JP2003525576A - NT2LP, a novel G protein-coupled receptor having homology to the neurotensin-2 receptor - Google Patents

Abstract

(57) Abstract The present invention relates to the identification of a novel G protein-coupled receptor (GPCR) and a nucleic acid molecule encoding a GPCR, mainly expressed in the brain, referred to herein as the NT2LP protein and the NT2LP gene, respectively. based on. The NT2LP protein has high homology to NT family receptors, particularly members of the NT2 subfamily. Based on this identification, the present invention provides: 1) an isolated NT2LP protein, 2) an isolated nucleic acid molecule encoding the NT2LP protein, 3) an antibody that selectively binds to the NT2LP protein, 4) an NT2LP protein And methods for isolating allelic variants of genes, 5) methods for identifying cells and tissues that express NT2LP protein / gene, 6) methods for identifying substances and cellular compounds that bind to NT2LP protein, 7) NT2LP A method for identifying a substance that modulates gene expression, 8) a method for modulating the activity of NT2LP protein in a cell or an organism, and 9) administering a substance that binds to and / or modulates NT2LP protein. Methods for treating a disease, condition or biological process, such as pain, including.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part filed on May 11, 1998, US patent application Ser. No. 09 / 076,313.

BACKGROUND G protein-coupled receptor of the present invention (GPCR) is one of the major class of proteins responsible for intracellular signaling. GPCR is a protein with a seven transmembrane domain. The binding of a ligand to the extracellular portion of the GPCR transmits a signal within the cell that alters the biological or physiological properties of the cell.

G protein-coupled receptors (GPCRs), along with G proteins and effectors (intracellular enzymes and channels regulated by G proteins), are modular signals that connect the state of intracellular second messengers with extracellular inputs. It is a component of the transmission system. These genes and gene products have the potential to cause disease (Spiegel et al., J. Clin. Invest. 92: 1119-1125 (1993); McKusick and Ambe.
rger, J. Med. Genet. 30: 1-26 (1993)). Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene are characterized by various types of autosomal dominant and autosomal recessive retinitis pigmentosa (Nathans et al., Annu. Rev. Genet. 26: 403-424 (1992). ), Renal diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2: 1201-1204 (1993) and references therein). These receptors are very important for both central nervous system and peripheral physiological processes. By evolutionary analysis,
It has been suggested that the ancestors of these proteins developed in concert with the complex organization and nervous system.

G protein-coupled receptors regulate many important physiological processes that lead to G
This is one of the reasons why protein-coupled receptors are common drug targets. For example,
There are at least three major classes of opioid G protein-coupled receptors that control pain: μ, κ, and δ. Endogenous ligands for these receptors include enkephalins, dinorphins, and endorphins. Opioid analgesics that have agonistic activity at opioid receptors and have been used to treat pain include morphine, levorphanol, and fentanyl (Reisine and Pasternak (1996) for more information). ,
"The pharmacological basis of Godman and Gilman's therapeutics (Goodman and Gilman's
The Pharmacological Basis of Therapeutics), 9th Edition, pp. 521-555).

The GPCP protein superfamily currently comprises more than 250 types of paralogs (par
alogues), receptors that represent variants caused by gene duplication (or other processes) in contrast to orthologues, identical receptors and homologues from different species, isolated from a single organism It contains different types of receptors. The superfamily can be classified into the following five families. Rhodopsin and β-
Family I, a receptor typified by adrenergic receptors and currently composed of over 200 unique members (Dohlman et al., Annu. Re.
v. Biochem. 60: 653-688 (1991) and references therein).
Family II, a recently characterized parathyroid hormone / calcitonin / secretin receptor family (Juppner et al., Science 254: 1024-1026 (1991); Lin et al., Science 254: 1022-1024 (1991)); NT receptor Family III, a metabolite-producing glutamate receptor family in mammals including the body (Nakanishi, Science 258
597: 603 (1992)); Family IV, a family of cAMP receptors important in chemotaxis and D. sidcoideum development (Klein et al., Science 241).
: 1467-1472 (1988)); and Family V, which is a fungal mating pheromone receptor such as STE2 (Kurjan, Annu. Rev. Biochem. 61: 1097-1129 (1992)).

In addition to these groups of GPCRs, there are a small number of other proteins that have seven putative hydrophobic segments and are considered to be unrelated to GPCRs. However, they have not been shown to be coupled to G proteins. Drosophila expresses a photoreceptor-specific protein, bride of sevenless (boss). This has been extensively studied and has not been shown to be a GPCR.
Protein (Hart et al., Proc. Natl. Acad. Sci. USA 90: 5047-5051 (1993).
). The Drosophila gene frizzled (fz) is also thought to be a protein with a seven transmembrane segment. Like boss, fz has not been shown to be coupled to G proteins (Vinson et al., Nature 338: 263-264 (1989)).

G proteins represent a family of heterotrimeric proteins consisting of α, β, and γ subunits that bind guanine nucleotides. These proteins are normally linked to cell surface receptors, for example 7-transmembrane domain containing receptors such as the NT receptor family. When the ligand binds to the GPCR, a conformational change is transmitted to the G protein, causing the α-subunit to exchange the bound GDP molecule for a GTP molecule and dissociate from the βγ-subunit. The GTP-bound form of the α-subunit typically functions as an effector modulator, cyclic AMP (eg, by activation of adenylate cyclase),
It results in the production of secondary messengers such as diacylglycerol, or inositol phosphates. In humans, over 20 different types of α-subunits are known that associate with relatively small pools of β- and γ-subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs, and Gt. G
Proteins are described in Lodish H. et al., Molecular Cell Biology, (
Scientific American Books Inc., New York, NY, 1995), the contents of which are incorporated herein by reference.

GPCRs are major targets for drug action and development. Therefore, identifying and characterizing previously unknown GPCRs is valuable to the field of drug development. One class of GPCRs of particular pharmaceutical importance is the neurotensin receptor protein (NTR).

Neurotensin (NT) is a tridecapeptide distributed in both the central nervous system and peripheral tissues. NT is high blood pressure, hyperglycemia, hypothermia, increased vascular permeability,
It is involved in mediating analgesia and acts as both a neurotransmitter and a neuromodulator in the brain and a cell-mediated factor and hormone in peripheral tissues. In particular, NT regulates dopamine transmission in the nitrostrial and mesolimbic pathways.

Previous studies have shown that NT binds to two different receptors.
A representative NT1 has been identified in rat and human (Tanaka et al., Neuron 4:84).
7-854 (1990) and Vita et al., FEBS Ltr. 317: 139-142 (1993)), a typical NT2 is
Identified from rat and mouse (Chalon et al. FEBS Ltrs. 366: 91-94 (199
6) and Mazella et al., J. Neuro. 16: 5613-5620 (1996)).

Recently, non-peptide inhibitors of NT1 have been identified (Labbe-Jullie et al., Mol. Pharm. 4
7: 1050-1056 (1995)). The identification of non-peptide inhibitors in NTR protein family members makes the identification of other family members important in the field of drug development.

The present invention aims to improve the state of the art by providing a previously unidentified GPCR, NT2LP protein, which is mainly expressed in the brain and whose low level expression is detected in the ovary. Make progress. The human and monkey NT2LP proteins show high sequence homology with members of the NT receptor family, particularly the NT2 receptor subfamily.

SUMMARY OF THE INVENTION The present invention is a novel G protein-coupled receptor (GPC) expressed mainly in the brain.
R), and the identification of nucleic acid molecules encoding GPCRs, which are referred to herein as the NT2LP protein and NT2LP gene, respectively. The NT2LP protein is a human or monkey protein with high sequence similarity to members of the NT receptor family, especially the NT2 receptor subfamily. Based on this identification, the present invention provides: 1) isolated NT2LP proteins and fragments, 2) isolated nucleic acids encoding NT2LP proteins and fragments, 3) antibodies that selectively bind to NT2LP proteins, 4) N.
Method for isolating allelic variants of T2LP protein and gene, 5) Method for identifying cells and tissues expressing NT2LP protein / gene, 6) Method for identifying agents and cellular compounds that bind to NT2LP protein, 7) A method of identifying an agent that modulates expression of the NT2LP gene, 8) a method of modulating the activity of the NT2LP protein in a cell or organism, and 9) administering an agent that binds to and / or modulates the NT2LP protein, Methods are provided for treating disorders, conditions, or biological processes, such as pain.

Preferred embodiments of the described invention is primarily novel G protein-coupled receptor expressed in the brain (GPC
R) is based on the discovery of a family of molecules and NT2LP protein-encoding nucleic acid molecules, NT2LP genes, or NT2LP nucleic acid molecules. The NT2LP protein is a GPCR with high sequence homology to members of the NT receptor family, especially the NT2 receptor subfamily.

Specifically, initially, ESTs were identified in public databases with low homology to G protein-coupled receptors. Next, based on this sequence, PCR primers were designed, and the cDNA was identified by screening a monkey fetal cDNA library (see Example 1). The positive clones were sequenced and contigs were constructed. Analysis of the constructed sequences revealed that the cloned cDNA molecule encodes a GPCR, referred to herein as the NT2LP protein, which has high sequence homology with members of the NT receptor family, particularly the NT2 receptor subfamily. It became clear.

The monkey NT2LP nucleic acid sequence was used to screen a variety of human cDNA libraries. Several clones were identified and sequenced. Two contigs were constructed representing two different splice forms of the full length human NT2LP gene and full length clones were represented (SEQ ID NOs: 3 and 5).

The NT2LP protein is a GPCR and plays a role in and functions in a signal transduction pathway in cells expressing the NT2LP protein, particularly brain cells.

[0018]   Various aspects of the invention are described in further detail below.

I. Isolated NT2LP Proteins The present invention provides isolated human and monkey NT2LP proteins, and peptide fragments of NT2LP proteins.

As used herein, the protein is substantially free of cellular material, or is isolated from recombinant and non-recombinant cells, or is a chemical precursor or other chemical entity. The protein is referred to as "isolated" or "purified" when it is free of, or chemically synthesized.
The term "substantially free of cellular material" includes preparations of NT2LP protein that have been separated from the cellular constituents of naturally or recombinantly produced cells.
In one embodiment, the term "substantially free of cellular material" refers to less than about 30% (on dry weight) of non-NT2LP protein (also referred to herein as "contaminant protein"), more preferably about. Less than 20% non-NT2LP protein, even more preferably less than about 10% non-NT2LP protein, most preferably less than about 5% non-NT2LP protein.
Includes preparations of NT2LP protein having NT2LP protein. If the NT2LP protein or fragment thereof is recombinantly produced, it is preferably substantially free of culture medium. That is, the culture medium is less than about 20%, more preferably less than about 10%, and most preferably less than about 5% by volume of the protein preparation. The term "substantially free of chemical precursors or other chemicals" includes preparations of NT2LP proteins that have been separated from chemical precursors or other chemicals involved in protein synthesis. In one embodiment, the term "substantially free of chemical precursors or other chemicals" means less than about 30% (by dry weight) chemical precursors or non-NT2LP chemicals, more preferably Less than about 20% chemical precursors or non-NT2LP chemicals, even more preferably less than about 10% chemical precursors or non-NT2LP chemicals, most preferably less than about 5% chemical precursors or non-NTs.
Includes a preparation of NT2LP protein with 2LP chemicals. In a preferred embodiment, the isolated protein or fragment thereof lacks contaminating proteins from the same animal from which the NT2LP protein was derived. Typically, such proteins are
For example, produced by recombinant expression of human or monkey NT2LP protein in non-human or non-monkey cells.

As used herein, the NT2LP protein is 1) the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, 2) a naturally occurring functional allele of the human or monkey NT2LP protein. Genetic variants and non-functional allelic variants, 3) recombinantly produced variants of the human or monkey NT2LP protein, and 4) NT2LP proteins isolated from organisms other than human or monkey (human Or ortholog of the NT2LP protein of monkey) is defined as a protein containing.

As used herein, an allelic variant of the human or monkey NT2LP protein refers to 1) a protein isolated from human or monkey cells or tissues, 2) a human or monkey NT2LP protein. Is defined as a protein encoded by the same locus that encodes H., and 3) proteins with substantial sequence similarity to the human or monkey NT2LP protein.

As used herein, the amino acid sequences of two proteins (or a region of these proteins) are at least about 60-65%, typically at least about 70-75%, more than each other. Two proteins are substantially similar if they typically have at least about 80-85%, and most typically at least about 90-95% or greater identity. To determine the identity of two amino acid sequences (eg, SEQ ID NO: 2 or SEQ ID NO: 4 and allelic variants thereof) or two nucleic acids, align the sequences for optimal comparison. (Eg gaps can be introduced in the sequence of one protein or nucleic acid to obtain optimal alignment with another protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in one sequence (eg SEQ ID NO: 2 or SEQ ID NO: 4) is occupied by the same amino acid residue or nucleotide as in the corresponding position in another sequence (eg human or Allelic variants of monkey NT2LP protein),
The molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie,% identity = number of identical positions / total number of positions x 100).

Allelic variants of the human or monkey NT2LP protein include both functional and non-functional NT2LP proteins. A functional allelic variant is a human or monkey NT2LP that retains the ability to bind a ligand (such as NT for the NT receptor) and transduce a signal in cells, preferably neurons.
It is a naturally occurring amino acid sequence variant of a protein. Functional allelic variants are typically conservative substitutions of one or more amino acids of SEQ ID NO: 2 or SEQ ID NO: 4 (allelic variants of the NT2LP protein), or within non-critical regions of the protein. It appears to contain only substitutions, deletions, or insertions at non-critical residues of.

A non-functional allelic variant is a human or monkey NT2LP protein that has no ability to bind a ligand (such as NT for the NT receptor) and / or transduce a signal intracellularly. Is a naturally occurring amino acid sequence variant of. Non-functional allelic variants typically include SEQ ID NO: 2 or SEQ ID NO: 4 (NT
2LP protein allelic variant) with one or more non-conservative amino acid substitutions, deletions, insertions, or premature truncations, or substitutions, insertions, or deletions of important residues or regions. It seems to contain.

The invention further provides non-human or non-monkey and non-human orthologs in the human or monkey NT2LP proteins of the invention. Human or monkey NT2LP
Protein orthologs are proteins isolated from non-human or non-human or non-monkey organisms and have the same ligand binding and signal transduction abilities (such as NT for NT receptors). An ortholog of the NT2LP protein can be readily identified as containing an amino acid sequence substantially homologous to SEQ ID NO: 2 or SEQ ID NO: 4 (NT2LP ortholog). However, the present invention does not include the known rat and mouse NT2 proteins (see Background of the Invention).

The NT2LP protein is a GPCR involved in a signal transduction pathway in cells expressing NT2LP, particularly brain cells. As used herein, a signal transduction pathway is a ligand and GPCR (NT2LP protein) similar to the binding between NT and NT receptor.
Modulation (eg, stimulation or inhibition) of cell function / activity in the binding of Examples of such functions include intracellular molecules involved in signaling pathways such as phosphatidylinositol 4,5-diphosphate (PIP2), inositol 1,4,5-triphosphate (
IP3), or the involvement of adenylate cyclase; polarization of the plasma membrane; production or secretion of molecules; modification of the structure of cell components; cell proliferation, eg DNA synthesis; cell migration; cell differentiation; cell survival; and Includes transmission of pain signals. Since the NT2LP protein is substantially expressed in the brain, examples of cells involved in the NT2LP signal transduction pathway include nerve cells such as peripheral nervous system cells and central nervous system cells such as brain cells. Include, for example, limbic cells, hypothalamic cells, hippocampal cells, nigral cells, cortical cells, brain stem cells, neocortical cells, brain stem ganglion cells, caudate putamen (
caudate putamen) cells, olfactory tuberculosis cells, dorsal root ganglion cells, trigeminal ganglion cells, sensory ganglion cells, nociceptive neurons, or superior colliculus cells.

Depending on the cell type, the response mediated by the NT2LP protein may be different.
For example, in some cells, binding of the ligand to the NT2LP protein, via phosphatidylinositol or cyclic AMP turnover,
Activities such as compound release, channel regulation, cell adhesion, migration, differentiation, etc. can be stimulated, but in other cells, binding of the ligand to the NT2LP protein results in
Different results are likely to occur. Regardless of the cellular activity / response regulated by the NT2LP protein, the NT2LP protein is a GPCR and is intracellularly a "G protein".
It is universal to interact with and give rise to one or more secondary signals in diverse intracellular signaling pathways, such as through phosphatidylinositol or cyclic AMP turnover, calcium channel regulation, and the like. G proteins represent a family of heterotrimeric proteins consisting of α, β, and γ subunits that bind guanine nucleotides. These proteins are normally linked to cell surface receptors, for example receptors with a seven transmembrane domain such as the NT receptor. When the ligand binds to the receptor, a conformational change G
Transferred to the protein, causing the α-subunit to exchange bound GDP molecules for GTP molecules and dissociate from βγ-subunits. The GTP-bound form of the α-subunit typically functions as an effector modulator and is a diamine such as cyclic AMP (eg, due to activation of adenylate cyclase), diacylglycerol, or inositol phosphate, or calcium. Next brings the generation of messenger. Over 20 different types of α-subunits are known in humans that associate with a relatively small pool of β- and γ-subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs, and Gt. G-protein is described in Lodish H. et al., Molecular Cell Biolo
gy), (Scientific American Books Inc., New York, NY, 1995), the contents of which are incorporated herein by reference.

As used herein, “phosphatidylinositol turnover” refers to molecules involved in phosphatidylinositol 4,5-diphosphate (PIP2) turnover, and the activity of these molecules. . PIP2 is a cytosolic plasma membrane
It is a phospholipid found in cytosolic leaflets. The binding of the ligand to NT2LP can activate the plasma membrane enzyme phospholipase C in some cells. And then it can hydrolyze PIP2 to give 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Once IP3 is formed, it can diffuse to the surface of the endoplasmic reticulum where it can bind to IP3 receptors, such as calcium channel proteins that contain IP3 binding sites. IP3
Binding induces the opening of the channel, causing calcium ions to be released into the cytoplasm. IP3 can also form inositol 1,3,4,5-tetraphosphate (IP4), which is phosphorylated by specific kinases and allows calcium to enter the cytoplasm from the extracellular medium. IP3 and IP4 can then be very rapidly hydrolyzed to the inactive products inositol 1,4-diphosphate (IP2) and inositol 1,3,4-triphosphate, respectively. These inactive products are recycled by the cells to synthesize PIP2. Another second messenger produced by hydrolysis of PIP2, 1,2-diacylglycerol (DAG), remains at the cell membrane,
There it acts to activate the enzyme protein kinase C. Protein kinase C is normally found in the cytoplasm of cells as a soluble form, but with increasing intracellular calcium concentration, this enzyme can translocate to the plasma membrane where it can be activated by DAG. Activation of protein kinase C in different cells causes different cellular responses such as phosphorylation of glycogen synthase or phosphorylation of different transcription factors such as NF-κB. As used herein, the term "phosphatidylinositol activity" refers to the activity of PIP2 or one of its metabolites.

Another signaling pathway in which the NT2LP protein may be involved is the cAMP turnover pathway. As used herein, a "cyclic AMP turnover" is a molecule involved in cyclic AMP (cAMP) turnover,
And the activity of these molecules. Cyclic AMP is a second messenger produced in response to stimuli induced by ligands of certain G protein-coupled receptors. In the cAMP signaling pathway, binding of GPCRs to ligands such as NT and NT receptors causes activation of the enzyme adenylate cyclase, which catalyzes the synthesis of cAMP. The newly synthesized cAMP, in turn, can activate a cAMP-dependent protein kinase. This activated kinase can phosphorylate voltage-gated potassium channel proteins or related proteins and block potassium channel opening during action potentials. Failure to open potassium channels results in a decrease in potassium efflux, which normally repolarizes the membrane of neurons, resulting in prolonged membrane depolarization.

The present invention further provides fragments of the NT2LP protein. As used herein, a fragment comprises at least 8 consecutive amino acids from the NT2LP protein. Preferred fragments are those that have one or more biological activities of the NT2LP protein, such as the ability to bind a G protein or ligand, as well as fragments that can be used as immunogens to raise anti-NT2LP antibodies. Is. The most preferred fragment is a fragment unique to NT2LP which is not present in other known proteins.

The biologically active fragment of NT2LP protein includes an amino acid sequence derived from the amino acid sequence of NT2LP protein, for example, the amino acid sequence shown in SEQ ID NO: 2 or NT2LP containing less amino acids than the full-length NT2LP protein. A peptide containing an amino acid sequence of a protein homologous to the protein, or a full-length protein homologous to the NT2LP protein and showing at least one activity in the NT2LP protein is included. Typically, biologically active fragments (peptides such as 5, 10, 1
5, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids long) are, for example, transmembrane domains, multiple extracellular domains, or G protein-binding domains. Contains the domain or motif of.

Preferred fragments include, but are not limited to, 1) the soluble peptide of SEQ ID NO: 2 or SEQ ID NO: 4, and 2) the peptide containing the G protein binding site of the NT2LP protein.

The isolated NT2LP protein is purified from cells that naturally express the protein, cells that have been modified to express the NT2LP protein, or synthesized using known protein synthesis methods. Can be done. Preferably, as follows:
The isolated NT2LP protein can be produced by recombinant DNA technology. For example, a nucleic acid molecule encoding a protein is cloned into an expression vector, the expression vector is introduced into a host cell, and the NT2LP protein is expressed in the host cell. The NT2LP protein can then be isolated from the cells by a suitable purification scheme using standard protein purification techniques. As an alternative to recombinant expression, the NT2LP protein or fragment may be chemically synthesized using standard peptide synthesis techniques. Finally, cells that naturally express the NT2LP protein (eg hippocampal cells or substantia nigra cells)
The native NT2LP protein can also be isolated from

The present invention further provides NT2LP chimeric or fusion proteins. As used herein, an NT2LP "chimeric protein" or "fusion protein" comprises an NT2LP protein operably linked to a non-NT2LP protein. "NT
"2LP protein" refers to a protein having an amino acid sequence corresponding to NT2LP protein, and "non-NT2LP protein" is a heterologous protein having an amino acid sequence corresponding to a protein that is not substantially homologous to NT2LP protein, such as NT2LP.
A protein different from a protein. The term "operably linked" in the context of a fusion protein is intended to indicate that the NT2LP protein and the non-NT2LP protein are fused in frame with each other. Non-NT2LP protein, NT2LP
It can be fused to the N-terminus or C-terminus of the protein. For example, in one embodiment, the fusion protein is a GST-N in which the NT2LP sequence is fused to the C-terminus of the GST sequence.
It is a T2LP fusion protein. Other types of fusion proteins include, but are not limited to, enzyme fusion proteins such as β-galactosidase fusions, yeast two-hybrid Gal fusions, polyHis fusions, and Ig fusions. Such fusion proteins, particularly polyHis fusions, facilitate purification of recombinant NT2LP proteins. In another embodiment, the fusion protein is an NT2LP protein containing a heterologous signal sequence at the N-terminus. In certain host cells (eg, mammalian host cells), NT2LP protein expression and / or secretion can be increased by using a heterologous signal sequence.

Preferably, the NT2LP chimera or fusion protein is made by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are digested, for example, with restriction enzymes to provide suitable ends, blunt ended, optionally cohesive ends, treated with alkaline phosphatase to avoid unwanted ligations, and enzymatically ligated. Both are ligated in-frame according to conventional techniques using blunt ends or cohesive ends for ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of a gene fragment is performed by using an anchor primer to create a complementary overhang between two consecutive gene fragments, which is then annealed to create a chimeric gene sequence and reamplified. Can be carried out (eg, Current Protocols in Molecular Biology).
), Edited by Ausubel et al., John Wiley & Sons, (1992)). Furthermore, the fusion substance (
Many expression vectors encoding, for example, the GST protein) are commercially available. The NT2LP-encoding nucleic acid can be cloned into such an expression vector such that the fusion agent is linked in-frame to the NT2LP protein.

The present invention also provides modified NT2LP proteins made with recombinant DNA or mutagenesis methods / substances. Modified NT2LP proteins can be made by mutagenesis methods well known in the art, eg, NT2LP protein point mutations or truncations, and recombinant DNA methods.

II . Antibodies that Bind NT2LP Proteins The present invention further provides antibodies that selectively bind NT2LP proteins. As used herein, an antibody is said to selectively bind the NT2LP protein if it binds the NT2LP protein and substantially no irrelevant proteins. One of skill in the art will readily recognize that an antibody may be considered substantially bound to the NT2LP protein even if it binds to a protein having homology to a fragment or domain of the NT2LP protein.

The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, antigens that specifically bind (in immunoreactivity) to an antigen such as the NT2LP protein. By a molecule containing the binding site.
Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ') ₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The present invention provides polyclonal and monoclonal antibodies that bind to the NT2LP protein. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a population of antigen molecules that comprises the only species of antigen binding site capable of immunoreacting with a particular epitope of the NT2LP protein. . A monoclonal antibody composition thus typically displays a single binding affinity for a particular NT2LP protein with which it immunoreacts.

To generate anti-NT2LP antibodies, immunization to generate antibodies that bind the isolated NT2LP protein or fragments thereof to NT2LP using standard techniques for the preparation of polyclonal and monoclonal antibodies. Used as a source. The full length NT2LP protein may be used, or an antigenic peptide fragment of NT2LP may be used as an immunogen. An antigenic fragment of an NT2LP protein typically comprises at least 8 consecutive amino acid residues of the NT2LP protein, eg 8 consecutive amino acids from SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the antigenic peptide is at least 10 amino acid residues of the NT2LP protein, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, most preferably at least 30 amino acids. Contains residues. A preferred fragment for making anti-NT2LP antibodies is a region of NT2LP located on the surface of the protein, eg the hydrophilic region, identified in the antigenicity plot provided in FIG.

The NT2LP immunogen is typically one that is suitable for the subject (eg, rabbit,
It is used to prepare antibodies by immunizing goats, mice, or other mammals. Suitable immunogenic preparations can contain, for example, recombinantly expressed NT2LP protein or chemically synthesized NT2LP peptides. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic NT2LP preparation induces a polyclonal anti-NT2LP antibody response.

Polyclonal anti-NT2LP antibodies can be prepared as described above by immunizing a suitable subject with an NT2LP peptide immunogen. Anti-NT2LP antibody titers in immunized subjects can be monitored over time by standard techniques such as enzyme-linked immunosorbent assay (ELISA) using immobilized NT2LP protein. If desired, antibody molecules raised against the NT2LP protein can be isolated from a mammal (eg, blood) and further purified using well-known techniques such as protein A chromatography to obtain an IgG fraction. You can At a suitable time after immunization, for example, when the anti-NT2LP antibody titer is at its highest, antibody-producing cells are obtained from the subject and used to study Kohler and Milstein.
(1975) Nature 256: 495-497 first described the hybridoma technology (Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al. (1980) J. Biol. Chem. 255: 4980.
~ 83; Yeh et al. (1976) PNAS 76: 2927-31; and Yeh et al. (1982) Int. Cancer 29: 2.
69-75), more recent human B cell hybridoma technology (Kozbor et al. (1
983) Immunol. Today 4:72), EBV-hybridoma technology (Cole et al. (1985) "Monoclonal Antibodies and Cancer Therapy"),
Monoclonal antibodies can be prepared by standard techniques, such as Alan R Lithink, pp. 77-96), or the trioma technique. Techniques for producing monoclonal antibody hybridomas are well known (generally, RH Kenneth "
Monoclonal Antibodies: A New Aspect in Biological Analysis (Monoclonal Antibodie
s: A New Dimension In Biological Analysis) '' Plenum Publishing Corp.,
New York, NY; EA Lerner (1981) Yale J. Biol. Med. 54:
387-402; ML Gefter et al. (1977) Somatic Cell Genet. 3: 231-36). Briefly, immortalized cell lines (typically myeloma) were
Fusion with lymphocytes (typically splenocytes) from mammals immunized with the NT2LP immunogen and screening of the resulting culture supernatants of hybridoma cells for hybridomas producing monoclonal antibodies that bind to NT2LP. Identify.

Any of the many well-known protocols for fusing lymphocytes with immortalized cell lines can be applied for the purpose of producing anti-NT2LP monoclonal antibodies (
For example, Galfre et al. (1977) Nature 266: 55052; Galfre et al. Somatic Cell Genet.
Above; Lerner, Yale J. Biol. Med., Above; Kenneth, "Monoclonal antibody (
Monoclonal Antibodies) ", see above). Moreover, one of skill in the art will recognize that there are many modifications to such methods that are also useful. Immortalized cell lines (eg, myeloma cell lines) are typically derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be made by fusing lymphocytes from mice immunized with the immunogen preparation of the invention to an immortalized mouse cell line. Preferred immortalized cell lines are sensitive to culture medium containing hypoxanthine, aminopterin, and thymidine (“HAT medium”),
It is a mouse myeloma cell line. For example P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 /
Many any myeloma cell line, such as the O-Ag14 myeloma cell line, can also be used as a fusion partner according to standard techniques. These myeloma cell lines are available from ATCC. Polyethylene glycol (“PEG”) is typically used to fuse HAT-sensitive mouse myeloma cells with mouse splenocytes. The hybridoma cells obtained by the fusion are then killed by unfused and nonproliferatively fused myeloma cells (unfused splenocytes die a few days after they have not been transformed) in HAT medium. Use to select. Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for the presence of NT2LP protein binding antibody, eg, using a standard ELISA assay.

Instead of preparing monoclonal antibody secreting hybridomas, recombinant anti-NT2LP proteins can be identified and isolated by screening recombinant combinatorial immunoglobulin libraries with NT2LP proteins, thereby binding to NT2LP. Immunoglobulin library members can be identified. Kits for preparing and screening phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and Stratagene's SurfZAP® Phage Display Kit). , Catalog number 240162). In addition, examples of methods and reagents that can be specifically used to generate and screen antibody display libraries are described in, for example, Ladner et al., US Pat. No. 5,223,409; Kang et al., WO 92/18619, Dower et al., WO 91/17271; Winter et al., WO 92/20791; Markland et al., WO 92/15679; Breitl
ing et al., WO 93/01288; McCafferty et al., WO 92/01047; Garrand et al., WO 92/09690; Ladner et al., WO 90/02809.
Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths
(1993) EMBO J. 12: 725-734; Hawakins et al. (1992) J. Mol. Biol. 226: 889.
~ 896; Clarkson et al. (1991) Nature 352: 624 ~ 628; Gram et al. (1992) PNAS 89: 3576.
~ 3580; Garrad et al. (1991) Bio / Technology 9: 1373 ~ 1377; Hoogenboom et al. (1991).
Nuc. Acid Res. 19: 4133-4137; Barbas et al. (1991) PNAS 88: 7978-7982; and McCafferty et al. (1990) 348: 552-554.

In addition, recombinant anti-NT2LP antibodies, such as chimeric, humanized monoclonal antibodies, which can be made using standard recombinant DNA techniques and which include both human and non-human fragments, are also included in the invention. Within the range of. Such chimeric humanized antibodies are described, for example, by Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Tani.
guchi, M. et al., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494.
Neuberger et al., WO 86/01533; Cabilly et al., US Pat. No. 4,186,56.
No. 7; Cabilly et al., European Patent Application No. 125,023; Better et al. (1988) Science 240: 10.
41-1043; Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987) J. Immunol. 139.
: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al. (1987) Canc. R.
es. 47: 999-1005: Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (198).
8) J. Natl. Cancer Inst. 80: 1553-1559; Morrison, SL (1985) Science.
229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; Winter, US Pat. No. 5,2.
25,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Scienc.
e 239: 1534; and Beidler et al. (1988) J. Immunol. 141: 4053-4060, can be used to make recombinant DNA techniques known in the art.

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies were generated using transgenic mice that are incapable of expressing the endogenous immunoglobulin heavy and light chain genes, but are capable of expressing human heavy and light chain genes. Can be done. Such transgenic mice can be immunized in the normal fashion with a selected antigen, eg, all or part of NT2LP. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutations. Therefore, using such mice, therapeutically useful human Ig
It is possible to produce G antibodies, IgA antibodies, and IgE antibodies. For an overview of this technology for making human antibodies, see Lonberg and Huszar (1995, Int. Rev.
Immunol. 13: 65-93). For a detailed discussion of this technique for making human antibodies and human monoclonal antibodies, as well as protocols for making such antibodies, see, for example, US Pat. No. 5,625,126, US Pat.
U.S. Patent No. 5,569,825, U.S. Patent No. 5,661,016, and U.S. Patent No. 5,545,
See issue 806.

Fully human antibodies that recognize selected epitopes are described by "guided s"
election) ”. In this approach, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope.

First, a non-human monoclonal antibody that binds to a selected antigen (epitope), for example, an antibody that inhibits NT2LP activity is identified. The heavy and light chains of the non-human antibody are cloned and used to generate phage display Fab fragments. For example, the heavy chain gene can be cloned into a plasmid vector so that the heavy chain can be secreted from the bacterium. The light chain gene can be cloned into the phage coat protein gene so that the light chain can be expressed on the surface of phage.
A repertoire of human light chains fused to phage (random assembly) is used to infect bacteria expressing non-human heavy chains. The resulting progeny phage represents a hybrid antibody (human light chain / non-human heavy chain). The selected antigen is used in a panning screen to select phage that bind to the selected antigen. Several rounds of selection may be necessary to identify such phage.
The human light chain gene is then isolated from the selected phage that binds to the selected antigen. These selected human light chain genes are then used to guide the selection of human heavy chain genes as follows. The selected human light chain gene is inserted into a vector for bacterial expression. Bacteria expressing selected human light chains are infected with a repertoire of human heavy chains fused to phage. The resulting progeny phages represent human antibodies (human light chain / human heavy chain). The selected antigen is then used in a panning screen to select phage that bind to the selected antigen. The phage selected in this step represent a fully human antibody that recognizes the same epitope recognized by the first selected non-human monoclonal antibody. Genes encoding both heavy and light chains are easily isolated,
It can be further engineered for the production of human antibodies. This technique is described in Jespers et al. (1994, Bio
technology 12: 899-903).

Anti-NT2LP antibodies (eg, monoclonal antibodies) can be used to isolate NT2LP proteins by standard techniques such as affinity chromatography or immunoprecipitation. Anti-NT2LP antibody facilitates purification of native NT2LP protein from cells and recombinantly produced NT2L expressed in host cells
Purification of P protein is facilitated. Moreover, anti-NT2LP antibodies can be used to detect NT2LP proteins (eg in cell lysates or cell supernatants) to assess the amount and pattern of NT2LP protein expression. Importantly, detection of circulating fragments of NT2LP protein is used to identify the metabolism of NT2LP protein in a subject. Anti-NT2LP antibodies can be used diagnostically as part of clinical trial techniques, for monitoring protein levels in tissues, for example to determine the efficacy of a given therapeutic regimen. Detection can be accomplished by coupling the antibody to a detectable substance (ie, physically binding). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish horseradish peroxidase, alkaline phosphatase, (-galactosidase or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin / biotin and avidin / biotin; suitable Examples of fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; Examples include luciferase, luciferin and aequorin; and examples of suitable radioactive materials are ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.
Is included.

III. Isolated NT2LP Nucleic Acid Molecules The present invention further provides isolated nucleic acid molecules encoding NT2LP proteins (hereinafter referred to as NT2LP genes or NT2LP nucleic acid molecules) and fragments of NT2LP genes.

The term “nucleic acid molecule” as used in the present invention is intended to include DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg mRNA) as well as DNA or RNA analogs made using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

An “isolated” nucleic acid molecule, as used herein, is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. An "isolated" nucleic acid is free from sequences that normally flank the nucleic acid in the genomic DNA of the organism from which it is derived (ie, sequences located at the 5'and 3'ends of the nucleic acid). preferable. For example, in various embodiments, an isolated NT2LP nucleic acid molecule can include about 5 kb, 4 kb of a nucleotide sequence that normally flanks the nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived (e.g., substantia nigra cells), It is less than 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or medium when made by recombinant methods, or precursor or other chemicals when chemically synthesized. May not be included in. However, the NT2LP nucleic acid molecule may be fused to coding or regulatory sequences for other proteins and still be considered isolated.

The isolated nucleic acid molecule of the present invention encodes the NT2LP protein. As mentioned above, NT2
The LP protein is a protein containing the amino acid sequence shown in SEQ ID NO: 2 (monkey NT2LP protein) or SEQ ID NO: 4 (human NT2LP protein), an allelic variant of the human or monkey NT2LP protein, and a human or monkey NT2LP protein. Defined as the ortholog of the NT2LP protein. A preferred NT2LP nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. The sequence of SEQ ID NO: 1 corresponds to monkey NT2LP cDNA. The sequence of SEQ ID NO: 3 corresponds to human NT2LP cDNA. This cDNA contains sequences encoding the human NT2LP protein (ie, the "coding region", start and stop codons are shown in Figures 1 and 2) and 5'untranslated and 3'untranslated sequences (Figure 1). And see 2).

Two types of human NT2LP cDNA have been found, both with identical putative coding regions. The difference between the two types is the result of different splicing in the 5'UTR. The human NT2LP amino acid sequence is shown in Figure 2. Human NT2LP protein is
It is mainly expressed in the brain and ovary.

One type of partial monkey NT2LP cDNA has been found. The monkey NT2LP amino acid sequence is shown in Figure 1. The monkey NT2LP protein is mainly expressed in brain and ovary.

The present invention differs from the nucleotide sequences (and fragments thereof) set forth in SEQ ID NOs: 1 and 3 (NT2LP) due to the degeneracy of the genetic code, and thus SEQ ID NO:
Further included are nucleic acid molecules that encode the same NT2LP protein as encoded by the nucleotide sequences shown in 1 and 3.

In another preferred embodiment, the isolated nucleic acid molecule of the invention is the complementary strand of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment of any of these nucleotide sequences. Contains nucleic acid molecules. A nucleic acid molecule complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 to form a stable duplex. To the extent possible, it is complementary to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

Orthologs and allelic variants of the human or monkey NT2LP gene can be readily identified using methods well known in the art. Human or monkey NT2L
Allelic variants and orthologs of the P gene may be synthesized with nucleotide sequences shown in SEQ ID NOs: 1 and 3 or fragments of these nucleotide sequences at least about 60-
65%, typically at least about 70-75%, more typically at least about 80-85%
, Most typically, will comprise nucleotide sequences having at least about 90-95% or greater homology. Such a nucleic acid molecule is capable of hybridizing to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a fragment of any of these nucleotide sequences, preferably under highly stringent conditions. Can be easily identified as

Furthermore, the nucleic acid molecule of the present invention may comprise only a fragment of the coding region of the NT2LP gene, such as the fragment of SEQ ID NO: 1 or SEQ ID NO: 3. Human or monkey N
Nucleotide sequences determined from the cloning of the T2LP gene are designed for use in identifying and / or cloning NT2LP gene homologues from other cell types, such as other tissues, and NT2LP gene orthologs from other mammals. Enables the production of probes and primers that The probe / primer typically comprises a substantially purified oligonucleotide. The oligonucleotide is typically at least about 12 of the sense strand, the antisense strand of SEQ ID NO: 1 or SEQ ID NO: 3, SEQ ID NO: 1 or SEQ ID NO: 3, or naturally occurring variants thereof. A region of nucleotide sequence that hybridizes under high stringency conditions with one, preferably about 25, more preferably about 40, 50, or 75 contiguous nucleotides. PCR based on the nucleotide sequence in SEQ ID NO: 1 or SEQ ID NO: 3 for cloning an NT2LP gene homolog
It can be used in the reaction. Probes based on the NT2LP nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In a preferred embodiment, the probe further comprises a labeling group attached to it. For example, the labeling group is a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such a probe may be used, for example, by measuring the level of NT2LP-encoding nucleic acid in a cell sample from a subject, for example, NT2LP levels or NT2LP m
As part of a diagnostic test kit for identifying cells or tissues that misexpress the NT2LP protein by detecting RNA levels or by determining whether the genomic NT2LP gene is mutated or deleted. Can be used.

In addition to the NT2LP nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, NT2
It will be appreciated by those skilled in the art that DNA sequence polymorphisms that alter the amino acid sequence of the LP protein may be present in a population (eg, a human or monkey population). Such NT2LP gene genetic polymorphisms may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to a nucleic acid molecule containing an open reading frame encoding an NT2LP protein, preferably a mammalian NT2LP protein. Such natural allelic diversity typically results in NT
It produces a 1-5% variance in the nucleotide sequence of the 2LP gene. All such amino acid polymorphisms in the NT2LP gene that result from nucleotide diversity and natural allelic diversity are intended to be within the scope of the present invention. Such allelic variations include both active allelic variants as well as inactive or diminished active allelic variants. The latter two types typically cause pathological disorders. Further, nucleic acid molecules encoding NT2LP proteins from other species and thus having a nucleotide sequence that differs from the human or monkey sequence of SEQ ID NO: 1 or SEQ ID NO: 3 are intended to be within the scope of the invention. To be done. Natural allelic variants and human or monkey NT2LP cDs of the invention
NA non-human orthologs or non-monkey orthologs are herein described according to standard hybridization techniques under high stringent hybridization conditions, using human or monkey cDNA or fragments thereof as hybridization probes. Can be isolated based on the homology with the human or monkey NT2LP nucleic acids disclosed in.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is a nucleic acid molecule that is at least 15 nucleotides in length and that, under high stringency conditions, comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Hybridize with. In other embodiments, the nucleic acid is at least 30, 50, 100, 250, or 500 nucleotides in length. In another preferred embodiment, the fragment of the NT2LP gene encodes the extracellular domain of the NT2LP protein, one or more transmembrane domains, or one or more intracellular domains. As used herein, the term "hybridize under conditions of high stringency" refers to at least each other.
It is intended to describe the hybridization and washing conditions under which nucleotide sequences having 60% identity typically remain hybridized to each other.
Preferably, the conditions are at least about 65%, more preferably at least about 70%, even more preferably at least about 75% or more, and sequences that are identical to each other typically remain hybridized to each other. It is a certain condition. Such highly stringent conditions are known to those of skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY.
. (1989), 6.3.1-6.3.6. Although not limited thereto, preferred examples of high stringent hybridization conditions include 6 at about 45 ° C.
X Hybridization in sodium chloride / sodium citrate (SSC) is followed by one or more washes in 0.2x SSC, 0.1% SSC at 50-65 ° C. Preferably, the isolated nucleic acid molecule of the present invention which hybridizes under high stringent conditions with the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 corresponds to a naturally occurring nucleic acid molecule. The term "naturally-occurring" nucleic acid molecule, as used herein, refers to an RNA or DNA molecule having a naturally-occurring nucleotide sequence (eg, encoding a native protein). In one embodiment, the nucleic acid is
Encodes the native human or monkey NT2LP protein.

In addition to naturally occurring allelic variants of the NT2LP nucleic acid sequences that may be present in a population, mutational changes may be introduced into the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, thereby altering the NT2LP protein. Those skilled in the art will further appreciate that it is possible to alter the amino acid sequence of the encoded NT2LP protein without altering its functional capacity. For example, at "non-essential" amino acid residues, nucleotide substitutions resulting in amino acid substitutions can be made within the sequence of SEQ ID NO: 2. The "non-essential" amino acid residue is a residue that can be changed from the wild-type sequence of the NT2LP protein (for example, the sequence of SEQ ID NO: 2) without changing the activity of NT2LP, and the "essential" amino acid residue Are residues necessary for the activity of the NT2LP protein. For example, conserved amino acid residues within the transmembrane domain of the NT2LP protein, such as aspartic acid, proline, threonine, and tyrosine, are most likely to be important for ligand binding and thus the essential residues of the NT2LP protein. Is. However, other amino acid residues (eg, non-conserved or semi-conserved residues within the transmembrane domain) may not be essential for activity, thus altering the activity of the NT2LP protein. No, it is likely to be changed.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding NT2LP proteins that contain changes in amino acid residues that are not essential for NT2LP activity. Such NT2LP proteins differ in amino acid sequence from SEQ ID NO: 2 or SEQ ID NO: 4, but retain at least one NT2LP activity described herein. In one embodiment, the isolated nucleic acid molecule is at least about 30-35%, preferably at least about 40-45%, more preferably at least about 50-55% with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, More preferably at least about 60-65%, more preferably at least about 70-75%, more preferably at least about 80-85%, most preferably at least about 90-95% or more homologous amino acid sequences. It includes a nucleotide sequence that encodes a protein that includes.

An isolated nucleic acid molecule encoding an NT2LP protein that is homologous to the protein of SEQ ID NO: 2 or SEQ ID NO: 4 has one or more amino acid substitutions, additions or deletions introduced into the encoded protein. Thus, one or more nucleotide substitutions, additions, or deletions can be made by introducing into the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Mutations can be introduced into SEQ ID NO: 1 or SEQ ID NO: 3 by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more putative nonessential amino acid residues. A "conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine,
Histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side Chain-bearing amino acids (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain amino acids (eg, threonine, valine, isoleucine), and aromatic side chain amino acids. (For example, tyrosine, phenylalanine, tryptophan,
Histidine) is included. Therefore, a putative nonessential amino acid residue in NT2LP is preferably replaced with another amino acid residue belonging to the same side chain family. Or, in another embodiment, for example, saturation mutagenesis
mutations were introduced randomly into all or part of the NT2LP coding sequence, resulting in
The resulting variants may be screened for NT2LP activity as described herein to identify variants that retain LP activity. Following mutagenesis of SEQ ID NO: 1 or SEQ ID NO: 3, the encoded protein is recombinantly expressed (eg, as described in Examples 3 and 4), eg, the assay methods described herein. Can be used to determine the activity of the protein.

In addition to the nucleic acid molecule encoding the NT2LP protein described above, another aspect of the invention relates to an isolated nucleic acid molecule antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence complementary to a "sense" nucleic acid encoding a protein, eg, complementary to the coding strand of a double stranded cDNA molecule or complementary to an mRNA sequence. Alternatively, the antisense nucleic acid molecule can hydrogen bond with the sense nucleic acid. Antisense nucleic acids can be complementary only to the full length NT2LP coding strand or fragments thereof. In one embodiment, the antisense nucleic acid molecule is antisense to the "coding region" of the coding strand of the nucleotide sequence encoding the NT2LP protein.

The term “coding region” refers to a region of a nucleotide sequence that contains codons translated into amino acid residues, eg, the full length coding region of SEQ ID NO: 1 or SEQ ID NO: 3 shown in FIGS. As expected. In another embodiment, the antisense nucleic acid molecule is antisense to the "non-coding region" of the coding strand of the nucleotide sequence encoding the NT2LP protein. The term "non-coding region" refers to the 5'and 3'sequences that flank the coding region that are not translated into amino acids (ie, 5'and 3 '.
Also called untranslated region).

Given the coding strand sequence encoding the NT2LP protein, Watson and Cri
The antisense nucleic acids of the invention can be designed according to the ck base pairing rules. The antisense nucleic acid molecule may be complementary to the entire coding region of NT2LP mRNA, but is more preferably an oligonucleotide that is antisense to only a fragment of the coding or non-coding region of NT2LP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NT2LP mRNA. Antisense oligonucleotides include, for example, about 5, 10, 15, 2
It can be 0, 25, 30, 35, 40, 45 or 50 nucleotides long. The antisense nucleic acids of the invention can be made using chemical synthesis and enzymatic ligation reactions known in the art. For example, a natural nucleotide, or to increase the biological stability of the molecule, such as phosphorothionate derivatives and acridine-substituted nucleotides, or a double-stranded physical chain formed between antisense and sense nucleic acids. Antisense nucleic acids (eg, antisense oligonucleotides) can be chemically synthesized using various modified nucleotides designed to increase stability. Examples of modified nucleotides that can be used to make antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-
Iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-
Galactosyl cueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6 -Adenine, 7-Methylguanine, 5-Methylaminomethyluracil, 5-Methoxyaminomethyl-2-thiouracil, β-D-Mannosyl cuocosine, 5'-Methoxycarboxymethyluracil, 5-Methoxyuracil, 2-Methylthio- N6-isopentenyladenine, uracil-5
-Oxyacetic acid (v), wybutoxosine, pseudouracil (ps)
eudouracil), cuocosine, 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-thiouracil, 3- (3-
Amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, the nucleic acid was subcloned in the antisense orientation (
That is, the RNA transcribed from the inserted nucleic acid will be in the antisense orientation to the target nucleic acid of interest as described in more detail in the subsections below). Can also be produced biologically.

The antisense nucleic acid molecules of the invention are typically administered to a subject, or they hybridize or bind to cellular mRNA and / or genomic DNA encoding the NT2LP protein, whereby, for example, Produced in situ to suppress protein expression by suppressing transcription and / or translation. Hybridization may be by conventional nucleotide complementation to form a stable duplex, or, for example, in the case of antisense nucleic acid molecules that bind to DNA duplexes, specificity in the major groove of the double helix. It may be through interaction. Examples of routes of administration of the antisense nucleic acid molecules of the invention include direct injection at the tissue site. Alternatively, the antisense nucleic acid molecule can be modified to target selected cells and administered systemically. For example, for systemic administration, the antisense nucleic acid molecule is linked to a peptide or antibody that binds to a cell surface receptor or antigen, to specifically bind to the receptor or antigen expressed on the selected cell surface. The antisense molecule can be modified to bind to.
The vectors described herein can also be used to deliver antisense nucleic acid molecules to cells. To achieve sufficiently high intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of the strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the present invention is a (-anomeric nucleic acid molecule. (-The anomeric nucleic acid molecule forms a specific double-stranded hybrid with a complementary RNA. However, unlike normal (-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625-6641).
This antisense nucleic acid molecule is a 2'-o-methyl ribonucleotide (Inoue et al. (1987)
Nucleic Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Let. 215: 327-330).

In yet another embodiment, the antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave single-stranded nucleic acids such as mRNAs that have complementary regions. Therefore, ribozymes (eg, hammerhead ribozymes (Haselhoff and Gerlach (1988) Nature 3
34: 585-591))) can be used to catalytically cleave NT2LP mRNA transcripts, thereby inhibiting the translation of NT2LP mRNA. Ribozymes with specificity for NT2LP-encoding nucleic acids are disclosed in NT2LP
It can be designed based on the nucleotide sequence of cDNA (for example, SEQ ID NO: 1 or SEQ ID NO: 3). For example, if the nucleotide at the active site encodes NT2LP
Derivatives of Tetrahymena L-19 IVS RNA can be made that are complementary to the nucleotide sequence to be cleaved in RNA. See, for example, Cech et al., US Pat. No. 4,987,071, and Cech et al., US Pat. No. 5,116,742. Alternatively, NT2LP mRNA can be used to select catalytic RNAs with specific ribonuclease activity from a pool of RNA molecules. For example, Bartel, D and Szostak,
See JW (1993) Science 261: 1411-1418.

Alternatively, by forming a triple helix structure that blocks transcription of the NT2LP gene in target cells by targeting to nucleotides complementary to the regulatory regions of NT2LP (eg, NT2LP promoter and / or enhancer), NT2LP
It can also inhibit the expression of the LP gene. For an overview, see Helene, C. (1991).
Anticancer Drug Des. 6 (6): 569-84, Helene, C. et al. (1992) Ann. NY Acad.
Sci. 660: 27-36, and Maher, LJ (1992) Bioassays 14 (12): 807-15.
Please refer to.

IV. Recombinant Expression Vectors and Host Cells Another aspect of the invention relates to vectors, particularly expression vectors containing a nucleic acid encoding an NT2LP protein (or fragment thereof). As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been attached. One type of vector is a "plasmid", which means a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector that is capable of ligating additional DNA segments into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such a vector is referred to herein as an "expression vector." In general, expression vectors used in recombinant DNA technology are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is most commonly used in the form of a vector. However, the invention is to be construed to include other forms of expression vectors such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) that have equivalent functions.

The recombinant expression vector of the invention comprises the nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which is based on the host cell in which the recombinant expression vector is used for expression. It is meant to include one or more regulatory sequences operably linked to the nucleic acid sequence to be selected and expressed. In the recombinant expression vector,
"Operably linked" means that the nucleotide sequence is linked to a regulatory sequence such that the nucleotide sequence is expressed (eg, in an in vitro transcription / translation system, or in the host cell if the vector is introduced into a host cell). Is interpreted as meaning that The term “regulatory sequence” is understood to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goddel "Gene Expression Technology" Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in many types of host cells, and sequences that direct expression of the nucleotide sequence only in particular host cells (eg, tissue-specific regulatory sequences). Be done. It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of host cell to be transformed, the desired level of protein expression and the like. The expression vector of the present invention can be introduced into a host cell, thereby providing a fusion protein or peptide encoded by the nucleic acids described herein (eg, NT2LP protein, altered forms of NT2LP protein, fusion protein, etc.). Proteins or peptides can be produced, including.

The recombinant expression vector of the present invention can be designed so that the NT2LP protein or a fragment thereof is expressed in prokaryotic cells or eukaryotic cells. For example
The NT2LP protein can be expressed in bacterial cells such as E. coli, insect cells (using, for example, baculovirus expression vectors) yeast cells, or mammalian cells. Suitable host cells include Goddel's Gene Expression Technology.
nology) "Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the amino terminus of the protein encoded by it, usually a recombinant protein. Such fusion vectors typically have three purposes: 1)
Increase the expression of the recombinant protein; 2) increase the solubility of the recombinant protein; and 3) aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site can be introduced at the junction of the fusion moiety and recombinant protein to separate the recombinant protein from the fusion moiety and subsequently purify the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include glutathione S-transferase (GST), maltose E binding protein,
Alternatively, pGEX (Pharmacia Biotech Inc .; Smith, DB and Johnson, KS (1988) Gene 67: 3, which fuses protein A and a target recombinant protein.
1-40), pMAL (New England Biolabs, Beverly, Mass.), And pRIT5 (Pharmacia, Piscataway, NJ). In one embodiment, the coding sequence of the NT2LP gene is cloned into a pGEX expression vector, NST to C-terminal to GST-thrombin cleavage site-NT2LP.
A vector encoding a fusion protein containing the protein is produced. The fusion protein may be purified by affinity chromatography using glutathione-agarose resin. Recombinant NT2LP protein not fused with GST can be recovered by cleaving the fusion protein with thrombin.

Examples of suitable inducible, non-fusion E. coli expression vectors include pTrc (Amann et al. (1988
) Gene 69: 301-315) and pET 11d (Studier et al., “Gene Expression Technology (Gene Ex)
Suppression Technology) "Methods in Enzymology 185, Academic Press, San Diego, CA (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from the T7 gn10-lac fusion promoter mediated by co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is transferred from a resident prophage carrying the T7 gn1 gene to the host strain BL under the transcriptional control of the lacUV 5 promoter.
21 (DE3) or HMS174 (DE3).

One strategy for maximizing recombinant protein expression in E. coli is to express the protein in host bacteria whose impaired ability to cleave the recombinant protein by proteolysis is compromised (Gottesman, S. "Gene expression technology (Gen
e Expression Technology) "Methods in Enzymology 185, Academic Press, San Diego, CA (1990) 119-128). Another strategy is to change the nucleic acid sequence of the nucleic acid inserted into the expression vector so that the individual codons for each amino acid are the codons that are selectively utilized in E. coli (Wa
da et al. (1992) Nucleic Acids Res. 20: 2111-2118). Such changes in the nucleic acid sequences of the invention can be made by standard DNA synthesis techniques.

In another embodiment, the NT2LP gene expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisae include pYepSec1 (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933. ~ 943), pJRY88 (Schultz et al. (1987) Gene 5
4: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA) and picZ (Invitrogen Corporation, San Diego,
California) is included.

Alternatively, the NT2LP gene can be expressed in insect cells using, for example, a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, Sf 9 cells) include pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and pVL series (Luckl).
ow and Summers (1989) Virology 170: 31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed,
B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:
187-195) are included. When used in mammalian cells, the expression vector's control functions 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 Sambrook, J., Fritsh, EF, and Mania.
tis, T., Molecular Cloning: Experimental Manual (Molecular Cloning: A Laborato
ry Manual) 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Labo
Ratory Press, Cold Spring Harbor, NY, 1989), chapters 16 and 17.

In another embodiment, the recombinant mammalian expression vector is capable of selectively directing the expression of nucleic acid in a particular cell type (eg, expressing nucleic acid using tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid cell-specific promoter (Calame and Eaton (1988) Adv. . Immnol. 43: 235
~ 275), especially the T cell receptor promoter (Winoto and Baltimore (1989) EM
BO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-
740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoter; Byrne and Ruddle (
1989) PNAS 86: 5473-5477), pancreas-specific promoter (Edlund et al. (1985) Sci.
ence 230: 912-916), mammary gland specific promoters (eg whey promoters;
U.S. Pat. No. 4,873,316 and European Patent Application No. 264,166). Developmentally regulated promoters, such as the mouse hox promoter (Kessel
And Gruss (1990) Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).

The invention further provides NT2LP cloned into the expression vector in the antisense orientation.
Provided is a recombinant expression vector containing a DNA molecule encoding a protein. That is, the DNA molecule is allowed to express an RNA molecule that is antisense to NT2LP mRNA (DN
(By transcription of the A molecule) is operably linked to regulatory sequences. It is possible to select a regulatory sequence functionally linked to the nucleic acid cloned in the antisense orientation, which directs the sustained expression of the antisense RNA molecule in a variety of cell types,
For example, viral promoters and / or enhancers, or regulatory sequences that direct constitutive, tissue-specific or cell-type specific expression of antisense RNA can be selected. An antisense expression vector is a recombinant plasmid in which antisense nucleic acid is produced under the control of a very efficient regulatory region, the activity of which can be determined by the cell type into which the vector is introduced, It can be in the form of a phagemid or an attenuated virus. For a discussion on the regulation of gene expression using antisense genes, see Antisense RNA as a molecular tool f as a molecular tool for genomic analysis by Weintraub, H. et al.
or genetic analysis), a review of Trends in Genetics, 1 (1), 1986.

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. It is understood that such terms refer not only to the particular test cell, but to the progeny or potential progeny of such a cell.
Such progeny may not actually be identical to the parental cell, although either mutations or environmental influences may cause changes in subsequent generations, but are still used herein. Within the scope of the term.

The host cell can be prokaryotic or eukaryotic. For example, the NT2LP protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (Chinese Hamster Ovary cells (CHO) or COS cells). Other suitable host cells are known to those of skill in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein,
The terms "transformation" and "transfection" introduce exogenous nucleic acid (eg, DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Are understood to mean a variety of art-recognized techniques for. Suitable methods for transforming or transfecting host cells are described by Sambrook et al. (Molecular Cloning: Experimental Manual (Molecular Cloning: A
Laboratory Manual) 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Ha
rbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.

It is known that for stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small fraction of the cells may incorporate exogenous DNA into their genome. is there. To identify and select for these integrants, a gene encoding a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest. Preferred selectable markers include genes that confer resistance to drugs such as G418, hygromycin and methotrexate. The nucleic acid encoding the selectable marker is
It can be introduced into the host cell on the same vector encoding the NT2LP protein, or it can be introduced into the host cell on a different vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive but other cells will die).

Host cells of the invention, such as cultured prokaryotic host cells or eukaryotic host cells, are capable of producing (ie, expressing) NT2LP protein. Accordingly, the invention further provides methods of producing NT2LP protein using the host cells of the invention. In one embodiment, the method comprises the step of culturing the host cell of the invention in a suitable medium until NT2LP protein (in which a recombinant expression vector encoding the NT2LP protein has been introduced) is produced. Including. In another embodiment, the method further comprises the step of isolating NT2LP protein from the medium or host cells.

The host cells of the invention can also be used to produce transgenic non-human animals. The non-human transgenic animal may ameliorate adverse effects of selected disorders or biological processes, such as mental disorders, disorders of the nervous system such as disorders or pain that impair the circadian rhythm and sleep-wake cycle. It can be used in screening assays designed to identify substances or compounds such as pharmaceuticals. For example, in one embodiment, a host cell of the invention has NT2L
A fertilized oocyte or embryonic stem cell into which the P protein coding sequence is introduced. Such host cells are then used to produce nonhuman transgenic animals in which the exogenous NT2LP gene sequence has been introduced into the genome, or homologous recombinant animals in which the endogenous NT2LP gene sequence has been altered. be able to. Such animals are NT2L
It is useful for examining the function and / or activity of P protein and for identifying and / or evaluating modulators of NT2LP gene activity. As used herein, a "transgenic animal" is a non-human animal in which one or more cells of the animal contains a transgene, preferably a mammal, more preferably a rat or mouse, etc. It is a tooth. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. The transgene is integrated into the genome of the cell from which the transgenic animal develops and remains in the mature animal's genome, thereby allowing the gene product encoded by one or more cell types or tissues of the transgenic animal. Exogenous DNA that directs expression. As used herein, a “homologous recombinant animal” has been introduced into an animal cell in which the endogenous NT2LP gene and the endogenous gene have been introduced, such as an embryonic cell of the animal prior to development into the animal. Extrinsic
It is a non-human animal, preferably a mammal, more preferably a mouse, which has been altered by homologous recombination with the DNA molecule.

In the transgenic animal of the present invention, a nucleic acid encoding the NT2LP protein is introduced into the male pronucleus of a fertilized egg by, for example, microinjection or retroviral infection to grow an oocyte in a pseudopregnant foster female animal. It can be produced by SEQ ID NO: 1 or SEQ ID NO: 3 human or monkey NT2LP
The cDNA sequence can be introduced as a transgene into the genome of a non-human animal. In addition, a non-human or non-monkey homologue of the human or monkey NT2LP gene, such as the mouse NT2LP gene, was isolated based on hybridization with a human or monkey NT2LP cDNA (described above) and It can be used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
Tissue-specific regulatory sequences can be operably linked to the NT2LP transgene to direct expression of the NT2LP protein to particular cells. Methods for regenerating transgenic animals, especially animals such as mice, through manipulation of embryos and microinjection are routine in the art and are described, for example, in Lede
r et al., US Pat. Nos. 4,736,866 and 4,870,009; Wagner et al., US Pat. No. 4,873.
, 191; and Hogan, B., "Manipulating the Mouse Embr.
yo) ”(Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, New York, 1986). Similar methods are used for the production of other transgenic animals. Transgenic probands can be identified based on the presence of the NT2LP transgene in their genome and / or the expression of NT2LP mRNA in animal tissues or cells. The transgenic progeny animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding the NT2LP protein can be further bred with other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, a deletion, addition, or substitution is introduced into the vector, thereby altering, eg, functionally disrupting, the NT2LP gene. Prepare a vector containing one fragment. NT2LP
The gene may be a human gene (eg, derived from a human genomic clone isolated from a human genomic library screened using the cDNA of SEQ ID NO: 1 or SEQ ID NO: 3), but more preferably the non-human NT2LP gene It is a human homolog. For example, the NT2LP cDNA of SEQ ID NO: 1 or SEQ ID NO: 3 can be used as a probe to isolate the mouse NT2LP gene from a mouse genomic DNA library. The mouse NT2LP gene can then be used to create a homologous recombination vector suitable for modifying the endogenous NT2LP gene in the mouse genome. In a preferred embodiment, the vector is designed such that upon homologous recombination the endogenous NT2LP gene is functionally disrupted (ie no longer encodes a functional protein; also referred to as a "knockout" vector). Alternatively, the vector may be such that upon homologous recombination, the endogenous NT2LP gene is mutated or otherwise encodes an altered but still functional protein (e.g., by altering upstream regulatory regions). Thereby altering the expression of the endogenous NT2LP protein). In the homologous recombination vector, the altered fragment of the NT2LP gene is flanked by additional nucleic acid of the NT2LP gene at its 5'and 3'ends, and the vector carries an exogenous NT2LP gene.
Allow homologous recombination between the gene and the endogenous NT2LP gene in embryonic stem cells. The flanking NT2LP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of adjacent DNA (5 '
And 3'at both ends) are included in the vector (see, eg, Thomas and Capecchi (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (eg, by electroporation) and selects for cells in which the introduced NT2LP gene has been homologously recombined with the endogenous NT2LP gene (eg, Li et al. (1992) Cell 69). : 915). The selected cells are then injected into animal (eg, mouse) embryonic cells to form aggregated chimeras (
For example, Bradley's “Teratocarcinomas and Embryonic Stem Cells: A Practical Approach”
bryonic Stem Cells: A Practical Approarch) ", edited by Robertson, (IRL, Oxford, 1987), pages 113-152). The chimeric embryo can then be implanted in a suitable foster mother, a pseudopregnant female animal, and the embryo allowed to grow to term. Progeny that have homologous recombinant DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologous recombinant DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are further described in Bradley (1991) Current Opinion in Biotechnology 2: 823-829 and International Publication No. 90/11354, International Publication No. 91/01140, International Publication No. 92/0
968 and WO 93/04169.

In another embodiment, transgenic non-human animals can be produced that contain a selection system that regulates and expresses 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) P.
See NAS 89: 6232-6236. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al. (1991) Science 251: 1351-1355). cre / lox
If the expression of the transgene is regulated using the P recombinase system, an animal containing the transgene encoding both the Cre recombinase and the selected protein is required. Such animals include the construction of “double” transgenic animals, eg, transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
It can be provided by crossing hens.

The non-human transgenic animal clones described herein are also Wilm.
ut, I. et al. (1997) Nature 385: 810-813, and WO 97/07668 and WO 97/07669. Briefly, a cell, for example a somatic cell from a transgenic animal, is isolated and
It can be induced to exit the growth cycle and enter G ₀ phase. Next, the resting cells
It can be fused with enucleated oocytes of an animal of the same species from which quiescent cells are isolated, for example by using electrical pulses. The reconstructed oocyte is then cultured so that it develops into a morula or germ cell and then transferred into a pseudopregnant female foster animal. The offspring born from this female foster animal is a clone of the animal from which the cells, eg somatic cells, were isolated.

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologs, modulators and antibodies described herein can be used in one or more of the following methods: a) drug screening assays, b) diagnostic assays. , Especially disease identification, allele screening and pharmacogenetic testing, c) treatment methods, d) pharmacogenomics, and e) observation of efficacy during clinical trials. The NT2LP protein of the present invention can be used as a drug target for developing a substance for modulating the activity of the NT2LP protein (NT receptor). As described in more detail below, the isolated nucleic acid molecule of the present invention may be used to express NT2LP protein (e.g., via a recombinant expression vector in a host cell,
Or in gene therapy applications), NT2LP mRNA (eg in a biological sample) or
It can be used to detect natural or recombinantly occurring gene mutations in the NT2LP gene and to modulate NT2LP activity. In addition, the NT2LP protein can be used for screening for drugs or compounds that modulate the activity of the NT2LP protein. Furthermore, the anti-NT2LP antibodies of the invention can be used to detect and isolate NT2LP proteins, especially fragments of NT2LP proteins present in biological samples, and to prepare NT2LP proteins.

A. Drug Screening Assays The present invention provides aberrant or abnormal forms of pain, for example.
Alternatively, methods are provided for identifying compounds or agents that can be used to treat disorders characterized by (or resulting from) expression of normal NT2LP nucleic acid and / or NT2LP protein activity. These methods are also referred to herein as drug screening assays, which typically involve, inter alia, specifically (eg, bind) the NT2LP protein, the interaction of the NT2LP protein with a target molecule (such as a ligand). And / or expression of NT2LP nucleic acid and / or N
A step of screening candidate / test compounds or agents to identify compounds that are agonists or antagonists of the NT2LP protein or fragments thereof with respect to modulating the activity of the T2LP protein is included. Candidate / test compounds or agents having one or more of these abilities are characterized by (or by) ectopic or abnormal or normal NT2LP nucleic acid expression and / or NT2LP protein activity, eg, pain. It can be used as a drug to treat disorders. Candidate / test compounds include, for example, 1) fusion peptides with an Ig tail, as well as random peptide libraries (eg, Lam et al. (1991) Nature 354: 82-84; H.
oughten et al. (1991) Nature 354: 84-86) and peptides, such as soluble peptides, containing members of a molecular library from combinatorial chemistry consisting of D- and / or L-amino acids, 2) phosphopeptides (eg. , Members of a directed and partially overlapping directed phosphopeptide library, eg Songya
ng et al. (1993) Cell 72: 767-778), 3) antibody (eg, polyclonal antibody, monoclonal antibody, humanized antibody, anti-idiotype antibody, chimeric antibody, and single chain antibody, and Fab, F ( ab ') ₂ , fragments of Fab expression libraries and epitope binding fragments of antibodies), and 4) small organic and inorganic molecules (eg, molecules obtained from combinatorial and natural product libraries).

In one embodiment, the invention provides an assay method for screening candidate / test compounds that interact with (eg, bind to) NT2LP protein or fragments thereof. Assays typically involve the presence of a candidate compound in a complex whereby the candidate compound interacts with an NT2LP protein or fragment thereof (eg,
The NT2LP protein or fragment thereof under conditions such that the interaction (eg, binding) of the candidate / test compound with the NT2LP protein or fragment thereof allows formation of a complex, such that the ability to Expressing cells or isolated NT2
A recombinant cell-based or cell-free assay comprising combining a candidate / test compound with an LP protein or fragment thereof, and detecting the formation of a complex. The formation of complexes between the NT2LP protein and candidate compounds can be detected using competitive binding assays and can be quantified using, for example, standard immunoassays.

In another embodiment, the present invention modulates the interaction between the NT2LP protein and the molecule with which the NT2LP protein normally interacts (target molecule), which is likely to be the activity of the NT2LP protein as well. Provide screening assays to identify candidate / test compounds that (eg, stimulate or suppress). Examples of such target molecules include ligands in the same signaling pathway as the NT2LP protein (NT
Such as NT to the receptor) and proteins, such as proteins that act upstream (including both stimulators and repressors of activity) or downstream of the NT2LP protein in cognitive function signaling pathways or pathways involving NT2LP protein activity, such as: Included are G proteins or other interactors involved in cAMP or phosphatidylinositol turnover and / or activation of adenylate cyclase or phospholipase C. Assays are typically performed on cells that express the NT2LP protein or fragment thereof, such as under conditions where the NT2LP protein or biologically active fragment thereof interacts with (eg, binds) the target molecule in the absence of the candidate compound. , A target molecule of the NT2LP protein (eg, a ligand or an NT2LP-binding signaling partner) and a candidate / test compound, and detecting complex formation involving the NT2LP protein and the target molecule or the NT2LP protein and the target molecule Recombinant cell-based assay comprising detecting interaction / reaction with. Detection of complex formation can include direct quantification of the complex, eg, by measuring the inducing effect of NT2LP protein. Interaction of the NT2LP protein with the target molecule in the presence of the candidate compound (compared to that detected in the absence of the candidate compound (eg, NT2LP
A statistically significant change (eg reduction) in the complex formation between the protein and the target molecule may modulate (eg stimulate or inhibit) the interaction between the NT2LP protein and the target molecule. Shown. Modulation of complex formation between the NT2LP protein and the target molecule can be quantified using, for example, immunoassay methods.

In order to perform a cell-free drug screening assay, the NT2LP protein or NT2LP protein or It is desirable to immobilize any of its target molecules. The interaction (eg, binding) between the NT2LP protein and the target molecule in the presence and absence of the candidate compound can be performed in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes and centrifuge tubes. In one embodiment, a fusion protein can be provided that has a domain added to allow binding of the protein to the substrate. For example, the glutathione-S-transferase / NT2L fusion protein
Glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates were adsorbed and subsequently mixed with cell lysates (eg, ³⁵ S-labeled) and candidate compounds to allow complex formation. The mixture is incubated under the conditions that result (eg physiological conditions for salt and pH). Following incubation, the beads are washed to remove unbound label and the immobilized substrate and radiolabel are measured directly or in the supernatant after dissociation of the complex. Alternatively, the complexes can be dissociated from the substrate, separated by SDS-PAGE using standard electrophoretic methods, and the level of NT2LP binding protein found in the bead fraction determined from the gel.

Other methods for immobilizing proteins on substrates can also be used in the screening assays of the invention. For example, binding to biotin and streptavidin can be used to immobilize the NT2LP protein or its target molecule. Biotinylated NT2LP protein was prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, IL) to obtain streptavidin. It can be fixed in the wells of coated 96-well plates (Pierce Chemical). Or NT2LP
It is also possible to derivatize an antibody to the wells of the plate that reacts with the protein, but does not interfere with the binding of the NT2LP protein to its target molecule, and the antibody binding can capture the NT2LP protein in the well. As described above, preparations of NT2LP binding protein and candidate compounds can be incubated in the wells of plates displaying NT2LP protein and the amount of complex trapped in the wells quantified. Methods for detecting such complexes include, in addition to those described above for GST-immobilized complexes, antibodies that react with the target molecule of the NT2LP protein, or antibodies that react with the NT2LP protein and compete with the target molecule. Enzyme-linked assays that rely on immunodetection of the complex used, as well as detection of enzyme activity associated with the target molecule are included.

In yet another embodiment, the present invention treats disorders characterized by (or resulting from) ectopic or aberrant or normal NT2LP nucleic acid expression or NT2LP protein activity, such as pain. Methods for identifying compounds that can be used to do so (eg, screening assays) are provided. This method typically assays the ability of a compound or substance to modulate the expression of NT2LP nucleic acid or the activity of NT2LP protein, and thereby ectopic or aberrant or normal NT2L.
Includes identifying compounds for treating disorders characterized by P nucleic acid expression or NT2LP protein activity. The method for assaying the ability of a compound or substance to modulate NT2LP nucleic acid expression or NT2LP protein activity is typically a cell-based assay. For example, cells that signal via a pathway involving the NT2LP protein can be induced to overexpress the NT2LP protein in the presence or absence of the candidate compound. It is possible to identify candidate compounds that result in a statistically significant change in the NT2LP protein dependent response (either stimulation or inhibition). In one embodiment, the expression of NT2LP nucleic acid or the activity of NT2LP protein in the cell is adjusted and the effect of the candidate compound on the reading of interest (such as cAMP or phosphatidylinositol turnover) is measured.
For example, the expression of genes that are upregulated or downregulated in response to the NT2LP protein dependent signal cascade can be assayed. In a preferred embodiment, the regulatory regions of such genes, such as the 5'flanking promoter and enhancer regions, are operably linked to a detection marker (such as luciferase) that encodes a readily detectable gene product. Phosphorylation of NT2LP protein or NT2LP protein target molecule can also be measured, for example, by immunoblotting.

Alternatively, modulators of NT2LP gene expression (eg, compounds that can be used to treat disorders or biological processes characterized by ectopic or aberrant or normal NT2LP nucleic acid expression or NT2LP protein activity). Can also be identified in a method of contacting cells with a candidate compound and measuring the expression of the NT2LP protein mRNA or protein in the cells. The expression level of NT2LP mRNA or protein in the presence of the candidate compound is compared to the expression level of NT2LP mRNA or protein in the absence of the candidate compound. Next, based on this comparison, the candidate compound was selected as NT2LP.
It can be identified as a modulator of nucleic acid expression and can be used in the treatment of disorders characterized by aberrant NT2LP nucleic acid expression. For example, if NT2LP mRNA or protein expression is higher (statistically significantly higher) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NT2LP nucleic acid expression. To be done. Alternatively, if the expression of the NT2LP nucleic acid is lower (statistically significantly lower) in the presence of the candidate compound than in the absence thereof, the candidate compound is identified as an inhibitor of the expression of the NT2LP nucleic acid. . The expression level of NT2LP nucleic acid in cells can be measured by the methods described herein for the detection of NT2LP mRNA or protein.

In yet another aspect of the invention, the NT2LP protein or fragment thereof modulates NT2LP protein activity with other proteins that bind or interact with the NT2LP protein (“NT2LP binding protein” or “NT2LP-bp”). Two-hybrid assays (see, for example, US Pat.
283,317; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem.
268: 12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (
1993) Oncogene 8: 1693-1696; and Brent, WO 94/10300). Such NT2LP binding proteins can also be labeled as NT2LP protein pathway upstream or downstream factors, for example.
Possibly involved in signal amplification by LP proteins.

The two-hybrid system is a modular nature of most transcription factors.
It is based on e), which consists of separable DNA binding and activation domains. B
artel et al., "Using the Two-Hybrid System to Detect Protein-Protein Intera"
ctions) ”, Cellar Interaction in Developmen
t), (Oxford University Press, Oxford, 1993) 153-179.
Briefly, this assay method uses two different DNA constructs. In one construct, the gene encoding the NT2LP protein is fused to the gene encoding the DNA binding domain of a known transcription factor (eg GAL-4).
In the other construct, an unidentified protein (“prey” or “sample”)
DNA sequences derived from a DNA sequence library coding for (1) are fused to a gene coding for the activation domain of a known transcription factor. When the "bait" and "prey" proteins interact to form an NT2LP protein-dependent complex in vivo, the DNA binding and activation domains of this transcription factor are:
It will be very close. This proximity allows transcription of a reporter gene (eg, LacZ) that is operably linked to transcriptional regulatory sites that respond to transcription factors. Expression of the reporter gene can be detected and can be used to isolate cell colonies containing functional transcription factors and to obtain a cloned gene encoding a protein that interacts with the NT2LP protein. it can.

Modulators of NT2LP protein activity and / or NT2LP nucleic acid expression identified according to these drug screening assays may be used, for example, for the treatment of nervous system disorders and processes or pain. These methods of treatment may include subjecting a subject in need of such treatment, such as a subject having a disorder or biological process described herein, to, for example, the pharmaceutical compositions described in subsection IV above. In some form, administering a modulator of NT2LP protein activity and / or nucleic acid expression is included.

B. Diagnostic Assays The present invention further provides methods for detecting the presence of NT2LP protein or NT2LP nucleic acid molecules or fragments thereof in a biological sample. This method applies to NT2L in biological samples.
Contacting the biological sample with a compound or substance capable of detecting NT2LP protein or mRNA such that the presence of the P protein / encoding nucleic acid molecule is detected. A preferred substance for detecting NT2LP mRNA was a labeled nucleic acid probe capable of hybridizing with NT2LP mRNA or a labelable nucleic acid probe. The nucleic acid probe is, eg, in a full-length NT2LP cDNA, eg, SEQ ID NO: 1 or 3, or a fragment thereof, eg, at least 15, 30, 50, 100, 250 or 500 nucleotides in length, and under stringent conditions. It can be, for example, an oligonucleotide that is sufficient to specifically hybridize to NT2LP mRNA below. NT
A preferred substance for detecting 2LP protein is a labeled antibody or a labelable antibody capable of binding to NT2LP protein. The antibody is a polyclonal antibody,
Or more preferably it may be a monoclonal antibody. Whole antibodies, or fragments thereof (eg, Fab or F (ab ′) ₂ ) can be used. The term "labeled or labelable" with respect to a probe or antibody, in addition to directly labeling the probe or antibody by binding (ie, physically linking) the probe or antibody to a detectable substance, directly labeled It is intended to include indirectly labeling the probe or antibody with its reactivity with another substance. Examples of indirect labeling include detection of the primary antibody with a fluorescently labeled secondary antibody, and end labeling of the DNA probe with biotin, which can be detected with fluorescently labeled streptavidin. 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 the subject. That is, the detection method of the present invention can be used to detect NT2LP mRNA or protein in a biological sample in vitro and in vivo.
For example, in vitro detection methods for NT2LP mRNA include Northern hybridizations and in situ hybridizations. In vitro detection methods for NT2LP protein include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation, and immunofluorescence. Alternatively, the NT2LP protein can be detected in vivo in a subject by introducing a labeled anti-NT2LP antibody into the subject. For example, the antibody can be labeled with a radioactive marker whose presence or location in a subject can be detected by standard imaging techniques. Methods of detecting allelic variants of NT2LP protein expressed in a subject and methods of detecting fragments of NT2LP protein in a sample are particularly useful.

The present invention also includes a kit for detecting the presence of NT2LP protein in a biological sample. The kit is eg a reagent such as a labeled compound or a labelable compound or substance capable of detecting NT2LP protein or mRNA in a biological sample; means for determining the amount of NT2LP protein in the sample; and Means for comparing the amount of NT2LP protein to a standard may be included. The compound or substance can be packaged in a suitable container. The kit may further include instructions for using the kit to detect NT2LP mRNA or protein.

The method of the present invention further detects a genetic mutation in the naturally occurring NT2LP gene, whereby a subject carrying the mutant gene is ectopic as described herein, eg, ectopic, such as pain. It can be used to determine whether at risk of a disorder characterized by the expression of a sex or aberrant NT2LP nucleic acid, or the activity of the NT2LP protein. In a preferred embodiment, the method comprises the presence or absence of a change in the cell sample from the subject that affects the integrity of the gene encoding the NT2LP protein or a gene mutation characterized by at least one in the misexpression of the NT2LP gene. Including detecting. For example, such gene mutations can be detected by confirming the presence of at least one of the following: 1) deletion of one or more nucleotides from the NT2LP gene; 2) NT2LP of one or more nucleotides. Addition to the gene; 3) substitution of one or more nucleotides of the NT2LP gene; 4) chromosomal rearrangement of the NT2LP gene; 5) alteration of the transcription level of the mRNA of the NT2LP gene; 6) abnormal modification of the NT2LP gene, eg Aberrant modification of methylation pattern of its genomic DNA; 7) Presence of non-wild type splicing pattern of transcription of mRNA of NT2LP gene; 8) Non-wild type level of NT2LP protein; 9) NT2
Loss of LP gene alleles; and 10) Inappropriate post-translational modification of NT2LP protein. As noted herein, there are numerous assays known in the art that can be used to detect mutations in the NT2LP gene.

In some embodiments, the detection of the aforementioned mutations can be accomplished by polymerase chain reaction (PCR), such as anchor PCR or RACE PCR (eg, US Pat. Nos. 4,683,195 and 4,683,
202) or ligation chain reaction (LCR) (eg Land
egran et al., Science 241: 1077-1080 (1988); and Nakazawa et al., PNAS 91: 360-364 (
1994)), the latter being particularly useful for detecting point mutations in the NT2LP gene (eg, Abravaya et al., Nucleic Acids Res. 23: 675). -682 (1995)). This method includes the steps of collecting a cell sample from a patient, nucleic acid (eg, genome,
mRNA or both), contacting this nucleic acid sample with one or more primers that specifically hybridize to the NT2LP gene under conditions which result in hybridization and amplification of the NT2LP gene (if present) And detecting the presence or absence of the amplification product, or detecting the size of the amplification product and comparing it to the length of the control sample.

In another embodiment, the mutation of the NT2LP gene derived from the sample cell can be determined by changing the restriction enzyme cleavage pattern. For example, sample and control DNA is isolated, amplified (optional), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length size between sample and control DNA indicate mutations in the sample DNA. In addition, with sequence-specific ribozymes (see, eg, US Pat. No. 5,498,531), the appearance or loss of ribozyme cleavage sites can be used as a score for the presence of specific mutations.

In yet another embodiment, the NT2LP gene is directly sequenced and the sample NT2LP gene sequence is sequenced using any of a variety of sequencing reactions known in the art.
Mutations can be detected by comparison with the corresponding wild type (control) sequence. Examples of sequencing reactions are the technology developed by Maxam and Gilbert (PNAS 74: 560 (1977)) or S
Including those based on the technology developed by anger (PNAS 74: 5462 (1977)). When performing diagnostic analysis, various automated sequencing methods (Biotechniques 19: 448 (1995)) can be used, which include mass spectrometric sequencing (eg PCT WO 9).
4/16101; Cohen et al., Adv. Chromatogr. 36: 127-162 (1996); and Griffin et al.,
Appl. Biochem. Biotechnol. 38: 147-159 (1993)).

Another method to detect mutations in the NT2LP gene is to use protection from the cleavage agent, R
Methods for detecting mismatched bases in NA / RNA or RNA / DNA duplexes (Myers et al., Science 230: 1242 (1985)); Cotton et al. (1988) PNAS 85: 4397; Saleeba et al. (19).
92) Meth. Enzymol. 217: 286-295), a method for comparing electrophoretic mobilities of mutant and wild type nucleic acids (Orita et al. (1989) PNAS 86: 2766; Cotton (1993) Mutat. Res.
285: 125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9: 73-79.
), A method of assaying migration of mutant and wild-type fragments in polyacrylamide gels containing denaturant gradients using denaturing gradient gel electrophoresis (Myers et al. (1985) Natur.
e 313: 495) are included. Examples of other techniques for detecting point mutations include:
Includes selective oligonucleotide hybridization, selective amplification and selective primer extension.

C. Method of Treatment Another aspect of the invention is a disease, disorder or disorder characterized by (or resulting from) ectopic or aberrant or normal NT2LP nucleic acid expression and / or NT2LP protein activity, such as pain. A method for treating a subject, such as a human, having a biological process. These methods include the step of administering to the subject a modulator (agonist or antagonist) of the NT2LP protein / gene such that treatment is effected. "Ectopic (aberrant) or abnormal (ab
The term "normal expression of NT2LP protein" means expression of non-wild type NT2LP protein, or expression of non-wild type NT2LP protein. Ectopic or aberrant NT2LP protein activity means non-wild type NT2LP protein activity or non-wild type levels of NT2LP protein activity. Since the NT2LP protein is involved in a pathway involved in intracellular signal transduction, the activity or expression of the ectopic or aberrant NT2LP protein is a function mediated by the NT2LP protein signaling,
It interferes with normal regulation, especially in brain cells.

As used herein, the term “treat” or “treatment” refers to disorders, diseases characterized by, or caused by, ectopic or aberrant or normal NT2LP protein activity or expression of NT2LP nucleic acid. Or, means reducing or alleviating at least one of the adverse effects or symptoms of a disorder, disease or biological process such as a biological process. Particularly useful is the treatment of disorders mediated by aberrant or normal NT2LP receptor interactions / signalling, such as pain. The term "treat" or "treatment" as used herein refers to a disorder, disease or biological process characterized by being able to be sedated by modulating the activity or expression of normal NT2LP nucleic acid or protein. It is also meant to reduce or alleviate at least one adverse effect or symptom.

As used herein, an NT2LP protein / gene modulator is a molecule capable of modulating the expression of NT2LP nucleic acid and / or NT2LP protein activity. For example, an NT2LP gene or protein modulator up-regulates (activates / agonizes) or down-regulates (suppresses / antagonizes) expression of, for example, an NT2LP nucleic acid. In another example, the NT2LP protein / gene modulator can modulate (eg, stimulate / agonize or suppress / antagonize) NT2LP protein activity. Inhibits NT2LP nucleic acid expression in disorders, diseases or biological processes characterized by (or caused by) ectopic or aberrant (non-wild-type) or normal NT2LP protein activity or NT2LP nucleic acid expression The NT2LP modulator may be an antisense molecule, such as a ribozyme described herein, if it is desired to treat it. Examples of antisense molecules that can be used to inhibit the expression of NT2LP nucleic acids include antisense molecules complementary to a fragment of the 5'untranslated region of SEQ ID NO: 1 or SEQ ID NO: 3, which also includes the start codon, And an antisense molecule complementary to a fragment of the 3'untranslated region of SEQ ID NO: 1 or SEQ ID NO: 3. Examples of antisense molecules complementary to a fragment of the 5'untranslated region of SEQ ID NO: 1 or SEQ ID NO: 3 including the start codon include nucleotides 1 to 616 of SEQ ID NO: 1 or SEQ ID NO: 3 Is a nucleic acid molecule containing a nucleotide complementary to.

An NT2LP modulator that inhibits expression of NT2LP nucleic acid may be, for example, a small molecule or other drug that inhibits expression of NT2LP nucleic acid, identified using the screening assays described herein. . Diseases, disorders or organisms characterized by (or resulting from) ectopic or aberrant (non-wild type) or normal NT2LP nucleic acid expression and / or aberrant or normal NT2LP protein activity, such as pain When it is desired to treat a biological process by stimulating the expression of an NT2LP nucleic acid, an NT2LP modulator is, for example, a nucleic acid molecule encoding the NT2LP protein (e.g., the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3). A nucleic acid molecule comprising a nucleotide sequence homologous thereto), or a small molecule such as a small molecule (peptide) or drug that stimulates expression of NT2LP nucleic acid or other drug identified using the screening assays described herein. Can be

Alternatively, a disease, disorder or organism characterized by (or resulting from) ectopic or aberrant (non-wild type) or normal NT2LP nucleic acid expression and / or NT2LP protein activity, such as pain. Where it is desired to treat a biological process by inhibiting NT2LP protein activity, an NT2LP modulator is, for example, a small molecule that inhibits NT2LP protein activity identified using the screening assays described herein. Or it can be an anti-NT2LP antibody, a small molecule or other drug, such as a drug, or a fragment of the NT2LP protein (eg, the extracellular domain). Ectopic or aberrant (non-wild type) or normal NT, eg pain
A disease, disorder or biological process characterized by (or resulting from) 2LP nucleic acid expression and / or normal or aberrant NT2LP protein activity is desired to be treated by stimulating NT2LP protein activity The NT2LP modulator is, for example, an activated NT2LP protein or a fragment thereof (for example, an NT2LP protein or a fragment thereof having an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof), or the present specification It can be a small molecule or other drug, such as a small molecule or drug that stimulates NT2LP protein activity, identified using the screening assay described in.

Another aspect of the invention pertains to methods for modulating NT2LP protein-mediated cellular activity. These methods modulate the cells with NT2LP protein activity or NT2LP nucleic acid expression such that NT2LP protein-mediated cellular activity is altered relative to normal levels (eg, cAMP or phosphatidylinositol metabolism).
Or a composition comprising an effective amount of a substance). As used herein, "NT2LP protein-mediated cellular activity" means normal or abnormal activity or function of cells. Examples of cellular activity via the NT2LP protein include phosphatidylinositol turnover, production or secretion of molecules such as proteins, contraction,
Involvement in proliferation, migration, differentiation, cell survival, and pain pathways. In one preferred embodiment, the cells are brain cells such as hippocampal cells. The term "altered" as used herein refers to changes associated with cell-related activities, in particular cAMP or phosphatidylinositol turnover and increased or diminished activation of adenylate cyclase or phospholipase C. In one embodiment, the substance is NT2LP
Stimulates protein activity or expression of NT2LP nucleic acids. In another embodiment, the agent inhibits NT2LP protein activity or expression of NT2LP nucleic acid. These methods of preparation may be performed in vitro (eg, by culturing cells with a substance) or in vivo (eg, by administering the substance to a subject). In one preferred embodiment, the method of adjustment is performed in vivo, i.e. the cells are present in a subject, such as a mammal, such as a human, and the subject is ectopic or aberrant or normal for NT2LP protein activity or NT2LP nucleic acid. Having a disorder, disease, or biological process characterized by or resulting from expression.

Nucleic acid molecules, proteins, modulators, compounds, etc., used in methods of treatment can be incorporated into suitable pharmaceutical compositions described below to provide nucleic acid molecules, proteins, NT2L.
The subject can be administered to the subject through a route by which the P modulator, compound, etc. can perform their intended functions.

D. Genomic pharmacology A test / candidate compound, or modulator, that has a stimulatory or inhibitory effect on NT2LP protein activity (eg, NT2LP gene expression), as identified by the screening analyzes described herein, is Disorders or biological processes associated with aberrant NT2LP protein activity (eg, CNS disorders and pain) can be administered to an individual to treat (prophylactically or therapeutically). In combination with such treatments, the genomic pharmacology of an individual (ie, the study of the relationship between an individual's genotype and the individual's response to foreign compounds or drugs) has been considered. Differences in the metabolism of therapeutic agents can lead to severe toxicity or treatment failure due to changes in the relationship between dose and blood concentration of the pharmaceutically active agent. Therefore, depending on the genomic pharmacology of an individual, an effective compound for prophylactic or therapeutic treatment based on consideration of the genotype of the individual (
For example, a drug) can be selected. Such genomic pharmacology can further be used to determine the appropriate dose and treatment regimen. Therefore, the activity of NT2LP protein, the expression of NT2LP nucleic acid, or the mutation content of NT2LP gene in an individual can be determined, which will select the appropriate compound for the therapeutic or prophylactic treatment of the individual.

Genomic pharmacology addresses clinically significant genetic variations in the response to drugs due to altered drug biokinetics and aberrant effects in affected individuals. About this, for example, Eichelbaum, M. (1996) Chin. Exp. P
haramacol. Physiol. 23 (10-11), Linder, MW, Clin. Chem. 43 (2): 254-266 (
1997). In general, two types of genetic pharmacological conditions can be distinguished. Transmitted as a single factor that modifies the way a drug acts on a living organism, transmitted as a single genetic factor (altered drug action) or as a single factor that modifies the way a living body acts on a drug It is a genetic condition (altered drug metabolism). These genetic pharmacological conditions can occur as either rare defects or polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) has major clinical complications as oxidants (antimalarial drugs, sulfonamides, analgesics, nitrofuran).
It is a common inherited enzymatic disease, such as hemolysis after ingestion and after eating fava beans.

In an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms in drug-metabolizing enzymes, such as N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2DG and CYP2Cl9, has shown that in some patients, after taking a standard safe dose of the drug, It explains why it is not possible to obtain the expected drug action, or there is excessive drug action and severe toxicity. These polymorphisms are manifested in the population in two phenotypes: excess metabolizer (EM) and poor metabolite (PM). The prevalence of PM varies among different populations. For example, the gene encoding CYP2D6 is highly polymorphic, and several mutations have been identified in PM, all of which lead to a loss of functional CYP2D6. Poor metabolites of CYP2D6 and CYP2Cl9 very often experience excessive drug reactions and side effects when taken at standard doses. If the metabolite is the therapeutically active moiety, PM does not show a therapeutic response, indicated by its analgesic effect of codeine mediated by its CYP2D6-generated metabolite morphine. Another excess metabolite is the so-called ultra-rapid metabolizers that do not respond at standard doses. More recently, it was established that the molecular basis of ultrarapid metabolism is due to gene amplification of CYP2D6.

Accordingly, the activity of NT2LP protein, expression of NT2LP nucleic acid, or NT in a subject.
The mutation content of the 2LP gene can be determined, which allows the selection of agents suitable for therapeutic or prophylactic treatment of an individual. Additionally, pharmacogenetic studies can be used in applying genotyping of polymorphic alleles encoding drug-metabolizing enzymes to determine the drug-responsive phenotype of a subject. When treating a subject with an NT2LP modulator, such as the modulator identified in one of the screening analyzes exemplified herein, if this knowledge is applied to dose determination or drug selection, an adverse reaction or treatment failure. Can be avoided, resulting in enhanced therapeutic or prophylactic efficacy.

E. Monitoring of effects during clinical trials Monitoring of the effects of compounds (eg drugs) on NT2LP protein / gene expression or activity can be applied not only in basic drug screening, but also in clinical trials. For example, a substance determined by screening analysis as described herein, NT2LP gene expression, the efficacy of increasing protein levels, or upregulating the activity of NT2LP, NT2LP gene expression,
It can be monitored in clinical trials in subjects exhibiting reduced protein levels or downregulation of NT2LP protein activity. Alternatively, a substance determined by screening analysis may also be effective in reducing NT2LP gene expression, protein levels, or down-regulating NT2LP protein activity, as well as increasing NT2LP gene expression, protein level, or NT2LP protein activity Can be monitored in clinical trials of subjects exhibiting In such clinical trials, the expression or activity of the NT2LP protein, and preferably other genes associated with diseases such as nervous system-related diseases, is used as a "read out" or marker for specific cells. be able to.

For example, a gene containing an NT2LP gene that is modulated in a cell by treatment with a compound (eg, drug or small molecule) that modulates NT2LP protein / gene activity (eg, identified by the screening analysis described herein). Can be identified, but is not limited to this method. Therefore, in clinical trials, etc., CNS
In order to study the effects of compounds on the process of injury or pain, cells are isolated,
RNA can be prepared and the expression levels of the NT2LP gene as well as other genes associated with the disorder or biological process can be analyzed. The level of gene expression (ie, gene expression pattern) can be determined as described herein by Northern blot analysis or RT-PCR as described herein, or by measuring the amount of protein produced. It can be quantified by one of the methods or by measuring the level of activity of the NT2LP protein or other gene. In this method, the gene expression pattern can be used as a marker or an indicator of the physiological response of cells to a compound. Thus, this response state can be determined prior to treatment of the individual with the compound, and at various times during treatment.

In certain preferred embodiments, the present invention provides compounds (eg, agonists, antagonists, pseudopeptides, proteins, peptides, nucleic acids, small molecules, or other drug candidates identified by the screening assays described herein). A method of monitoring the efficacy of treatment of a subject by: (i) obtaining a pre-dose sample from the subject prior to administering the compound; (ii) NT2LP protein, mRNA in the pre-dose sample,
Or detecting the expression level of genomic DNA; (iii) obtaining one or more post-administration samples from the subject; (iv) NT2LP protein, mRNA, or genome D in the post-administration sample
Detecting the level of NA expression or activity; (v) the level of expression or activity of NT2LP protein, mRNA, or genomic DNA in the pre-dose sample, and the level of NT2LP in the post-dose sample
Providing a method comprising comparing to protein, mRNA or genomic DNA; and (vi) modifying the administration of the compound to the subject accordingly. For example, NT2L
In order to increase the P protein / gene expression or activity to a higher level than the time point detected, ie to increase the efficacy of the substance, it is desirable to increase the dose of the compound. Alternatively, it may be desirable to reduce the dose of the compound in order to reduce the expression or activity of NT2LP to a level lower than the time point detected, ie to reduce the efficacy of the substance.

VI. Pharmaceutical Compositions NT2LP nucleic acid molecules of the invention, NT2LP proteins (particularly NT-like extracellular domains).
2LP fragments), modulators of NT2LP proteins, and anti-NT2LP antibodies (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration to a subject, eg, a human. Such compositions typically include a nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption agents that are compatible with pharmaceutical administration. Interpreted to include retarders and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Such vehicles can be used in the compositions of the present invention unless the conventional vehicle or material is incompatible with the active compound. Supplementary active agents can be incorporated into the compositions as well.

The 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 (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous application may include the following components:
Sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; such as ascorbic acid or sodium bisulfite. Antioxidants; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and isotonic modulators 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 enclosed in ampoules, 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 sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Suitable carriers for intravenous administration include saline, bacteriostatic water, Cremophr EL® (BASF; Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and
It should be fluidized to the extent that it facilitates syringe actuation. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof.
Proper fluidity can be maintained, for example, by maintaining the required particle size in the case of dispersion, and by using surfactants, by using coatings such as lecithin. Protection against the action of microorganisms can be obtained, for example, by various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, ascorbic acid and thimerosal. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be obtained by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound (eg, NT2LP protein or anti-NT2LP antibody) in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required. It can be prepared by sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying,
This gives a powder of the active ingredient and further desired ingredients from the previously filter-sterilized solution.

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 with a liquid carrier that is used as a mouthwash in which the compound in the liquid carrier is orally applied to rinse, exhale, or swallow. Pharmaceutically compatible binders and / or auxiliary materials can be included as part of the composition. Tablets, pills,
Capsules, troches and the like may contain any of the following ingredients or compounds of similar properties; binders such as microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch or lactose; alginic acid. , Disintegrants such as primogel or corn starch; lubricants such as magnesium stearate or sterotes; moisturizers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or peppermint, methyl salicylate or orange flavors. Fragrance. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated 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. Transmucosal administration can be performed using nasal sprays or suppositories. For transdermal administration, the active compound is formulated in an ointment, salve, gel or cream as is generally known in the art.

The compositions 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 compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Ethylene, vinyl acetate, polyanhydrides
Biodegradable biocompatible polymers can be used, such as, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations will be apparent to those of skill in the art. The material is Alza Corpo
ration) and Nova Pharmaceuticals, Inc.
Can also be obtained commercially from. Liposomal suspensions, including liposomes directed to infected cells by monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These are according to methods known to those skilled in the art,
For example, it can be prepared as described in US Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage units for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units, which are suitable as unit doses for the subject to be treated. Here, each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention depend on the unique characteristics of the active compounds, and the particular therapeutic effect to be achieved, and limitations inherent in the field of synthesis of such active compounds for the treatment of individuals. Directed and directly dependent on them.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors include, for example, intravenous injection, local administration (US Pat.
No. 8,470) or by stereotactic injection (see, eg, Chen et al., (1994) PNAS 91: 3054-3057) to the subject. The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where a complete gene transfer vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical preparation can include one or more cells that produce the gene transfer system. .

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

VII. Uses of NT2LP Subsequences The fragments or fragments of the cDNA sequences identified herein (and corresponding full-length gene sequences) can be used in a variety of ways as polynucleotide reagents. For example, these sequences (a) map its individual genes onto chromosomes, thereby locating the genetic regions associated with genetic diseases, and (b) identifying individuals from a small amount of biological sample ( Tissue typing), and (c) to aid forensic assessment of biological samples. These applications are described in the subsections below.

Chromosomal Map Generation Once a gene sequence (or fragment of a sequence) is isolated, this sequence can be used to map the location of the gene on the chromosome. This process is called chromosome mapping. Therefore, fragments of the NT2LP nucleic acid sequence can be used to map the position of the NT2LP gene on each chromosome. Mapping the NT2LP sequences to the chromosome is an important first step in associating these sequences with genes associated with disorders.

Briefly, the NT2LP gene can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp in length) from the NT2LP gene sequence. Computer analysis of the NT2LP gene sequence can be used to rapidly select primers that do not span multiple exons in genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing human individual chromosomes. Only hybrids containing the human gene corresponding to the NT2LP gene sequence give rise to amplified fragments.

Somatic cell hybrids are prepared by the fusion of somatic cells of different mammalian origin (eg human and mouse cells). As human and mouse cell hybrids grow and divide, they lose human chromosomes in an increasingly chaotic order, but mouse chromosomes continue to be maintained. By using a medium in which mouse cells are unable to grow but human cells are able to grow due to the lack of a particular enzyme, one human chromosome containing the gene encoding the required enzyme is maintained. It is thought to be. A panel of hybrid cell lines can be established by using various media. Each cell line in the panel contains either a single human chromosome or a small number of human chromosomes, and a complete set of mouse chromosomes, facilitating the mapping of individual genes to specific human chromosomes. (D'Eustachio
Et al., Science 220: 919-924 (1983)). Somatic cell hybrids containing only human chromosome fragments can also be made using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid method of assigning specific sequences to specific chromosomes. One heat cycle can allocate more than two sequences per day. Using NT2LP gene sequences to design oligonucleotide primers, sublocalization with a panel of fragments from a particular chromosome
It can be performed. Another mapping strategy that can also be used to map the NT2LP gene sequence to its chromosomes is in situ hybridization (Fan et al., PNAS 87: 6223-27 (1990)), labeled flow-sorted chromosomes. And preselection by hybridization to a chromosome-specific cDNA library.

Additionally, fluorescence in situ hybridization (FISH) of DNA sequences to metaphase chromosome spreds can be used to provide the correct chromosomal location in one step. Chromosome smears can be made using cells whose division has been arrested at metaphase by chemicals such as colcemid that disrupt the spindle. These chromosomes can be briefly trypsinized and then Giemsa stained. A light-dark band pattern appears on each chromosome, so that these chromosomes can be individually identified. The FISH technique can be used with DNA sequences as short as 500-600 bases. However, clones larger than 1,000 bases are more likely to bind to unique chromosomal locations with sufficient signal strength for easy detection. Preferably 1,000 bases, more preferably 2,000 bases, are considered to be sufficient to obtain good results in a reasonable time. To verify this technique, Verma et al., "Human Chromosomes: A Manual of Basic Techni
ques) ”(Pergamum Publishing Company, New York, 1988).

Use reagents for chromosome mapping individually and mark one chromosome or one site on a chromosome, or use a panel of reagents to mark multiple sites and / or polychromosomes be able to. In fact, reagents that correspond to the non-coding regions of the gene are preferred for mapping purposes. The coding sequences are probably conserved in gene families, resulting in an increased chance of cross-hybridization during chromosome mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be related to genetic map data (such data is
For example, V. McKusick's book, "Mendelian Inheritance in
Man) ”(available online from Welch Medical Library at John Hopkins University). The relationship between genes and diseases that have been mapped to the same chromosomal region can then be identified by linkage analysis (physically adjacent genes are linked and inherited) for which Egeland, J. et al., Nature, 325: 783-787 (198
It is written in 7).

In addition, DNA sequence differences between individuals with and without a disease associated with the NT2LP gene can be determined. If some or all of the affected individuals have a mutation, but none at all, then
This mutation is probably the causative agent of this particular disease. A comparison of affected and unaffected individuals generally involves a structural alteration of the chromosome that is initially visible, for example, a deletion that is observable from a chromosome smear or is detectable by PCR based on its DNA sequence. Or it is related to looking for translocation. Eventually, complete sequencing of genes from several individuals will be performed, confirming the presence or absence of mutations and distinguishing mutations from polymorphisms.

B. Tissue Typing The NT2LP gene sequences of the invention can also be used to identify individuals from microbiological samples. For example, the US military is considering using Restricted Fragment Length Polymorphism (RFLP) to identify soldiers. In this technique, individual genomic DNA is digested with one or more restriction enzymes and probed by Southern blotting to obtain unique bands for recognition. This method does not have the drawbacks of the current law of "dog tags", which can be lost, replaced or stolen and are difficult to clearly identify. The sequence of the present invention is an addition of RFLP.
It is also useful as a DNA marker (disclosed in US Pat. No. 5,272,057).

In addition, the sequences of the invention can be used to provide an alternative method for determining the actual base-by-base DNA sequence of selected fragments of an individual's genome. Therefore, the N
The T2LP sequence can be used to prepare two PCR primers from the 5'and 3'ends of this sequence. Then use these primers to amplify the individual's DNA,
It can be subsequently sequenced.

Since each individual has its own set of DNA sequences due to allelic differences, a panel of corresponding DNA sequences derived from individuals prepared by the method described above,
A unique individual identification can be provided. The sequences of the invention can be used to obtain such discriminating sequences from individuals and tissues. The NT2LP gene sequence of the present invention uniquely represents a fragment of the human genome. Allelic variation occurs to some extent in the coding regions of these sequences and more in non-coding regions. Allelic variation between human individuals is estimated to occur at a frequency of approximately 1 per 500 bases. Each of these sequences described herein is derived from an individual for identification purposes.
It can be used considerably as a standard for comparing DNA. Fewer sequences are needed to distinguish individuals, as more polymorphisms occur in the non-coding regions.
The non-coding regions of SEQ ID NO: 1 or SEQ ID NO: 3 were each non-coding amplified
A clear identification of individuals, perhaps with a panel of 10-1,000 primers that yields a sequence of 100 bases, can be adequately provided. When a putative coding sequence such as SEQ ID NO: 1 or SEQ ID NO: 3 shown in Figure 1 and Figure 2 is used, a more suitable number of primers for unambiguous individual identification is considered to be 500-2,000. To be

When a panel of reagents derived from the NT2LP gene sequences described herein is used to create a unique identification database for an individual, the same reagents can then be used to identify tissues from that individual. Using a unique individual identification database, clear identification can be performed from a very small amount of tissue sample regardless of life or death.

C. Use of partial NT2LP sequences in forensic biology DNA-based discrimination methods can also be used in forensic biology. Forensic biology is a scientific discipline that uses genotyping of biological evidence found at crime scenes, for example, as a means of unequivocal identification of perpetrators of crime. To make such discrimination, PCR technology has been used, obtained from tissues such as hair or skin found at crime scenes, or from very small biological samples such as body fluids such as blood, saliva or semen. DNA is amplified. The amplified sequence is then compared to a standard, which allows identification of the origin of the biological sample.

The sequences of the invention can be used to provide polynucleotide reagents targeted to specific loci in the human genome, such as PCR primers, which may, for example, be another "discriminating marker" (ie, a particular individual). (Provided with another DNA sequence that is unique to), the reliability of DNA-based forensic identification can be increased. As mentioned above, the actual nucleotide sequence information can be used for identification as an accurate substitute for the pattern formed by the restriction enzyme generated fragments. Sequences targeted to the non-coding region of SEQ ID NO: 1 or SEQ ID NO: 3 are particularly suitable for such use as more polymorphisms occur in the non-coding region and using this method Individual identification becomes easier. Examples of polynucleotide reagents include the NT2LP sequence, or a fragment thereof, eg, a fragment derived from the non-coding region of SEQ ID NO: 1 or SEQ ID NO: 3, which is at least 20 bases, preferably at least 30 bases in length.

The NT2LP sequences described herein may further be labeled or labelable probes that may be used in, for example, in situ hybridization methods to identify specific tissues such as brain tissue. , Can be used to provide polynucleotide reagents. This is very useful for forensic pathologists to describe tissues of unknown origin. Such NT2LP probe panels can be used to identify tissues by type and / or organ.

In a similar manner, these reagents, such as NT2LP primers or probes, can be used to screen tissue culture for contaminants (ie, screening for the inclusion of different cell types in culture).

The present invention is explained in more detail by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited herein are hereby incorporated by reference.

Examples Example 1: Identification of human and monkey NT2LP cDNAs In this example, human and monkey NT2LP nucleic acid molecules were identified. Analysis of the EST database based on a search designed to identify sequences showing low levels of homology with the GPCR (GenBank® dbEST database search) revealed that the unannotated EST (GenBank (GenBank The registered trademark) Accession No. T00621) was first identified. Subsequently, primers were designed based on the EST sequence and used for screening a human or monkey fetal cDNA library. Several positive clones were identified, sequenced and sequence assembled (FIG. 1 and SEQ ID NO: 1 and SEQ ID NO: 2). BLAS for nucleic acid databases in the public domain
T-analysis revealed homology with members of the NT family of receptors and glutamate receptors.

This monkey NT2LP DNA sequence was used as a probe for human sequences obtained from various libraries. Several clones from human brain cDNA library
It was identified as containing a sequence that is highly homologous to the 2LP sequence. The sequences were assembled into contig groups, leading to the identification of two splice forms of human NT2LP. These two splice variants (Fig. 2) have the same coding region,
The only difference was the 5'UTR.

Example 2: Northern Blot Analysis on Tissue Expression of NT2LP Gene Human brain multiple tissue Northern containing 2 μg of poly A + RNA per lane.
ssue northern (MTN) blots, human MTN I, II and III blots (Clontec
h, Palo Alto, CA) were probed with monkey NT2LP-specific probes.
The filters were prehybridized in 10 ml of Express Hyb hybridization solution (Clontech; Palo Alto, Calif.) At 68 ° C. for 1 hour and then 100 ng of ³² P-labeled probe was added. The probe is Stratagene Prime-It kit, Catalog No. 300392 (Clontech, Pa
lo Alto, CA). Hybridization was allowed to proceed at 68 ° C for about 2 hours. After washing the filter with 0.05% SDS / 2xSSC solution at room temperature for 15 minutes,
Washed twice with 0.1% SDS / 0.1xSSC solution at 0 ° C for 20 minutes, followed by exposure to autoradiographic film at -80 ° C overnight using one screen. The human tissues examined included: heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, colon (mucosal layer) and Peripheral white blood cells.

Strong hybridization to the whole human brain was observed, with weaker signals in the ovary. No signal was observed in placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus, small intestine, colon (mucosal layer) and peripheral blood leukocytes. Hybridization within the brain was observed in all subregions.

Example 3 Expression of Recombinant NT2LP Protein in Bacterial Cells In this example, NT2LP was expressed as a recombinant glutathione-S-transferase (GST) fusion protein in E. coli, and isolation and characterization of the fusion protein. I do. Specifically, NT2LP is fused with GST, and this fusion protein is expressed in Escherichia coli such as PEB199 strain. Expression of GST-NT2LP fusion protein in PEB199 is I
Induce with PTG. The recombinant fusion protein is purified from the induced crude bacterial lysate of strain PEB199 by affinity chromatography on glutathione beads. Polyacrylamide gel electrophoretic analysis of proteins purified from bacterial lysates is used to determine the molecular weight of the resulting proteins.

Example 4 Expression of Recombinant NT2LP Protein in COS Cells To express the NT2LP gene in COS cells, Invitrogen (Invitrog) was used.
en Corporation) (San Diego, CA). 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. The HA tag (at the 3'end of the DNA fragment encoding the entire NT2LP protein)
An in-frame fusion of Wilson et al. (1984) Cell 37: 767) is cloned into the polylinker region of the vector, thereby placing the recombinant protein under the control of the CMV promoter.

To construct the plasmid, the NT2LP DN was constructed by PCR using two different primers.
Amplify the A sequence. The 5'primer contains the restriction enzyme site of interest followed by an NT2LP coding sequence of about 20 nucleotides starting from the start codon, and the 3'terminal sequence contains the other restriction enzyme site of interest, translation stop codon, HA. It contains the tag and the complementary sequence to the last 20 nucleotides of the NT2LP coding sequence. PCR amplified fragment and p
CDNA / Amp vector was cleaved with appropriate restriction enzymes and CIAP enzyme (New England Biolabs
, Beverly, MA). The two restriction enzyme sites selected are preferably different so that the NT2LP gene is inserted in the correct orientation. This ligation mixture was used to transform E. coli cells (Stratagene Cloning Systems (Stra
Tagene Cloning Systems), HB101, DH5a, SURE available from La Jolla, CA can be used) and the transformed culture is plated on ampicillin plate medium to select resistant colonies. Plasmid DNA is isolated from the transformants and the presence of the correct fragment is confirmed by restriction analysis.

COS cells are then transfected with NT2LP-pcDNA / Amp plasmid DNA using calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells are described by Sambrook et al., Molecular Cloning: Experimental Manual (Molecular Cloning: A
Laboratory Manual), 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. N
Expression of the T2LP protein was achieved by radiolabelling ( ³⁵ S-methionine or ³⁵ S-cysteine available from NEN, Boston, MA can be used) and immunoprecipitation using HA-specific monoclonal antibodies (Harlow and Lane, antibody : Experiment manual (
Antibody: A Laboratory Manual), Cold Spring Harbor Laboratory Press, Co
ld Spring Harbor, NY, 1988). Briefly, ³ cells
Label with ⁵ S-methionine (or ³⁵ S-cysteine) for 8 hours. Next, the medium was collected and detergent (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50%
Solubilize the cells with mM Tris, pH 7.5). Both cell lysates and media are precipitated with HA-specific monoclonal antibodies. Then, the precipitated protein is SD
Analyze by S-PAGE.

Alternatively, the DNA containing the NT2LP coding sequence was transformed into pCDNA / A using the appropriate restriction enzyme sites.
Clone directly into the polylinker of the mp vector. The resulting plasmid is transformed into COS cells in the above manner and the expression of NT2LP protein is detected by radioprecipitation and immunoprecipitation using NT2LP-specific monoclonal antibodies.

Example 5: Characterization of Human NT2LP Protein In this example, the amino acid sequence of human NT2LP protein was compared to the amino acid sequences of known proteins to identify various motifs.

Hydrophobicity analysis showed that the human NT2LP protein contains seven transmembrane domains (amino acids 34-58, 72-96, 113-131, 154-175, 212-236). , 298-314, and 339-358; Figure 4). As shown in FIG. 5, human NT2LP has a region (amino acids 49 to 358) homologous to the consensus sequence of the 7-transmembrane receptor family obtained by the hidden Markov model (PF0001). For general information on PFAM identifiers, see Sonnhammer et al. (1997) Protein 28: 405-420 and http: // www.
See .psc.edu / general / software / packages / pfam / pfam.html. BLASTN
The nucleotide sequences of human and monkey NT2LP were used for database queries using the program (BLASTN1.3MP, Altschul et al. (1990) J. Mol. Biol. 215: 403). Figure 7
Part of NT2LP (sbjct) and mouse neurotensin receptor type 2 (P70310).
) Is a series of aligned products.

Example 6: Tissue distribution of NT2LP mRNA 10μ of selected tissues for in situ hybridization analysis.
m-thick sections were fixed in DEPC-treated 1x phosphate buffered saline containing 4% formaldehyde for 10 minutes at room temperature, then twice with DEPC 1x phosphate buffered saline, 0.1M triethanolamine-HCl. It was rinsed once with (pH 8.0). After incubating in 0.25% acetic anhydride-0.1M triethanolamine-HCl for 10 minutes, the sections are twice with DEPC, PBS.
Rinse once. The sections were then dehydrated by the ethanol series, incubated in 100% chloroform for 5 minutes (twice), rinsed in 100% ethanol for 1 minute, followed by 95% ethanol for 1 minute and air dried.

Hybridization was performed with a ³⁵ S-radiolabeled (5 × 10 ⁷ cpm / ml) cRNA probe designed to specifically hybridize with NT2LP messenger RNA. The probe is 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% yeast tRN.
A, 0.05% yeast total RNA type X1, 1 x Denhardt's solution, 50% formaldehyde, 10
Incubated at 55 ° C. for 18 hours in the presence of a solution containing% dextran sulfate, 100 mM dithiothreitol, 0.25% sodium dodecyl sulfate (SDS).

After hybridization, the slide glass was washed twice with 2 × SSC. The sections were then placed in TNE (10 mM Tris-HCl (pH 7.6), 500 mM NaCl and 1 mM EDTA) for 10 min, 40 μg RNase A per ml in TNE for 30 min, and finally TNE.
For 10 minutes at room temperature. Next, rinse the slide glass with 2 x SSC at room temperature, wash with 2 x SSC at 60 ° C for 30 minutes, 0.2 x SSC at 65 ° C for 30 minutes, and 0.2 x SSC at 65 ° C for 30 minutes. did. The sections were then rapidly dehydrated with an ethanol series, air-dried, exposed to Kodak Biomax MR imaging film for scientific research for 24-48 hours, then immersed in NTB-2 photoemulsion solution at room temperature. After exposure for 14 days, development and counterstaining were performed.

This analysis revealed that human NT2LP is expressed in neurons of the central and peripheral nervous system. NT2LP was detected in dorsal root ganglia, trigeminal ganglia and superior cervical ganglion.

Example 7: Genomic mapping of human NT2LP Genebridge 4 Radiation Hybrid Panel was used for mapping of human NT2LP gene. NT2LP is the Whitehead Institute framework marker on chromosome 2.
WI-6565 to 19.5cR ₃₀₀₀ telomere side, Whitehead Institute framework marker D25359 to 90cR ₃₀₀₀ centromeric side. NT2LP is located at the cytoplasmic gene position 2p24pTEL at the telomeric end of the P arm of chromosome 2.

Equivalents One of ordinary skill in the art would be able to ascertain or ascertain, from routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to fall within the scope of the appended claims.

[Brief description of drawings]

FIG. 1 Monkey NT2LP nucleotide (SEQ ID NO: 1) and NT2LP amino acid (
This shows the sequence of SEQ ID NO: 2). Start and stop codons are shown in bold.

FIG. 2 Two kinds of human NT2LP nucleotide splice variants (SEQ ID NO:
3 and 5) are shown. Start and stop codons are shown in bold.

FIG. 3 shows the amino acid (SEQ ID NO: 4) sequence of human NT2LP.

FIG. 4 shows a hydropathy plot of human NT2LP. Putative transmembrane (TM),
The positions of the cytoplasmic (IN) and extracellular (OUT) domains are indicated along with the position of cysteine (cys; vertical bar just below the plot). Relative hydrophilicity is shown above the dotted line and relative hydrophobicity is shown below the dotted line.

FIG. 5 is a sequence comparison between a portion of human NT2LP and the consensus sequence of 7-transmembrane receptor obtained by the hidden Markov model.

FIG. 6 is a series of alignment products of a portion of human NT2LP (sbjct) and a portion of rat neurotensin receptor type 2 (reference; Q63384).

FIG. 7 is a series of alignments of a portion of NT2LP (sbjct) and mouse neurotensin receptor type 2 (reference; P70310).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 33/50 Z 4H045 G01N 33/15 33/53 D 33/50 M 33/53 33 / 566 C12N 15/00 ZNAA 33/566 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), AE, AL, AM, AT, AU, AZ, BA ， B, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, 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, US, UZ, VN, YU, ZA, ZW F Term (reference) 2G045 AA34 AA35 BB07 BB14 BB29 BB48 BB50 BB51 CB01 DA13 DA36 FB02 FB03 FB05 FB06 FB08 4B024 AA11 BA43 BA44 BA63 CA04 DA02 DA06 EA04 GA13 GA14 HA14 HA15 4B063 QA18 QQ53 QR56 QR62 QS24 QS25 QS34 QX01 QX07 4B064 AG20 CA02 CA10 CA19 CC24 DA13 4B065 AA26X AA90X AA91Y AA93Y AB01 AC14 CA24 CA46 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA50 DA76 DA86 EA50 FA72 FA74

Claims (22)

[Claims] 1. A) a nucleic acid molecule encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, b) comprising at least 15 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4, A nucleic acid molecule encoding a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and c) hybridizing under stringent conditions with a nucleic acid molecule comprising SEQ ID NO: 2 or SEQ ID NO: 4, An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules encoding naturally occurring allelic variants of a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 2. The nucleic acid molecule of claim 1, further comprising a vector nucleic acid sequence. 3. The nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding a heterologous protein. 4. A host cell containing the nucleic acid molecule of claim 1. 5. The host cell of claim 4, which is a mammalian host cell. 6. A non-human mammalian host cell comprising the nucleic acid molecule of claim 1. 7. The isolated nucleic acid of claim 1 selected from the group consisting of the coding region of SEQ ID NO: 1 or SEQ ID NO: 3 and the extracellular domain encoded by SEQ ID NO: 1 or SEQ ID NO: 3. molecule. 8. A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; b) SEQ ID NO: 2 or comprising at least 15 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2, encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 4. Or an isolated protein selected from the group consisting of naturally occurring allelic variants of the protein comprising the amino acid sequence of SEQ ID NO: 4. 9. The protein of claim 8, further comprising a heterologous amino acid sequence. 10. An antibody that selectively binds to the protein of claim 8. 11. A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; b) SEQ ID NO: 2 or comprising at least 15 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2, encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 4. Or a method for producing a protein, selected from the group consisting of naturally occurring allelic variants of a protein comprising the amino acid sequence of SEQ ID NO: 4, under conditions in which the nucleic acid molecule is expressed, A method comprising culturing a host cell according to claim 4. 12. A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, b) SEQ ID NO: 2 or comprising at least 15 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2, encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 4. Or a method for detecting the presence of a protein in a sample, which is selected from the group consisting of naturally occurring allelic variants of a protein comprising the amino acid sequence of SEQ ID NO: 4, wherein in the sample: A method comprising the steps of: i) contacting the sample with a compound that selectively binds a protein; and ii) the compound binds to a protein in the sample Determining whether or not. 13. The method of claim 12, wherein the compound that binds to the protein is an antibody. 14. A kit containing the reagent used in the method according to claim 12, wherein the reagent is a) a protein containing the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and b) SEQ ID NO: 2 Or a peptide comprising at least 15 contiguous amino acids of SEQ ID NO: 4, and c) encoded by a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3, A kit comprising a compound that selectively binds to a protein selected from the group consisting of naturally occurring allelic variants of the protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 15. A) a nucleic acid molecule encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, b) comprising at least 15 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4, A nucleic acid molecule encoding a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and c) hybridizing under stringent conditions with a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3, Method for detecting the presence of a nucleic acid molecule in a sample selected from the group consisting of nucleic acid molecules encoding naturally occurring allelic variants of a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 A method comprising the following steps in the sample: i) contacting the sample with a nucleic acid probe or primer that selectively hybridizes to a nucleic acid molecule. Step is; and ii) the step of nucleic acid probes or primers to determine whether binding to a nucleic acid molecule in the sample. 16. The method of claim 15, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe. 17. A kit comprising the reagents used in the method of claim 15, wherein the reagents are: a) a nucleic acid molecule encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, b ) A nucleic acid molecule encoding a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 comprising at least 15 contiguous amino acids of SEQ ID NO: 2 or SEQ ID NO: 4, and c) stringent A nucleic acid molecule encoding a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, which hybridizes under conditions to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3. A kit comprising a compound that selectively hybridizes with a nucleic acid molecule selected from the group consisting of: 18. A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, b) SEQ ID NO: 2 or comprising at least 15 consecutive amino acids of SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2, encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions, and a fragment of a protein comprising the amino acid sequence of SEQ ID NO: 4. Or a method for identifying a compound that binds to a protein selected from the group consisting of naturally occurring allelic variants of the protein comprising the amino acid sequence of SEQ ID NO: 4, the method comprising the steps of: i Contacting the protein, or cells expressing the protein, with a test compound; and ii) whether the protein binds to the test compound. Stage to be constant. 19. The binding between a test compound and a protein comprises a) Detection of binding by direct detection of test compound / protein binding; b) Detection of binding using a competitive binding assay; c) Detection of binding using an assay for NT2LP activity 19. The method of claim 18, detected by a method selected from the group consisting of: 20. A nucleic acid that hybridizes to a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and b) a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions. A method for modulating the activity of a protein selected from the group consisting of naturally occurring allelic variants of the protein encoded by the molecule and comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. A method comprising the steps of: i) contacting the protein-expressing cells with a protein-binding compound in a concentration sufficient to modulate the activity of the protein. 21. The activity is phosphatidylinositol activity.
The method described in 0. 22. The method of claim 20, wherein the activity is cAMP activity.