Dominant-negative inhibition of pheromone receptor signaling by a single point mutation in the G protein alpha subunit.
In yeast, two different constitutive mutants of the G protein alpha subunit have been reported. Gpa1(Q323L) cannot hydrolyze GTP and permanently activates the pheromone response pathway. Gpa1(N388D) was also proposed to lack GTPase activity, yet it has an inhibitory effect on pheromone responsiveness. We have characterized this inhibitory mutant (designated Galpha(ND)) and found that it binds GTP, interacts with G protein betagamma subunits, and exhibits full GTPase activity in vitro. Although pheromone leads to dissociation of the receptor from wild-type G protein, the same treatment promotes stable association of the receptor with Galpha(ND). We conclude that agonist binding to the receptor promotes the formation of a nondissociable complex with Galpha(ND), and in this manner prevents activation of the endogenous wild-type G protein. Dominant-negative mutants may be useful in matching specific receptors and their cognate G proteins and in determining mechanisms of G protein signaling specificity.
Wu
Yuh-Lin
YL
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260, USA.
Hooks
Shelley B
SB
Harden
T Kendall
TK
Dohlman
Henrik G
HG
D004926:Escherichia coli; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D017354:Point Mutation; D043563:Receptors, Pheromone / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
D044385:GTP-Binding Protein alpha Subunits; D043563:Receptors, Pheromone; D029701:Saccharomyces cerevisiae Proteins; C084293:GPA1 protein, S cerevisiae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11