JP2002523091A - Method for altering the function of a heterologous G protein-coupled receptor - Google Patents

Abstract

  (57) [Summary] The present invention provides a mutant G protein-coupled receptor with altered G protein binding and receptor response, a yeast cell expressing such receptor, a vector useful for producing such a cell, and Regarding how to use.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION

The present invention relates to mutant G protein-coupled receptors with altered G protein binding and receptor response, host cells expressing such receptors, vectors useful for producing such cells, and And methods for producing and using them.

[0002] neurotransmitters, hormones, effects of many extracellular signals, such as odor substances, and the light is mediated by the protein triad, which have been identified in organisms ranging in mammals from yeast. This triad consists of a receptor linked to a trimeric guanine nucleotide binding regulatory protein (G protein), which in turn binds to the effectors of the cell. These receptors have seven transmembrane domains and have been termed "G protein-coupled receptors"("GPCRs") for their association with G proteins.

The regulatory G protein consists of three subunits: a guanyl nucleotide binding α subunit; a β subunit; and a γ subunit (BR Conklin and
HRBourne (1993)). G proteins circulate between the two forms depending on whether GDP or GTP is bound to the α subunit. When GDP is bound, the G protein exists as a heterotrimeric Gαβγ complex.
If GTP is bound, the α subunit dissociates, leaving the Gβγ complex. Importantly, the Gαβγ complex is operatively associated with the activated G protein-coupled receptor at the cell membrane and increases the rate of exchange of GTP for bound GDP, thereby increasing the bound Gα subunit from the Gβγ complex. The dissociation rate of the unit also increases. Free Gα subunits and Gβγ complexes can transmit signals to downstream elements of various signal transduction pathways. Examples of these downstream cell effector proteins include adenylate cyclase, ion channels and phospholipase, among others. This basic scheme of phenomena underlies the diversity of various cell signaling phenomena (HG Dohlman et al. (1991)).

[0004] Due to their ubiquity in key biochemical pathways, G protein-coupled receptors represent important targets for novel therapeutic agents. Second, the discovery of such agents will necessarily require high specificity screening assays and high throughput screening (HTS) assays in processing terms. Screening assays that utilize microorganisms such as yeast strains that have been genetically modified to provide functional expression of G protein-coupled receptors offer significant advantages for studies of ligand binding to a number of receptors involved in various disease states. give.

[0005] However, microorganisms transformed with the wild-type receptor show, for example, inability to interact with heterotrimeric G proteins, inappropriate localization and / or desensitization in growth assays, Sometimes it doesn't work very well. Many GPs
CR is phosphorylated in response to chronic and sustained agonist stimuli, which often results in desensitization followed by receptor sequestration or internalization. Desensitization of the GPCR causes release from interaction with the heterotrimeric G protein. This process is mediated by a variety of regulatory receptor protein kinases, including G protein-coupled receptor kinase (GPK), protein kinase A (PHA), protein kinase C (PKC) and casein kinase (CK). Internalization requires removal of the GPCR from the plasma membrane by receptor-mediated endocytosis. The internalized receptor can recirculate back to the cell surface or be delivered to the lysosome / vacuum compartment for degradation. Degradation pathways mediated by ubiquitin are also involved in this process. The end result of receptor phosphorylation and sequestration / internalization is often the inhibition of cell growth, which makes genetically modified microorganisms significantly less useful in screening assays.

[0006]

Summary of the Invention

It is an object of the present invention to provide modified G protein-coupled receptors that function well in high-throughput screening assays performed on any eukaryotic cell, preferably a yeast cell. Thus, the first aspect of the present invention provides for more effective binding of the receptor to heterotrimeric G protein and / or cell desensitization and / or
Or encodes a G protein-coupled receptor that has been modified to enhance GPCR function by rendering it incapable of interacting with the sequestration / internalization mechanism and / or by appropriately localizing to the plasma membrane Directed to the nucleotide sequence. According to a preferred embodiment, such modification results in a modified agonist-stimulated growth promoting ability. One particular modification of the nucleotide sequence encoding a G protein-coupled receptor encompassed by the present invention is a mutation in the membrane region adjacent to any intracellular or internal domain. The mutation can be, for example, a deletion including a point mutation.

The present invention also provides heterologous G proteins that provide advantageous G protein binding properties or domains that are not subject to yeast cell desensitization and / or sequestration / internalization mechanisms.
The intracellular domain of the PCR is used to direct a chimeric GPCR in which a comparable domain in the GPCR of interest has been replaced. The present invention also relates to a modified nucleotide sequence encoding a chimeric GPCR, an expression vector comprising the modified nucleotide sequence, and host cells transformed therewith.

A further aspect of the invention is an improved method of assaying a compound for determining the effect of a ligand that binds to a mutant or chimeric GPCR of the invention by measuring the effect of a test compound on cell proliferation. Mutant GPCRs reduce or reduce the rate of cell proliferation arrest by chronic and persistent agonist stimulation, thereby reducing the number of false negatives that occur in prior art screening methods and / or increasing the sensitivity of bioassays.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Nucleotide sequences encoding modified G protein-coupled receptor G protein-coupled receptors can more efficiently bind the receptor to heterotrimeric G proteins and / or cell desensitization And / or can be modified to enhance GPCR function by rendering it incapable of interacting with the sequestration / internalization mechanism. Such modification results in a modified agonist-stimulated yeast cell growth promoting ability. GPCR-G in host cells
Modification of protein binding and / or elimination of receptor phosphorylation and / or sequestration / internalization provide a means to alter the function of a wild-type heterologous GPCR that cannot stimulate a useful yeast cell growth response. Thus, GPCRs that cannot function in their wild-type form may become operative by the methods of the present invention.

[0010] Modification of GPCR-G protein binding and / or elimination of receptor phosphorylation and / or sequestration / internalization in host cells may be as described in the Examples set forth below, or known to those of skill in the art. What is necessary is just to evaluate by using usual techniques, such as. For example, an alteration in the function of the mutant GPCR relative to the wild type can be quantified as an increase in signal rate to noise and / or an increase in sensitivity of the liquid bioassay. The signal rate to noise is determined by comparing the agonist-induced growth rate with the growth rate observed in the absence of agonist. A statistically significant increase in the signal rate to noise due to agonist stimulation of the mutant GPCR over similar values obtained from cells containing the wild-type receptor indicates that the function of the mutant GPCR has been altered. .

[0011] The sensitivity of a liquid bioassay is defined as the agonist concentration required to produce a half-maximal growth rate (ED50 or EC50). The sensitivity of the bioassay is increasing if the mutant GPCR exhibits a half-maximal growth rate at lower agonist concentrations than required by the wild-type GPCR.

Similarly, more quantitative agar-based bioassays reflect an increased signal rate and / or sensitivity to noise upon agonist stimulation of mutant GPCRs. In agar-based bioassays, the increase in signal to noise is determined by comparing the degree of proliferation in the area of agonist-induced growth due to stimulation of mutant and wild-type receptors. The sensitivity of the bioassay is proportional to the radius of the growth area. As the compound applied spreads radially from the application site of the agar, the agonist concentration varies with the square of the radius of the growth area. Therefore, the increase in the growth area in response to the agonist activation of the mutant GPCR reflects an increase in sensitivity.

In practicing the present invention, any G protein-coupled receptor may be used. Examples of such receptors include, but are not limited to, adenosine receptor, somatostatin receptor, dopamine receptor, cholecystokinin receptor, muscarinic cholinergic receptor, α-adrenergic receptor, β Adrenergic receptor,
These include opiate receptors, cannabinoid receptors, growth hormone releasing factors, glucagon, serotonin receptors, vasopressin receptors, melanocortin receptors, and neurotensin receptors. According to certain preferred embodiments, the receptor is a muscarinic acetylcholine receptor, more preferably the muscarinic acetylcholine receptor is of the M3 subtype.

[0014] Similarly, any suitable host cell may be transformed with a nucleotide sequence encoding a modified G protein-coupled receptor of the invention. Examples of suitable host cells include yeast cells, mammalian cells, insect cells, and bacterial cells. Preferably, the host cell is a yeast cell.

[0015] One generalizable method of altering the function of a GPCR expressed in a host cell is to modify or eliminate the intracellular domain of the GPCR, such as the third intracellular domain (IC3) sequence of a G protein-coupled receptor. There is something. Since the desensitization and internalization mechanisms act on the intracellular domain of the GPCR,
Elimination of the intracellular domain of the CR results in more stable receptor expression.
This has been demonstrated in experiments performed on mammalian cells. Muscarinic acetylcholine receptors, including the M3 subtype, lacking the domain of their third intracellular loop, which are thought to be involved in receptor internalization, are to a greater extent than their wild-type counterparts. (Moro et al. (19
93)).

[0016]

【Example】

Hereinafter, typical embodiments of the present invention will be described in more detail with reference to Examples. Example 1 Mutant rat M3 muscarinic acetylcholine receptor in yeast (M
AR) Functional expression The third intracellular loop of the GPCR is thought to interact with the G protein and participate in G protein activation upon agonist binding (J. Wess (1997)). Mutations in IC3 of the yeast mating pheromone receptors Ste2 and Ste3 are G
Significant effects on protein binding (C. Boone et al. (1993) and C
Clark et al. (1994)). Importantly, mammalian MARs, especially rat M3
Deletion of the IC3 portion of the MAR correlates with altered functional expression in mammalian cells that retains sufficient capacity to bind the heterotrimeric G protein Gq (Gαβγ). Mutant M3 MAR retains all outer loops. Internal domains other than the transmembrane domain (TMD) and IC3 remain unchanged. This IC3 seen between the spirals that span the fifth and sixth membranes
Was the only domain modified. Most of this domain lacked the central 96 amino acids of IC3 (Ala273-Lys469), leaving only 22 amino acids adjacent to both the fifth and sixth transmembrane helices. Thus, while the wild-type M3 MAR has 240 IC3 amino acids, the third intracellular loop of the MAR containing the IC3 deletion (IC3Δ) is 44 amino acids long. Modification of functional expression may be due to the elimination of domains known to interact with the desensitization mechanism of the cell and to have a more functional MAR on the cell surface.

To determine whether this IC3Δ mutation can also alter functional expression in yeast, wild-type and IC3Δ rat M3 M
The DNA sequence encoding the AR was cloned proximal to the glycerol-phosphate dehydrogenase promoter in the yeast expression plasmid p426GPD. The rat M3 MAR sequence was replaced with 5'BglII (AAAAGATCT AAA ATG T
AC CCC TAC GAC GTC CCC) (SEQ ID NO: 1) and 3'X
hoI (AAA CTCAGAG CTA CAA GGC CTG CTC C
Amplification was performed by PCR using an oligonucleotide containing a GG CAC TCG C) (SEQ ID NO: 2) site. The resulting PCR product was digested with the appropriate restriction endonuclease, purified and ligated into the appropriate site of p426GPD. Rat M3
To form IC3Δ, three M3 MAR fragments were amplified by PCR. The fragment encoding the amino terminus was ligated to 5'BgII (AAAAGATCT AAA
ATG TAC CCC TAC GAC GTC CCC) (SEQ ID NO: 1) and 3 'AgeI (ATAGTCATGATGGGTG ACCGGGT ATGT
(AAAAGGCAGCGATC) (SEQ ID NO: 3). The fragment encoding the carboxy terminus is 5'PmII (GCCT
TCATCAT CACGTG GACCCCCTACACC) (SEQ ID NO: 4)
And 3 'XhoI (AAA CTCAGAG CTA CAA GGC CTG
Amplification was performed using an oligonucleotide containing a CTC CGG CAC TCG C) (SEQ ID NO: 2) site. The fragment coding for IC3 was prepared using the M3 IC3Δ sequence and using 5 ′ AgeI (CGATCGCTGCCTTTTACTT
CATCATCATGACTAT) (SEQ ID NO: 5) and 3'PmII (GT
Amplification was performed using an oligonucleotide containing a TGTAGGGGGTC CACGTG ATGATGAAGGC (SEQ ID NO: 6) site (J. Wess (1997)). The resulting PCR product is digested with appropriate restriction endonucleases, purified and
Ligation was performed at an appropriate site in 26GPD. Plasmids were confirmed by restriction endonuclease mapping and DNA sequencing. The resulting plasmids can be transformed using conventional lithium acetate transformation methods, such as those described in US Pat. No. 5,691,188, which is incorporated herein by reference, specifically Paush et al. al.
(1998) introduced into yeast cells useful for performing GPCR agonist-stimulated proliferation assays, including the MPY578fc cells.

[0018] Yeast cells containing MAR are prepared using the MAR agonist carbachol (CCh).
Assayed in liquid culture. The cells were cultured in 2 ml of SC-glucose-ura medium.
Cultured overnight in the ground. 5 mM 3-aminotri to reduce basal growth rate
Cells are grown in SC-glucose-ura-his medium containing azole pH 6.8.
Diluted 500-fold. Serially diluted samples of muscarinic receptor agonists (10 ^-1 -10^-8M) A sterile 96-well microtiter tube containing 20 μl
A cell suspension sample (180 μl) was dispensed into the wells of the tissue. This plate
Was incubated at 30 ° C. for 18 hours with shaking (600 rpm). Proliferation
OD using a microplate reader₆₂₀Due to the increase in recorded values in
Monitored. The assay was performed twice and the growth rate was measured during the logarithmic growth phase of the yeast cells.
Was. Analyze the measured optical density using GraphPad Prism and calculate the mean ± standard error.
And plotted against agonist concentration. As shown in FIG.
 Yeast cells containing MAR IC3Δ have an agonist-dependent proliferative response and M3
MAR IC3Δ was shown to function, but was dependent on yeast cell agonists.
Wild-type MAR is not functioning because no sexual growth was observed. M3 MAR
The proliferative response of cells containing IC3Δ is dose-dependent and depends on carbachol (CCh).
EC against₅₀Corresponded to 3 μM. This value corresponds to the C obtained in HEK cells.
K for Ch_DValue (7.9 μM) and CCh-induced IP₃(Inositol 3
EC for phosphoric acid) accumulation₅₀(4.0 μM), but this was
MAR IC3Δ retains expected pharmacological properties when expressed in yeast cell membrane
It suggests that In addition, the proliferative response is a MAR-specific antagonis
Inhibited by atropine (At).

Alternatively, the response to CCh by yeast cells containing M3 MAR IC3Δ can be observed by measuring the increase in agonist-induced increase in fluorescent emission of a green fluorescent protein reporter gene whose expression is stimulated by a MAR agonist. . Green fluorescent protein (GFP) is a protein derived from jellyfish (Aequorea) and has endogenous fluorescence when expressed in yeast cells. GFP-derived fluorescence is detectable in live yeast and its expression level can be measured without damaging the yeast cells. This feature is particularly advantageous in reporter gene assays because no additional steps are required for its detection. Heterologous GPCR expressed in yeast
An inducible reporter gene useful for detecting the activation of an agonist utilizes a transcription promoter activated by crossing the signal transduction pathway. One such promoter is the FUS2 promoter. In the absence of agonist stimulation, F
Little or no expression of the us2 protein or any other protein whose expression is driven by the FUS2 promoter is detected. After agonist treatment, transcription from the FUS2 promoter was induced up to 700-fold,
2 or a substantial increase in the expression of any of the gene products whose expression is under the control of the FUS2 promoter. Thus, the fluorescence of yeast cells resulting from GFP expression under the control of the FUS2 promoter derived from the FUS2-GFP reporter gene is only observed after agonist activation of the heterologous GPCR.

In order to produce a GFP reporter gene, enhanced GFP (EGFP, Cl
DNA sequence encoding the FUS2 promoter and FUS2 transcription terminator sequence was amplified by PCR. These fragments are converted into plasmid p
EGFP expression was integrated into the centromere containing RS414 to transform plasmid pRS414.
PMP under the control of a centromeric pheromone-responsive FUS2 promoter containing
241 was created. The resulting plasmid is of the type described in US Pat. It was introduced into yeast cells. Specifically, this plasmid was introduced into MPY578fc cells described in Pausch et al (1998).

Yeast Cells Containing M3 MAR IC3Δ and FUS2-EGFP Receptor Plas
Sumids were prepared in liquid culture using the MAR agonist carbachol (CCb).
I did it. The cells were cultured overnight in 2 ml of SC-glucose-ura medium.
Was. Cells were washed and 5 mM 3-aminotria to reduce basal growth rate.
5-fold dilution in SC-glucose-ura-his medium containing sol, pH 6.8
did. The cell suspension sample (180 μl) was mixed with a serially diluted sample of CCh (10 ^-1 ~ 10^-8M) A sterile 96-well microtiter dish containing 20 μl
Was dispensed into the wells. The plate is shaken for 6 hours at 30 ° C. (600
rpm) and incubated. Stimulation of FUS2-EGFP reporter gene expression
Is 53% after excitation with 480 nm light using a fluorescence microplate reader.
The increase in emission at 0 nm was monitored by recording. Two assays
Measurements were taken during the exponential growth phase of the yeast cells. The measured fluorescence was measured using GraphPad P
Analyzed using rism, expressed as mean ± standard error,
I did it. As shown in FIG. 1B, yeast cells containing M3 MAR IC3Δ
It shows a dose-dependent increase in fluorescence emission, which indicates agonist-induced FUS2-
Consistent with increased EFGP expression from the GFP reporter gene construct. CC
EC for h-stimulated fluorescence₅₀Is 4 μM, which is
Was the same as the value obtained.

Thus, the deletion of the IC3 part in rat M3 MAR resulted in the production of a functional GPCR when expressed in yeast, which means that the modification of the internal domain has a functional modification of the heterologous GPCR expressed in yeast. Suggest that this may be a generalizable method for

Example 2 Mutant D. in Yeast Melanogaster (D. melanogaster) Muscari
Agonists down-like acetylcholine receptor functionally expressed G protein coupled insect muscarinic acetylcholine receptors (MAR) has a substantial insecticidal activity and acaricidal force (MR Dick et al. (1997
)). Based on these findings, the development of a yeast-based high-throughput screening method (HTS) for agonists effective against insect MARs would be useful in identifying lead compounds that could evolve into novel modes of action insecticides. It is suggested that there is. Preliminary experiments show that wild-type D. elegans, an insect G protein-coupled receptor (GPCR). Melanogaster MAR (DMAR) has been shown not to function in yeast.
Thus, efforts were undertaken to develop a method to improve the function of DMAR in yeast by replacing DMAR IC3 with a functional IC3 from M3 MAR IC3Δ.

In insect cells, DMAR interacts with heterotrimeric Gq proteins and increases intracellular calcium in response to muscarinic agonists. DM in yeast
One possible explanation for the inactivity of AR is that it cannot bind efficiently to the heterotrimeric G protein of yeast. Therefore, in order to devise a method for altering the DMAR function in yeast, selective mutations in GPCRs that function in altering functional expression and binding to heterotrimeric G proteins were examined.

To construct IC3 substitutions, PCR fragments encoding three domains were prepared by standard methods. Fragment 1 is Drosophila MA in the fifth TMD
An oligonucleotide (AAA AGATCT AAA ATG TACGGAAACCAGACGA) consisting of the amino terminal coding portion of R to the AgeI site
AC) (SEQ ID NO: 7) and (CCA GTA GAG GAA GCACAT
GATGGCTC AGCCT AAG TAG AAG GCG GCC A
(GT GC) (SEQ ID NO: 8). The second of DMAR
Fragment consists of the carboxy-terminal coding sequence starting at the PmII site in the 6th TMD and has an oligonucleotide (TTCATCATCACGTGGACTCCCG
TACAACATC) (SEQ ID NO: 9) and (AAA CTCGAGTTATC
Amplification was performed by PCR using TAATTGTAGACCGCGGC) (SEQ ID NO: 10). This M3 MAR IC3Δ domain was amplified as an AgeI-PmII fragment having the coding sequence in frame with fragments 1 and 2 described in Example 1. These fragments were incorporated into plasmid p426GPD to obtain the mutant DMA
R was under the control of the GPD promoter. This wild type DMAR was cloned into the expression vector pMP3 described in US Pat. No. 5,691,188.
The resulting plasmid was purified using conventional lithium acetate transformation methods in US Pat.
No. 691,188, especially MPY578f described in Pausch et al. (1998).
Introduced into yeast cells useful for performing GPCR agonist stimulated proliferation assays, including c cells.

Yeast cells containing DMAR and plasmids containing wild-type DMAR were assayed in liquid cultures using the MAR agonist carbachol (CCh). The cells were cultured overnight in 2 ml of SC-glucose-ura medium. The cells were transformed with SC containing 5 mM 3-aminotriazole to reduce basal growth rate.
-Diluted 500-fold in glucose-ura-his medium (pH 6.8). Cell suspension samples (180 μl) were dispensed into wells of a sterile 96-well microtiter dish containing 20 μl of serially diluted samples of muscarinic receptor agonists (10 ^-1 to 10 ^-8 M). The plate was incubated at 30 ° C. for 18 hours with shaking (600 rpm). Assays were performed twice and growth rate measurements were made during the logarithmic growth phase of the yeast cells. Optical density measurements were analyzed using GraphPad Prism and expressed as mean ± sem and plotted against agonist concentration.
As shown in FIG. 2, yeast cells containing the mutated DMAR, ie, M3 MAR IC3Δ, exhibited an agonist-dependent proliferative response, indicating that DMAR-M3 MAR I
C3Δ was shown to be functioning. Wild-type DMAR did not function as indicated by the lack of agonist-dependent yeast cell growth.

[0027] Thus, when the IC3 of DMAR is replaced with a functionally deleted IC3 derived from the rat M3 MAR product, a functional chimeric GPCR is produced when expressed in yeast. In cell-based assays such as assays, it has been shown that this may be a generalizable method for altering the functional expression of a heterologous GPCR.

EXAMPLE 3 Functional Expression of Mutant Rat Cholecystokinin CCKB Receptor in Yeast
As shown in present Examples 1 and 2, the IC of mammalian MAR, especially rat M3 MAR
The three-part deletion correlates with an alteration in the functional expression of mammalian and yeast cells that retains its full ability to bind heterotrimeric G proteins. To determine whether this IC3Δ mutation could also alter the functional expression of other GPCRs in yeast, the DNA sequence encoding rat wild-type and IC3Δ cholecystokinin CCKB receptor was amplified by PCR. And in a standard way
It was cloned proximal to the glycerol phosphate dehydrogenase promoter in the yeast expression plasmid p426GPD. The wild-type CCKBR is an oligonucleotide (ACTTAGATCAAAAAATGGAGCGCTCAAGCTGAACC).
G) (SEQ ID NO: 11) and (TCCCGTCGACTCAGCCAGGCCC
(CAGTGTGCTG) (SEQ ID NO: 12) and amplified by PCR. I
The C3Δ cholecystokinin CCKB receptor was prepared by fusing two overlapping fragments. Fragment 1 contains an amino-terminal coding sequence containing 22 amino acids adjacent to the fifth TMD, which is an oligonucleotide (ACTTAGA).
TCAAAAAATGGAGGCCTCAAGCTGAACCG) (SEQ ID NO: 1
1) and (CGAGGGCCAGGGACTGGCCCCGGCCGGGCCC
Amplification was carried out by PCR using CGGCTTTGGGTCTCG) (SEQ ID NO: 13). Fragment 2 contains a carboxy-terminal coding sequence that includes the 22 amino acids adjacent to the 6th TMD, which contains the oligonucleotide (TCCCGTCCGA).
CTCAGCCAGGCCCCAGTGTGCTG) (SEQ ID NO: 12) and (
CGAGACCCAAAGCCGGGCCCGGCCGGGGCCAGTCCC
TGGCCCTCG) (SEQ ID NO: 14) and amplified by the PCR method. This 2
The two fragments were amplified and fused by PCR using oligonucleotides at the 5 'and 3' ends of the full length CCKB receptor. The resulting plasmid was purified using conventional lithium acetate transformation techniques, including those described in US Pat. No. 5,691,188, and in particular, GPCRs including MPY578fc cells described in Pausch et al. (1998). Introduced into yeast cells useful for performing agonist-stimulated proliferation assays.

A yeast strain containing wild-type and IC3Δ cholecystokinin CCKB receptor was
l complete glucose medium (2%) and uracil-deficient (SCD-
ura) The medium was grown overnight. In this agar-based plate assay, 0.
Melted (50 ° C.) S containing 5 mM AT (3-aminotriazole)
CD-ura-his agar medium (35 ml, concentrated KO before autoclaving)
H or NH ₄ OH to adjust to pH 6.8) and cultured overnight (2 × 1
0 ⁴ cells / ml) were seeded and poured into Petri plates square (9 × 9cm).
CCK agonist solution in DMSO (1 mM, 10 μl) was applied to the surface of the solidified agar (top left, CCK8S; top right, CCK8US; bottom left, CCK5;
Lower right, CCK4). The compound applied to the plate surface diffuses radially from the applied site and binds to the CCKB receptor expressed on the cell surface embedded in the agar, resulting in the induction of FUS1-HIS3 expression. Responding cells form areas of high density growth and are easily distinguishable from the limited cell proliferation seen in response to basal FUS1-HIS3 expression. Plates were incubated at 30 ° C. for 3 days (FIG. 3). FIG. 3A shows the strong proliferative response of yeast cells containing the IC3Δ cholecystokinin CCKB receptor, while FIG. 3B shows only limited growth of yeast cells containing the wild-type CCKB receptor. Thus, the third of the CCKB receptor
It is shown that deletion of the intracellular loop portion of E. coli modified its function in yeast.

EXAMPLE 4 Functionality of Mutant Rat Somatostatin Receptor (SSTR) in Yeast
The third intracellular loop of expression is involved in many GPCR functions, including G protein coupling, desensitization and interaction with various modified proteins. The somatostatin receptor is encoded by five subtypes designated SSTR1-5. SSTR
Some amino acids are found in the third subcellular loop of the three subtypes,
It is not found in the same region of the SSTR2 subtype. Since SSTR2 functions effectively in yeast, deletion of these amino acids from IC3 is believed to confer SSTR3 with this functional effectiveness. Thus, the eight amino acids Gln-Tr
p-Val-Gln-Ala-Pro-Cys (SEQ ID NO: 15) is rSSTR3
It has been deleted from the third intracellular loop of the cDNA, allowing more efficient receptor signaling in yeast.

The rat SSTR3 sequence was amplified by a PCR method using an oligonucleotide containing a 5 ′ BglII and 3 ′ XhoI site. The obtained PCR of about 1.3 kb
The product was purified by digestion with BglII and XhoI and the BamHI of p426GPD.
And the plasmid p426GPD-rSSTR3 was inserted between the and XhoI sites. Recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing.

The standard PCR reaction was used to amplify the rSSTR3 cDNA, and two PCR fragments having the following 36 bp overlap were obtained. 5 ′ BgI oligonucleotide (AAAAAAGATCT AAAATGGCCA CTGTTACCT T
) (SEQ ID NO: 16) and 3 ′ oligonucleotide (CTCAGAGCGG C
GTCGCCGCT GACCGAGGG CGCCG (SEQ ID NO: 17))
Was used to create a PCR insert A with an approximate size of 750 bp. 5
'Oligonucleotides GCGCCCTCGT GTCAGCGGCG ACGC
CGCTCT GAG (SEQ ID NO: 18) and 3 ′ XhoI oligonucleotide (AAAACTCGAG TTACAGATGGCTCAGTGTGTCT) (
Using SEQ ID NO: 19), a PCR insert B with an approximate size of 530 bp
Was created. The PCR fragments A and B were gel-purified, annealed, and amplified by PCR using flanking 5'BgIII and 3'XhoI oligonucleotides to obtain -1.3 kb rSSTR3ΔIC3 PCR product. Elaborate, B
After digestion with gIII-XhoI, the rSST3ΔIC3 insert was inserted into p426GP.
D, ligated into the BamHI-XhoI site and plasmid p426GPD-rSST
R3ΔIC3 was created. The appropriate reading frame and sequence were confirmed by restriction mapping and DNA sequencing.

[0033] Yeast cells of the type useful for expression of GPCRs described in US Pat. No. 5,691,188 were transformed with p426GPD-rSSTR3 and p426GPD-rSSTR3ΔIC3 using standard methods. This cell (ie,
LY296 cells described in Price et al. (1995)) were assayed using the agar-based bioassay described in Example 3. Somatostatin (S-14, 1 mM)
Samples (10 μl) were spread on the surface of a selective agar medium containing yeast cells expressing SSTR3. The plate was incubated at 30 ° C. for 3 days. p4
Yeast cells transformed with pLP82 together with 26GPD-rSSTR3 (Gp
al / Gai2 chimeric G protein expression plasmid) showed a weak proliferative response to S-14 (FIG. 4A), but p426GPD-rSSTR3ΔIC3
Showed a stronger response when assayed under the same conditions (FIG. 4B). These results indicate that deletion of the IC3 portion alters the function of SSTR3 in yeast.

Example 5 IC3 Deletion Human α2A Adrenergic Receptor As shown in Examples 1-4, deletion of the IC3 portion of a mammalian GPCR retains sufficient capacity for binding to heterotrimeric G proteins. Correlated with the functional expression of the modified form in selected mammalian and yeast cells. Mutant MAR, CCKBR, and SSTR3 retain the entire outer loop. The transmembrane main and internal domains other than IC3 have not changed. Fifth and sixth spirals across the membrane
The IC3 found in the middle is the only domain modified. Fifth and sixth
Most of this domain was deleted, leaving only the 22 amino acids adjacent to both of the second transmembrane helices. Thus, the IC3 deletion (IC3Δ
) Is 44 amino acids long. Modification of functional expression
This may be due to the removal of domains known to interact with the cell desensitization mechanism and the retention of more functional MARs on the cell surface. To examine whether other IC3Δ mutations also alter the functional expression of other GPCRs in yeast, the DNA sequence encoding the IC3Δ human α2A adrenergic receptor was amplified by PCR and subjected to standard methods. Was cloned into the yeast expression plasmid p426GPD, proximal to the glycerol-phosphate dihydrogenase promoter. This IC3Δ human α2A adrenergic receptor comprises 2
Created by fusing two overlapping fragments. Fragment 1 contains an amino-terminal coding sequence containing the 39 amino acids adjacent to the fifth TMD, which contains the oligonucleotide (GGCCAGGATCCAAAAATGGGCTCCCTG).
CACGCCGGACGC) (SEQ ID NO: 20) and (CGGGCCCCGCGG
Amplification was performed by PCR using GCGCTCGGGGCCCAGACCGTTGGGC) (SEQ ID NO: 21). The second fragment contains the carboxy-terminal coding sequence containing the 41 amino acids adjacent to the 6th TMD, comprising the oligonucleotides (CGGGCGACAGCCTGCCGCCGGC) (SEQ ID NO: 22) and (AGCGGTCGACTCACACGATCCCCTTCCTGTCCCC).
(SEQ ID NO: 23) and amplified by the PCR method. The two fragments are called the full length α
Oligonucleotides were used at the 5 'and 3' ends of the 2A adrenergic receptor for amplification by PCR and fusion. The resulting plasmid was transformed into a plasmid using the conventional lithium acetate transformation method, including those described in U.S. Pat. No. 5,691,188, especially the MPY578fc cells described in Pausch et al. (1998).
PCR agonists-introduced into yeast cells useful for performing stimulated proliferation assays.

Yeast cells containing the wild-type IC3Δ human α2A adrenergic receptor were assayed in liquid culture using the α-adrenergic full agonist UK14304 (RB1) and the partial agonist chloridine. 2 ml of these cells
Was cultured overnight in SC-glucose-ura medium. The cells were converted to SC-glucose-
The ura-his medium was diluted 500-fold with pH 6.8. The cells were diluted 500-fold in SC-glucose-ura-his medium pH 6.8 containing 5 mM 3-aminotriazole to reduce basal growth rate. Cell suspension samples (180 μl) were dispensed into wells of a sterile 96-well microtiter dish containing 20 μl of serially diluted samples (10 ^{−3 -10 −10} M) of the adrenergic receptor agonist UK14304. The plate was incubated at 30 ° C. for 18 hours with shaking (600 rpm). Proliferation was monitored by increasing recorded values at OD ₆₂₀ using a microplate reader. The assay was performed twice and growth rate measurements were obtained during the logarithmic growth phase of yeast cell growth. Optical density measurements were analyzed using GraphPad Prism and expressed as mean ± sem and plotted against agonist concentration.

Yeast cells containing the wild-type IC3Δ human α2A adrenergic receptor showed a dose-dependent proliferative response, indicating that this IC3 deletion is functioning (FIG. 5).

Example 6 Truncation of the rat neurotensin NT1 receptor increases agonist sensitivity
In Examples 1-4, where the third intracellular loop was modified, the functional expression of various heterologous GPCRs expressed in yeast was improved. Agonist-induced desensitization of GPCRs is also mediated, in part, by GPCR internal domains other than a third intracellular loop, such as the intracellular carboxy-terminal tail.

[0038] Removal of the carboxy-terminal domain from GPCRs has been shown to enhance functional expression in yeast and mammalian cells. Truncation of the carboxy-terminal tail of the G protein-coupled α-conjugated pheromone receptor expressed in α-conjugated yeast cells causes hypersensitivity to the presence of the conjugated pheromone (Reneke et al. (198
8); Konopka et al. (1988)). Consistent with these findings, mutant rat neurotensin NT1 receptor (rNTR1) lacking its carboxy-terminal tail is resistant to agonist-induced internalization when expressed in mammalian cells (Hermans et al. (1996)).

To investigate whether truncation of the carboxy terminus modifies the functional response of a heterologous GPCR expressed in yeast, the 52 amino acids that make up the carboxy terminal tail were deleted, and the receptor was reduced to 372 amino acids. Rat N by shortening to long
TR1 was modified. Wild-type and truncated neurotensin receptors (rNTR
1C-termΔ) is replaced with the 5 ′
Oligonucleotide primers (AGTCAGATCTAAGCTT AAAA
ATG CAC CTC AAC AGC TCC (SEQ ID NO: 24) and a separate oligonucleotide defining the wild type (AGTC AGATCT CTA)
GTA CAG GGTCTCCC) (SEQ ID NO: 25), and truncated carboxy terminus (AGAG AGATCT TTAGCGCCACCCAGGAGAC)
AAAGGC) (SEQ ID NO: 26). These fragments were cloned proximal to the PGK promoter in the yeast expression vector pPGK by standard methods (YS. Kang et al. (1990)). The obtained plasmid is
Using conventional lithium acetate transformation methods, in particular, the method described in Pausch et al. (1998)
Introduced into yeast cells of the type described in US Pat. No. 5,691,188 useful for performing assays of GPCR agonist-stimulated proliferation, including PY578fc cells.

Acetyl neurotensin 8-13 (AcNt8
Using -13), yeast cells containing NTR1 were assayed in liquid culture. This
Cells were cultured overnight in 2 ml of SC-glucose-ura medium. Cell-based
SC- containing 2 mM 3-aminotriazole to reduce growth rate
It was diluted 500-fold with a glucose-ura-his medium (pH 6.8). Cell suspension
The liquid sample (180 μl) was mixed with a serially diluted sample of AcNT8-13 (10^-3 ~ 10^-10M) Sterile 96-well microtiter dish containing 20 μl
Was dispensed into the wells of the menu. The plate is shaken at 30 ° C. for 18 hours (600
rpm) and incubated. Proliferation was performed using a microplate reader at OD ₆₂₀ Was monitored by the increase in the recorded value. Growth rate measurements are increased in yeast cells
Obtained during the logarithmic growth phase of the breeding. Analyze the measured optical density using GraphPad Prism,
Expressed as mean ± standard error and plotted against agonist concentration. Shown in FIG.
As noted, yeast cells containing NTR1 elicit an agonist-dependent proliferative response.
This indicates that both wild-type and truncated NTR1 at the carboxy terminus
Is working. Increase of cells containing rNTR1 C-termΔ
The reproductive response is dose-dependent and the ECNT8-13 EC₅₀Is equivalent to 520 nM
Was. This value is the value observed in cells expressing wild-type NTR1 (2.1
μM). Lower concentrations of NT due to deletion of the carboxy terminus
RNTR1 responsive to R agonists is produced, increasing the sensitivity of the yeast bioassay.
Improved.

Thus, the deletion of the portion of the intracellular domain at the carboxy terminus of rat NTR1 produced a functional GPCR whose agonist sensitivity was enhanced when expressed in yeast, indicating that this internal domain has It suggests that the modification is a generalizable method for modifying the function of a heterologous GPCR expressed in yeast.

Example 7 Mutant C. in Yeast The mechanism of C. elegans serotonin receptor
Functionally expressed G protein conjugate Agonists of the elegance serotonin receptor (Ce 5HTR) are believed to have intrinsic nematicidal activity. From these findings, the development of yeast-based HTSs for agonists that are effective at the Ce5HTR could be useful in identifying lead compounds that could evolve into a novel mode of action nematicide. Preliminary experiments have shown that wild-type Ce5HTR does not function in yeast. Therefore, Ce 5HTR IC3 is replaced by M3 MAR IC3
Efforts have begun to develop a method to modify the function of Ce5HTR in yeast by substituting functional IC3 from Δ.

To construct IC3 substitutions, PCR fragments encoding three domains were prepared by standard methods. Fragment 1 contains the amino-terminal coding portion of Ce5HTR for the fifth intracellular interface of TMD, and the oligonucleotide (
AAAAGATCTAAAAATGACGACGAGACGCTTC (SEQ ID NO: 27) and (CCGCTTGGTGGATCTGACTTCTGGTTTCT
GTCCCAGAGGCC TGTAGGCCCAGCCAGCTCTTTGG
TACGCTTCTCAGTTTCCTTATAGATCTTCCCAGTACA
CGCAAATTATTGC) (SEQ ID NO: 28). The second fragment of the Ce5HTR contains the carboxy-terminal coding sequence beginning proximal to the intracellular interface of the sixth TMD, the oligonucleotides (AAAACTCGAGTCAAATAATCGTGGAATAAGGCA) (SEQ ID NO: 29) and (GGCCTACAGGCCTCTGGGACAGAAACC)
AGAAGTCAGATCACCAAGCGGAAGAGGATGTCGCTC
ATCAAGGAGAAGAAGGCCGCCAGAACGCTAGCAATT
(ATTACGGTAC) (SEQ ID NO: 30) and amplified by PCR.
The M3 MAR IC3Δ domain was amplified by the PCR method as described in Example 1.

These fragments were isolated and mixed, and 5 ′ (AAAAGATCTAAAATGAT
CGACGAGACGCTTC) (SEQ ID NO: 27) and 3 '(AAAACTTC
GAGTCAATAATCGTGAATAAGGCA) (SEQ ID NO: 29) was amplified by a PCR method using an oligonucleotide. The resulting fragment was digested with appropriate restriction endonucleases and incorporated into p426GPD to place the mutated Ce5HTR under the control of the GPD promoter. The resulting plasmid was transformed using conventional lithium acetate transformation techniques, such as those described in US Pat. No. 5,691,188.
In particular, GPYs such as MPY578fc cells described in Pausch et al. (1998)
Introduced into yeast cells useful for performing assays of CR agonist-stimulated growth.

[0045] Yeast cells containing the Ce 5HTR were assayed in liquid culture. 2m cells
Cultured overnight in 1 SC-glucose-ura medium. The cells were diluted 500-fold in SC-glucose-ura-his medium (pH 6.8) containing 2 mM 3-aminotriazole to reduce the basal growth rate. A cell suspension sample (200 μl) was mixed with a serially diluted sample of serotonin (5HT) (10 ⁻² to 1 ⁻² ).
0 ^-9 M) was dispensed 2.0μl to wells of sterile 96-well microtiter dishes containing minute. In a similar reaction, the serotonergic antagonists lisuride and mianserin were added at 10 μM per well. The plate was incubated at 30 ° C. for 18 hours with shaking (600 rpm). Proliferation was monitored by increasing recorded values at OD ₆₂₀ using a microplate reader. The assay was performed twice and growth rate measurements were obtained during the logarithmic growth phase of yeast cell growth. Optical density measurements were analyzed using GraphPad Prism and expressed as mean ± sem and plotted against agonist concentration. Cheng and Pruesoff (Y. Ch
The Ki value was determined using the equation of Eng et al. (1973). As shown in FIG.
Yeast cells containing the mutant Ce5HTR containing the M3 MAR IC3Δ showed an agonist-dependent proliferative response, indicating that Ce5HTR, when expressed in yeast, exhibits the expected pharmacological properties. Is shown.

Thus, Ce with functionally deleted IC3 from rat M3 MAR
Functional chimeric GPC when expressed in yeast by substitution of IC3 in 5HTR
R was produced, suggesting that this method of replacing internal domains may be a generalizable method for altering the functional expression of heterologous GPCRs in cell-based assays such as yeast assays. Is what you do.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. The references cited herein are hereby incorporated by reference in their entirety.

References

[Table 1]

[Table 2]

[Brief description of the drawings]

FIG. 1A shows the results of a liquid culture assay on yeast cells containing rat M3 muscarinic acetylcholine receptor (MAR) using the MAR agonist, carbachol (CCh). Yeast cells containing M3MAR with a deletion in the third intracellular loop (IC3Δ) showed an agonist-dependent proliferative response, whereas wild-type MAR did not, indicating that M3MARIC3Δ is a functional GPCR. It is shown that it is.

FIG. 1B: Rat M3MARIC using a MAR agonist, carbachol (CCh)
Figure 3 shows the results of a liquid culture fluorescence induction assay on yeast cells containing 3Δ and FUS2-GFP receptor plasmids. C of M3MARIC3Δ expressed in yeast
There is a dose-dependent increase in green fluorescent protein expression in response to Ch activation.

FIG. 2 shows the results of a liquid culture assay on yeast cells containing a Drosophila muscarinic acetylcholine receptor using the MAR agonist, carbachol (CCh). Mutant Drosophila melanogaster MA containing M3MARIC3Δ
R-containing yeast cells showed an agonist-dependent proliferative response, whereas wild-type Drosophila MAR did not.

FIG. 3A shows the results of an agar baseplate bioassay. Figure 4 shows a strong proliferative response of yeast cells containing the IC3Δ cholecystokinin CCKB receptor.

FIG. 3B shows the results of an agar base plate bioassay. Proliferation by yeast cells containing the wild-type CCKB receptor is only limited, indicating that in yeast, deletion of part of the third intracellular loop of the CCKB receptor alters its function. It is shown.

FIG. 4A shows rSSTR3 and yeast cells transformed with rSSTR3ΔIC3. Yeast cells containing p426GPD-rSSTR3 were somatostatin (S-14)
It shows that it shows only a weak response to

FIG. 4B shows rSSTR3 and yeast cells transformed with rSSTR3ΔIC3. It demonstrates a much stronger response by yeast cells containing p426GPD-rSSTR3ΔIC3 assayed under similar conditions.

FIG. 5 shows the results of a liquid culture assay on yeast cells containing the wild-type IC3Δ human α2A adrenergic receptor using the α-adrenergic receptor full agonist UK14304. Yeast cells containing wild-type and IC3Δ human α2A adrenergic receptors show a dose-dependent proliferative response, indicating that IC3 deletion is functional.

FIG. 6 shows the results of a liquid culture assay using yeast neurotensin receptor agonist AcNT8-13 on yeast cells containing wild-type and carboxy-truncated rat NT1-neurotensin receptor. Truncated rat NT1-neurotensin receptor results in a more sensitive agonist-dependent proliferative response than found in the wild-type receptor.

FIG. 7 illustrates the use of serotonin (5HT) to stimulate yeast cell growth. Figure 4 shows the results of a liquid culture assay on yeast cells containing the C. elebans serotonin receptor. Mutant C. containing M3MARIC3Δ. Yeast cells containing the elegance serotonin receptor showed a 5HT-dependent proliferative response. This proliferative response was prevented by the addition of the serotonin receptor antagonists lisuride and mianserin.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/50 G01N 33/566 33/566 33/68 33/68 (C12N 1/19 // (C12N 1 / 19 C12R 1: 865) C12R 1: 865) C12N 15/00 ZNAA (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 , A , BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW Jurgen, Wes Maryland, USA Bethesda, Rockville, Pike, 9000, Room, B1A-05, Building, 8A, NIH-NDC, Laboratory, Ob, Bioorganic, Chemistry Of which F-term (reference) 2G045 AA40 BB20 CB21 DA36 DA77 FB02 FB07 FB12 GC30 4B024 AA01 AA11 BA63 CA01 DA12 EA04 FA02 FA10 GA11 GA19 4B063 QA01 QA18 QQ61 QR33 QR60 QR76 QR80 QS05 QS36 QA02ABAA AA86A48B AA10 AA30 CA20 CA40 CA51 DA50 EA50 FA74

Claims (37)

[Claims] 1. An isolated nucleic acid sequence having a sequence encoding a modified G protein-coupled receptor (GPCR), wherein the modification comprises a mutation in the intracellular domain of the G protein-coupled receptor, Providing a modified functional response in a cell-based assay, and wherein the modified G protein-coupled receptor is a muscarinic acetylcholine receptor;
A nucleic acid sequence selected from the group consisting of cholecystokinin CCKB receptor, somatostatin receptor, α2A adrenergic receptor, and serotonin receptor. 2. The nucleic acid sequence of claim 1, wherein the modification promotes agonist-stimulated growth, wherein the agonist is a G protein-coupled receptor agonist. 3. The modification results in altered binding between the receptor and the heterotrimeric G protein or prevents the receptor from interacting with a cell desensitization or sequestration-internalization mechanism; Or the nucleic acid of claim 2, which provides for proper plasma membrane localization. 4. The nucleic acid of claim 1, wherein the mutation is a deletion. 5. The nucleic acid according to claim 4, wherein the deletion is a point mutation. 6. The nucleic acid of claim 4, wherein the deletion is in a third intracellular loop of a G protein-coupled receptor. 7. The nucleic acid of claim 6, wherein the G protein-coupled receptor is selected from the group consisting of a muscarinic acetylcholine receptor, a cholecystokinin CCKB receptor, and an α2A adrenergic receptor. 8. The nucleic acid sequence according to claim 1, wherein the serotonin receptor is Ce5HTR. 9. The muscarinic acetylcholine receptor may be rat M3 muscarinic acetylcholine receptor or D. muscarinic acetylcholine. 2. The nucleic acid sequence according to claim 1, wherein the nucleic acid sequence is a melanogastamscarin-like acetylcholine receptor. 10. The nucleic acid sequence of claim 1, wherein the cholecystokinin CCKB receptor is the rat cholecystokinin CCKB receptor. 11. The somatostatin receptor is rat somatostatin receptor subtype 3.
A nucleic acid sequence according to claim 1. 12. The nucleic acid sequence of claim 1, wherein the α2A adrenergic receptor is a human α2A adrenergic receptor. 13. A modified G protein-coupled receptor encoded by the nucleic acid sequence according to claim 1. 14. The G protein-coupled receptor according to claim 13, wherein the deleted third intracellular loop is 44 amino acids long. 15. The sequence Gln-Trp encoded by the nucleic acid sequence according to claim 10
-Val-Gln-Ala-Pro-Ala-Cys (SEQ ID NO: 15) is deleted from the third intracellular loop of the mutant G protein-coupled receptor.
Protein-coupled receptor. 16. An isolated nucleic acid having a sequence encoding a chimeric G protein-coupled receptor,
A nucleic acid, wherein the chimeric G protein-coupled receptor comprises a modified intracellular domain of the G protein-coupled receptor that confers a functional response modification to the chimeric G protein-coupled receptor in a cell-based assay. 17. The nucleic acid of claim 16, wherein the modified intracellular domain is a third intracellular loop. 18. A vector comprising the nucleic acid sequence according to claim 1 or 16. (19) A host cell transformed with the vector according to (8). 20. The host cell according to claim 19, further comprising a plasmid containing an inducible receptor gene. 21. The host cell of claim 19, which is a eukaryotic cell. 22. The host cell according to claim 21, wherein the eukaryotic cell is a yeast cell. 23. The host cell according to claim 22, further comprising a plasmid containing an inducible receptor gene. 24. The host cell according to claim 23, wherein the receptor gene is a green fluorescent protein. 25. The host cell of claim 24, wherein the green fluorescent protein is operably linked to the FUS2 promoter. 26. A method for screening a compound capable of binding to a G protein-coupled receptor, comprising: (a) exposing the host cell according to claim 19 to a test compound; and (b) exposing the test compound to cell proliferation. Measuring the effect. 27. A host cell comprising a heterologous G protein-coupled receptor, wherein the G protein-coupled receptor has a modification that results in an altered functional response of the G protein-coupled receptor in a cell-based assay. Host cells. 28. The host cell of claim 27, wherein the modification promotes agonist-stimulated growth and the agonist is a G protein-coupled receptor agonist. 29. The modification results in altered binding between the receptor and the heterotrimeric G protein, or prevents the receptor from interacting with a cell desensitization or sequestration-internalization mechanism, 29. The host cell of claim 28, wherein said host cell provides for proper plasma membrane localization. 30. The host cell according to claim 27, which is a eukaryotic cell. 31. The heterologous G protein-coupled receptor is modified in an intracellular domain.
The host cell according to item 1. 32. The host cell according to claim 31, wherein the intracellular domain is a third intracellular domain. 33. The host cell of claim 27, wherein the heterologous G protein-coupled receptor has been modified at the carboxy terminal tail of the G protein-coupled receptor. 34. The host cell according to claim 31, which is a yeast. 35. The modified G protein-coupled receptor is a neurotensin receptor.
The host cell according to item 1. 36. The host cell of claim 35, wherein the neurotensin receptor is a rat neurotensin NT1 receptor. 37. A method for screening a compound capable of binding to a G protein-coupled receptor, comprising: (a) exposing the host cell according to claim 27 to a test compound; and (b) testing for cell growth of yeast. A method comprising measuring the effect of a compound.