AFR1P REGULATES THE SACCHAROMYCES-CEREVISIAE ALPHA-FACTOR RECEPTOR BYA MECHANISM THAT IS DISTINCT FROM RECEPTOR PHOSPHORYLATION AND ENDOCYTOSIS

The alpha-factor pheromone receptor activates a G protein signaling pa thway that induces the conjugation of the yeast Saccharomyces cerevisi ae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the recepto r is regulated by Afr1p. To investigate the mechanism of this regulati on, we tested AFR1 for a role in the two processes that are known to r egulate receptor signaling: phosphorylation and down-regulation of lig and-bound receptors by endocytosis. AFR1 overexpression diminished sig naling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylati on. The effects of AFR1 overexpression were weaker in strains that wer e defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to th e number of receptors. These results indicate that AFR1 acts independe ntly of endocytosis, and that the weaker effects of AFR1 in strains th at are defective in receptor endocytosis were probably an indirect con sequence of their increased receptor number caused by the failure of r eceptors to undergo ligand-stimulated endocytosis. Analysis of the lig and binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity fo r alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activ ating G protein signaling by a mechanism that is distinct from other k nown pathways.