Random Mutagenesis of the M3 Muscarinic Acetylcholine Receptor Expressed in Yeast
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m411623200
ISSN1083-351X
AutoresBo Li, Nicola M. Nowak, Soo‐Kyung Kim, Kenneth A. Jacobson, Ali Bagheri, Clarice Schmidt, Jürgen Wess,
Tópico(s)Neuropeptides and Animal Physiology
ResumoThe M3 muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M3 receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M3 receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M3 muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M3 receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation. The M3 muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M3 receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M3 receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M3 muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M3 receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation. The superfamily of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; GPD, glyceraldehyde-3-phosphate dehydrogenase; i3 loop, the third intracellular loop of G protein-coupled receptors; IP1, inositol monophosphate; M1–M5, muscarinic acetylcholine receptors 1–5; [3H]NMS, N-[3H]methylscopolamine; PI, phosphatidylinositol; SC medium, synthetic complete medium; TM, transmembrane; WT, wild-type.1The abbreviations used are: GPCR, G protein-coupled receptor; GPD, glyceraldehyde-3-phosphate dehydrogenase; i3 loop, the third intracellular loop of G protein-coupled receptors; IP1, inositol monophosphate; M1–M5, muscarinic acetylcholine receptors 1–5; [3H]NMS, N-[3H]methylscopolamine; PI, phosphatidylinositol; SC medium, synthetic complete medium; TM, transmembrane; WT, wild-type. represents the largest group of cell surface receptors found in nature (1Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. Kann L. Lehoczky J. LeVine R. McEwan P. McKerman K. Meldrim J. Mesirov J.P. Miranda C. Morris W. Naylor J. Raymond C. Rosetti M. Santos R. Sheridan A. Sougnez C. Stange-Thomann N. Stojanovic N. Subramanian A. Wyman D. Rogers J. Sulston J. Ainscough R. Beck S. Bentley D. Burton J. Clee C. Carter N. Coulson A. Deadman R. Deloukas P. Dunham A. Dunham I. Durbin R. French L. Grafham D. Gregory S. Hubbard T. Humphray S. Hunt A. Jones M. Lloyd C. McMurray A. Matthews L. Mercer S. Milne S. Mullikin J.C. Mungall A. Plumb R. Ross M. Shownkeen R. Sims S. Waterston R.H. Wilson R.K. Hillier L.W. McPherson J.D. Marra M.A. Mardis E.R. Fulton L.A. Chinwalla A.T. Pepin K.H. Gish Chissoe S.L. Wendl M.C. Delehaunty K.D. Miner T.L. Delehaunty A. Kramer J.B. Cook L.L. Fulton R.S. Johnson D.L. Minx P.J. Clifton S.W. Hawkins T. Branscomb E. Predki P. Richardson P. Wenning S. Slezak T. Doggett N. Cheng J.F. Olsen A. Lucas S. Elkin C. Uberbacher E. Frazier M. Gibbs R.A. Muzny D.M. Scherer S.E. Bouck J.B. Sodergren E.J. Worley K.C. Rives C.M. Gorrell J.H. Metzker M.L. Naylor S.L. Kucherlapati R.S. Nelson D.L. Weinstock G.M. Sakaki Y. Fujiyama A. Hattori M. Yada T. Toyoda A. Itoh T. Kawagoe C. Watanabe H. Totoki Y. Taylor T. Weissenbach J. Heilig R. Saurin W. Artiguenave F. Brottier P. Bruls T. Pelletier E. Robert C. Wincker P. Smith D.R. Doucette-Stamm L. Rubenfield M. Weinstock K. Lee H.M. Dubois J. Rosenthal A. Platzer M. Nyakatura G. Taudien S. Rump A. Yang H. Yu J. Wang J. Huang G. Gu J. Hood L. Rowen L. Madan A. Qin S. Davis R.W. Federspiel N.A. Abola A.P. Proctor M.J. Myers R.M. Schmutz J. Dickson M. Grimwood J. Cox D.R. Olson M.V. Kaul R. Raymond C. Shimizu N. Kawasaki K. Minoshima S. Evans G.A. Athanasiou M. Schultz R. Roe B.A. Chen F. Pan H. Ramser J. Lehrach H. Reinhardt R. McCombie W.R. de la Bastide M. Dedhia N. Blocker H. Hornischer K. Nordsiek G. Agarwala R. Aravind L. Bailey J.A. Bateman A. Batzoglou S. Birney E. Bork P. Brown D.G. Burge C.B. Cerutti L. Chen H.C. Church D. Clamp M. Copley R.R. Doerks T. Eddy S.R. Eichler E.E. Furey T.S. Galagan J. Gilbert J.G. Harmon C. Hayashizaki Y. Haussler D. Hermjakob H. Hokamp K. Jang W. Johnson L.S. Jones T.A. Kasif S. Kaspryzk A. Kennedy S. Kent W.J. Kitts P. Koonin E.V. Korf I. Kulp D. Lancet D. Lowe T.M. McLysaght A. Mikkelsen T. Moran J.V. Mulder N. Pollara V.J. Ponting C.P. Schuler G. Schultz J. Slater G. Smit A.F. Stupka E. Szustakowski J. Thierry-Mieg D. Thierry-Mieg J. Wagner L. Wallis J. Wheeler R. Williams A. Wolf Y.I. Wolfe K.H. Yang S.P. Yeh R.F. Collins F. Guyer M.S. Peterson J. Felsenfeld A. Wetterstrand K.A. Patrinos A. Morgan M.J. de Jong P. Catanese J.J. Osoegawa K. Shizuya H. Choi S. Chen Y.J. Szustakowki J. the International Human Genome Sequencing Consortium Nature. 2001; 409: 860-921Crossref PubMed Scopus (17302) Google Scholar, 2Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. Zhang Q. Kodira C.D. Zheng X.H. Chen L. Skupski M. Subramanian G. Thomas P.D. Zhang J. Miklos G.L.G. Nelson C. Broder S. Clark A.G. Nadeau J. McKusick V.A. Zinder N. Levine A.J. Roberts R.J. Simon M. Slayman C. Hunkapiller M. Bolanos R. Delcher A. Dew I. Fasulo D. Flanigan M. Florea L. Halpern A. Hannenhalli S. Kravitz S. Levy S. Mobarry C. Reinert K. Remington K. Abu-Threideh J. Beasley E. Biddick K. Bonazzi V. Brandon R. Cargill M. Chandramouliswaran I. Charlab R. Chaturvedi K. Deng Z. Di Francesco V. Dunn P. Eilbeck K. Evangelista C. Gabrielian A.E. Gan W. Ge W. Gong F. Gu Z. Guan P. Heiman T.J. Higgins M.E. Ji R.-R. Ke Z. Ketchum K.A. Lai Z. Lei Y. Li Z. Li J. Liang Y. Lin X. Lu F. Merkulov G.V. Milshina N. Moore H.M. Naik A.K. Narayan V.A. Neelam B. Nusskern D. Rusch D.B. Salzberg S. Shao W. Shue B. Sun J. Wang Z.Y. Wang A. Wang X. Wang J. Wei M.-H. Wides R. Xiao C. Yan C. Yao A. Ye J. Zhan M. Zhang W. Zhang H. Zhao Q. Zheng L. Zhong F. Zhong W. Zhu S.C. Zhao S. Gilbert D. Baumhueter S. Spier G. Carter C. Cravchik A. Woodage T. Ali F. An H. Awe A. Baldwin D. Baden H. Barnstead M. Barrow I. Beeson K. Busam D. Carver A. Center A. Cheng M.L. Curry L. Danaher S. Davenport L. Desilets R. Dietz S. Dodson K. Doup L. Ferriera S. Garg N. Gluecksmann A. Hart B. Haynes J. Haynes C. Heiner C. Hladun S. Hostin D. Houck J. Howland T. Ibegwam C. Johnson J. Kalush F. Kline L. Koduru S. Love A. Mann F. May D. McCawley S. McIntosh T. McMullen I. Moy M. Moy L. Murphy B. Nelson K. Pfannkoch C. Pratts E. Puri V. Qureshi H. Reardon M. Rodriguez R. Rogers Y.-H. Romblad D. Ruhfel B. Scott R. Sitter C. Smallwood M. Stewart E. Strong R. Suh E. Thomas R. Tint N.N. Tse S. Vech C. Wang G. Wetter J. Williams S. Williams M. Windsor S. Winn-Deen E. Wolfe K. Zaveri J. Zaveri K. Abril J.F. Guigo ́ R. Campbell M.J. Sjolander K.V. Karlak B. Kejariwal A. Mi H. Lazareva B. Hatton T. Narechania A. Diemer K. Muruganujan A. Guo N. Sato S. Bafna V. Istrail S. Lippert R. Schwartz R. Walenz B. Yooseph S. Allen D. Basu A. Baxendale J. Blick L. Caminha M. Carnes-Stine J. Caulk P. Chiang Y.-H. Coyne M. Dahlke C. Mays A.D. Dombroski M. Donnelly M. Ely D. Esparham S. Fosler C. Gire H. Glanowski S. Glasser K. Glodek A. Gorokhov M. Graham K. Gropman B. Harris M. Heil J. Henderson S. Hoover J. Jennings D. Jordan C. Jordan J. Kasha J. Kagan L. Kraft C. Levitsky A. Lewis M. Liu X. Lopez J. Ma D. Majoros W. McDaniel J. Murphy S. Newman M. Nguyen T. Nguyen N. Nodell M. Pan S. Peck J. Peterson M. Rowe W. Sanders R. Scott J. Simpson M. Smith T. Sprague A. Stockwell T. Turner R. Venter E. Wang M. Wen M. Wu D. Wu M. Xia A. Zandieh A. Zhu X. Science. 2001; 291: 1304-1350Crossref PubMed Scopus (10363) Google Scholar, 3Takeda S. Kadowaki S. Haga T. Takaesu H. Mitaku S. FEBS Lett. 2002; 520: 97-101Crossref PubMed Scopus (309) Google Scholar). All GPCRs contain a bundle of seven transmembrane (TM) helices (TM I–VII) that are connected by alternating intracellular and extracellular loops (4Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2033) Google Scholar, 5Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 6Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1214) Google Scholar, 7Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (366) Google Scholar; Fig. 1). Based on sequence similarity, mammalian GPCRs can be grouped into three major receptor subfamilies (A, B, and C). Family A contains by far the largest number of receptors including, for example, the receptors for light (rhodopsin), a very large number of odorant receptors, and the classical biogenic amine neurotransmitter receptors including the five muscarinic acetylcholine receptors (M1–M5) (4Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2033) Google Scholar, 5Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 6Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1214) Google Scholar, 7Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (366) Google Scholar). The hallmark of class A GPCRs is a set of about 20 amino acids that is highly conserved only within this GPCR subfamily (5Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 7Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (366) Google Scholar, 8Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar). The majority of these amino acids are located within the cytoplasmic half of the TM receptor core. Mutagenesis data suggest that these highly conserved residues are required for protein stability and/or for mediating the conformational changes that accompany receptor activation (5Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 7Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (366) Google Scholar, 8Baldwin J.M. Schertler G.F. Unger V.M. J. Mol. Biol. 1997; 272: 144-164Crossref PubMed Scopus (632) Google Scholar). At present, bovine rhodopsin, in its inactive state, is the only GPCR for which high resolution structural information is currently available (9Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4966) Google Scholar, 10Teller D.C. Okada T. Behnke C.A. Palczewski K. Stenkamp R.E. Biochemistry. 2001; 40: 7761-7772Crossref PubMed Scopus (624) Google Scholar, 11Li J. Edwards P.C. Burghammer M. Villa C. Schertler G.F. J. Mol. Biol. 2004; 343: 1409-1438Crossref PubMed Scopus (667) Google Scholar). Attempts to obtain a high resolution structure of a GPCR in its active form have not been successful so far. In the absence of this information, different mutagenesis techniques have been employed, often combined with molecular modeling approaches, to gain insight into the functional roles of specific receptor domains/amino acids in GPCR function (5Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 7Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (366) Google Scholar, 12Ballesteros J.A. Shi L. Javitch J.A. Mol. Pharmacol. 2001; 60: 1-19Crossref PubMed Scopus (399) Google Scholar). During the past decade, we have used the M3 muscarinic receptor as a model system to study the molecular mechanisms underlying the function of class A GPCRs activated by small diffusible ligands. The M3 muscarinic receptor preferentially activates G proteins of the Gq/11 family which mediate the activation of phospholipase Cβ (13Wess J. Crit. Rev. Neurobiol. 1996; 10: 69-99Crossref PubMed Scopus (414) Google Scholar). To facilitate structure-function studies of the M3 muscarinic receptor, we recently developed a heterologous expression system that allows the functional expression of the M3 muscarinic receptor in yeast (Saccharomyces cerevisiae) (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Specifically, we expressed the M3 muscarinic receptor in a genetically modified yeast strain that requires agonist-dependent receptor/G protein coupling for cell growth. This strain, referred to as MPY578q5, harbors a mutant version of the GPA1 gene coding for a hybrid yeast/mammalian G protein α subunit in which the last five amino acids of Gpa1p were replaced with the corresponding mammalian Gαq residues (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). We demonstrated previously that the M3 muscarinic receptor can activate this hybrid G protein with high efficiency and selectivity (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). One major advantage of the yeast expression system is that powerful genetic screens can be employed to isolate mutant receptors endowed with novel phenotypes from large receptor libraries generated by random mutagenesis (15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 16Erlenbach 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, 17Celic A. Connelly S.M. Martin N.P. Dumont M.E. Methods Mol. Biol. 2004; 237: 105-120PubMed Google Scholar, 18Geva 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, 19Baranski 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, 20Sommers C.M. Dumont M.E. J. Mol. Biol. 1997; 266: 559-575Crossref PubMed Scopus (43) Google Scholar, 21Konopka J.B. Margarit S.M. Dube P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6764-6769Crossref PubMed Scopus (106) Google Scholar, 22Stefan C.J. Blumer K.J. Mol. Cell. Biol. 1994; 14: 3339-3349Crossref PubMed Scopus (71) Google Scholar). Another advantage is that the results obtained by the use of this strategy (receptor random mutagenesis followed by yeast genetic screens) do not rely on preconceived notions of GPCR function. Using this approach, we recently identified a point mutation (Q490L) in the M3 muscarinic receptor which leads to robust agonist-independent receptor signaling in both yeast and mammalian cells (15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We then applied a secondary yeast genetic screen to isolate second-site mutations that were able to suppress the activating effects of the Q490L mutation. This screen led to the identification of 12 amino acids predicted to play key roles in M3 receptor activation and/or receptor/G protein coupling (15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Previous studies with different classes of GPCR have shown that the identification of second-site suppressor mutations can provide important new insights into the structural and functional roles of specific amino acids (20Sommers C.M. Dumont M.E. J. Mol. Biol. 1997; 266: 559-575Crossref PubMed Scopus (43) Google Scholar, 23Prioleau C. Visiers I. Ebersole B.J. Weinstein H. Sealfon S.C. J. Biol. Chem. 2002; 277: 36577-36584Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 24Ward R.J. Milligan G. Cell. Signal. 2002; 14: 139-144Crossref PubMed Scopus (8) Google Scholar, 25Donnelly D. Maudsley S. Gent J.P. Moser R.N. Hurrell C.R. Findlay J.B. Biochem. J. 1999; 339: 55-61Crossref PubMed Google Scholar, 26Sealfon S. Chi L. Ebersole B.J. Rodic V. Zhang D. Ballesteros J.A. Weinstein H. J. Biol. Chem. 1995; 270: 16683-16688Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 27Zhou W. Flanagan C. Ballesteros J.A. Konvicka K. Davidson J.S. Weinstein H. Millar R.P. Sealfon S.C. Mol. Pharmacol. 1994; 45: 165-170PubMed Google Scholar). The present study was designed to learn more about the functional role of a TM II Asp residue that is highly conserved among class A GPCRs (Asp2.50 according to the Ballesteros/Weinstein amino acid numbering system (28Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2383) Google Scholar); corresponding to Asp-1132.50 in the rat M3 muscarinic receptor; Fig. 1). Studies with many different class A GPCRs have shown that replacement of this residue by other amino acids, such as Gln, abolishes or drastically reduces receptor-mediated G protein activation (29Chung F.Z. Wang C.D. Potter P.C. Venter J.C. Fraser C.M. J. Biol. Chem. 1988; 263: 4052-4055Abstract Full Text PDF PubMed Google Scholar, 30Strader C.D. Sigal I.S. Candelore M.R. Rands E. Hill W.S. Dixon R.A. J. Biol. Chem. 1988; 263: 10267-10271Abstract Full Text PDF PubMed Google Scholar, 31Fraser C.M. Wang C.D. Robinson D.A. Gocayne J.D. Venter J.C. Mol. Pharmacol. 1989; 36: 840-847PubMed Google Scholar, 32Wang C.D. Buck M.A. Fraser C.M. Mol. Pharmacol. 1991; 40: 168-179PubMed Google Scholar, 33Neve K.A. Cox B.A. Henningsen R.A. Spanoyannis A. Neve R.L. Mol. Pharmacol. 1991; 39: 733-739PubMed Google Scholar, 34Ji I. Ji T.H. J. Biol. Chem. 1991; 266: 14953-14957Abstract Full Text PDF PubMed Google Scholar, 35Surprenant A. Horstman D.A. Akbarali H. Limbird L.E. Science. 1992; 257: 977-980Crossref PubMed Scopus (146) Google Scholar, 36Ho B.Y. Karschin A. Branchek T. Davidson N. Lester H.A. FEBS Lett. 1992; 312: 259-262Crossref PubMed Scopus (177) Google Scholar, 37Bihoreau C. Monnot C. Davies E. Teutsch B. Bernstein K.E. Corvol P. Clauser E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5133-5137Crossref PubMed Scopus (143) Google Scholar, 38Nussenzveig D.R. Heinflink M. Gershengorn M.C. Mol. Endocrinol. 1993; 7: 1105-1111PubMed Google Scholar, 39Wang C.D. Gallaher T.K. Shih J.C. Mol. Pharmacol. 1993; 43: 931-940PubMed Google Scholar, 40Rose P.M. Krystek Jr., S.R. Patel P.S. Liu E.C. Lynch J.S. Lach D.A. Fisher S.M. Webb M.L. FEBS Lett. 1995; 361: 243-249Crossref PubMed Scopus (34) Google Scholar, 41Prossnitz E.R. Schreiber R.E. Bokoch G.M. Ye R.D. J. Biol. Chem. 1995; 270: 10686-10694Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 42Tao Q. Abood M.E. J. Pharmacol. Exp. Ther. 1998; 285: 651-658PubMed Google Scholar, 43Fanelli F. Barbier P. Zanchetta D. de Benedetti P.G. Chini B. Mol. Pharmacol. 1999; 56: 214-225Crossref PubMed Scopus (91) Google Scholar, 44Ascoli M. Fanelli F. Segaloff D.L. Endocr. Rev. 2002; 23: 141-174Crossref PubMed Scopus (500) Google Scholar). Consistent with these previous reports, we initially demonstrated in the present study that the D113N mutant M3 receptor was unable to interact productively with G proteins in yeast or mammalian cells (COS-7 cells). To learn more about the functional role of the conserved Asp-113 residue in M3 receptor function, we decided to use yeast expression technology to identify second-site suppressor mutations that are able to restore function to the D113N mutant receptor. Specifically, we subjected the Asp-113 mutant receptor to random mutagenesis and then used a yeast genetic screen to recover second-site suppressor mutations. This screen led to the identification of three point mutations, R165W3.50, R165M3.50, and Y250D5.58, which were able to restore function to the D113N mutant receptor when expressed in yeast. To examine whether these results could be reproduced in a mammalian expression system, we also characterized the recovered mutant M3 receptors in transfected COS-7 cells. Moreover, we carried out additional site-directed mutagenesis studies to examine the allele specificity of the recovered second-site suppressor mutations and the ability of other amino acid substitutions at positions Arg-165 and Tyr-250 to rescue the function of the D113N receptor. Interestingly, the three recovered second-site suppressor mutations involved the mutational modification of two amino acids which, like Asp-113, are highly conserved among class A GPCRs. It is likely that these three amino acids (Asp-1132.50, Arg-1653.50, and Tyr-2505.58) participate in a network of interactions that is critical for converting the inactive state of the M3 receptor into its active conformation. Given the conserved nature of the amino acids targeted in the present study, our results should be of broad general relevance. Materials—Media for mammalian cell culture were from Invitrogen. Yeast media components were purchased from Qbiogene. Carbamylcholine chloride (carbachol), atropine sulfate, 3-amino-1,2,4-triazole, phenylmethylsulfonyl fluoride, glass beads (425–600 μm, acid-washed), and Tween 20 were obtained from Sigma. N-[3H]Methylscopolamine ([3H]NMS; 81 Ci/mmol) and myo-[3H]inositol (20 Ci/mmol) were from American Radiolabeled Chemicals. The BCA protein assay kit was purchased from Pierce. All enzymes used for molecular cloning were from New England Biolabs. Construction of Plasmids—For yeast expression studies, all mutations were introduced into a modified version of the rat M3 muscarinic receptor lacking the central portion of the i3 loop (Ala-274 to Lys-469) and containing an N-terminal, nine-amino acid hemagglutinin epitope tag (YPYDVPDYA) inserted after the initiating methionine codon (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We demonstrated previously that these modifications have little effect on the ligand binding and G protein coupling properties of the M3 receptor (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 45Scho ̈neberg T. Liu J. Wess J. J. Biol. Chem. 1995; 270: 18000-18006Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 46Maggio 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, 47Zeng F.-Y. Soldner A. Scho ̈neberg T. Wess J. J. Neurochem. 1999; 72: 2404-2414Crossref PubMed Scopus (61) Google Scholar). For the sake of simplicity, this i3 loop-deleted, epitope-tagged version of the M3 receptor is referred to as 'WT' M3 receptor. The coding sequences of the 'WT' M3 receptor or 'WT' M3 receptor-based mutant constructs were inserted into the polylinker of the yeast expression plasmid, p416GPD, as described previously (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar). Site-directed mutagenesis was performed by using standard PCR mutagenesis techniques or the QuikChange site-directed mutagenesis kit (Stratagene), according to the manufacturer's instructions. The identity of all constructs was verified by DNA sequencing. Yeast Strains, Growth, and Transformation—The haploid yeast strain MPY578q5 (MATa gpa1::Gαq5 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 the 'WT' M3 receptor and all 'WT' M3 receptor-based mutant constructs (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The specific features of this strain have been described previously (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In brief, the MPY578q5 strain contains inactive versions of the FAR1, SST2, and STE2 genes. Moreover, it harbors a mutant version of GPA1 coding for a G protein α subunit in which the last five amino acids of Gpa1p were replaced with the corresponding sequence derived from mammalian αq. Importantly, the genomic incorporation of a 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 (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Yeast cells were grown at 30 °C in synthetic complete medium (SC) (48Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2526) Google Scholar) unless noted otherwise. The lithium acetate method was used to transform yeast with plasmid DNAs coding for the different receptor constructs (49Agatep R. Kirkpatrick R.D. Parchaliuk D.L. Woods R.A. Gietz R.D. Technical Tips Online. 1998; 1 (tto.trends.com): P01525Google Scholar). Transformants were selected and maintained in SC medium lacking uracil (SC-Ura). Yeast Liquid Bioassays—Yeast liquid bioassays were performed essentially as described previously (14Erlenbach I. Kostenis E. Schmidt C. Hamdan F.F. Pausch M.H. Wess J. J. Neurochem. 2001; 77: 1327-1337Crossref PubMed Scopus (50) Google Scholar, 15Schmidt C. Li B. Bloodworth L. Erlenbach I. Zeng F.Y. Wess J. J. Biol. Chem. 2003; 278: 30248-30260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In brief, mid-log phase cell cultures (1–4 × 107 cells/ml) were washed with phosphate-buffered saline and diluted to 105 cells/ml in SC medium lacking uracil and histidine (pH 7). 3-Amino-1,2,4-triazole (5 mm) was added to the medium to suppress background growth. Cell suspensions were incubated in 96-well microtiter dishes at 25 °C for 72 h in the presence of increasing concentrations of carbachol (10–13 to 10–4m). Receptor-mediated yeast growth was assessed by measuring increases in the optical density of the yeast cultures at 630 nm. Assays were conducted in triplicate, using three in
Referência(s)