To understand how GPCRs function at the molecular level, it is important to identify the amino acids that are critical for ligand binding, receptor activation, and productive receptor/G protein coupling. We recently described a novel experimental strategy that allows the rapid identification of functionally critical amino acids in the M3 muscarinic receptor (M3R), a prototypic class I GPCR (Li et al., Nat. Methods 4, 169-74, 2007). This method involves random mutagenesis of the entire M3R coding sequence (except for the central portion of the third intracellular loop), followed by a yeast genetic screen that allows the recovery of mutant M3Rs containing single point mutations that disrupt receptor function. This approach clearly differs from previously used yeast genetic screens which were designed to recover mutant receptors that retain functional activity.[unreadable] We generated mutant M3R libraries via error-prone PCR using conditions to maximize the occurrence of single nucleotide changes. All mutant M3Rs were expressed in the MPY578q5 strain which expresses a chimeric yeast/mammalian G protein alpha subunit and contains several other modifications that allow efficient activation of the yeast MAP kinase/pheromone pathway by functional M3Rs. We took advantage of the fact that the MPY578q5 strain contains the pheromone-sensitive FUS2-CAN1 reporter gene. As a result, only yeast clones expressing functionally inactive mutant M3Rs will grow in the simultaneous presence of the cytotoxin, canavanine (this agent requires the expression of Can1p to enter the yeast cell), and the muscarinic agonist, carbachol. [unreadable] All mutations were introduced into a modified version of the M3R that contained a C-terminal EGFP tag. As a result, yeast clones that survived the canavanine/carbachol selection procedure but failed to display robust GFP fluorescence were eliminated early during the screen. These yeast clones contained either truncated receptors caused by nonsense or frameshift mutations, or mutant M3Rs which were expressed at low levels. Thus, this strategy allowed us to recover full-length mutant M3Rs containing missense mutations that did not lead to major reductions in protein stability.[unreadable] Application of this screening strategy resulted in the recovery of 174 single point mutations that abolished M3R signaling in yeast. Functional studies with transfected mammalian (COS-7) cells showed that the vast majority of these mutant M3Rs (159 out of 174) were either unable or significantly impaired in their ability to induce carbachol-mediated increases in intracellular calcium levels. [unreadable] Previous site-directed mutagenesis studies have identified many amino acids in the M3R and other muscarinic receptor subtypes that play critical roles in muscarinic receptor function. The vast majority of these functionally critical amino acids identified previously were also identified in the present study. These include, for example, amino acids that are highly conserved among class I GPCR and residues known to be important for acetylcholine binding. Taken together, these observations convincingly validated the usefulness of this novel yeast screening strategy.[unreadable] We also recovered many novel point mutations that interfered with M3R function that had not been described previously. Interestingly, the majority of these substitutions involved amino acids located within transmembrane domains I and II (TM I and II), two M3R regions that have not been subjected to systematic site-directed mutagenesis studies in the past. Molecular modeling studies suggested that most of the mutagenized TM I and II residues project into the interior of the receptor protein or towards other TM helices. We hypothesize that these residues are part of network of intramolecular H-bonds and/or hydrophobic interactions predicted to be critical for M3R activation. [unreadable] Recent studies suggest that the second extracellular loop (o2 loop) of class I GPCRs is in close contact with the transmembrane receptor core. In the past, the potential role of the o2 loop in agonist-dependent GPCR activation has not been studied systematically. Using a similar experimental strategy as described by Li et al. (Nat. Methods 4, 169-74, 2007), we found that several residues in the o2 loop of the M3R play important roles in regulating the efficiency of agonist-induced receptor activation (Scarselli et al., J. Biol. Chem. 282, 7385-96, 2007). In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. [unreadable] Given recent advances in yeast expression technology, it should be possible to express most GPCRs in yeast in a coupling-competent form. Thus, the novel experimental strategy that we developed (Nat. Methods 4, 169-74, 2007) should be applicable to many or most mammalian GPCRs. Since GPCRs are known to represent excellent targets for drug therapy, this strategy should be of broad general relevance and considerable clinical interest.