| Popis: |
G-protein coupled receptor (GPCR) signaling is fundamental to various cellular processes such as cardiac contractility, immune response and gradient sensing. Budding yeast, Saccharomyces cerevisiae, detect gradients of mating pheromone as shallow as 1-5% across their diameter and generate appropriate morphological response. Receptor polarization has been implicated in pheromone gradient sensing and recent work has suggested that endocytosis is important in establishing polarity of membrane proteins. However, the precise mechanisms that regulate the endocytosis of the receptor Ste2 to establish a polarized receptor distribution have not been fully characterized. The Regulator of G-protein Signaling (RGS) protein, Sst2, negatively regulates G-protein activity and facilitates mating pathway desensitization in yeast. It has been previously suggested that Sst2 interacts with the receptor Ste2 through its DEP domains. In the chapters that follow, we use an integrated approach that combines computational and experimental methods to demonstrate that following pheromone stimulation, Sst2 inhibits Ste2 endocytosis and stabilizes it at the plasma membrane. We also show that disrupting the binding interaction between Sst2 and Ste2 by a point mutation Q304N in Sst2 leads to loss of Ste2 polarization. Our mathematical model predicts pheromone-induced synthesis of Sst2 as a key factor in establishing Ste2 polarization. We verify this prediction by replacing the endogenous promoter of SST2 with a promoter that is not responsive to pheromone. Thus, in this work, we show that by inhibiting Ste2 endocytosis, Sst2 plays a positive role in the yeast pheromone response. By discovering a novel positive role for a negative regulator, this work has provided an improved understanding of how RGS proteins and GPCRs interact in yeast. GPCR signaling is a highly conserved pathway and RGS proteins bind GPCRs even in mammalian systems. Therefore, the inhibition of GPCR internalization by RGS proteins could be of a general occurrence. Since RGS proteins are potential drug targets, in addition to GPCRs, an improved understanding of RGS-GPCR interaction, as provided in this work, will greatly benefit drug discovery. |
