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J. Biol. Chem., Vol. 280, Issue 7, 5664-5675, February 18, 2005

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Random Mutagenesis of the M₃ Muscarinic Acetylcholine Receptor Expressed in Yeast

IDENTIFICATION OF SECOND-SITE MUTATIONS THAT RESTORE FUNCTION TO A COUPLING-DEFICIENT MUTANT M₃ RECEPTOR^*

Bo Li, Nicola M. Nowak, Soo-Kyung Kim, Kenneth A. Jacobson, Ali Bagheri, Clarice Schmidt, and Jürgen Wess¶

From the Departments of Molecular Signaling and Molecular Recognition, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

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.

Received for publication, October 12, 2004 , and in revised form, November 12, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Molecular Signaling, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes, Bldg. 8A, Rm. B1A-05, 8 Center Dr., MSC 0810, Bethesda, MD 20892-0810. Tel.: 301-402-3589; Fax: 301-480-3447; E-mail: jwess{at}helix.nih.gov.

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