To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G s -coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein ␣ subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G␣ q sequence

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... . In contrast, the V2 receptor efficiently coupled to a Gpa1p/G␣ s hybrid subunit containing the C-terminal G␣ s sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met 145 in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met 145 led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met 145 had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.