A limited spectrum of mutations causes constitutive activation of the yeast alpha-factor receptor

Activation of G protein coupled receptors (GPCRs) by binding of ligand is t he initial event in diverse cellular signaling pathways. To examine the fre quency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant r eceptor alleles that exhibit signaling in the absence of added ligand, 14 d ifferent point mutations were isolated. All of these 14 mutants could be fu rther activated by alpha-factor. Ten of the mutants also acquired the abili ty to signal in response to binding of desTrp(1)[Ala(3)]alpha-factor, a pep tide that acts as an antagonist toward normal alpha-factor receptors. Of th ese 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signalin g. The remaining alleles are hypersensitive to alpha-factor rather than con stitutive. They can be activated by low concentrations of endogenous alpha- factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and ex tensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limi ted number of mutations is capable of causing constitutive activation of th is receptor. Constitutive and hypersensitive signaling by the mutant recept ors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.