Scanning mutagenesis of regions in the Galpha protein Gpa1 that are predicted to interact with yeast mating pheromone receptors.
The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Galpha protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Galpha are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (beta2-beta3, alpha2-beta4, alpha3-beta5, and alpha4-beta6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gbetagamma. However, the constitutive activity caused by the F344C and E335C mutations in the alpha2-beta4 loop and F378C in the alpha3-beta5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gbetagamma. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the beta2-beta3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Galpha contribute to activation of signaling.
Gladue
Douglas P
DP
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794-5222, USA.
Konopka
James B
JB
D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics* / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D016296:Mutagenesis; D010675:Pheromones / Q000378:metabolism*; D043563:Receptors, Pheromone / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
D044385:GTP-Binding Protein alpha Subunits; D010675:Pheromones; D043563:Receptors, Pheromone; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins