepes, pH 7.5, wash 3 times in HBS (20 mM Hepes, pH 7.5, 150 mM NaCl) and heat at 65 ° C. for 30 minutes to inactivate endogenous alkaline phosphatase activity It has become. The bound AP fusion protein was added to a BCIP / NBT substrate solution (1
00 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl,
Detected by developing sections in 0.17 mg / ml BCIP and 0.33 mg / ml NBT).

[0238] AP-hFDRG was expressed in epidermis (epi) in both E14.5 and P1.5 sections.
dermis) and adipose tissue of P1.5 sections.
), But not the AP protein. It was also observed that APhFDRG bound but not AP to epidermis and adipose tissue of adult mice. These results suggest that cells in the epidermis and adipose tissue harbor the binding site for FDRG.

Example 4: Isolation and characterization of murine FDRG cDNAs. Murine FDRG cDNA (also referred to herein as "BK89") was
Cloning was performed by the subtractive hybridization method based on R. NIH
3T3 fibroblasts were stably infected with a virus having pBABE-PPARγ or a virus having an empty pBABE vector. Two cell lines were purified at 10 μg / ml pioglitazone (pio
glitazone), treated with PPARγ ligand for 3 hours, and Pol
yA + RNA was isolated from the cells. The double-stranded cDNA resulting from the reverse transcription is then digested and modified modified representational analysis protocols (modified rep)
presentational analysis proto
col) -based subtractive hybridization and multiple rounds of PCR amplification. Next, the subtracted library was differentiated from a differentially regulated gene.
Screened by slot blot analysis for the rated gene and candidates were confirmed by Northern blotting. FDRG was one of the first ten or so clones to be isolated from this screen.

The full-length clone of murine FDRG was 3
T3-F442A adipocyte λZAPII cDNA library (3T3-F44
2A adipocyte λZAPII cDNA library). An open reading frame encoding 410 amino acid residues was identified. A hydrophobic sequence is present in the N-terminal region of murine FDRG, which is consistent with a secretory signal peptide. A search for sequence homology was performed using the murine FDRG C
The terminal half (C-terminal half) is an angiopoietin 1,2
, Fibrinogen α, β and γ chains, tenascin, ficollin and pT49, indicating strong similarity to a family of proteins sharing a so-called fibrinogen-like motif. N-terminal half (N-termi
nal Half) is expected to form a coiled-coil domain. The murine FDRG protein has three potential N-glycosylation sites (potentia).
It also contains two cysteines that may be available for lN-glycosylation sites) and intramolecular disulfide bonds.

Example 5: Secretion of murine FDRG from COS7 cells Vectors encoding untagged murine FDRG and tagged murine FDRG were constructed as follows. Briefly, forward primer and reverse primer (fo
backward and reverse primers) and PCR
Used to amplify the full length open reading frame of FDRG from pBluescriptSK (+/-). The 5 'primer is a native 5' untranslated region (untransl) upstream of the initiator methionine.
The incorporated region is incorporated and the 3 'primer comprises a nucleotide sequence encoding a YPYDVPDYA (hemagglutinin, HA) epitope (SEQ ID NO: 26) followed by a stop codon.

[0242] For untagged FDRG, the following primers were used:

5 ′ Primer-AATTAACCCTCACTAAAGGG (SEQ ID NO: 18) 3 ′ Primer-ACGCGTCGACTAATACGACTCACTATA GGGCG (SEQ ID NO: 19) The following primers were used in the tagged FDRG.

5'primer-AATTAACCCTCACTAAAGGG 3'primer-TACGCGTCGACCTAAGCGTAGTCTGGGACGTC (SEQ ID NO: 20) GTATGGGGTAAGAGGCTCTGTAGCCTC CAT (SEQ ID NO: 21)
It was cloned into I / SalI cut pcDNA3.

HA-tagged murine FDRG (in pcDNA) was transformed into COS using transient transfection.
The protein was expressed in 7 cells and detected in conditioned media by immunoblotting. To increase the visibility of the signal, the conditioned medium was 10 kDa
Load by centrifugation on MW cut-off spin column (MSI)
before ing). Epitope-tagged proteins produced by cells have slightly higher molecular weights than in vitro translates, and are inconsistent with post-translational modifications such as glycosylation. Not observed.

Example 6: Generation and characterization of epitope-tagged FDRG protein Expression of epitope-tagged human FDRG Human FDRG flag epitope-tagged protein (huFDRG: flag) vector (human FDRG flag epitope-tagg)
The ed protein (huFDRG: flag) vector was constructed by PCR followed by ligation to an expression vector, pMET stop. The full-length open reading frame incorporates a Kozak sequence upstream of the initiator methionine and a 5 'primer containing the initiator methionine, and a nucleotide sequence encoding the DYKDDDDK flag epitope (SEQ ID NO: 23) and PCR amplification was performed using the 3 'primer containing the subsequent termination code. The primer sequences are shown below.

5 'primer: 5' TTTTTCAATTGACCGCCATGAGCGG TGCTCCGACG3 '(SEQ ID NO: 24) 3' primer: 5 'TTTTTGTCGACTTATCACTTGTCGTc GTTCTC e. ) (GIBCO / BRL)
Short transfected HEK 293T cells in mm plates.
72 hours after transfection, serum-free conditioned medium (serum-
free conditioned medium) (OptiMEM, Gib
(co / BRL) was collected and centrifuged.

The conditioned medium was purified by SDS-PAGE on a 4-20% gradient gel followed by PVD.
Electroblotting on F membrane (Novex) and probing with M2 anti-flag polyclonal antibody (1: 500, Sigma) followed by horseradish peroxidase (HRP) conjugated sheep anti-mouse antibody (1: 5000, Am
probed with a chemiluminescent reagent (Renaissance, D.
and an autoradiograph film (Bi).
omax MR2 film, Kodak). Flag immunoreactivity was measured using MultiMark molecular weight markers (Novex) by SDS-PAGE.
As a major band migrated to an apparent molecular weight of 42 kDa as determined by

Expression of Epitope-Tagged Murine FDRG A murine HA-tagged FDRG expression construct was made as described in Example 5. The DNA construct was sequenced and then HEK in 150 mm plates using Lipofectamine (GIBCO / BRL) according to the manufacturer's protocol.
293T cells were transiently transfected. 72 of transfection
After hours, conditioned medium without serum (OptiMEM, Gibco / BRL) was collected and spun.

The conditioned media was separated by SDS-PAGE on a 4-20% gradient gel followed by PVD
F on a membrane (Novex) and the anti-HA monoclonal antibody, HA. 11 (1: 500, Babco) followed by H
Probed with RP-conjugated sheep anti-mouse antibody (1: 5000, Amersham), developed using chemiluminescent reagents (Renaissance, Dupont), and autoradiographed (Biomax MR2 film)
, Kodak). HA immunoreactivity was determined by MuS by SDS-PAGE.
Appeared as the main band migrated at 60 kDa as determined by the ltiMark marker (Novex).

A second, weaker band was seen at 42 kDa.

Mock transfected supernatants. Transfecting 293T cells with a pMET stop vector without insert insert results in mock supernatants.
) Manufactured. Seventy-two hours after transfection, conditioned medium without serum (OptiMEM, Gibco / BRL) was collected and spun. Conditioned media was electroblotted, developed and exposed as described above. No specific band was detected on the anti-flag or anti-HA blot.

Example 7 Characterization of Anti-Human TANGO115 Polyclonal Antibody A synthetic peptide corresponding to the C-terminal of human TANGO115 was prepared according to the following sequence. KTWRRGRYYPLQATTMLIQPMAA (SEQ ID NO: 27). This is converted to KLH (keyhole limpet hemocyanin (key) at the N-terminal lysine.
hole limpet hemocyanin (conjuga)
ted), and standard immunization protocols (eg, Harlow and L
ane, 1987) were used to inject two rabbits for the production of polyclonal antisera.

A synthetic peptide corresponding to the N-terminus of human TANGO115 was prepared using the following sequence. PVQSKSPRFASWDEMN (SEQ ID NO: 28). This is referred to as the MAP core at C-terminal asparagine (Research Genetics, Ro
ckville MD). It was injected into two rabbits for production of a polyclonal antiserum using a standard immunization protocol (eg, Harlow and Lane, 1987).

Antiserum samples were taken 10 weeks after the first immunization. Immunoreactivity was tested by ELISA against the immunizing peptide and pre-immune serum bleeding (pre-
immune serum bleeds). Recombinant human FDRG: flag and mouse FDRG: HA protein supernatants and mock transfected (mock transfected) prepared as described above.
ed) Replicate samples of the negative control supernatant were tested on SDS-PAGE and electroblotted onto PVDF membrane (Millipore). A 1: 1000 dilution of all four rabbit antisera was used for the replication blot as the first antiserum, followed by an anti-rabbit HRP-conjugated secondary antibody. Blots were run on a chemiluminescent reagent (Renaisense, Dupont).
t) and autoradiographic film (Biomax)
(MR2 film, Kodak).

The immunospecificity of the rabbit antiserum was tested by standard Western blot. Antisera from two rabbits immunized with the C-terminal peptide were labeled with a C-terminally tagged human FDRG that was also immunoreactive with the anti-flag M2 antibody in a replication blot.
The band in the lane containing the flag was labeled. The approximate molecular weight of this band was determined using the MultiMark standard (Novex) to be 40 (+/-
5) It was kDa. This band was not seen in lanes containing supernatants from mock or mouse FDRG HA transfected cells.

Antisera from rabbits immunized with the N-terminal peptide were compared to human or mouse FDR when compared to lanes containing mock transfected samples.
No specific band was shown in the lane containing any of the G proteins. This indicates that these antibodies do not interact with the FDRG protein by Western blot analysis.

Example 8: Tissue expression of murine FDRG Total RNA was obtained from 9 week old male C57BKFJ mice using mouse liver, muscle, heart, spleen, white fat, brown fat, kidney, testis, brain, And isolated from lung tissue samples and analyzed for FDRG expression by Northern blotting. The tissue distribution of FDRG mRNA is highly adipose tissue selective, with at least 10-20 in white and brown fat relative to the other tissues examined.
Expression was twice as high. Low levels of expression could be detected in other tissues (eg, liver), which may be due to fat contamination.

Example 9 In Vitro Regulation of Murine FDRG in PPARγ-Expressing Cells NIH3T3 fibroblasts stably expressing PPARγ were treated with 10 μg / ml pioglitazone for 0, 2, and 4 hours. Northern blot analysis performed on cellular RNA shows that FDRG mRNA is hardly detected in untreated cells, but undergoes at least a 10-fold increase within 2 hours after pioglitazone administration.
Suppression of protein synthesis by pretreatment with 5 μg / ml cycloheximide does not prevent this induction. Cycloheximide itself is often associated with immediate early genes and is a phenomenon commonly attributed to increased mRNA stability, FD
Causes an increase in RG mRNA. The increase in FDRG message levels caused by pioglitazone and cycloheximide is additive, possibly reflecting an independent regulatory mechanism.

Example 10: Bioregulation of murine FDRG in Zucker diabetic fat rats Male Zucker diabetic (fa / fa) rats
ic fatty (fa / fa) rats) at 6 mg / k mixed with food
g / day troglitazone, which treatment is 6 g / day.
Departed at the age of week and continued for up to 26 weeks. By this time, untreated littermates (li
termates) cause some hyperglycemia and pancreatic β-cell dysfunction, while the treated animals have normoglycemia (normogl).
cemic). Northern blots were performed on RNA isolated from white adipose tissue of 5 treated and untreated animals from each group. In vivo FDRG expression was observed to be consistently upregulated 3-5 fold in treated animals compared to controls. Glut4mR
NA has also been found to be increased, consistent with reports published by other groups. Expression of the control mRNA, 36B4, was not affected by troglitazone treatment.

Example 11: Regulation of FDRG in a murine model of obesity 9 week old male obese (ob / ob) and diabetic (db / db) mice (obes)
FDRG expression was examined in white and brown adipose tissue from e (ob / ob) and diabetic (db / db) mice). Northern analysis showed that in both white and brown fat, lean controls of the congenic lines of each of the ob / ob and db / db animals.
ols) compared to FDRG mRNA in ob / ob and db / db animals
Showed a uniform rise. This may reflect the correlation between obese conditions and increased FDRG expression in general, and raises the possibility that FDRG may contribute to some of the pathophysiological features of such conditions. Has raised. Alternatively, FDRG mRNA levels indicate genetic defects specific to these two models, namely leptin and the leptin receptor (leptin re).
may be directly or indirectly regulated by the Example 12: Regulation of murine FDRG by nutrition and leptin administration Lean mice were divided into three experimental groups and fed food ad libitum or
Food fasted for 7.5 hours or fasted for 7.5 hours
ing) (4.5 hours). Animals were killed at the end of 12 hours. Northern blots show a 2-3 fold increase in FDRG mRNA in white adipose tissue after a short period of fasting, which is later prevented by feeding. This suggests that FDRG levels are responding to acute metabolic changes in vivo.

In the second study, 10 week old lean or ob / ob mice were divided into three groups. The first group was controls and was intraperitoneally injected twice daily with 1.5 ml of PBS. The second group was infused twice daily with 12.5 mg / kg leptin in PBS, with a concomitant reduction in dietary intake. A third group, the pair-fed group,
Diet was restricted to meet the dietary intake by the leptin injected group and PBS was also injected. Animals were killed at the end of the week. FD in brown fat
RG expression was seen to increase substantially with restriction of caloric intake, consistent with the findings in the first study, but not in leptin-treated animals. This may reflect a fundamental difference between leptin-induced dietary restrictions versus externally imposed restrictions, or additional downstream effects, such as usually reduced food intake May be consistent with leptin-mediated suppression of FDRG up-regulation that would result from E. coli.

Example 13: Tissue expression of human FDRG Northern blot analysis of RNA isolated from human placenta, heart, kidney, liver and adipose tissue samples shows high levels in white fat consistent with murine FDRG tissue expression data And high expression in the placenta. Placental expression is interesting.
Because it is known to undergo extensive vascular remodeling in adults (eg, during development during pregnancy) and in tissues involved in the transport of metabolic substrates for the fetus Because there is. Leptin, a secreted protein from other fats with important metabolic and endocrine functions, is also produced in large amounts in the placenta.

Example 14 Expression of Recombinant FDRG Protein in Bacterial Cells FDRG can be expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli, and the fusion polypeptide Can be isolated and characterized. In particular, FDRG is GST
And the fusion polypeptide is expressed in E. coli, eg, strain PEB199. Expression of the GST-FDRG fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide was derived from PEB19
Nine strains of crude bacterial lysate
es) is purified by affinity chromatography on glutathione beads. Polyacrylamide gel electrophoresis analysis of the polypeptide purified from bacterial lysate is used to determine the molecular weight of the resulting fusion polypeptide.

Example 15: Expression of recombinant FDRG protein in COS cells To express the FDRG gene in COS cells, Invitron C
pcDNA / Amp by corporation (San Diego, CA)
Use vectors. This vector contains the SV40 origin of replication, the ampicillin resistance gene, the E. coli origin of replication, the CMV promoter, followed by a polylinker region and the SV40 intron and polyadenylation site. An HA tag (Wilson et al. (1984) Cell 37: 767) fused in-frame to the 3 'end of the entire FDRG protein and fragment.
) Is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

To construct a plasmid, the FDRG DNA sequence is amplified by PCR using two primers. The 5 'primer contains about 20 nucleotides of the FDRG encoding sequence starting from the initiation codon, followed by the restriction site of interest, and the 3' end sequence is complementary to the other restriction sites of interest. Sequence, the translation stop codon, the HA tag, and the last 20 nucleotides of the FDRG coding sequence. The PCR amplified fragment and the pCDNA / Amp vector are digested with appropriate restriction enzymes and the vector is digested with the CIAP enzyme (New England).
d Biolabs, Berly, MA). The two restriction sites selected were such that the FDRG gene had the correct orientation (correct or
Preferably, they are different so that they are inserted in the event. The ligation mixture was transferred to E. coli cells (Stratagene Cloning System).
s, strains available from La Jolla, HB101, DH5a, SURE can be used), the transformed culture is plated on ampicillin medium plates, and resistant colonies are selected. Plasmid DNA is isolated from the transformants and tested by restriction analysis for the presence of the correct fragment.

Then, the COS cells were subjected to calcium phosphate or calcium chloride coprecipitation, DEAE
-Transfect with FDRG-pcDNA / Amp plasmid DNA using dextran-mediated transfection, lipofection or electroporation. Other suitable methods for transfecting host cells are described in Sambrook, J .; Fritsh, E .; F. , And Mania
tis, T .; Molecular Cloning: A Laboratory
Manual. 2nd, ed. , Cold Spring Harbor L
laboratory, Cold Spring Harbor Laboratory
ory Press, Cold Spring Harbor, NY, 1989.
Can be found in Expression of the FDRG protein was determined by radiolabeling (NEN,
³⁵ S-methionine or ³⁵ S-cysteine available from Boston, MA can be used) and by immunoprecipitation (Harlow, E. and La).
ne, D.E. Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY, 1988) using an HA-specific monoclonal antibody. Briefly, cells are labeled with ³⁵ S-methionine (or ³⁵ S-cysteine) for 8 hours. The culture medium is then collected and the cells are washed with detergent (RIPA buffer, 150 mM NaCl,
1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH7.
Dissolve using 5). Both cell lysate and culture medium are precipitated with an HA-specific monoclonal antibody. The precipitated protein is then analyzed by SDS-PAGE.

Alternatively, the DNA containing the FDRG coding sequence is cloned directly into the pCDNA / Amp vector polylinker using appropriate restriction sites. The resulting plasmid is transfected into COS cells as described above, and FD
RG protein expression is detected by radiolabeling and immunoprecipitation using an FDRG-specific monoclonal antibody.

Example 16: Retroviral delivery of FDRG (Retroviral D
elivery) The entire open reading frame of the FDRG is defined as Hawley et a.
l. (1994) Gene Therapy 1: 136-138, subcloning into the retroviral vector MSCVneo. The cells (293Ebna, Invitrogen) are then short-term transfected with the FDRG construct and a construct containing viral regulatory elements to produce a high titer retrovirus containing the FDRG gene. The virus is then used to transfect mice. These mice are then tested for gross pathology and for changes in biological response, eg, cell migration, using standard assays.

Equivalents The skilled artisan will recognize many equivalents to the specific embodiments of the invention described herein, or will be able to ascertain using only routine experimentation. Such equivalents are intended to be encompassed by the following claims.

[0271]

[Sequence list]

[Brief description of the drawings]

FIG. 1. cD of human FDRG (also called “hFDRG” or “hT115”)
Draw the NA sequence and deduced amino acid sequence. The nucleotide sequence corresponds to nucleic acids 1-1894 of SEQ ID NO: 1. The amino acid sequence corresponds to amino acids 1 to 406 of SEQ ID NO: 2.

FIG. 2 depicts the cDNA sequence and deduced amino acid sequence of mature human FDRG. The nucleotide sequence corresponds to nucleic acids 1-1143 of SEQ ID NO: 4. The amino acid sequence corresponds to amino acids 1 to 381 of SEQ ID NO: 5.

FIG. 3. Human FDRG (corresponding to amino acids 1 to 406 of SEQ ID NO: 2), mouse F
DRG (corresponding to amino acids 1-410 of SEQ ID NO: 12), human angiopoietin 2 (Genbank ™ accession number AF004327)
) (SEQ ID NO: 8), mouse angiopoietin 2 (Genbank [Genbank
] (TM) accession number AF004326) (SEQ ID NO: 9) and porphycoline (
Genbank accession number L12345 (SEQ ID NO: 1)
Draw multiple amino acid sequences of 0). The alignment is based on the megualine [Me
gAlign] ™ sequence alignment software. The first paired alignment step was performed using the Lipman-Pearson algorithm with a K-tuple of 1, a gap penalty of 3, a window of 5, and a diagonal saved to 5. . Multiple alignment steps were performed using the Clustal algorithm with a PAM250 residue weight table, a gap penalty of 10, and a gap length penalty of 10.

FIG. 4 depicts the cDNA sequence and deduced amino acid sequence of mouse FDRG (also called “mFDRG”, “mT115” or BK89). The nucleotide sequence corresponds to nucleic acids 1 to 1893 of SEQ ID NO: 11. The amino acid sequence is SEQ ID NO: 12
Corresponds to amino acids 1-410 of

FIG. 5 depicts the cDNA sequence and deduced amino acid sequence of mature mouse FDRG. The nucleotide sequence corresponds to nucleic acids 1 to 1161 of SEQ ID NO: 14. The amino acid sequence corresponds to amino acids 1-387 of SEQ ID NO: 15.

FIG. 6 depicts a paired alignment of human and mouse FDRG nucleotide sequences (corresponding to SEQ ID NO: 1 and SEQ ID NO: 11, respectively). The alignment is based on the ALIGN program, version 2.0 (Myers and Miller (
1989) CAIBOS), scoring ma
trix) = PAM120, gap penalty: -12 / -4.

FIG. 7 depicts a paired alignment of the human and mouse FDRG amino acid sequences (corresponding to SEQ ID NO: 2 and SEQ ID NO: 12, respectively). The alignment occurred as described in the description of FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61P 3/10 4C085 A61P 3/04 C07K 14/47 4H045 3/10 16/18 C07K 14 / 47 C12Q 1/68 A 16/18 G01N 33/53 M C12N 5/10 D C12Q 1/68 33/566 G01N 33/53 33/68 C12N 15/00 ZNAA 33/566 A61K 37/02 33/68 C12N 5 / 00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL , SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Spiegelman, Bruce M, Massachusetts, United States ) Inventor Joan, Clifford Hyyunsqua Rica, Massachusetts, U.S.A. 02138 Cambridge Bridge, Peabo Day Terrace 24, Apartment 10, F-Term (Reference) 2G045 AA34 AA35 AA40 DA12 DA13 DA14 DA36 FB02 FB03 4B024 AA01 AA11 BA80 CA04 DA03 EA04 GA11 HA17 4B063 QA01 Q13 QA01 QAQ1 AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA06 AA07 AA17 BA01 BA08 BA22 BA23 CA53 CA56 ZA362 ZB262 ZC352 4C085 AA13 AA14 BB11 EE01 4H045 AA10 AA11 BA10 CA40 DA01 DA76 EA20 EA50 FA72 FA74

Claims (26)

[Claims] 1. a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11,
The nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 14, accession no.
A nucleic acid molecule comprising a nucleotide sequence that is at least 60% homologous to the DNA insert of the plasmid deposited at the TCC, or their complement; b) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, At least 5 of the nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 14, the DNA insert of the plasmid deposited at the ATCC as accession number 98632, or their complement
A nucleic acid molecule comprising a 55 nucleotide fragment; c) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or encoded by the DNA insert of the plasmid deposited with the ATCC as accession number 98632. A nucleic acid molecule encoding a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid sequence; d) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15 Or a nucleic acid molecule encoding a fragment of the polypeptide encoded by the DNA insert of the plasmid deposited with the ATCC under accession number 98632, wherein the fragment comprises SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, The amino acid sequence of No. 15 or the accession number 98632 A nucleic acid molecule comprising at least 15 contiguous amino acid residues of the polypeptide encoded by the DNA insert of the plasmid deposited at the ATCC; and e) SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15 Or a nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as accession number 98632. A nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14, The isolated nucleic acid molecule selected. 2. a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11,
SEQ ID NO: 13, nucleotide sequence of SEQ ID NO: 14, or accession number 98632
A nucleic acid molecule comprising the DNA insert of the plasmid deposited with the ATCC, or the complement thereof; and b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or accession number 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as 98632. 3. The nucleic acid molecule of claim 1, further comprising a vector nucleic acid sequence. 4. The nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding a heterologous polypeptide. 5. A host cell containing the nucleic acid molecule of claim 1. 6. The host cell of claim 5, which is a mammalian host cell. 7. A non-human mammalian host cell containing the nucleic acid molecule of claim 1. 8. a) SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15
Or a fragment of the polypeptide encoded by the DNA insert of the plasmid deposited at the ATCC under accession number 98632, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, No. 15 or D of the plasmid deposited with the ATCC under accession number 98632
A fragment comprising at least 15 contiguous amino acids of the amino acid sequence encoded by the NA insert; b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or the ATCC as accession number 98632 A naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the DNA insert of the deposited plasmid, wherein the polypeptide has SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 1
And c) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or accession number 98632. A polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 60% homologous to the nucleic acid comprising the nucleotide sequence of the DNA insert of the plasmid deposited with the ATCC. d) an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15 or an amino acid which is at least 60% homologous to the polypeptide encoded by the DNA insert of the plasmid deposited at the ATCC under accession number 98632 An isolated polypeptide selected from the group consisting of a polypeptide comprising a sequence. 9. comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as accession number 98632, 9. The isolated polypeptide of claim 8. 10. The polypeptide of claim 8, further comprising a heterologous amino acid sequence. 11. An antibody that selectively binds to the polypeptide of claim 8. 12. A method comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed, comprising: a) the amino acids of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15. A polypeptide comprising the sequence or the amino acid sequence encoded by the DNA insert of the plasmid deposited at the ATCC as accession number 98632; b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12 or SEQ ID NO: 15 Or the DNA of the plasmid deposited with the ATCC under accession number 98632
A DNA fragment of a polypeptide comprising the amino acid sequence encoded by the insert, wherein the DNA insert is a plasmid deposited with the ATCC as SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or Accession No. 98632. A fragment comprising at least 15 contiguous amino acids of the amino acid sequence encoded by the product; and c) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 15, or deposited with the ATCC as accession number 98632 A naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the DNA insert of the selected plasmid, wherein the polypeptide has SEQ ID NO: 1, SEQ ID NO: 3 under stringent conditions. , SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14. A method for producing a polypeptide selected from the group consisting of encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule comprising 14. 13. A method comprising: a) contacting a sample with a compound that selectively binds to the polypeptide of claim 8; and b) determining whether the compound binds to the polypeptide in the sample. ,
Detecting the presence of the polypeptide of claim 8 in said sample, thereby detecting the presence of said polypeptide in the sample. 14. The compound according to claim 1, wherein the compound that binds to the polypeptide is an antibody.
Method 3. 15. A kit comprising a compound that selectively binds to the polypeptide of claim 8 and instructions for use. 16. A) contacting a sample with a nucleic acid probe or primer that selectively hybridizes to the nucleic acid molecule of claim 1; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample. Determining whether the nucleic acid molecule of claim 1 is present in said sample, thereby detecting the presence of said nucleic acid molecule in said sample. 17. The method of claim 16, wherein said sample comprises mRNA molecules and is contacted with a nucleic acid probe. 18. A kit comprising a compound that selectively hybridizes to the nucleic acid molecule of claim 1 and instructions for use. 19. a) contacting the polypeptide of claim 8 or a cell expressing said polypeptide with a test compound; and b) determining whether said polypeptide binds to said test compound; A method for identifying a compound that binds to the polypeptide, comprising: 20. Binding of the test compound to the polypeptide can be determined by: a) detecting binding by direct detection of the test compound / polypeptide binding; b) detecting binding using a competitive binding assay; and c) detecting FDRG activity. 20. The method of claim 19, wherein said binding is detected by a method selected from the group consisting of: detecting binding using said assay. 21. A method comprising contacting the polypeptide of claim 8 or a cell expressing said polypeptide with a compound that binds to said polypeptide at a concentration sufficient to modulate the activity of said polypeptide. A method for modulating the activity of said polypeptide. 22. a) contacting the polypeptide of claim 8 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide, thereby modulating the activity of the polypeptide. 9. A method for identifying a compound that modulates the activity of the polypeptide of claim 8, comprising identifying the compound. 23. An FDR such that obesity or diabetes in the subject is treated.
A method for treating obesity or diabetes in a subject, comprising administering to the subject an agent selected from the group consisting of a small molecule modulator of G, a FDRG nucleic acid molecule and an FDRG antibody. 24) a) isolating a sample from the subject; b) contacting the sample with a nucleic acid probe or primer that selectively hybridizes to an FDRG nucleic acid molecule; and c) the nucleic acid probe or primer is Determining whether or not it binds to an FDRG nucleic acid molecule in the sample, thereby detecting the presence of obesity or diabetes in the subject. 25. A method comprising: a) contacting cells expressing the FDRG receptor with a compound to form an assay mixture; b) contacting the assay mixture with FDRG; Determining whether the compound modulates FDRG activity. A method of identifying a compound that modulates a metabolic disorder. 26. Administering to the subject an agent selected from the group consisting of a small molecule modulator of FDRG, an FDRG nucleic acid molecule and an FDRG antibody such that angiogenesis in the subject is modulated. A method of modulating angiogenesis in said subject.