Random Mutagenesis of the M3 Muscarinic Acetylcholine Receptor Expressed in Yeast
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m304991200
ISSN1083-351X
AutoresClarice Schmidt, Bo Li, Lanh M. Bloodworth, Isolde Erlenbach, Fu‐Yue Zeng, Jürgen Wess,
Tópico(s)Neuropeptides and Animal Physiology
ResumoThe M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V–VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V–VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function. The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V–VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V–VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; ADH, gene encoding alcohol dehydrogenase 1; AT, 3-amino-1,2,4-triazole; GPD, gene coding for glyceraldehyde-3-phosphate dehydrogenase; HA tag, hemagglutinin epitope tag; IP1, inositol monophosphate; [3H]NMS, N-[3H]methylscopolamine; i3 loop, the third intracellular loop of G protein-coupled receptors; PBS, phosphate-buffered saline; SC medium, synthetic complete medium; TEF, gene encoding translation elongation factor 1α; TM I–VII, the seven transmembrane domains of GPCRs; WT, wild type; PI, phosphatidylinositol.1The abbreviations used are: GPCR, G protein-coupled receptor; ADH, gene encoding alcohol dehydrogenase 1; AT, 3-amino-1,2,4-triazole; GPD, gene coding for glyceraldehyde-3-phosphate dehydrogenase; HA tag, hemagglutinin epitope tag; IP1, inositol monophosphate; [3H]NMS, N-[3H]methylscopolamine; i3 loop, the third intracellular loop of G protein-coupled receptors; PBS, phosphate-buffered saline; SC medium, synthetic complete medium; TEF, gene encoding translation elongation factor 1α; TM I–VII, the seven transmembrane domains of GPCRs; WT, wild type; PI, phosphatidylinositol. constitute the largest class of cell surface receptors found in nature, and the individual members of this receptor superfamily are involved in an extraordinarily large number of signaling functions (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2064) Google Scholar, 2Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 3Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1218) Google Scholar, 4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar). Despite the remarkable structural diversity of ligands acting on specific GPCRs, all GPCRs are thought to share a conserved three-dimensional structure characterized by a bundle of seven transmembrane helices (TM I–VII) that are connected by three extracellular and three intracellular loops (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2064) Google Scholar, 2Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 3Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1218) Google Scholar, 4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar). The molecular mechanisms involved in ligand-dependent GPCR activation are not well understood at present. However, recent studies suggest that agonist ligands trigger distinct changes in the relative orientations of individual TM helices (2Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar, 5Meng E.C. Bourne H.R. Trends Pharmacol. Sci. 2001; 22: 587-593Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (718) Google Scholar, 7Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), leading to secondary structural changes on the intracellular receptor surface allowing the receptor to interact productively with G proteins.Classical mutagenesis approaches have led to many important insights into the molecular mechanisms governing GPCR function. However, a major limitation of such classical mutagenesis strategies is that the number of mutant proteins that can be generated and characterized in a single study is usually relatively small. To facilitate the rapid screening of large numbers of mutant GPCRs, we recently established a heterologous expression system that allows the functional characterization of various muscarinic acetylcholine (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar) and vasopressin (9Erlenbach I. Kostenis E. Schmidt C. Serradeil-Le Gal C. Raufaste D. Dumont M.E. Pausch M.H. Wess J. J. Biol. Chem. 2001; 276: 29382-29392Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) receptor subtypes in yeast (Saccharomyces cerevisiae). The muscarinic and vasopressin receptors are prototypical members of the class I GPCR subfamily that is particularly large and contains most classical neurotransmitter and hormone receptors (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2064) Google Scholar, 2Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 3Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1218) Google Scholar, 4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar).One major advantage of the yeast expression system is that powerful yeast genetic screens can be employed to isolate mutant receptors endowed with novel phenotypes from large receptor libraries generated by random mutagenesis (9Erlenbach I. Kostenis E. Schmidt C. Serradeil-Le Gal C. Raufaste D. Dumont M.E. Pausch M.H. Wess J. J. Biol. Chem. 2001; 276: 29382-29392Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10Martin N.P. Celic A. Dumont M.E. J. Mol. Biol. 2002; 317: 765-788Crossref PubMed Scopus (33) Google Scholar, 11Geva A. Lassere T.B Lichtarge O. Pollitt S.K. Baranski T.J. J. Biol. Chem. 2000; 275: 35393-35401Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 12Baranski T.J. Herzmark P. Lichtarge O. Gerber B.O. Trueheart J. Meng E.C. Iiri T. Sheikh S.P. Bourne H.R. J. Biol. Chem. 1999; 274: 15757-15765Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 13Sommers C.M. Dumont M.E. J. Mol. Biol. 1997; 266: 559-575Crossref PubMed Scopus (43) Google Scholar, 14Konopka J.B. Margarit S.M. Dube P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6764-6769Crossref PubMed Scopus (106) Google Scholar, 15Stefan C.J. Blumer K.J. Mol. Cell. Biol. 1994; 14: 3339-3349Crossref PubMed Scopus (71) Google Scholar). This approach (receptor random mutagenesis followed by a yeast genetic screen) also offers the advantage that it does not rely on preconceived notions of GPCR function. For example, by using this strategy, we recently identified a series of mutant V2 vasopressin receptors that displayed altered G protein coupling properties (9Erlenbach I. Kostenis E. Schmidt C. Serradeil-Le Gal C. Raufaste D. Dumont M.E. Pausch M.H. Wess J. J. Biol. Chem. 2001; 276: 29382-29392Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Structural analysis of the recovered mutant receptors provided novel information regarding the structural elements that govern the G protein coupling selectivity of this receptor subtype (9Erlenbach I. Kostenis E. Schmidt C. Serradeil-Le Gal C. Raufaste D. Dumont M.E. Pausch M.H. Wess J. J. Biol. Chem. 2001; 276: 29382-29392Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).For the present study, we used the rat M3 muscarinic receptor as a model system to study the molecular mechanisms involved in the activation of class I GPCRs. We recently described a strategy that allows the functional expression of the M3 muscarinic receptor, as well as other muscarinic receptor subtypes, in yeast (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). To facilitate structure-function relationship studies, the M3 receptor was expressed in genetically modified yeast strains that required ligand-dependent receptor/G protein coupling for cell growth (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 16Pausch M.H. Price L.A. Kajkowski E.M. Strnad J. dela Cruz F. Heinrich J. Ozenberger B.A. Hadcock J.R. Lynch K.R. Identification and Expression of G Protein-coupled Receptors. Wiley-Liss, New York1998: 196-212Google Scholar, 17Price L. Strand J. Pausch M. Hadcock J. Mol. Pharmacol. 1996; 50: 829-937PubMed Google Scholar, 18Price L.A. Kajkowski E.M. Hadcock J.R. Ozenberger B.A. Pausch M.H. Mol. Cell. Biol. 1995; 15: 6188-6195Crossref PubMed Scopus (122) Google Scholar). In this heterologous expression system, deletion of the large central portion of the third intracellular loop (i3 loop) of the M3 receptor led to a dramatic increase in receptor density, probably due to the removal of sequences that promote degradation of the M3 receptor in yeast (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). The M3 receptor did not couple well to G proteins containing the endogenous yeast G protein α subunit, Gpa1p, or a mutant version of Gpa1p containing five amino acids of mammalian αs sequence at its C terminus (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). However, the M3 receptor coupled efficiently to G proteins containing a mutant version of Gpa1p in which the last five amino acids of Gpa1p were replaced with the corresponding sequence of mammalian αq (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). These findings indicated that the M3 muscarinic receptor, which selectively interacts with G proteins of the Gq family in mammalian cells (4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (507) Google Scholar), retains the proper G protein coupling selectivity in yeast.Constitutively active mutant GPCRs have served as useful research tools to study the molecular mechanisms involved in GPCR activation (19Parnot C. Miserey-Lenkei S. Bardin S. Corvol P. Clauser E. Trends Endocrinol. Metab. 2002; 13: 336-343Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 20Lu Z.L. Saldanha J.W. Hulme E.C. Trends Pharmacol. Sci. 2002; 23: 140-146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21Pauwels P.J. Wurch T. Mol. Neurobiol. 1998; 17: 109-135Crossref PubMed Scopus (78) Google Scholar, 22Scheer A. Cotecchia S. J. Recept. Signal Transduct. Res. 1997; 17: 57-73Crossref PubMed Scopus (94) Google Scholar, 23Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar). In addition, activating GPCR mutations are also of considerable clinical relevance (19Parnot C. Miserey-Lenkei S. Bardin S. Corvol P. Clauser E. Trends Endocrinol. Metab. 2002; 13: 336-343Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 21Pauwels P.J. Wurch T. Mol. Neurobiol. 1998; 17: 109-135Crossref PubMed Scopus (78) Google Scholar, 22Scheer A. Cotecchia S. J. Recept. Signal Transduct. Res. 1997; 17: 57-73Crossref PubMed Scopus (94) Google Scholar, 23Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 24Seifert R. Wenzel-Seifert K. Naunyn-Schmiedeberg's Arch. Pharmacol. 2002; 366: 381-416Crossref PubMed Scopus (500) Google Scholar). In most cases, activating GPCR mutations appear to involve the mutational modification of amino acids predicted to be engaged in interhelical (intramolecular) interactions critical for stabilizing the inactive receptor conformation (19Parnot C. Miserey-Lenkei S. Bardin S. Corvol P. Clauser E. Trends Endocrinol. Metab. 2002; 13: 336-343Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 20Lu Z.L. Saldanha J.W. Hulme E.C. Trends Pharmacol. Sci. 2002; 23: 140-146Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 21Pauwels P.J. Wurch T. Mol. Neurobiol. 1998; 17: 109-135Crossref PubMed Scopus (78) Google Scholar, 22Scheer A. Cotecchia S. J. Recept. Signal Transduct. Res. 1997; 17: 57-73Crossref PubMed Scopus (94) Google Scholar, 23Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar).In the present study, we describe an activating point mutation (Q490L) that results in robust ligand-independent signaling of the M3 muscarinic receptor in both yeast and mammalian cells. To learn more about the molecular mechanisms by which point mutations can render GPCRs constitutively active, we employed receptor random mutagenesis followed by a yeast genetic screen to identify second-site mutations that could restore wild-type (WT) receptor function to the Q490L mutant receptor. A considerable body of work indicates that the isolation of intragenic second-site suppressor mutations represents a useful approach to the structural and functional analysis of different membrane proteins including the yeast α-factor receptor (13Sommers C.M. Dumont M.E. J. Mol. Biol. 1997; 266: 559-575Crossref PubMed Scopus (43) Google Scholar).This screen led to the identification of 12 point mutations that were able to "silence" the Q490L mutant M3 receptor and enabled it to function in a fashion similar to the WT receptor. The recovered mutations were clustered either around the ligand binding pocket or mapped to distinct sites on the intracellular surface of the receptor. Interestingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling.These findings highlight the usefulness of yeast expression technology to study the molecular mechanisms involved in GPCR function. Since the M3 muscarinic receptor is a prototypical class I GPCR, the findings obtained here should also be applicable to other members of this receptor family.EXPERIMENTAL PROCEDURESMaterials—Carbamylcholine chloride (carbachol), atropine sulfate, 3-amino-1,2,4-triazole (AT), phenylmethylsulfonyl fluoride, glass beads (425–600 μm, acid-washed), and Tween 20 were obtained from Sigma. N-[3H]Methylscopolamine ([3H]NMS; 70 Ci/mmol), AmpliTaq® DNA Polymerase, and Western blot Chemiluminescence Reagent Plus were from PerkinElmer Life Sciences. myo-[3H]Inositol (20 Ci/mmol) was from American Radiolabeled Chemicals. Yeast media components were purchased from Qbiogene, Inc. Media for mammalian cell culture, precast Novex® Tris-glycine polyacrylamide gels, polyvinylidene difluoride membranes, and buffers used for Western blotting were from Invitrogen. The BCA™ protein assay kit was purchased from Pierce. The mouse anti-hemagglutinin (HA) monoclonal antibody (clone 12CA5) was obtained from Roche Applied Science. The anti-mouse IgG antibody conjugated to horseradish peroxidase was from Roche Applied Science. All enzymes used for molecular cloning were from New England Biolabs. The p416GPD, p416TEF, and p416ADH yeast expression plasmids were purchased from American Type Culture Collection (ATCC).Construction of Plasmids—All mutations were introduced into a modified version of the rat M3 muscarinic receptor that lacked the central portion of the i3 loop (Ala274–Lys469) and contained a 9-amino acid HA epitope tag (YPYDVPDYA) inserted after the initiating methionine codon (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 25Schöneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 26Maggio R. Barbier P. Fornai F. Corsini G.U. J. Biol. Chem. 1996; 271: 31055-31060Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 27Zeng F.-Y. Soldner A. Schöneberg T. Wess J. J. Neurochem. 1999; 72: 2404-2414Crossref PubMed Scopus (61) Google Scholar). Previous studies showed that these modifications have little effect on the ligand binding and G protein-coupling properties of the M3 muscarinic receptor (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 25Schöneberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 26Maggio R. Barbier P. Fornai F. Corsini G.U. J. Biol. Chem. 1996; 271: 31055-31060Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 27Zeng F.-Y. Soldner A. Schöneberg T. Wess J. J. Neurochem. 1999; 72: 2404-2414Crossref PubMed Scopus (61) Google Scholar). For the sake of simplicity, this i3 loop-shortened, epitope-tagged version of the M3 receptor is referred to as 'WT'-M3 receptor throughout the paper.The 'WT'-M3 receptor coding sequence was inserted into the polylinker of the yeast expression plasmid, p416GPD, as described previously (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). p416GPD is a single copy plasmid containing the CEN/ARS element, the URA3 gene as selectable marker for maintenance in yeast, and the Amp gene for propagation in Escherichia coli (28Mumberg D. Müller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1569) Google Scholar). In an analogous fashion, the 'WT'-M3 receptor coding sequence was also cloned into two related yeast expression plasmids, p416TEF and p416ADH (28Mumberg D. Müller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1569) Google Scholar). These three expression plasmids contain the yeast GPD (glyceraldehyde-3-phosphate dehydrogenase), TEF (translation elongation factor 1α), and ADH (alcohol dehydrogenase 1) promoters, respectively. Introduction of the activating Q490L point mutation into 'WT'-M3-p416GPD resulted in plasmid 'WT'-M3(Q490L)-p416GPD. Point mutations isolated during the yeast genetic screen (see below) were introduced de novo into 'WT'-M3-p416GPD and 'WT'-M3(Q490L)p416GPD by standard PCR mutagenesis techniques (29Higuchi R. Erlich H.A. PCR Technology. Stockton Press, New York1989: 61-70Crossref Google Scholar). The identity of all constructs was verified by DNA sequencing.Yeast Strains, Growth, and Transformation—The haploid yeast strain MPY578q5 (MAT a GPA1 far1::LYS2 fus1::FUS1-HIS3 sst2::SST2-G418r ste2::LEU2 fus2::FUS2-CAN1 ura3 lys2 ade2 his3 leu2 trp1 can1) was used as a host for the expression of all receptor constructs. This strain is isogenic to strain MPY578fc (16Pausch M.H. Price L.A. Kajkowski E.M. Strnad J. dela Cruz F. Heinrich J. Ozenberger B.A. Hadcock J.R. Lynch K.R. Identification and Expression of G Protein-coupled Receptors. Wiley-Liss, New York1998: 196-212Google Scholar) except for the G protein α subunit. Whereas the fc strain carries the WT GPA1 gene coding for the native yeast Gα, the q5 strains used in the present study harbors a mutant version of GPA1 coding for a Gα subunit in which the last 5 amino acids of Gpa1p (KIGII) were replaced with the corresponding sequence derived from mammalian αq (EYNLV) (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 16Pausch M.H. Price L.A. Kajkowski E.M. Strnad J. dela Cruz F. Heinrich J. Ozenberger B.A. Hadcock J.R. Lynch K.R. Identification and Expression of G Protein-coupled Receptors. Wiley-Liss, New York1998: 196-212Google Scholar). In these strains, deletion of the FAR1 gene allows yeast to grow despite G protein-mediated activation of the pheromone pathway that normally leads to growth arrest (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 16Pausch M.H. Price L.A. Kajkowski E.M. Strnad J. dela Cruz F. Heinrich J. Ozenberger B.A. Hadcock J.R. Lynch K.R. Identification and Expression of G Protein-coupled Receptors. Wiley-Liss, New York1998: 196-212Google Scholar). Moreover, the presence of the FUS1-HIS3 reporter construct makes the production of His3 protein dependent on receptor-mediated activation of the yeast pheromone pathway, allowing auxotrophic (his3) yeast strains expressing coupling-competent receptors to grow in histidine-deficient media. The SST2 gene was disrupted to prevent attenuation of G protein signaling mediated by the GTPaseactivating protein activity of Sst2p. The STE2 gene encoding the yeast α-factor (pheromone) receptor was deleted to prevent the sequestration of co-expressed G proteins.Yeast cells were grown at 30 °C in synthetic complete medium (SC) (30Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2530) Google Scholar) unless noted otherwise. Cells were transformed with plasmid DNA coding for the different receptor constructs by using a lithium acetate method (31Agatep, R., Kirkpatrick, R. D., Parchaliuk, D. L., Woods, R. A., and Gietz, R. D. (1998) Technical Tips Online 1:51:P01525, http://tto.trends.com.Google Scholar). Transformants were selected and maintained in SC medium lacking uracil (SC-Ura).Yeast Functional Assays—Mid-log phase cell cultures (1–4 × 107 cells/ml) were washed with PBS and diluted to 105 cells/ml in SC medium lacking uracil and histidine (pH 7). Unless stated otherwise, growth assays were carried out using 96-well microtiter dishes essentially as described (8Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). Cell suspensions were incubated at 25 °C for 72 h in the presence of carbachol (10–4 to 10–13m serial dilutions). Basal growth was measured in the absence of ligand, and cell viability was monitored in the presence of histidine (20 μg/ml). Background growth was suppressed by the addition of 5 mm of AT. Receptor-mediated yeast growth was assessed by measuring increases in the absorbance of the yeast cultures at 630 nm. Assays were conducted in triplicate, using three independent transformants. Growth rate measurements were obtained during the logarithmic phase of yeast cell growth. Carbachol concentration-response curves were analyzed using the KaleidaGraph™ program (Synergy Software).Mutagenesis of the Q490L Mutant M 3 Muscarinic Receptor—We recently used PCR-based receptor random mutagenesis (32Fromant M. Blanquet S. Plateu P. Ann. Biochem. 1995; 224: 347-353Crossref Scopus (234) Google Scholar) and a gaprepair protocol (33Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (416) Google Scholar, 34Oldenburg K.R. Vo K.T. Michaelis S. Paddon C. Nucleic Acids Res. 1997; 25: 451-452Crossref PubMed Scopus (405) Google Scholar, 35Sommers C.M. Dumont M.E. Wess J. Structure-Function Analysis of G Protein-coupled Receptors. Wiley-Liss, New York1999: 141-166Google Scholar) to generate libraries of yeast (MPY578q5) clones expressing mutant M3 muscarinic receptors containing point mutations throughout the entire coding sequence of the 'WT'-M3 receptor. A subsequent yeast screen (this screen will be described in detail elsewhere) 2C. Schmidt and J. Wess, manuscript in preparation. yielded a mutant receptor, 'WT'-M3(Q490L), which displayed robust agonist-independent signaling.To identify secondary mutations that restore WT receptor function to the 'WT'-M3(Q490L) mutant receptor, a C-terminal region of the 'WT'-M3(Q490L) receptor that included TM V–VII (Arg213–Cys562) was subjected to random mutagenesis (Fig. 1). In the first step, two separate receptor regions, coding for Arg213–Ile508 and Thr475–Cys562 (Fig. 1 and Table I), were amplified under mutagenic PCR conditions (32Fromant M. Blanquet S. Plateu P. Ann. Biochem. 1995; 224: 347-353Crossref Scopus (234) Google Scholar). In each case (receptor region), four independent PCRs were carried out (10 mm Tris-HCl (pH 8.3), 50 mm KCl, 9.5 mm MgCl2, 0.5 mm MnCl2, 0.001% (w/v) gelatin, 8 units of AmpliTaq® DNA polymerase, 500 nm of each primer, and 100 ng of 'WT'-M3(Q490L)-p416GPD as template, in 100-μl reaction mixtures), each containing 3.4 mm of one "forcing" dNTP and 0.2 mm of the three other dNTPs. After 30 PCR cycles (94 °C for 1 min, 42 °C for 1 min, and 72 °C for 1 min), the products were purified from an agarose gel and mixed at equivalent concentrations. The primer sequences and the sizes of the amplified PCR segments are given in Table I.Table IPCR strategy used to amplify regions of the Q490L mutant M3 muscarinic receptor under mutagenic conditionsReactionAmplified receptor regionOligonucleotides usedaThe PCR primer sequences are as follows: ON1-S, 5′-AGA ACT GTG CCC CCA GGA GAA TGT TTC ATT CAG TTT CTG AGT GAG CCC-3′ (corresponding nucleotides/amino acids: 637-685/Arg213-Pro228); ON1-AS, 5′-GAT GTT GTA GGG GGT CCA CGT GAT GAT GAA GGC TAG CAA GAT GGC ACT GAG CGT CAG-3′ (corresponding nucleotides/amino acids: 1468-1524/(Gln490-Ile508); ON2-S, 5′-ACC AAG CGG AAG AGG ATG TCG CTC ATC AAG GAG AAG AAG GCC GCC CTG-3′ (corresponding nucleotides/amino acids: 1423-1470/Thr475-Gln490); ON2-AS, 5′-ACA CTG GCA CAA GAG GAG CGT CTT GAA GGT GGT TCT GAA TGT TTT GTT-3′ (corresponding nucleotides/amino acids: 1639-1686/Asn574-Cys562). The Gln490 codon is shown underlined.Size of PCR segmentEnzymes used for gapping M3(Q490L)-p416GPDbp1Arg213-Ile508ON1-S and ON1-AS300BstXI + NheI2Thr475-Cys562ON2-S and ON2-AS264NheI + PmlIa The PCR primer sequences are as follows: ON1-S, 5′-AGA ACT GTG CCC CCA GGA GAA TGT TTC ATT CAG TTT CTG AGT GAG CCC-3′ (corresponding nucleotides/amino acids: 637-685/Arg213-Pro228); ON1-AS, 5′-GAT GTT GTA GGG GGT CCA CGT GAT GAT GAA GGC TAG CAA GAT GGC ACT GAG CGT CAG-3′ (corresponding nucleotides/amino acids: 1468-1524/(Gln490-Ile508); ON2-S, 5′-ACC AAG CGG AAG AGG ATG TCG CTC ATC AAG GAG AAG AAG GCC GCC CTG-3′ (corresponding nucleotides/amino acids: 1423-1470/Thr475-Gln490); ON2-AS, 5′-ACA CTG GCA CAA GAG GAG CGT CTT GAA GGT GGT TCT GAA TGT TTT GTT-3′ (corresponding nucleotides/amino acids: 1639-1686/Asn574-Cys562). The Gln490 codon is shown underlined. Open table in a new tab To generate libraries of yeast clones expressing randomly mutagenized mutant M3 muscarinic receptors, we used a gap-repair
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