IE83419B1 - Expression of G protein coupled receptors in yeast - Google Patents

Description

PATENTS ACT, 1992 3242/91 EXPRESSION OF G PROTEIN COUPLED RECEPTORS IN YEAST DUKE UNIVERSITY Field of the invention This invention relates to yeast cells expressing heterologous G protein coupled receptors, vectors useful for making such cells, and methods of using the same.

Background of the Invention The actions of many extracellular signals (for example, neurotransmitters, hormones, odorants, light) are mediated by receptors with seven transmembrane domains (G protein coupled receptors) and heterotrimeric guanine nucleotide—binding regulatory proteins (G proteins). gee H. Dohlman, M. Caron, and R.

Lefkowitz, Biochemistry gg, 2657 (1987); L. Stryer and Cell Biol. g, 391 (1986). Such G protein-mediated signaling systems have been identified H. Bourne, Ann. Rev. in organisms as divergent as yeast and man. ggg H. prototype of the seven—transmembrane—segment class of Stryer and H. Bourne, G protein—coup1ed culminates in mating (fusion) of a and a haploid cell types to I Herskowitz, Mjgzgpjgl, Rey. 51, 536 (1988).

The present invention is based on our continued research into the expression of heterologous G protein coupled receptors in yeast. um In a. first aspect of the present invention provides a transformed yeast cell lacking an endogenous G protein a subunit gene (yeast Ga) and comprising a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein a subunit (mammalian Ga), wherein said first and second heterologous DNA sequences are capable of expression in said cell, and wherein upon ligand binding to said mammalian receptor a functional interaction with G proteins is produced in said cell. sequence, with the third heterologous DNA sequence comprising a The cell optionally contains a third heterologous DNA pheromone-responsive promoter and an indicator gene positioned downstream from the pheromone-responsive promoter and operatively associated therewith.

In a second aspect of the present invention provides a method of testing a compound for the ability to affect the rate of dissociation of Ga from GM.in a cell, comprising providing a ’transformed yeast cell lacking an endogenous G protein a subunit (yeast Ga) and comprises a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA for a mammalian G“, wherein said first and second heterologous DNA sequence which codes sequences are capable of expression in said cell, and wherein upon ligand binding to said mammalian receptor a functional interaction with G proteins is produced in said cell contacting cell; the dissociation of G“ from Gm in said cell. said compound to said and detecting rate of aqueous solution, and the contacting step carried out by adding the compound to the aqueous solution. third transformed yeast cell as described herein wherein the first In a invention aspect the present provides a heterologous DNA sequence which codes for a mammalian G protein coupled receptor is carried by a DNA expression vector capable of expressing a transmembrane protein into the cell membrane of yeast cell, the DNA expression vector comprising: a first segment comprising at least a'.fragment. of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor; and a second segment downstream from said first segment and therewith, comprising a DNA sequence encoding ea heterologous (3 protein in a correct reading frame said second segment coupled receptor.

Brief Description of the Drawings Figure 1 illustrates the construction of the yeast human 62 Adrenergic Receptor expression plasmid, pYBAR2.

Figure 2 illustrates hfiAR ligand binding to membranes from pYBAR2-transformed yeast cells.

Figure 3 shows a comparison of B-adrenergic agonist effects on pheromone—inducible gene .activity. a-MP, (‘) I50, 50 MM (‘) (’) alprenolol; (+) ISO, 100 uM (+) isoproterenol. uM a-mating factor; ALP, 50 uM (-) isoproterenol; Detailed Description of Embodiments of the Present Invention Nucleotide bases are abbreviated herein as follows: A=Adenine G=Guanine C=Cytosine T=Thymine Amino acid residues are abbreviated herein to either three letters or a single letter as follows: Ala; A=Alanine Leu; L=Leucine Arg; R=Arginine Lys; K=Lysine Asn; N=Asparagine Met; M=Methionine Asp; D=Aspartic acid Phe; F=Phenylalanine Cys; C=Cysteine Pro; P=Proline Gln;Q=Glutamine Glu;E=Glutamic acid Ser;s=Serine Thr;T=Threonine Gly;G=Glycine Trp;W=Tryptophan His;H=Histidine Tyr;Y=Tyrosine .Val;V=Valine The term "mammalian" as used herein refers to Ile;I=Isoleucine any mammalian species (e.g., human, mouse, rat, and monkey).

The term "heterologous" is used herein with respect to yeast, and hence refers to DNA sequences, proteins, and other materials originating from organisms other than yeast (e.g., mammalian, avian, amphibian), or combinations thereof not naturally found in yeast.

The terms "upstream" and "downstream" are used herein to refer to the direction of transcription and translation, with a sequence being transcribed or translated prior to another sequence being referred to as "upstream" of the latter.

G proteins are comprised of three subunits: a guanyl—nucleotide binding a subunit; a B subunit; and a 7 subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound thereto. When GDP is bound the G protein exists as an inactive fly when GTP is bound the a subunit dissociates, leaving a G” complex. heterotrimer, the G complex.

Importantly, when a Gd” complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and, hence, the rate of dissociation of the bound the a subunit from the GM complex increases.

This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena. ggg generally stryer and Bourne, supra.

Any mammalian G protein coupled receptor, and the DNA sequences encoding these receptors, may be employed in practicing the present invention. Examples of such receptors include, but are not limited to, dopamine receptors, muscarinic cholinergic receptors, a-adrenergic receptors, B-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors. The term receptor as used herein is intended to encompass subtypes of the named receptors, and mutants and homologs thereof, along with the DNA sequences encoding the same.

The human D1 dopamine receptor cDNA is reported in A. Dearry et al., Nature ggz, 72-76 (1990).

Muscarinic cholinergic receptors (various subtypes) are disclosed in E. Peralta et al., Nature ;g;, 434 (1988) and K. Fukuda et al., Nature 327, 623 (1987).

Serotonin receptors (various subtypes) are disclosed in S. Peroutka, Ann. Rev. Neurosci. 1;, 45 (1988).

A cannabinoid receptor is disclosed in L.

Matsuda et al., Nature gig, 561 (1990).

Any DNA sequence which codes for a mammalian G a subunit (Ga) may be used to practice the present invention. Examples of mammalian G a subunits include G5 a subunits, Gi-a subunits, Go a subunits, G2 a subunits, and transducin a subunits. See generally Stryer and Bourne, supra. G proteins and subunits useful for practicing the present invention include subtypes, and mutants and homologs thereof, along with the DNA sequences encoding the same.

Heterologous DNA sequences are expressed in a host by means of an expression vector. An expression vector is a replicable DNA construct in which a DNA sequence encoding the heterologous DNA sequence is operably linked to suitable control sequences capable of effecting the expression of a protein or protein subunit coded for by the heterologous DNA sequence in the intended host. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and (optionally) sequences which control the termination of transcription and translation.

Vectors useful for practicing the present invention include plasmids, viruses (including phage), and integratable DNA fragments (i.e., fragments integratable into the host genome by homblogous recombination). The vector may replicate and function independently of the host genome, as in the case of a plasmid, or may integrate into the genome itself, as in the case of an integratable DNA fragment. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. For example, a promoter operable in a host cell is one which binds the RNA polymerase of that cell, and a ribosomal binding site operable in a host cell is one which binds the endogenous ribosomes of that cell.

DNA regions are operably associated when they are functionally related to each other. For example: a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

Generally, operably linked means contiguous and, in the case of leader sequences, contiguous and in reading phase. ._7_ Transformed host cells of the present invention are cells which have been transformed or transfected with the vectors constructed using recombinant DNA techniques and express the protein or protein subunit coded for by the heterologous DNA sequences. In general, the host cells are incapable of expressing an endogenous G protein a—subunit (yeast Ga).

The host cells do, however, express a complex of the G protein 3 subunit and the G protein 7 subunit (GM).

The host cells may express endogenous G”, or may optionally be engineered to express heterologous Gm (e.g., mammalian) in the same manner as they are engineered to express heterologous G“.

A variety of yeast cultures, and suitable expression vectors for transforming yeast cells, are See, e.g., U.S. Patent No. 4,745,057; U.S.

Patent No. 4,797,359; U.S. Patent No. 4,615,974; U.s.

Patent No. 4,880,734; U.s. Patent No. 4,711,844; and U.S. Patent No. 4,865,989. the most commonly used among the yeast, although a known.

Saccharomvces cerevisiae is number of other strains are commonly available. gee‘ g;g;, U.S. Patent No. 4,806,472 (Kluveromvces lactis and expression vectors therefor); 4,855,231 (Pichia pastoris and expression vectors therefor). Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the heterologous DNA sequences, sequences for polyadenylation and transcription termination, and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979): Tschemper et al., Gene 10, 157 (1980)). provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4—l (Jones, Genetics 85, 12 (1977)).

This plasmid contains the TRP1 gene, which The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for metallothionein, ’ 3~phosphoglycerate kinase (Hitzeman et al., J. Biol.

Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzvme Reg. 1, 149 (1968); and Holland et al., Biochemistry ll, 4900 (1978)), such as enolase, glyceraldehyde—3—phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose—6-phosphate isomerase, 3—phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in R.

Hitzeman et al., EPO Publn. No. 73,657."Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3éphosphate dehydrogenase, as well as enzymes responsible for maltose and galactose utilization.

In constructing suitable expression plasmids, the termination sequences associated with these genes may also be ligated into the expression vector 3' of the heterologous coding sequences to provide polyadenylation and termination of the mRNA.

A novel-DNA expression vector described herein which is particularly useful in the present invention contains a first segment comprising at least a fragment of the extreme amino- terminal coding sequence of a yeast G protein coupled receptor and a second segment downstream from said Any of a variety of means for detecting the dissociation of Ga from Gm can be used in connection with the present invention. The cells could be disrupted and the proportion of these subunits and complexes determined physically (i.e., by chromatography). The cells could be disrupted and the quantity of Ga present assayed directly by assaying for the enzymatic activity possessed by Ga in isolation (i.e., the ability to hydrolyze GTP to GDP). Since whether GTP or GDP is bound to the G protein depends on whether the G protein exists as a G” or Gd” complex, dissociation can be probed with radiolabelled GTP. As explained below, morphological changes in the cells can be observed. A particularly convenient method, however, is to provide in the cell a third heterologous DNA sequence, wherein the third heterologous DNA sequence comprises a pheromone-responsive promotor and an indicator gene positioned downstream from the pheromone—responsive promoter and operatively associated therewith. This sequence can be inserted with a vector, as described in detail herein. With such a sequence in place, the detecting step can be carried out by monitoring the expression of the indicator gene in the cell. Any of a variety of pheromone responsive promoters could be used, examples being the 553; gene promoter and the Egg; gene promoter. Likewise, any of a broad variety of indicator genes could be used, with examples including the gig; gene and the Lag; gene.

As noted above, transformed host cells of the present invention express the protein or protein subunit coded for by the heterologous DNA sequence. when expressed, the G protein coupled receptor is located in the host cell membrane (i.e., physically positioned therein in proper orientation for both the stereospecific binding of ligands on the extracellular side of the cell membrane and for functional interaction with G proteins on the cytoplasmic side of the cell membrane).

The ability to control the yeast pheromone response pathway by expression of a heterologous adrenergic receptor and its cognate G protein a-subunit has the potential to facilitate structural and functional characterization of mammalian G protein- coupled receptors. By scoring for responses such as growth arrest or fi—galactosidase induction, the functional properties of mutant receptors can now be rapidly tested. Similarly, as additional genes for putative G protein—coupled receptors are isolated, numerous ligands can be screened to identify those with activity toward previously unidentified receptors. ggg F. Libert gt al., Science 244, 569 (1989); M. S. Chee gg al., Nature 354, 774 (1990). Moreover, as additional genes coding for putatiVe G protein a- subunits are isolated, they can be expressed in cells of the present invention and screened with a variety of G protein coupled receptors and ligands to characterize these subunits. These cells can also be used to screen for compounds which affect receptor-G protein interactions.

Cells of the present invention can be deposited in the wells of microtiter plates in known, predetermined quantities to provide standardized kits useful for screening compounds in accordance with the various screening procedures described above.

The following Examples are provided to further illustrate various aspects of the present invention. They are not to be construed as limiting the invention.

EXAMPLE1 Construction of the Human B2-Adrenerqic Expression Vector pYBAR2 and Expression in Yeast To attain high level expression of the human fi2—adrenergic receptor (hBAR) in yeast, a modified hBAR gene was placed under the control of the QAL; promoter in the multicopy vector, YEp24 (pYBAR2).

Figure 1 illustrates the construction of yeast expression plasmid pYBAR2. In pYfiAR2, expression of the hBAR sequence is under the control of the gggl promoter. Figure 1A shows the 5'—untranslated region and the first 63 basepairs (bp) of coding sequence of the hBAR gene in pTZNAR, B. O'Dowd gt al., Q. pigl. ghgm. ggg, 15985 11988), which was removed by Aat II cleavage and replaced with a synthetic oligonucleotide corresponding to 11 bp of noncoding and 42 bp of coding sequence from the gig; gene. gee N. Nakayama et al., gggg Q. 2643 (1985); A. Burkholder and L. Hartwell, .

Nucleic Acids Res. lg, 8463 (1985). The resulting plasmid, pTZYNAR, contains the modified hfiAR gene flanked by Hind III sites in noncoding sequences. The Hind III—Hind III fragment was isolated from pTZYNAR and inserted into pAAH5 such that the 3'— untranslated sequence of the modified hflAR gene was followed by 450 bp containing termination sequences from the yeast gggl gene. (1983).

See G. Ammerer, Methods. Enzvmol. 101, 192 Cell. Biol. 9, 2950 (1989).

J. Salmeron et al., 59;.

Cells grown to late exponential phase were induced in medium containing 3% galactose, supplemented with about 10 uM alprenolol, and grown for an additional 36 hours. Standard methods for the maintenance of cells were used. ggg F.

Sherman et al., Methods in Yeast Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1986).

Maximal expression required (i) expression of a transcriptional transactivator protein (gggg), (ii) replacement of the 5' untranslated and extreme NHf- terminal coding sequence of the hfiAR gene with the corresponding region of the yeast sggg (a-factor receptor) gene, (iii) induction with galactose when cell growth reached late exponential phase, and, (iv) inclusion of an adrenergic ligand in the growth medium during induction.

The plasmid pYfiAR2 was deposited in accordance with the provisions of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn ..]_3..

Drive, Rockville, MD 20852 USA, on September 11, 1990, and has been assigned ATCC Accession No. 40891.

EXAMPLE2 Binding Affinity of hBAR Ligands in Yeast Transformed with pYBAR2 A primary function of cell surface receptors is to recognize only appropriate ligands among other extracellular stimuli. Accordingly, ligand binding affinities were determined to establish the functional integrity of hBAR expressed in yeast. As discussed in detail below, an antagonist, 151-labeled cyanopindolol (‘5I—CYP), bound in a saturable manner and with high affinity to membranes prepared from pYBAR2-transformed yeast cells. By displacement of ‘”I—CYP with a series of agonists, the order of potency and stereospecificity expected for hBAR was observed. 7 Figure 2 illustrates hBAR ligand binding to membranes from pYBAR2—transformed yeast cells. (A) Bmx (maximum ligand bound) and Kd (ligand dissociation constant) values were determined by varying 125,-CYP concentrations (5 — 400 pM). Specific binding was _14_. defined as the amount of total binding (circles) minus nonspecific binding measured in the presence of 10 pM (—) alprenolol (squares). A Kd of 93 pM for l25,—CYP binding was obtained and used to calculate agonist affinities (below). (3) Displacement of 18 pM l25wCYP with various concentrations of agonists was used to determine apparent low affinity Kivalues (non G protein coupled, determined in the presence of 50 uM GTP) for receptor binding, squares; (—) isoproterenol, circles; (—) epinephrine, downward—pointing triangles; (+) isoproterenol, upward pointing triangles; (—) norepinephrine.

COMPARATIVE EXAMPLE A Ligand Bindinq_Affinitv for hBAR Expressed in Yeast and Mammalian Cells The binding data of Figures 2 }A) and (B) were analyzed by nonlinear least squares regression, gee A. DeLean et al., flgl. Pharmacol. g;, (1982), and are presented in Table I. Values given are averages of measurements in triplicate, and are representative of 2 — 3 experiments. Binding affinities in yeast were nearly identical to those observed previously for hfiAR expressed in mammalian cells. -15..

Table 1 Comparison of ligand Binding Parameters for High Level Expression of Human p—Adrenergic Receptor in Yeast and COS-7 Cellst Yeast SC261 (pYfiAR2, pMTL9) Monkey cos-7 (pBC12 :hpAR) l—CYP: ‘Kd 0.093 nn 1 0.013 0.110 nM 10.009 28“ 115 pmol/mg 24 pmol/mg Ki(M): (-) isoproterenol 103 1 26 130 1 15 (+) isoproterenol 3670 t 420 5000 t 184 (-) epinephrine 664 t 123 360 1 30 (-) norepinephrine 6000 i 1383 5800 t 373 *Values derived from Fig. 2 and H. Dohlman gt gl., Biochemistry gg, 2335 (1990).; i S.E.

K , ligand dissociation constant 2 d . .

B X, maximum ligand bound Ki, inhibition constant EXAMPLE13 Aqonist-Dependent Activation of Mating Signal Transduction in Yeast Expressing hBAR A second major function of a receptor is agonist-dependent regulation of downstream components in the signal transduction pathway. Because the pheromone—responsive effector in yeast is not known, indirect biological assays are the most useful indicators of receptor functionality. gee K. Blumer and J. Thorner, AQgg.ggy. Physiol. in press; I.

Herskowitz, Microbiol. Rev. gg, 536 (1988). In yeast _16_ cells expressing high concentrations of hfiAR, no agonist-dependent activation of the mating signal transduction pathway could be detected by any of the typical in vivo assays; for example, imposition of G1 arrest, induction of gene expression, alteration of morphology (so—called "shmoo" formation) or stimulation of mating. A likely explanation for the absence of responsiveness is that hBAR was unable to couple with the endogenous yeast G protein.

EXAMPLE4 coexpression of hEAR and Mammalian G9 a—Subunit in Yeast Expression of a mammalian Gs a—subunit can correct the growth defect in yeast cells lacking the corresponding endogenous protein encoded by the Q25; gene. ggg C. Dietzel and J. Kurjan, Qgll gg, 1001 (1987). mammalian cells is conferred by the a-subunit of Moreover, specificity of receptor coupling in (NNYI9) coexpressing hBAR and rat Ggz, bu+ containing See C.

In yeast wild—type GPA1, no adrenergic agonist-induced shmoo formation, a characteristic morphological change of yeast in response to mating pheromone, was observed.

EXAAAPLEi5 coexpression of hBAR and Mammalian qt-subunit in Yeast Lacking an Endogenous G Protein a-Subunit To prevent interference by the endogenous yeast G protein a—subunit, gpa; mutant cells (strain _17_ 8c) were used.

Morphologies of yeast cells cotransformed with pYfiAR2, pMTL9, and pYSK136Gas were examined after incubation with (A) no adrenergic agentf (B) 100 uM (-) isoproterenol; (C) 100 pM (-) isoproterenol and 50 uM (-) alprenolol; and (D) 100 uM (+) isoproterenol.

Results showed that treatment of 8c cells coexpressing hfiAR and rat Gsa with the B-adrenergic agonist isoproterenol indeed induced shmoo formation, and that this effect was blocked by the specific antagonist alprenolol.

EXAMPLES Coexpression of hfiAR and Mammalian Gga-subunit in - Yeast Containing a B-Galactosidase siqnal sequenae The isoproterenol-induced morphological response of 8c cells coexpressing hBAR and rat G51 suggested that these components can couple to each other and to downstream components of the pheromone response pathway in yeast lacking the endogenous G a—subunit. To confirm that the pheromone signaling pathway was activated by hBAR and rat Gsa, agonist induction of the pheromone-responsive Egg; gene promoter was measured in a strain of yeast derived from -18 .... c cells (8cl) in which a FUSl-lacZ gene fusion had been stably integrated into the genome. et a1., EMBO Q. g, 691 (1990).

See S. Nomoto Strains 8c (Fig. 3, legend) and NNYl9 (MAT; ura3 leu2 his3 trp1 lvsz FUS1-LacZ::LEU2) were modified by integrative transformation with YIpFUSl02 (gggg), S.

Nomoto et al., su ra, and designated 8cl and NNYl9, respectively. These strains were transformed with pYBAR2 and pYSKl36Gas and maintained on minimal selective plates containing glucose and 50 uM CuS0,.

Colonies were inoculated into minimal selective media (3% glycerol, 2% lactic acid, 50 uM CuSO,), grown to early log phase (ODwD = 1.0), and induced for 12 hours by addition of 3% galactose. Cells were washed and resuspended in induction media (ODwo = 5.0) containing 0.5 mM ascorbic acid and the indicated ligands. After a 4 hour incubation at 30°C, cells were harvested, resuspended into 1 ml of Z-buffer, sgg J. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972), supplemented with 0.0075% SDS, and B—galactosidase activities were determined in 3 — 4 independent experiments as described previously. ggg J. Miller, ggpra.

Figure 3 shows a comparison of fl-adrenergic agonist effects on pheromone—inducible gene activity. a—MF, 10 uM a—mating factor; (—) ISO, 50 uM (—) isoproterenol; (—) ALP, 50 pM (—) alprenolol; (+) ISO, 100 uM (+) isoproterenol. In 8c1 (gpgl) cells coexpressing hfiAR and rat Ggz, a dramatic isoproterenol- stimulated induction of B-galactosidase activity was observed. Agonist stimulation was stereoselective and was blocked by addition of a specific antagonist.

Agonist responsiveness was dependent on expression of both hBAR and rat Ggz, and required a strain in which the endogenous G protein a-subunit was disrupted. The final fi—galactosidase activity achieved in response to isoproterenol in transformed 8C1 cells was comparable ._19.. to that induced by a-factor in nontransformed cells that express QEA; (NNYl9), although basal fi—galactosidase activity in NNY19 cells was considerably lower than in 8c1 cells. Taken together, our results indicated that coexpression of hfiAR and rat Gka was sufficient to place under catecholamine control key aspects of the mating signal transduction pathway in yeast. However, the adrenergic agonist did not stimulate mating in either 8c cells or NNY19 cells coexpressing hfiAR and rat Gga, in agreement with recent observations that yeast pheromone receptors, in addition to binding pheromones, participate in other recognition events required for mating. ggg A. Bender and G. Sprague, Genetics ;g;, 463 (1989).

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims (1)

1. CLAIMS A transformed yeast cell lacking an endogenous G protein a subunit (yeast Ga} and comprising a first heterologous DNA sequence which codes for a mammalian G protein Coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein a subunit (mammalian Ga), wherein said first and second heterologous DNA sequences are capable of expression in said cell, and wherein upon ligand binding to said mammalian receptor a functional interaction with G proteins is produced in said cell. A transformed yeast cell according to claim 1, wherein said first heterologous DNA sequence is carried by a plasmid. A transformed yeast cell according to either of claims 1 or 2, wherein said second heterologous DNA sequence is carried by a plasmid. A transformed yeast cell according to any preceding claim, wherein said mammalian G protein a subunit is selected from the group consisting of G5 a subunits, G1 a subunits, Go a subunits, G, d subunits, and transducing d subunits. A transformed yeast cell according to any preceding claim which expresses a complex of the G protein 8 subunit and the G protein v subunit (GM). A transformed yeast cell according to claim» 5 which expresses endogenous GM. A transformed yeast cell according to any preceding claim, wherein said first heterologous DNA sequence codes for a mammalian G protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors d-adrenergic receptors, B-adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors. A transformed yeast cell according to any preceding claim further comprising a third heterologous DNA sequence, wherein said third heterologous DNA sequence comprises a pheromone-responsive promoter and an indicator gene positioned downstream from said pheromone-responsive promoter and operatively associated therewith. A transformed yeast cell according to claim 8, wherein said pheromone responsive promoter is selected from the group consisting of the BAR; gene promoter and the £351 gene promoter, and wherein said indicator gene is selected from the group consisting of the E151 gene and the Lagz gene. A method of testing a compound for the ability to affect the rate of dissociation of Ga from GW in a cell, comprising: providing a transformed yeast cell lacking an endogenous G protein a subunit gene (yeast Ga) and comprises a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian Ga, wherein said first and second heterologous DNA sequences are capable of expression in said cell, and wherein upon ligand binding to said mammalian receptor a functional interaction with G proteins is produced in said cell; contacting said compound to said cell; and detecting the rate of dissociation of Ga from G“ in said cell. A method according to claim 10, wherein said yeast cells are provided in an aqueous solution and said contacting step is carried out by adding said compound to said aqueous solution. A method according to either one of claims 10 or 11, wherein said mammalian G protein K subunit is selected from the group consisting of G, a subunits, G; d subunits, Go a subunits, G, a subunits, and transducing a subunits. A method according_to any one of claims 10 to 12, wherein said yeast cell expresses endogenous GM. A method according to any one of claims 10 to 13, wherein said first heterologous DNA sequence codes for a mammalian protein-coupled receptor selected from the group consisting of dopamine receptors, muscarinic cholinergic receptors, d-adrenergic receptors, B—adrenergic receptors, opiate receptors, cannabinoid receptors, and serotonin receptors. A method according to any one of claims 10 to 14, said yeast cell further comprising a third heterologous DNA sequence, wherein said third heterologous DNA sequence comprises a pheromone—responsive promoter and an indicator gene positioned downstream from said pheromone-responsive promoter and operatively associated therewith; and wherein said detecting step is carried out by monitoring the expression of said indicator gene in said cell. A transformed yeast cell according to claim 1 wherein the first heterologous DNA sequence which codes for a mammalian G protein coupled receptor is carried by a DNA expression vector capable of expressing a transmembrane protein into the cell membrane of yeast cells, the DNA expression vector comprising: a first segment comprising at least a fragment of the extreme amino—termina1 coding sequence of a yeast G protein coupled receptor; second and a segment downstream from said first segment and in a correct reading frame therewith, said second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor. A transformed yeast cell according to claim 16, wherein a fragment of the extreme amino-terminal coding sequence of said heterologous G protein coupled receptor is absent. A transformed yeast cell according to claim 16, wherein said first and second segments are operatively associated with a promoter operative in a yeast cell. A transformed yeast cell according to claim 18, wherein said promoter is the QALL promoter. A transformed yeast cell according to claim 16, wherein said first segment comprises at least a fragment of the extreme amino-terminal coding sequence of a yeast pheromone receptor. A transformed yeast cell according to claim 20, wherein the yeast pheromone receptor is selected from the $132 gene or the SIB} gene. A transformed yeast cell according to claim 16, further comprising at least a fragment of the 5'—untranslated region of a yeast G protein coupled receptor gene positioned upstream from said first segment and operatively associated therewith. A transformed yeast cell according to claim 16, further comprising at least a fragment of the 5'-untranslated region of a yeast pheromone receptor gene positioned upstream from said first segment and operatively associated therewith. A transformed yeast cell according to claim 23, wherein said yeast pheromone receptor gene is the $232 gene or the EIE; gene. A transformed yeast cell according to claim 16, wherein said vector comprises a plasmid. A transformed yeast cell according to claim 16, wherein said second segment comprises a DNA sequence encoding a mammalian G protein coupled receptor. A transformed yeast cell according to claim 16, wherein said second segment comprising a DNA sequence encoding a mammalian G protein—coup1ed receptor is selected from the group consisting’ of dopamine receptors, muscarinic cholinergic receptors, a-adrenergic receptors, B-adrenergic receptors, and opiate receptors, cannabinoid receptors, serotonin receptors. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.