Molecular basis of V2 vasopressin receptor/Gs coupling selectivity.

The molecular mechanisms governing the coupling selectivity of G protein-coupled receptors activated by peptide ligands are not well understood. To shed light on this issue, we have used the Gq/11-linked V1a and the Gs-coupled V2 vasopressin peptide receptors as model systems. To explore the structural basis underlying the ability of the V2 receptor to selectively recognize Gs, we systematically substituted distinct V2 receptor segments (or single amino acids) into the V1a receptor and studied whether the resulting hybrid receptors gained the ability to mediate hormone-dependent cAMP production. This strategy appeared particularly attractive since hormone stimulation of the V1a receptor has virtually no effect on intracellular cAMP levels. Functional analysis of a large number of mutant receptors transiently expressed in COS-7 cells indicated that the presence of V2 receptor sequence at the N terminus of the third intracellular loop is critical for efficient activation of Gs. More detailed mutational analysis of this receptor region showed that two polar V2 receptor residues, Gln225 and Glu231, play key roles in Gs recognition. In addition, a short sequence at the N terminus of the cytoplasmic tail was found to make an important contribution to V2 receptor/Gs coupling selectivity. We also made the novel observation that the efficiency of V2 receptor/Gs coupling can be modulated by the length of the central portion of the third intracellular loop (rather than the specific amino acid sequence within this domain). These findings provide novel insights into the molecular mechanisms regulating peptide receptor/G protein coupling selectivity.

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast.

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.

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 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 alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(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.