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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• This invention covers protein fusions between the C- terminus of any G-protein coupled receptor and the N- terminus of the Saccharomyces cerevisiae G-alpha protein Gpalp, the DNA constructs encoding the same, yeast strains expressing the same, methods to ensure that the fusion protein is coupled to the yeast mating pathway, and assays for such coupling.
• G-protein-coupled receptors seven transmembrane domain receptors or serpentine receptors, is characterized by their interaction with heterotrimeric G-protein complexes comprised of alpha, beta and gamma subunits (Watson and Arkinstall, The G-Protein Linked Receptors Facts Book, c. 1994 by Academic Press) .
• Mammalian receptors of this class include the alpha- and beta-adrenergic, muscarinic cholinergic, cannabinoid, dopamine, opiate, serotonin, thrombin, platelet activating factor and thromboxane A2 receptors. Agonists and antagonists of several of these receptors are important therapeutic agents, and many members of this class of receptors are involved in various disease processes.
• Ligands for many of these receptors have been identified by binding assays using membrane preparations from tissue culture cells or heterologous systems such as insect cells overexpressing the relevant receptor. Ligands identified thus, however, may be agonists, antagonists or neutral in terms of receptor function, since only binding and not activation is measured by these assays. Moreover, even binding assays cannot be used to study the so-called "orphan" receptors, which were identified by DNA homology methods, and whose physiological ligands and functions are unknown.
• these proteins traverse the membrane seven times, giving rise to one free end and three loops on both sides of the membrane. Potentially, all three loops and the end could contribute to forming the ligand binding pocket on the outside and the recognition site for G-proteins on the inside. These factors render it difficult to study these protein by X-ray crystallography and molecular modeling. In addition, dividing these proteins into domains of specific function that can be analyzed separately, either by proteolysis or expression of gene fragments, is not feasible because of the loops. This also renders this class of receptors less suited to rational drug design. Therefore, there is a need in the art for new and convenient assays to identify agonists and antagonists of these receptors.
• the yeast Saccharomyces cerevisiae has already proven useful in developing such assays.
• Two endogenous G- protein coupled receptors and one heterotrimeric G- protein complex have been characterized from this organism, all of which are involved in a developmental pathway leading to the formation of a diploid yeast cell from fusion of two haploid cells of the a and alpha mating type.
• the two receptors, the a-factor receptor have been characterized from this organism, all of which are involved in a developmental pathway leading to the formation of a diploid yeast cell from fusion of two haploid cells of the a and alpha mating type.
• thrombin pathway in response to mating factor, and their similarity to mammalian signal transduction components (the thrombin pathway is chosen as an example) are represented in Fig. 1. (Jones, Pringle and Broach, The Molecular and Cellular Biology of Yeast Saccharomyces, Vol. 2., c. 1992 by Cold Spring Harbor Laboratory Press) . Activation of heterotrimeric G-proteins requires a specific interaction between the receptor and the G- protein complex that is mediated primarily by the G- alpha subunit. Unactivated receptors are normally bound to a trimeric complex with inactive GDP-bound G-alpha.
• Receptor activation by the ligand stimulates GDP release from G-alpha followed by GTP binding and the dissociation of the beta and gamma subunits from the alpha subunit. In mammalian cells, this renders both G- alpha and G-beta-gamma "active" and capable of activating downstream signaling elements such as adenylyl cyclase. Hydrolysis of GTP to GDP switches G-alpha back to the inactive state, where it reassociates with G-beta-gamma to regenerate the inactive complex, which then associates with a receptor.
• the entity that propagates the signalling cascade is the released complex of G-beta and G-gamma subunits; however, dissociation of that complex from G- alphais still the crucial activation step.
• G-alpha is the subunit that interacts primarily with the receptor, the affinity of a particular G-alpha for a given receptor largely determines which of the many heterotrimeric complexes in mammalian cells is associated with the receptor, and therefore determines the efficiency of coupling between a receptor and a given G-protein complex.
• BAR beta-2-adrenergic receptor
• yeast G-alpha protein such that it can be coupled to heterologous receptor activation.
• the invention described here is a means toward such adaptation of the yeast G-alpha protein.
• a critical and novel feature of our invention is the creation of a covalent linkage between a mammalian receptor and the endogenous yeast G-alpha protein, which is achieved by an in-frame gene fusion between the C-terminal end of the heterologous receptor gene and the N-terminal end of the yeast GPA1 gene.
• the presence of the yeast G-alpha protein as a linked moiety should greatly increase its local concentration and thus facilitate its interaction with the receptor and its response to activation of the receptor, as shown schematically in Fig. 2.
• Our invention also provides for the possibility that such facilitation is insufficient to overcome the lack of recognition between the two components. This is achieved by selection schemes used along with mutagenesis of the Gpalp domain of such fusion proteins, thereby identifying mutants in this domain in which activation of the receptor moiety is coupled to activation of the Gpalp moiety, adn therefore to the yeast mating pathway.
• the presence of the linked G-alpha may impede desensitization of the ligand response either by masking receptor determinants that mediate desensitization or by protecting G-alpha from degradation.
• the article does not envision the use of the potentiated response to facilitate interactions between components that may not interact or only interact weakly, nor does it envision applications where the receptor and G-alpha protein are from different species.
• patent 5,030,576 covers the fusion of the ligand binding domain of a receptor to a reporter polypeptide that undergoes a conformational change upon ligand binding to the binding domain, but the application to G-protein-coupled receptors mentioned in the '576 patent describes the relevant reporter polypeptide as the cytoplasmic domain of such a receptor that is capable of interaction with G-proteins.
• U.S. patent application WO 91/12273 covers hybrid proteins created by replacing domains other than the ligand-binding domain of a G-protein coupled receptor with corresponding domains of a yeast G-protein coupled receptor. In contrast to and as distinct from U.S.
• the present invention embodies the idea of using covalent linkage between two proteins created by gene fusion to potentiate their mutual interaction.
• the invention provides DNA constructs that encode and express a fusion protein with a peptide bond between the
• the invention further provides yeast strains expressing these fusion proteins.
• the invention also provides methods to ensure that these fusion proteins are synthesized and localized to the plasma membrane such that the Gpalp domain of the fusion protein can interact with yeast G-beta and G-gamma proteins.
• the invention further provides methods that can be used to select, from a collection of mutants of the G-alpha domain of such fusion constructs, individual mutants demonstrating coupling of receptor activation to the mating pathway of yeast through the fusion protein.
• the invention further provides for use of the said strains to identify small molecule agonists and antagonists of these receptors.
• the invention further provides for use of the said strains to identify peptide agonists and antagonists of receptor activation by transformation with a combinatorial peptide library, which is created by expressing a randomized DNA sequence in yeast such that the individual peptides are secreted into the medium via gene fusions to the signal peptide of the yeast alpha-factor.
• Figure 1 shows G-protein signaling pathways in mammals and humans.
• Figure 2 shows a map of plasmid pRMHBT4.
• Figure 3 shows a map of plasmid pRMHBTlO.
• Figure 4 shows a map of plasmid pRMHBT18-NG.
• Figure 5 shows a map of plasmid pRMHBT20-NG.
• Figure 6 shows a map of plasmid pRMHBT26.
• Figure 7 shows a map of plasmid pRMHBT41.
• Figure 8 shows a map of plasmid pRMHBT43.
• Figure 9 shows a map of plasmid pRMHBT44.
• Figure 10 shows a map of plasmid pRMHBT45.
• Figures 11A-11G show the nucleotide sequence encoding the STE2-GPA1 fusion protein and its amino acid sequence. The sequence starts at position 520 in STE2, and extends through position 1850 in GPA1 . GenBank accession numbers for sequences are provided below. The extra amino acids generated at the junction are underlined.
• Figures 12A-12G show the nucleotide sequence encodng ThR-GPAl fusion protein and its amino acid sequence. The sequence starts at position 288 in ThrR, and extends through position 1850 in GPA1. GenBank accession numbers for sequences are provided below. The extra amino acids generated at the junction are underlined. The STE2 leader sequence that proceeds and is linked in- frame to the ThrR sequence is also shown below.
• MS2288 mat a his3D200, Ieu2-3,U2, trplDl, Dr. M. Rose, Princeton ura3-52 Univ.
• HBSIO mat a ade2-101, farl-x200, his3D200, Heartland BioTechnologies leu2-3,112, lys2DS738, trplDl, ura3-52
• HBS10::pFFLZ same as HBSIO except farl ::URA3-FUSlp- Heartland BioTechnologies LACZ
• HBS12 mat a farl-x200, his3D200, Ieu2-3,U2, Heartland BioTechnologies ste2, trplDl, ura3-S2
• HBS12LZ same as HBS12 except leu2::LEU2-FUSlp- Heartland BioTechnologies LACZ
• TMHY3D a/a ADE2/ade2, FARllfarl, Heartland BioTechnologies GPAl/gpal.'.TRFl, his3/his3, LEU2/leu2, Iys2llys2, trplltrpl, ura3lura3
• HBS14 same as TMHY3D Heartland BioTechnologies 9A mat a, ade2-101, farl-x200, gpa .TRPl, Heartland BioTechnologies his3D200, leu2-3,112, lys2DS738, trplDl, ura3-52
• 9ALZ ⁇ GS same as 9ALZ except also ste2 Heartland BioTechnologies Gene names are italicized (GPA1) , and are in upper case (GPA1) when indicating a functional and dominant gene, and in lower case (gpal) when indicating a non ⁇ functional recessive mutant gene.
• the corresponding proteins are in plain text (Gpalp) .
• An agonist is defined as a molecule that binds to a receptor protein, and activates the receptor by inducing conformational or other changes in it such that the heterotrimeric G- protein complex that is bound to the receptor is disrupted, leading to release of the beta-gamma complex from the alpha subunit.
• This invention embodies the idea of using covalent linkage between two proteins created by gene fusions to potentiate the interaction between G-protein-coupled receptors from other species and a protein homologous in function to the Gpalp protein of the yeast Saccharomyces cerevisiae .
• the experiments of Bertin et al have shown that there is a potentiation of the downstream response to receptor activation when the human beta-2-adrenergic receptor and its cognate G-alpha protein are linked in this manner.
• potentiation could also result from: a) more efficient coupling (which is considered in present models as transmission of a conformational change in the receptor to the G-protein complex) due to proximity of the interacting molecules brought about by covalent linkage; b) more efficient coupling because of the great increase in local concentration of Gpalp brought about by covalent linkage, thereby overcoming the effects of an unfavorable equilibrium binding constant for a heterologous receptor and yeast Gpalp; c) the presence of stoichiometric amounts of the two components, leaving no molar excess of either component to dilute the effects of ligand-mediated activation; d) better membrane anchoring of G-alpha by covalent attachment to the receptor compared to the normal situation of anchoring via N-terminal myristoylation, which may be reversible. Regardless of the precise reason, it is likely that covalent coupling ameliorates the lack of recognition specificity between a given mammalian receptor and the yeast
• the DNA constructions needed for the present invention can be made in vectors that can replicate independently in yeast cells, including the YCp or the YEp class of vectors or in vectors that are designed for integration into the yeast chromosome such as the YIp class. Most preferred vector are those which autonomously replicate in yeast.
• G-protein-coupled receptors used in the present invention may be from animal species, including both vertebrates and invertebrates, plants or fungi other than S. cerevisiae .
• Preferred receptors are those from mammals, especially humans.
• Mammalian receptors of this class that are encompassed by the present invention include, but are not limited to the following, whose nucleotide sequences are disclosed in the listed references:
• Adenosine receptor Al Libert F. et al, Biochem. Biophys. Res. Commun. 187:919 (1992) .
• Adenosine receptor A2B Pierce K. D. et al, Biochem. Biophvs. Res. Commun. 187:86 (1992) .
• Adrenergic receptor alpha-lA Bruno, J. F. et al, Biochem Biophvs. Res. Comm. 179: 1485 (1991) .
• Adrenergic receptor alpha-IB Ramarao, C. S. et al, J. Biol. Chem. 267:21936 (1992) .
• Adrenergic receptor alpha-2A Kobilka B. K. et al, Science 238:650 (1987) .
• Adrenergic receptor alpha-2B Weinshank et al, Mol. Pharmacol. 38:681 (1990) .
• Adrenergic receptor alpha-2C Regan, J. W. et al, Proc. Natl. Acad. Sci. USA 85:6301 (1988) .
• Adrenergic receptor beta-1 Frielle, T. et al Proc. Natl. Acad. Sci. USA 84:7920 (1987)
• Adrenergic receptor beta-2 Kobilka B. K. et al, Proc. Natl. Acad. Sci. USA 84:46 (1987) .
• Adrenergic receptor beta-3 Emorine, L. J. et al, Science 245:1118 (1989) .
• Amyloid protein precursor Kang, J. et al, Nature 325:733 (1987) .
• Angiotensin II receptor type 1 Furuta H. et al, Biochem. Biophys. Res. Commun. 183:8 (1992) .
• Antidiuretic hormone receptor Birnbaumer, M. et al, Nature 357:333 (1992) .
• Bradykinin receptor Hess J. -F. et al, Biochem. Biophvs. Res. Commun. 184:260 (1992) .
• Cannabinoid receptor Gerard C. M. et al, Biochem. J. 279:129 (1991) .
• Chemokine C-C (mip-1/RANTES) receptor Neote K. et al, Cell 72:415 (1993) .
• Dopamine receptor Dl Dearry A. et al, Nature 347: 7276 (1990) .
• Dopamine receptor D2 Grandy D. K. et al, Proc. Natl. Acad. Sci. USA 86:9762 (1989) .
• Dopamine receptor D4 van Tol, H. H. et al, Nature 350:610 (1991) .
• Endothelin receptor A Hayzer D. J. et al, Am. J. Med. Sci. 304:231 (1992) .
• Endothelin receptor B Nakamuta M. et al, Biochem. Biophvs. Res. Commun. 177:34 (1991) .
• f-met-leu-phe receptor Boulay F. et al, Biochemistry 29:11123 (1990) .
• Gonadotropin-releasing factor receptor Chi L. et al, Mol. Cell. Endocrinol. 91.R1 (1993) .
• GHRH Growth hormone releasing hormone
• Histamine H2 receptor Gantz, I. et al, Biochem. Biophvs. Res. Commun. 178:1386 (1991) .
• Hydroxytryptamine (serotonin) receptor IA Kobilka B. K. et al, Nature 329:75 (1987) .
• Hydroxytryptamine (serotonin) receptor IB Jin H. et al, J. Biol. Chem. 267: 5735 (1992) .
• Hydroxytryptamine (serotonin) receptor IC 34. Hydroxytryptamine (serotonin) receptor IC:
• Hydroxytryptamine (serotonin) receptor IE McAllister G. et al, Proc. Natl. Acad. Sci. USA 8_9:5517 (1992) .
• IGF II Insulin-like growth factor
• Interleukin 8 receptor B Murphy P. M. and Tiffany H. L. Science 253 : 1280 (1991) .
• Lutenizing hormone/chorionic (LH/CG) gonadotropin receptor Minegish T. et al, Biochem. Biophvs. Res.
• MSH Melanocyte stimulating hormone
• Muscarinic acetylcholine receptors m5 Bonner T. I. et al, Neuron 1:403 (1988) .
• Neuropeptide Y receptor Herzog, H. et al, Proc. Natl. Acad. Sci. USA 89:5794 (1992) .
• Oxytocin receptor Kimura T. et al, Nature 356:526 (1992) .
• Platelet activating factor receptor Kunz D. et al, J. Biol. Chem. 267:9101 (1992) .
• Rhodopsin and related pigments Nathans J. and Hogness D. S. Proc. Natl. Acad. Sci. USA 81:4851 (1984) ; Nathans J. and Hogness, D. S. Science 232:193 (1986) .
• Somatostatin receptor 3 Yamada Y. et al, Mol. Endocrinol. 6:236 (1992) .
• Somatostatin receptors 1 and 2 Yamada Y. et al, Proc. Natl. Acad. Sci. USA 89:251 (1992) .
• Substance K neurokinin A receptor: Gerard N. P. et al, J. Biol. Chem. 265: 20455 (1990) .
• Substance P (NK1) receptor Gerard N. P. et al, Nature 349: 614 (1991) .
• Thrombin receptor Vu T. K. et al, Cell 64:1057 (1991) .
• Thromboxane A2 receptor Hirata M. et al, Nature 349:617 (1991) .
• Thyroid stimulating hormone (TSH) receptor Nagayama Y. et al, Biochem. Biophvs. Res. Commun. 165:1184 (1989) .
• Vasoactive intestinal peptide receptor 59. Vasoactive intestinal peptide receptor:
• Human acetylcholine muscarinic receptor 2098bp M35128 Human activin type I receptor, 1518bp U14722 Human activin type II receptor, 2382bp M93415 Human adenosine receptor (Al) 2900bp L22214 Human adenosine receptor (Al) brain hippocampus, 1267bp S45235 Human adenosine receptor (A2) 2383bp M97370
• A2 Human adenosine receptor
• Human B-cell antigen receptor 681bp M74721 Human bombesin receptor subtype-3, 1413bp L08893 Human bradykinin Bl receptor 1082bp U12512 Human bradykinin Bl receptor, 4168bp U22346
• CNTFR Human ciliary neurotrophic factor receptor
• D3 receptor Human dopamine D3 receptor (DRD3) gene, 1727bp U25441
• EBI2 EBV induced G-protein coupled receptor
• EPCR Human endothelial cell protein C/APC receptor
• GPR12 Human G protein coupled-receptor
• AJ Human G protein-coupled receptor
• Human G protein-coupled receptor (EBI1) 2139bp L31581 Human G protein-coupled receptor (EBI1) , 2215bp L31584 Human G protein-coupled receptor (GPR1) 1438bp L35539 Human G protein-coupled receptor (GPR1) 1438bp U13666 Human G protein-coupled receptor (GPR19) 2932bp U21051 Human G protein-coupled receptor (GPR3) 1262bp L32831 Human G protein-coupled receptor (GPR3) 3542bp U18550 Human G protein-coupled receptor (GPR4) 1365bp L36148 Human G protein-coupled receptor (GPR5) 1265bp L36149 Human G protein-coupled receptor (GPR6) 1477bp L36150 Human G protein-coupled receptor (GPR6) 2699bp U18549 Human G protein-coupled receptor (V28) 3100bp U20350 Human G-binding regulatory protein-coupled receptor, M28269 Human galanin receptor, 1050bp U23854 Human G
• HBGR2 Human glutamate receptor 2
• Human interleukin 8 receptor B 1750bp M94582 Human interleukin 8 receptor beta (IL8RB) 2856bp
• IL8RBA Human interleukin 8 receptor type A
• GLVR2 Human leukemia virus receptor 2
• Human lymph node homing receptor 2354bp M25280 Human macrophage inflammatory protein-1-alpha/RANTES receptor, L10918 Human major group rhinovirus receptor (HRV) 3003bp M24283
• NKIR Human neurokinin 1 receptor
• NMB-R Human neurokinin receptor
• NPYR Human neuropeptide Y receptor
• TR3 Human orphan receptor
• PAFR Human platelet activating factor receptor
• PTAFR Human platelet activating factor receptor
• Human prostaglandin receptor (EP3A1) 1652bp U13216 Human prostaglandin receptor (EP3D) 154Obp U13217 Human prostaglandin receptor (EP3E) 1429bp U13215 Human prostaglandin receptor (EP3F) 1456bp U13214 Human prostaglandin receptor (PGE-2) , 1515bp L26976 Human prostanoid receptor EP3-I, 1870bp L27490 Human prostanoid receptor EP3-II, 1682bp L27488 Human prostanoid receptor EP3-III, 1379bp L27489 Human prostanoid receptor FP, 2494bp L24470 Human prostanoid receptor IP, 1417bp L29016 Human RMLP-related receptor I (RMLP RI) 1062bp M76673 Human RPE-retinal G protein coupled receptor (rgr) 694bp U15790
• Human RPE-retinal G protein-coupled receptor 1415bp U14910 Human secretin receptor precursor, 1650bp U20178 Human secretin receptor, 1616bp U13989 Human serotonin IB receptor, (5-HT1B) 2635bp D10995 Human serotonin IC receptor, 2733bp M81778 Human serotonin ID receptor (5-HT1D) 120Obp M81589 Human serotonin ID receptor (5-HT1D) 1260bp M81590 Human serotonin ID receptor, 1348bp L09732 Human serotonin ID receptor, 1506bp M89955 Human serotonin lDb receptor (HTRlDb) 1959bp M75128 Human serotonin IE receptor 5HTR1E, 1221bp M92826 Human serotonin IE receptor, 1930bp M91467
• Human substance P receptor (short form) 1268bp M84426
• TSHR Human thyroid stimulatory hormone receptor
• V1RG Human vasoactive intestinal peptide receptor type 1 (V1RG) U11087 Human vasoactive intestinal peptide receptor, 2754bp
• VIPR2 Human vasoactive intestinal polypeptide receptor 2 L40764
• V2 Human vasopressin receptor 2282bp L22206 Human vasopressin receptor V3, 1869bp L37112
• the protein analogous in function to the Gpalp of Saccharomyces cerevisiae can be, of course, the Gpalp of S. cerevisiae.
• the GPA2 gene of S. cerevisiae (Nakafuku et al. , Proc. Natl . Acad. Sci USA £5.-1374 (1988) .
• the Gpa2p protein is not able to complement defective Gpalp function, but nevertheless the Gpa2p protein might interact with G- beta-gamma complexes to couple a seven-transmembrane receptor to a biochemically selectable pathway. It is expected that other species of yeasts, for example Schizosaccharomyces pombe, will also have proteins that can be used for the Gpalp protein in practicing the present invention.
• the seven- transmembrane protein is operatively linked to the protein having an activity analogous to the Gpalp of Saccharomyces cerevisiae .
• the two proteins can be directly fused; the carboxy-terminus of the seven- transmembrane protein being joined to the amino- terminus of the protein having Gpalp activity.
• a short linker peptide can be used to join the two proteins.
• the linker is preferably from 1-25 amino acids long, more preferably from 1-20 amino acids long, still more preferably from 1-10 amino acids long and most preferably from 3-10 amino acids long.
• a "reporter" gene is operatively linked to the promoter of a gene analogous in function to the FUS1 gene of S . cerevisiae.
• a reporter gene is one which signals the function of the expression cassette, typically of the promoter function, into which the reporter gene is inserted.
• the amount of the gene product of the reporter gene can be measured by immunoassay, by enzyme activity (if the reporter gene encodes an enzyme) or by a metabolic selection strategy.
• Preferred reporter genes encode a protein that is not made by the yeast strain into which they are inserted, to avoid a high background result.
• Preferred reporter genes in implementing the present invention encode enzymes whose activity can be measured colorimetrically or by a luminescence assay and include 0-galactosidase, glucuronidase (GUS) , green fluorescence protein, and luciferase. If a yeast strain in which the endogenous genes for them have been knocked out is used, genes encoding alkaline phosphatase and invertase (SUC2) are also useful reporter genes.
• a yeast cell expressing a fusion protein comprising the seven- transmembrane protein of interest and a Gpalp that functionally couples to the mating-type pathway with the compound to be tested and with a ligand for said receptor. Then, the level of expression of a reporter gene, which measures the activity of a promoter that depends upon the activation of the mating-type pathway, for example, the FUS1 promoter, is measured. The level of reporter gene expression is compared in the presence and absence of the compound to be tested for antagonist activity.
• Antagonist activity is considered to be observed if the level of reporter gene expression, and thus activity of the mating-type activation-dependent promoter, is lower in the cell contacted with the compound being tested together with the ligand than in the cell contacted with the ligand, but not contacted with the compound being tested.
• “lower” is meant a degree of difference between the reporter gene expression in the cells treated with the test compound together with ligand of at least 1/3. The larger the degree of difference, the greater the antagonist activity.
• a range of differences between 1/3 and 1/10 is expected. Preferably the range is 1/5 to 1/25. More preferably, the range is 1/5 to 1/50. Most preferably, the range is 1/50 to 1/200.
• a method for testing a compound for receptor agonist activity is similar to the test for receptor antagonist activity.
• the level of expression of a reporter gene which measures the activity of a promoter that depends upon the activation of the mating-type pathway, for example, the FUS1 promoter, is measured.
• the level of reporter gene expression is compared in the presence and absence of the compound to be tested for agonist activity.
• Agonist activity is considered to be observed if the level of reporter gene expression, and thus activity of the mating-type activation-dependent promoter, is higher in the cell contacted with the compound being tested than in the cell not contacted with the compound being tested.
• “higher” is meant a degree of difference between the reporter gene expression in the cells treated with the test compound together with ligand of at least 3-fold. Greater degrees of difference are preferred.
• An expected range is from 3 to 10-fold higher.
• An acceptable range is 3 to 8-fold higher.
• the degree of difference is 10 to 25-fold. More preferably, the degree of difference is 20 to 100-fold.
• a plasmid construct is made that expresses the Receptor-Gpalp fusion protein, and this plasmid is transformed into diploid yeast cells having one mutant and one wild type copy of the essential yeast G-alpha protein gene GPA1 .
• Sporulation of the diploid should give two viable and two non-viable spores because GPA1 is essential for haploid growth, unless the fusion protein contains a functional Gpalp domain. If so, more than two viable segregants will be obtained, providing a simple genetic complementation assay for appropriate expression and activity of the Gpal domain of the fusion protein.
• assays based on mating pathway activation are performed, using known activators of the receptor domain of the fusion protein, to test whether the receptor domain of the fusion protein is functional and capable of transmitting the ligand-binding signal to the fused Gpal domain. If so, the fusion molecule is fully functional in both of its domains. If not, the same assays can be adapted, in conjunction with mutagenesis of the Gpal domain, to select for mutants in which the intact receptor domains can signal to the mutant Gpal domain to activate the mating pathway upon activator binding. A detailed description of the procedure is given below.
• Yeasts can be tranformed with vectors encoding the recombinant DNA molecules of the present invention by means well-known in the art.
• membranes from yeasts expressing the recombinant DNA molecules of the present invention can be prepared and stored by methods well-known in the art.
• Step 1 engineering a covalent linkage between the full length receptor (excluding the cleaved signal peptide, for reasons given in step 2) and Gpalp at their respective carboxy and amino terminal ends.
• the fusion construct includes in addition the endogenous 3' processing signals of the GPA1 gene for proper termination of transcription and polyadenylation.
• the construct can be made in a vector that can either replicate autonomously in yeast cells, or that integrates into the yeast chromosome.
• the vector additionally includes a transformation marker gene so that the final construct can be transformed into yeast cells and transformant selected by using the marker.
• Step 2 engineering the fusion protein for yeast plasma membrane expression. This is achieved by replacing part of the signal sequence of the receptor in question with part of the N-terminal signal sequence of the yeast G-protein-coupled receptor
• N-terminal signal sequence that directs co-translational insertion across the rough endoplasmic reticulum membrane may also be used; examples include N-terminal signal sequences of the a- factor receptor STE3 or secreted proteins such as invertase, and alpha mating factor precursors ⁇ fFal and
• Attachment of the signal seguence is done by an in-frame fusion of a DNA fragment encoding the signal peptide, preferably from Ste2, with the DNA fragment encoding the construct from step 1 by standard methods of molecular biology, as illustrated in example 2.
• Step 3 placing the construct under the control of a yeast promoter. This is achieved by cloning in an appropriate promoter fragment contiguous to the 5' of the construct .
• Preferred promoters are those which can replicate autonomously in yeast.
• Example 1 demonstrates how this can be done using inducible and moderately strong GAL promoter.
• Example 3 describes constructions using the strong and constitutive PGK promoter. Codon usage in yeast is biased such that genes expressed at high levels use only one or two of the several possible degenerate codons to encode amino acids. (Jones, Pringle and Broach, The Molecular and Cellular Biology of Yeast Saccharomyces, Vol. 2, c.
• a strong promoter such as the PGK promoter may therefore be required to generate sufficient RNA levels to overcome the lack of codons preferred by yeast in receptor genes from other species.
• the receptor-encoding DNA can be engineered to utilize preferred yeast codons.
• Step 4 mutating the FARl gene.
• Farlp is required for growth arrest induced by activation of the mating pathway.
• the farl mutation is needed to enable haploid cells with an activated mating pathway to grow while retaining other features of mating pathway activation.
• the FARl gene can be mutated by replacement with another auxotrophic marker gene (Scherer and Davis, Proc. Natl . Acad. Sci . USA 76.:4951) , or by the two- step mutation strategy (Rothstein, Methods in Enzymology, 101:202) . The latter method is described in Example 11.
• Step 5 constructing diploid yeast cells with one wild type and one mutant copy of GPA1. Because GPA1 is an essential gene for haploid cell growth and cannot be mutated in haploid cells directly, the mutation has to be made in a diploid strain preferably a mutant strain having several auxotrophic marker genes on both copies of its chromosomes. Diploid cells of this genotype are constructed by disruption of one of the two GPA1 copies by integration of an auxotrophic marker gene, as in example 5 where the TRPl gene is used. In subsequent segregation, the mutant copy can be followed by the TRPl marker.
• GPA1 is essential, sporulation of each tetrad should give two large colonies, and two small or undetectable colonies, and both of the large colonies should require tryptophan for growth, i.e. lack the TRPl gene. This is illustrated in example 5.
• Step 6 transforming the construct of Step 3 into the diploid strain of Step 5.
• the construct of Step 3 is cloned into a yeast vector that can replicate as a plasmid, and carries a gene that complements one of the auxotrophic mutations present in the diploid strain used to create grpal and farl mutations in Steps 4 and 5.
• Replicating vectors based on either a yeast centromere sequence, exemplified by the YCp series of vectors, or the 2-micron plasmid origin, exemplified by the YEp series of vectors can be used. (Rose and Broach, Methods in Enzymol . 194 :195.
• the plasmid is then cloned into the diploid strain of Step 5, and transformants carrying the plasmid are selected on the basis of a marker present in the plasmid, preferably an auxotrophic marker, which is URA3 in the case of YEp and YCp vectors.
• a marker present in the plasmid preferably an auxotrophic marker, which is URA3 in the case of YEp and YCp vectors.
• Step 7 genetic complementation method for testing function of Gpal domain of the receptor-Gpal fusion.
• Sporulation of the diploid strain of step 6 carrying the fusion construct provides a convenient way to test if the Gpalp domain in the fusion construct can functionally replace the Gpalp gene product.
• Segregation of GPA1 and FARl in the diploid strain from Step 5, of genotype GPAl/gpal ; FARl/ farl should yield the following four haploid genotypes: (i) GPA1 ;FAR1 (ii) GPAl ;farl (iii) gpal ; FARl and (iv) gpal/ farl .
• Haploids with genotypes i and ii should give viable colonies, those with genotype iii should not give a detectable colony and those with genotype iv should give very small colonies because of incompleteness of growth arrest due to farl . If random spores from this population are analyzed, each of these genotypes should occur at equal frequency. However, because of independent assortment in each tetrad, the two spores that carry gpal from a single meiotic event may be both FARl, both farl , or one of each.
• dissection of any tetrad should always yield two large colonies, and two others which may be both very small (genotype gpal ; farl) , both invisible (genotype gpal , FARl) or one of each.
• segregation is illustrated in examples 5, 6, 7 and 8, where tryptophan prototrophy is used to follow segregation of gpal : : TRPl , and a PCR assay is used to follow segregation of FARl.
• the initial diploid cell carried a plasmid, it should be present in all four spores of the tetrad with equal probability. This probability is always less than one since plasmids can be lost at some frequency in the mitotic divisions preceding meiosis where selection for the marker carried on the plasmid is relaxed, and also in the two divisions of meiosis. If this plasmid carried a gene capable of fully complementing the gpal mutation, then dissection of each tetrad would yield two large colonies as before due to the presence of GPA1, and of the two remaining spores of genotype gpal, some would yield large colonies due to complementation.
• example 6a for the GPA1 gene expressed from its own promoter
• example 6b for the GPA1 gene expressed from the PGK1 promoter
• example 7 for a STE2-GPA1 in-frame fusion protein expressed from the PGJF1 promoter
• example 8 for an in-frame fusion protein between the thrombin receptor and GPA1 expressed from the PGK1 promoter.
• Step 8 confirmation of the functionality of the Gpal domain of fusion proteins.
• Step 7 describes how simple segregation analysis of genetic complementation can provide a good indication of the function of the Gpal domain.
• other genetic phenomena can also give rise to deviations from 2:2 segregation.
• gene conversion of the disrupted gpal by the wild type copy either in meiosis or in the mitotic divisions preceding mitosis could give rise to 3:1 or 4:0 segregation respectively.
• gene conversion could also occur between the complete coding sequence of GPAl present on the plasmid construct and the disrupted chromosomal copy.
• diploid cells that sporulate might not carry the plasmid since it is lost at some frequency in mitosis unless selection for the plasmid is maintained. Diploid cells can undergo several mitotic divisions without selection prior to meiosis in the sporulation medium, which may lead to loss of the plasmid and thus give rise to 2:2 segregation. In the analysis of Step 7, this would be incorrectly interpreted as an inability of the plasmid to complement gpal .
• the four colonies from each tetrad are tested for growth on media that detects the presence of the marker that disrupts the GPAl gene ( TRPl in example 5) , and the plasmid marker ( URA3 in examples 6, 7 and 8) .
• TRPl the marker that disrupts the GPAl gene
• URA3 the plasmid marker
• Step 9 tests for function of the receptor domain.
• Binding assays provide a sensitive assay for proper expression of the receptor fusion protein, its targeting to the yeast plasma membrane and appropriate folding and generation of transmembrane domains to generate the extracellular binding site.
• Scatchard analysis of binding data can provide measurements of binding affinity, which can be compared to the affinity in mammalian cells expressing wild-type receptor to obtain a further measure of appropriate expression. Scatchard analysis also provide measurements of the number of binding sites for ligand per cell, which is a good measure of expression levels. In the examples cited here, however, we have used the more stringent alternate approach described in step 10, which not only requires binding to the receptor domain of the fusion protein, but also requires transmission of the binding signal through the linked Gpal domain to the mating factor pathway.
• Step 10 mating and shmoo formation assay for coupling of receptor domain activation to mating pathway activation.
• Activation of the mating pathway in haploid cells leads to a distinct morphological change from the typical ovoid cells of vegetatively growing yeast to a pear-shaped "shmoo" which enables mating with cells of the opposite mating type if they are present.
• shmoo formation assay
• mating assay to detect functional coupling between the covalently linked domains.
• the mating assay can only be used with the endogenous yeast receptors Ste2p and Ste3p, because this requires a response to mating pheromones secreted by another yeast cell of the opposite mating type, but the shmoo formation assay can be adapted to other receptors from heterologous organisms.
• Step 11 beta-galactosida ⁇ e induction assay for coupling of receptor domain activation to mating pathway activation.
• This method uses a mating pathway-inducible promoter operatively linked to the bacterial beta-galactosidase gene ( lacZ) as a reporter. For example, transcription from the FUS1 promoter is stimulated by activation of the mating pathway, and therefore, in cells carrying FUSl-lacZ constructs, induction of beta-galactosidase becomes a sensitive indicator for receptor activation.
• lacZ bacterial beta-galactosidase gene
• Examples 9b, lOd, 12, 13 and 14 describe this assay for wild type cells to characterize the method (9b) , Ste2p- Gpalp protein fusions (lOd, 12) and thrombin receptor- Gpalp fusions (13, 14) . Because expression of beta- galactosidase is easily quantitiated by spectrophotometry, a quantitative measure of coupling is obtained by means of this assay.
• the FUSl-beta-galactosidase construct can be transformed into the haploid strain from Step 8 and maintained on a replicating plasmid of the YEP type. This gives higher basal values of /3-galactoridase due to the 50-100 copies of the plasmid present in each cell, as shown in example 9b. Alternately, the basal expression level can be reduced by integration of the construct into the chromosome, as shown in examples 9b, lOd, 12, 13, and 14 for integration into the FARl locus and the LEU2 locus.
• Step 12 growth assay for coupling of receptor domain activation to mating pathway activation.
• a mating pathway-inducible promoter such as FUS1 is operatively linked to a an auxotrophic marker gene that is mutated in the cells to be tested.
• activation of the mating pathway leads to expression of the auxotrophic marker gene, conferring the ability to grow in appropriate media that lacks the final end product of the marker enzyme.
• LYS2 gene in this manner in example 10c.
• the particular advantage of LYS2 (and also URA3) is that expression of this gene can be selected for in lysine deficient media as well as selected against in media containing the reagent alpha-aminoadipate.
• Step 13 mutagenesis of the Gpal domain to increase coupling efficiency.
• the G-alpha domain of the fusion can be mutagenized by the standard methods, including those described below, and mutants which are created thereby that confer increased coupling efficiency can be selected using the methods described in steps 10 and 11.
• Mutagenesis can preferably be effected using one or a combination of the following methods: a) random mutagenesis by PCR amplification (Cadwell and Joyce, PCR and Its Applications, c. 1994 by Cold Spring Harbor Laboratory Press, esp. pp.
• Plasmid DNA from a bulk plate growth of the entire transformation mix will be used to transform yeast and select for mutants with functional coupling as described by the selection procedure of step 11 or the screening procedure of step 10.
• the primer extension mixture will be transformed into E. coli such that sufficient individual transformants are recovered to ensure adequate representation of the pool of mutants.
• Regions to be mutagenized would include the carboxy terminal, which has been implicated in binding to the receptor, and other regions of weak homology.
• Comparison of the amino acid sequence of GPAl to human Gs-alpha shows that several large regions of the GPAl sequence are non-homologous to the human protein, and would be good candidates for loop- out mutagenesis (e.g. amino acids 1-61, 75-110, 142- 188, 217-237 of the GPAl sequence.
• EXAMPLE 1 CONSTRUCTION OF A FUSION BETWEEN THE YEAST ALPHA FACTOR RECEPTOR Ste2p AND G-ALPHA PROTEIN Gpalp UNDER TRANSCRIPTIONAL CONTROL OF THE GALI PROMOTOR a) Ligating the GALI promoter into the yeast vector
• YCp50 the yeast vector YCp50 was digested with BamHl and EcoRI, and the resulting 7572 bp fragment was purified from an agarose gel using GeneCleanTM (Bio 101) .
• GeneCleanTM GeneCleanTM (Bio 101) .
• Gall promoter (position #1-810 of GenBank accession number K02115, where a BamHl site was added to the 3' end) was ligated into this YCp50 fragment and the resulting plasmid is designated pRMHBTl.
• oligos anneal to form a polylinker with BamHl, NotI, Mlul and PflMI sites, in that order.
• the annealed oligo fragment was then cloned into the 7574 pRMHBTl BamHl/PflMI fragment to make pRMHBT2.
• GPAl was amplified by PCR from a Saccharomyces cerevisiae genomic DNA prep using the following two primers: oligo c) 5 GACACGCGTGTAATGGGGTGTACAGTGAGTACGC 3' oligo d) 5 CGTCCAAGGGATGGACCTTTTTTTTCTCATGCG 3'
• Oligo "c” contains bp 200 to 223 of the GPAl seguence (GenBank accession number M15867) and 10 additional nucleotides containing a Mlul restriction site. Oligo “d” contains bases complementary to residues 1829 to 1850 of the GPAl sequence and additional nucleotides creating a PflMI site homologous to the PflMI site at position 1610 in YCp50. PCR amplification of yeast genomic DNA with these oligos yields a GPAl fragment that contains nucleotides 200-1850 of the GPAl sequence.
• the Mlul site is immediately upstream of the ATG start codon, and the PflMI site is 232 bp downstream of the TGA stop codon.
• the amplified GPAl fragment was digested with Mlul and PflMI and ligated to the 7583 bp MluI/PflMI fragment of pRMHBT2 to make pRMHBT3.
• STE2 was amplified by PCR from Saccharomyces cerevisiae genomic DNA using the following 2 primers: oligo e) 5 CGGGATCCAAGAATCAAAAATGTCTGATG 3' oligo f) 5 GAACGCGTTAAATTATTATTATCTTCAGTCC 3' Oligo "e" contains nucleotides 520 to 544 of the STE2 sequence (GenBank accession number M24335) and 4 additional nucleotides which create a BamHl restriction site.
• Oligo "f” contains bases complementary to nucleotides 1804 to 1827 of the STE2 sequence and eight additional nucleotides which include a Mlul site. PCR amplification yields a STE2 fragment containing nucelotides 520-1827 of the STE2 seguence, and includes the entire coding seguence from the ATG start codon (pos. 535, underlined in the oligonucleotide sequence "e” above) to the last base of the Ste2p C-terminal leucine codon (pos. 1827, underlined in the oligonucleotide sequence "f” above) .
• the STE2 PCR product was cut with BamHl and Mlul and ligated to the 9224 bp BamHl/MluI fragment of pRMHBT3 to make pRMHBT4.
• the Mlul junction forms an in-frame fusion between STE2 and GPAl ; the resulting chimera codes for all of Ste2p, a tripeptide thr-arg-val orginating from the oligonucleotides used, and all of Gpalp.
• the STE2-GPA1 fusion construct in pRMHBT4 is transcriptionally regulated by the GALI promoter.
• EXAMPLE 2 CONSTRUCTION OF A FUSION BETWEEN THE HUMAN THROMBIN RECEPTOR AND THE YEAST G-ALPHA PROTEIN Gpalp a) PCR-amplifying thrombin receptor cDNA: a portion of the thrombin gene was PCR amplified from a human lung fibroblast lambda GT10 cDNA library using the following two oligonucleotides: oligo g) CGGGATCCATAAGCGGCCGCACCCGGGCCCGCAGGCC oligo h) GAACGCGTAGTTAACAGCTTTTTGTATATGC Oligo "g” contains nucelotides 290 to 312 of the thrombin receptor (ThrR) cDNA sequence (GenBank accession # M62424) and sixteen additional bases coding for a BamHl and a NotI restriction site.
• ThrR thrombin receptor
• Oligo "h” contains bases complementary to nucleotides 1477 to 1499 of the ThrR sequence and eight additional nucleotides which include a Mlul site. Regions of homology to the ThrR cDNA are in bold type.
• the PCR product contains bp 291 to 1499 of the human ThrR cDNA sequence, coding for amino acids 22 (arginine) to the COOH-terminal threonine.
• EXAMPLE 3 TRANSFER OF FUSION CONSTRUCTS OF EXAMPLES 1 AND 2 TO HIGH-COPY VECTORS CONTAINING THE CONSTITUTIVELY ACTIVE PGK PROMOTOR
• the fusion constructs in Examples 1 and 2 were placed under the transcriptional control of the PGK1 promoter carried on a yeast 2-micron-plasmid-based vector.
• a BamHl/Ncol fragment of pRMHBT4 containing the fusion construct and part of the URA3 marker was ligated into the BamHI/NcoI digested pPGK (Kang et al, 1990, Mol. Cell. Biol., 10:2582) .
• the PGK1 promoter carried on a yeast 2-micron-plasmid-based vector.
• ThrR/Gpal fusion and part of the URA3 marker was ligated into the BamHI/NcoI digested pPGK.
• the resulting plasmids were designated pRMHBTl ⁇ NG and pRMHBT20NG, respectively.
• EXAMPLE 4 DISRUPTION OF THE CHROMOSOMAL FARl GENE
• the FARl gene was amplified from yeast genomic DNA using the following primers : oligo i) CAACATGCAGCCATTTCACCG oligo j) CGCGAGCTCGCCAATAGGTTCTT CTTAGG
• Oligo "i” contains the sequence from residues 34 to 54 of FARl (GenBank accession # M60071)
• Oligo "j” contains the seguence complementary to nucleotides 2959 to 2980 of the FARl gene and eight additional nucleotides which create a SacI restriction site. The amplified seguence extended from nucleotides 34 to 2980.
• the FARl PCR product was digested with Kpnl and SacI, and ligated into those same sites in the yeast integrating vector pRS306 (Sikorski and Hieter, 1989, Genetics 122:19-27) . The resulting plasmid was designated pFARl.
• the farl mutation was constructed by deleting an internal 700 bp Xbal fragment from pFARl, which removed bp 1917 to 2616, and results in a protein that is missing 153 of its 781 amino acids.
• the resulting plasmid was designated pFARX.
• the pFARX plasmid was used to introduce the farl mutation into the chromosome of the haploid yeast strain MS2288 (mat a, ura3-52, leu2-3,112, his3D200, trplDl; M. Rose, Princeton University) .
• pFARX was linearized at its single EcoRI site (position 2771 of FARl) and used to transform competent MS2288 cells to uracil prototrophy, thereby integrating the pFARX plasmid at the FARl locus.
• Strains in which the plasmid had recombined back out of the chromosome were identified using 5-FOA selection, and ura ' derivatives were screened for retention of the farl mutation by PCR analysis using oligos "i" and "j”.
• EXAMPLE 5 DISRUPTION OF THE CHROMOSOMAL GPAl GENE a) Construction of a FARl/farl diploid strain: Strain HBS10 (mat a, ura3-52, leu2-3,112, his3 ⁇ 200, trpl ⁇ l, lys2 ⁇ S738, farl-X200) was mated to MS16 (mat a, trpl ⁇ l, ade2-101) and the resulting strain was sporulated. Segregants from this cross included TMHY2-14A (mat a, ura3-52, his3 ⁇ 200, trpl ⁇ l, lys2 ⁇ S738, ade2-101) .
• TMHY2-14A was then mated to HBSIO and diploids were selected.
• the resulting diploid strain was designated TMHY2-223D (a/a, ura3/ura3 , Ieu2/LEU2, his3/his3, trpl/trpl , Iys2/lys2, farl /FARl , ADE2/ade2) .
• the TRPl gene was amplified from the vector pRS304 (Sikorski and Hieter, 1989, Genetics 122:19-27) by PCR using the following oligos, both with SphI sites (underlined) , to yield a 1134 bp fragment containing a functional TRPl gene: oligo k) GAATGCATGCGGCATCAGAGCAG oligo 1) GAATGCATGCGGTATTTTCTCCTTACGC This PCR product was digested with SphI and ligated into the 8386 bp SphI fragment of pRMHBT3.
• the two SphI sites are present within the coding seguence of the GPAl gene in this plasmid. Replacement of this fragment with the TRPl gene yielded the plasmid pRMHBTIO in which the TRPl gene is flanked by GPAl sequences.
• pRMHBTl0 Disrupting the chromosomal GPAl locus: pRMHBTl0 was digested with Mlul and PflMI to liberate a 1887 bp fragment containing the TRPl gene flanked by GPAl sequences as described in 5b. This fragment used to transform the diploid strain TMHY2-223D to tryptophan prototrophy. The deletion was confirmed by PCR analysis of several transformants using the GPA1- specific oligos "c" and "d” of Example 1.
• TMHY3D strains were given the designation TMHY3D (genotype a/a, ura3/ura3, his3/his3 , Iys2/lys2, trpl/trpl , ADE2/ade2, FARl/farl , LEU2/leu2, GPAl /gpal : : TRPl) .
• TRP + transformants Five different TRP + transformants (TMHY3D-1, TMHY3D-2, TMHY3D-3, TMHY3D-5, and TMHY3D-6) were sporulated and tetrads dissected. Representative data for one of the transformants is given below. Four of seven tetrads produced two normal colonies, one small colony, and one non-viable spore (2:1:1) . Two tetrads produced two normal colonies and two non-viable spores (2:0:2) . One tetrad produced two normal colonies and two small ones (2:2:0) . Similar data was obtained for the other four sporulations. Each tetrad, on non-selective plates, is expected to give only two normally growing colonies (both
• EXAMPLE 6 COMPLEMENTATION OF THE gpal MUTATION BY CLONED GPAl a) Complementation of opal with a full length GPAl gene: a 1924 bp EcoRI fragment including the entire GPAl gene (Dietzel and Kurjan, 1987, Cell 50:1001- 1010) was amplified from yeast genomic DNA using the following oligos: oligo m) GGAATTCCACCAATTTCTTTACG oligo n) GGAATTCGAGATAATACCCTGTCC The resulting PCR product was ligated into the EcoRI site of the vector pRS316 (Sikorski and Heiter) and the 2-micron vector YEp352 (Hill et . al .
• plasmids were designated p316GPAl and p352GPAl, respectively.
• Strain TMHY3D-1 was transformed with both plasmids to uracil prototrophy, and the strains were sporulated. Complementation of the gpal mutation by a plasmid carrying GPAl should result in 4:0 segregation for viable vs. small or non-viable colonies (assuming the plasmid segregates to all four spores) . However, the theoretical 4:0 segregation expected for full complementation would not be always realized since plasmids are lost at some frequency in both the mitotic divisions in the sporulation medium, and in meiosis.
• EXAMPLE 7 COMPLEMENTATION OF THE gpal MUTATION BY EXPRESSION OF A STE2-GPA1 CHIMERIC PROTEIN
• the plasmid pRMHBT18NG encoding a Ste2p-Gpalp fusion protein was used to transform HBS14 to uracil prototrophy.
• the resulting strain was sporulated and 20 tetrads were dissected.
• EXAMPLE 8 COMPLEMENTATION OF THE gpal MUTATION BY EXPRESSION OF A THROMBIN RECEPTOR-GPAl FUSION PROTEIN
• the plasmid pRMHBT20NG was used to transform strain
• Ste2p chimera complements the gpal mutant phenotype.
• EXAMPLE 9 REPORTER ASSAYS FOR ACTIVATION OF THE MATING PATHWAY a) Construction of a lacZ gene transcriptionally regulated by the mating pathway-specific FUS1 promoter: The FUS1 promoter was amplified from yeast genomic DNA by PCR using oligos "o" and "p” shown below : oligo o) GCATGCTGCAGGATCGCCCTTTTTGACG oligo p) GACGTCGACAGAAACTTGATGGCTTATATCCTGC Oligo "o” contains the sequence of nucleotides 1 to 23 of FUS1 (GenBank accession # M17199) and five additional nucleotides creating a SphI restriction site.
• Oligo "p” contains the seguence complementary to residues 232 to 258 of the FUS1 gene and eight additional nucleotides which create a Sail restriction site.
• the amplified seguence encompasses nucleotides 1 to 258, and includes a Pstl site at residue 1 in FUS1 .
• the FUS1 promotor was digested with Sail and Pstl, and ligated into those same sites in the vector pUC19 (Yanisch-Perron et. al. , 1985, Gene 33 :103-119) .
• the resulting plasmid was designated pUFS.
• the LacZ coding sequence was cut from pON831
• pFus-Lac contained the lacZ coding sequence under transcriptional control of the FUS1 promoter.
• the Fusl-lacZ gene was then moved into three different yeast vectors: 1) pFus-Lac was digested with SphI and the resulting FUS-lacZ segment was cloned into the SphI site within the coding seguence of the FARl gene in the plasmid pFARl.
• the resulting plasmid (which is an integrating vector containing URA3 as its selectable marker) was designated pFFLz. 2) pFus-Lac was digested with Hindlll and Kpnl, and the resulting FUS-lacZ segment was cloned into the 2-micron vector YEp352. The resulting plasmid which uses C7RA3 as a selectable marker was designated pYFL3. 3) pFus-Lac was digested with Pstl and Kpnl, and the resulting FUS-lacZ segment was cloned into the integrating vector YIp351 (Hill et. al., 1986, Yeast 2:163-167) . The resulting plasmid which uses LEU2 as a selectable marker was designated pLZ351.
• the above plasmids were transformed into yeast strains, and the cells were analyzed for their ability to induce beta-galactosidase in response to alpha factor addition to the growth medium.
• Strain HBSIO transformed with pYFL3 exhibited an alpha factor- independent beta-galactosidase specific activity of 212 nmol/mgmin, and alpha factor-induced activity of 2023 nmol/mgmin, representing a 9.5 fold induction.
• pFFLz was digested with EcoRI which linearized the plasmid within the FARl coding seguence, and this DNA was used to transform strain HBSIO to uracil prototrophy. This integrated the plasmid at the chromosomal FARl locus.
• HBSIO: :pFFLz exhibited an alpha factor-independent beta-galactosidase activity of 23 nmol/mgmin and alpha factor-induced activity of 735 nmol/mgmin, representing a 32.0 fold induction.
• LYS2 was PCR-amplified (only coding sequence and 3' untranslated region) from yeast genomic DNA using the following oligonucleotides: oligo q) CGGCGGTCGACTAATGACTAACGAAAAGG oligo r) CCCGGGCGCAAGTATTCATTTTAGACCCATGGTGG Oligo "q” contains the sequence of nucelotides 299 to 312 of LYS2 (GenBank accession # M36287, M14967) and nine additional nucleotides creating a Sail restriction site. Oligo "r” contains the sequence complementary to nucleotides 4822 to 4850 of the LYS2 gene and six additional nucleotides which create a Smal restriction site.
• the 4566 bp LYS2 PCR product was digested with Sail and Smal, and ligated into the Sail-Nrul sites of pRMHBT25 to generate pRMHBT26, which contains the LYS2 coding seguence under transcriptional control of the FUS1 promoter.
• HBSIO cells were transformed to uracil prototrophy by pRMHBT26.
• Transformants were grown to mid-log in ura " media, and cells were back-diluted into ura'lys " media with or without 5.8mM alpha factor. Growth was measured by OO 600 , but the initial measurement was taken using a Coulter Counter, yielding a starting cell count of 5.18 X 10 5 /ml .
• the time point readings (ODgoo) of the cultures were as follows:
• HBS10 (without pRMHBT26) did not grow in lys- media. These results clearly demonstrate that strain HBS10/pRMHBT26 exhibits alpha factor-dependent lysine prototrophic growth, which confirms that expression of Lys2p is dependent upon mating pathway activation by alpha factor.
• EXAMPLE 10 ALPHA FACTOR-DEPENDENT ACTIVATION OF THE MATING PATHWAY BY THE STE2-GPA1 FUSION IN gpal CELLS
• Examples 7 and 8 show that the Gpalp domain of the Ste2-Gpal fusion construct functionally complements the chromosomal gpal mutation.
• LYS2 prototrophy assay To change the selectable marker from URA3 to IS3, the 6159 bp Apal-Clal fragment of pRMHBT26 containing the FUS1 promotor -LYS2 gene fusion was ligated into the Apal-Clal sites in pRS313 (Sikorski and Hieter) to generate pRMHBT41.
• 9A/pRMHBT18NG/pRMHBT41 Cells were grown to mid-log in ura ' his " media, at pH 6.5 and 4.0. 9A/pRMHBT18NG controls were grown similarly in ura " media. The cells were washed three times with sterile water before being diluted into the experimental (lys-) media.
• strain 9A/pRMHBT18NG/pRMHBT41 exhibits alpha factor-dependent lysine prototrophic growth at pH 4.0 and pH 6.5, which confirms that expression of Lys2p is dependent upon mating pathway activation (by alpha factor) .
• the very slow growth seen in "1” is most likely due to basal activity of the FUS1 promotor. That we did not see slow growth in "3” probably reflects the fact that the yeast pH optima for growth is less than 4, and at 6.5 they are sufficiently stressed as to be unable to support lysine prototrophy from the basal activity of the FUS1 promotor.
• Alpha factor-dependent lysine-prototrphic growth demonstrates that the Ste2p-Gpalp fusion protein activates the mating pathway in a gpal background.
• the mating pathway is not constitutively activated in the gpal strain 9A/pRMHBT18NG/pRMHBT41 since lysine prototrophy is alpha factor-dependent. This further supports the conclusion that the Gpalp domain of the Ste2-Gpalp fusion protein correctly associates with the Gb and Gg subunits.
• the mating pathway can be effectively activated at pH 6.5, which more closely resembles physiological conditions for mammalian receptors.
• the higher pH preferably pH 6 to 7.5 reduces background prototrophy due to basal activity of the FUS1 promotor ("1" vs. "3") .
• Lac Z reporter assay pLZ351 (see 9 above) was digested with BstEII to linearize the plasmid within the LEU2 sequence, and then this DNA was used to transform strain 9A/pRMHBT18NG to leucine prototrophy.
• Strain 9A/pRMHBT18NG is a haploid segregant of HBS14 carrying the plasmid pRMHBT18NG, whose genotype is mat a, ade2-101, ura3-52, leu2, 3-112, his3D200, trplDl, lys2-DS738, farl-X200, gpal::TRPl (the gpal mutation is complemented by the URA3-containing plasmid pRMHBT18NG) .
• the resulting strain is designated 9ALZ/pRMHBTl8NG. Cells were grown to mid-log in ura ' media, and diluted to an ODgoo of approximately 0.3 in the same media.
• EXAMPLE 11 ALPHA FACTOR-DEPENDENT ACTIVATION OF THE YEAST MATING PATHWAY BY THE STE2-GPA1 FUSION IN Ste2 CELLS
• Oligonucleotide "s” contains the seguence from bp 534 to 557 of STE2 (GenBank accession # M24335) and seventeen additional nucleotides creating NotI and Hindlll restriction sites. Oligonucleotide "t” contains the sequence complementary to bp 1800 to 1832 of the STE2 gene and ten additional nucleotides which create Mlul and Xbal restriction sites. The resulting 1295 bp PCR product was digested with NotI and Xbal and ligated into pBluescript (Stratagene) cut with the same enzymes.
• Nsil fragment of the STE2 gene (at positions 1148 and 1436) was deleted by digestion with Nsil and religation, creating a frameshift mutation in addition to the deletion.
• the resulting plasmid was digested with Hindlll and Xbal and the 1005 bp fragment with the STE2 deletion mutation was ligated into the yeast integrating vector pRS306 (Sikorski and Hieter Genetics 122 :19 (1989)) .
• This mutant gene was used to replace wild type STE2 by the two step method.
• the deletion plasmid was linearized within STE2 at the Hpal site and integrated into HBS32 by selection for uracil prototrophy.
• HBS12LZ leu2: :LEU2-FUS1-LACZ
• the Ste2p domain of the Ste2p-Gpal chimera is functional : To determine if the Ste2p domain of the Ste2p-Gpalp fusion protein is functional (able to bind alpha factor and transmit the binding signal to Gpal) , the following experiment was performed. Strain HBS12LZ was transformed to uracil prototrophy with pRMHBT18NG, which carries the fusion construct, and the resulting strain, HBS12LZ/pRMHBT18NG was examined for alpha factor-dependent shmoo formation. Cells were grown to mid-log phase in ura " media, and alpha factor was added to 5.8 mM.
• EXAMPLE 12 ENHANCED ACTIVATION OF THE MATING PATHWAY BY THE STE2-GPA1 FUSION IN ste2, gpal CELLS
• a) Deletion of chromosomal STE2 in strain 9ALZ The strain 9ALZ/pRMHBTl8NG with a chromosomal gpal mutation was transformed to lysine prototrophy with pRMHBT44 to remove the URA3 marker of pRMHBTl ⁇ NG and replace it with a LYS2 marker.
• pRMHBT44 is functionally equivalent to pRMHBT43 (GPAl under PGK promotor transcriptional control) , except it has a LYS2 marker.
• the strain 9ALZ/pRMHBT44 was identified by 5-FOA counter-selection against the URA3-containing plasmid pRMHBT18NG.
• This strain was used for disruption of the STE2 gene by the two step method using URA3 , as in example 11.
• the new ste2, gpal strain was designated 9ALZ ⁇ GS/pRMHBT44.
• a "plasmid shuffle" was then performed to replace pRMHBT44 with pRMHBT18NG carrying the STE2-GPA1 fusion construct.
• Strain 9ALZ ⁇ GS/pRMHBT44 was transformed to uracil prototrophy with pRMHBT18NG.
• URA + cells were then grown to saturation in ura" media, and cells that had lost pRMHBT44 were selected for by growth on ura " plates with 5.0% a-aminoadipic acid (a-aminoadipic acid is lethal to LYS + cells, and selects against pRMHBT44) .
• a similar plasmid shuffle was also performed to replace pRMHBT44 with pRMHBT20NG. The resulting strains were designated 9ALZ ⁇ GS/pRMHBTl8NG and 9ALZ ⁇ GS/pRMHBT20NG, respectively.
• Ste2p and Gpalp were expressed separately from the same promoter and vector in the same yeast strain used to express the fusion protein.
• 9ALZ ⁇ GS/pRMHBT44/pRMHBT45 cells were grown to mid-log phase in ura " media, ⁇ -factor was added to 5.8 mM, and the cells were assayed for beta- galactosidase activity after incubation for four hours at 30°C on a roller drum. Cells were also observed via light microscopy after 4.0 hours of incubation, and no shmoos were detectable in either the treated or non- treated control cultures.
• the beta-galactosidase assay data is shown below:
• EXAMPLE 14 THROMBIN-DEPENDENT ACTIVATION OF THE YEAST MATING PATHWAY BY THE HUMAN THROMBIN RECEPTOR- GPAl FUSION PROTEIN IN ste2 gpal ' CELLS
• 9ALZ ⁇ GS cells were transformed with the thrombin construct pRMHBT2O ⁇ Gby the plasmid shuffle method described in Example 12a. These cells were grown to mid-log phase in ura " media buffered at pH 7.0. Human thrombin was added to 71.4 units/ml media, and the cells were incubated for four hours at 30°C on a roller drum. Crude extracts were then prepared as described and beta-galactosidase assays were performed. The results are shown below:
• Example 12b these results are consistent with the results in Example 13, and show that the two components of the Thrombin Receptor-Gpalp fusion protein can measurably couple with each other and to the yeast mating pathway. Further modification of the Gpalp domain of the chimera by methods such as that described in Example 15 should enable greater efficiency of coupling.
• the host strain can also be modified by random mutagenesis to reduce the background activation and to enhance the induction of the mating pathway. Mutants that provide hypersensitivity to mating factor are known (e.g. Chan et al, 1982, Mol. Cell. Biol. 2:21)
• EXAMPLE 15 MUTAGENESIS OF THE Gpalp DOMAIN OF A FUSION PROTEIN TO ENABLE COUPLING OF THROMBIN RECEPTOR ACTIVATION TO THE MATING PATHWAY aj constructing a library of mutations: oligonucleotides "c" and "d” described in example 1 are used to amplify the GPAl gene from a wild type plasmid copy under PCR conditions shown to introduce mutations at a frequency of 6.6 per 1000 bases
• the amplification product that contains individual molecules with single or multiple mutations is digested at the Mlul and PflMI sites present in the two primers.
• the plasmid pRMHBT20 which encodes the thrombin receptor fused in frame with Gpalp, is digested with Mlul and PflMI to release GPAl, and the fragment with the thrombin receptor is purified and ligated to the digested PCR product, and transformed into competent E.
• a diploid yeast strain of genotype gpal/gpal is constructed by mating haploid gpal strains of opposite mating type in which the gpal mutation is complemented by a plasmid carrying GPAl.
• the strain 9ALZ carrying pRMHBT44 (with a LYS2 selectable marker) is mated to any of the C7RA+ segregants in Example 7a or 7b that are of the alpha mating type.
• Both strains carry the reporter construct FUS1-LACZ integrated at the LEU2 locus (leu2 : :LEU2-FUSlp-LACZ) .
• Diploids are selected on ura-, lys- plates. The two plasmids are then eliminated by counterselection with 5-FOA and alpha- aminoadipate, which is possible because GPAl is required for growth only in haploids and not diploids.
• This strain is transformed with the mutant library so that at least IO 6 URA+ transformants are obtained, if necessary by repeated transformation experiments.
• the population of transformants is then sporulated, and random spores are germinated to yield at least 10 s individual colonies by standard genetic or chemical methods for random spore analysis (Rose et al, Methods in Yeast Genetics : A Labora tory Course Manual , c. 1990 by Cold Spring Harbor Laboratory Press.) .
• Only spores in which the Gpalp domain of the fusion can complement the gpal mutation can grow, thus selecting for mutants with Gpalp domains that can interact with G- beta/gamma.
• c) screening for functional coupling of thrombin receptor activation to the mating pathway is achieved by growing the mutants selected from the previous step in the presence of thrombin and the dye X-gal, which is a substrate of the reporter lacZ gene. Functional coupling is selected for by induction of beta galactosidase, and consequent blue color formation. Note that because the reporter gene is present at both LEU2 loci in the diploid, all haploid segregants will have a functional reporter construct. Growth of such cells on plates containing thrombin and the dye X-gal causes blue color formation in colonies in which functional coupling is present between the receptor and Gpal domains. Others will remain white.

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