Loss of sustained Fus3p kinase activity and the G₁ arrest response in cells expressing an inappropriate pheromone receptor

The yeast pheromone response pathway is mediated by two G protein- linked receptors, each of which is expressed only in its specific cell type. The STE3(DAF) mutation results in inappropriate expression of the a-factor receptor in MATa cells. Expression of this receptor in the inappropriate cell type confers resistance to pheromone-induced G₁ arrest, a phenomenon that we have termed receptor inhibition. The ability of STE3(DAF) cells to cycle in the presence of pheromone was found to correlate with reduced phosphorylation of the cyclin-dependent kinase inhibitor Far1p. Measurement of Fus3p mitogen- activated protein (MAP) kinase activity in wild-type and STE3(DAF) cells showed that induction of Fus3p activity was the same in both strains at times of up to 1 h after pheromone treatment. However, after 2 or more hours, Fus3p activity declined in STE3(DAF) cells but remained high in wild-type cells. The level of inducible FUS1 RNA paralleled the changes seen in Fus3p activity. Short-term activation of the Fus3p MAP kinase is therefore sufficient for the early transcriptional induction response to pheromone, but sustained activation is required for cell cycle arrest. Escape from the cell cycle arrest response was not seen in wild-type cells treated with low doses of pheromone, indicating that receptor inhibition is not simply a result of weak signaling but rather acts selectively at late times during the response. STE3(DAF) was found to inhibit the pheromone response pathway at a step between the G(β) subunit and Ste5p, the scaffolding protein that hinds the components of the MAP kinase phosphorylation cascade. Overexpression of Ste20p, a kinase thought to act between the G protein and the MAP kinase cascade, suppressed the STE(DAF) phenotype. These findings are consistent with a model in which receptor inhibition acts by blocking the signaling pathway downstream of G protein dissociation and upstream of MAP kinase cascade activation, at a step that could directly involve Ste20p.