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.
The M(3) 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 M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) 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 M(3) 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 M(3) 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.
Li
Bo
B
Department of Molecular Signaling, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, 8 Center Dr., Bethesda, MD 20892, USA.
Nowak
Nicola M
NM
Kim
Soo-Kyung
SK
Jacobson
Kenneth A
KA
Bagheri
Ali
A
Schmidt
Clarice
C
Wess
Jürgen
J
D000483:Alleles; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D006868:Hydrolysis; D008024:Ligands; D016296:Mutagenesis / Q000235:genetics*; D010716:Phosphatidylinositols / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011869:Radioligand Assay; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D010716:Phosphatidylinositols; D043587:Receptor, Muscarinic M3; D014443:Tyrosine; D001120:Arginine