Single Amino Acid Substitutions and Deletions That Alter the G Protein Coupling Properties of the V2 Vasopressin Receptor Identified in Yeast by Receptor Random Mutagenesis*

To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical Gs-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 α subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal Gαq sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/Gαs hybrid subunit containing the C-terminal Gαs 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 Met145 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 Met145 led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met145 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.