Feedback Phosphorylation of the Yeast a-Factor Receptor Requires Activation of the Downstream Signaling Pathway from G Protein through Mitogen-Activated Protein Kinase

The two yeast pheromone receptors, the a and α-factor receptors, share many functional similarities: both G protein-coupled receptors couple to the same downstream signal transduction pathway, and both receptors undergo feedback regulation involving increased phosphorylation on their C-terminal domains in response to ligand challenge. The present work, which focuses on the signaling mechanism controlling this feedback phosphorylation, indicates one striking difference. While the α-factor-induced phosphorylation of the α-factor receptor does not require activation of the downstream G protein-directed signaling pathway (B. Zanolari, S. Raths, B. Singer-Kruger, and H. Riezman, Cell 71:755–763, 1992), the a-factor-induced phosphorylation of the a-factor receptor (Ste3p) clearly does. Induced Ste3p phosphorylation was blocked in cells with disruptions of various components of the pheromone response pathway, indicating a requirement of pathway components extending from the G protein down through the mitogen-activated protein kinase (MAPK). Furthermore, Ste3p phosphorylation can be induced in the absence of the a-factor ligand when the signaling pathway is artificially activated, indicating that the liganded receptor is not required as a substrate for induced phosphorylation. While the activation of signaling is critical for the feedback phosphorylation of Ste3p, pheromone-induced gene transcription, one of the major outcomes of pheromone signaling, appears not to be required. This conclusion is indicated by three results. First,ste12 Δ cells differ from cells with disruptions of the upstream signaling elements (e.g.,ste4 Δ,ste20 Δ,ste5 Δ,ste11 Δ,ste7 Δ, orfus3Δ kss1 Δ cells) in that they clearly retain some capacity for inducing Ste3p phosphorylation. Second, while activated alleles ofSTE11 andSTE12 induce a strong transcriptional response, they fail to induce a-factor receptor phosphorylation. Third, blocking of new pheromone-induced protein synthesis with cycloheximide fails to block phosphorylation. These findings are discussed within the context of a recently proposed model for pheromone signaling (P. M. Pryciak and F. A. Huntress, Genes Dev. 12:2684–2697, 1998): a key step of this model is the activation of the MAPK Fus3p through the Gβγ -dependent relocalization of the Ste5p-MAPK cascade to the plasma membrane. Ste3p phosphorylation may involve activated MAPK Fus3p feeding back upon plasma membrane targets.