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.
Erlenbach
I
I
Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kostenis
E
E
Schmidt
C
C
Serradeil-Le Gal
C
C
Raufaste
D
D
Dumont
M E
ME
Pausch
M H
MH
Wess
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D001483:Base Sequence; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D003001:Cloning, Molecular; D017931:DNA Primers; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015723:Gene Library; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D044385:GTP-Binding Protein alpha Subunits; D009838:Oligodeoxyribonucleotides; D021122:Protein Subunits; D017483:Receptors, Vasopressin; D029701:Saccharomyces cerevisiae Proteins; D001127:Arginine Vasopressin; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins