Regulation of Saccharomyces cerevisiae Mating Pheromone Response: G-protein-coupled Receptor Ste2 Down-modulation by Specific alpha-Arrestins

The yeast G-protein-coupled receptor (GPCR) Ste2 is an integral plasma membrane protein that initiates response to an extracellular stimulus (the peptide mating pheromone α-factor) by mediating ligand-dependent activation of a cognate heterotrimeric G-protein. Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest. In the research described in this dissertation, I found that three α-arrestins — Rod1/Art4, Rog3/Art7 and Ldb19/Art1 — serve as adaptors to promote the ubiquitinylation-dependent internalization of Ste2 and block its ability to signal, thereby desensitizing the cells to continued stimulation. Deleting the genes encoding these three α-arrestins increases the sensitivity of a MATa haploid cell to mating pheromone and results in an increase in Ste2 abundance at the plasma membrane. Conversely, overexpression of either Rod1 or Rog3 enhances the rate of adaptation. To contribute to negative regulation of the mating pheromone response pathway, Ldb19 requires binding of a HECT domain class of ubiquitin ligase, Rsp5, and most likely clears misfolded Ste2 from the PM. I found that Rod1 and Rog3 contribute to clearance of the pheromone-bound state of Ste2 and negatively regulate the mating pheromone response pathway by both Rsp5-dependent and Rsp5-independent mechanisms. In addition, I identified two classes of protein kinases (Snf1/AMPK and Ypk1/SGK) that phosphorylate and inactivate Rod1. Conversely, I showed that the phosphoprotein phosphatase responsible for dephosphorylating and re-activating Rod1 is calcineurin / PP2B. Because the S. cerevisiae genome does not encode any β-arrestins, the findings I made and present in this dissertation are the first to show that α-arrestins alone are capable of negatively regulating a GPCR.