MECHANISMS OF SIGNAL TRANSDUCTION
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*

https://doi.org/10.1074/jbc.M103203200Get rights and content
Under a Creative Commons license
open access

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.

Cited by (0)

Published, JBC Papers in Press, May 25, 2001, DOI 10.1074/jbc.M103203200

*

This work was supported by a grant from the Deutscher Akademischer Austauschdienst (NATO) (to I. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.