Effects of mutations in the N terminal region of the yeast G protein alpha-subunit Gpa1p on signaling by pheromone receptors.
The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the Galpha subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of Galpha as well. We screened for mutations in the N-terminal region of the Galpha subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast alpha-mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones alpha-factor and a-factor without affecting cellular levels of the subunit or its ability to interact with beta and gamma subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast Galpha subunit does not undergo extensive interactions with the alpha-factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.
Roginskaya
M
M
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, PO Box 712, Rochester, NY 14642, USA.
Connelly
S M
SM
Kim
K S
KS
Patel
D
D
Dumont
M E
ME
D000483:Alleles; D005453:Fluorescence; D005656:Fungal Proteins / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D015151:Immunoblotting; D008565:Membrane Proteins; D016296:Mutagenesis; D020125:Mutation, Missense / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D043563:Receptors, Pheromone / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics* / Q000502:physiology
C490024:FUS1 protein, S cerevisiae; D005656:Fungal Proteins; D044385:GTP-Binding Protein alpha Subunits; D008565:Membrane Proteins; D043563:Receptors, Pheromone; D029701:Saccharomyces cerevisiae Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11