Date of Award

Summer 8-23-2019

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

Advisor

Joshua B. Kelley

Second Committee Member

Dorothy E. Croall

Third Committee Member

Robert E. Gundersen

Abstract

G protein-coupled receptors (GPCRs) are involved in numerous signaling processes ranging from neuronal growth to immune cells tracking invaders. GPCR signaling plays a role in many human diseases and thus GPCRs are important drug targets. Yeast respond to mating pheromone using a GPCR signaling system homologous to those used in humans to polarize their cytoskeleton toward the pheromone source. This is accomplished by initializing a MAPK signaling cascade to arrest the cells in mitosis and upregulate expression of chemotropic proteins. Pathway desensitization is accomplished by the Regulator of G-protein Signaling (RGS). RGS abrogates signaling by binding to the active GPCR, accelerating hydrolysis of GTP bound to G-proteins, bringing them to an inactive state. Previous studies have found the yeast RGS, Sst2, undergoes feedback phosphorylation by the MAPK, though the effect of this modification on RGS function was not determined. We examined the spatiotemporal dynamics of Sst2 using fluorescent live cell imaging in a microfluidics gradient chamber and performed computational image analysis of single cells. We have found that changes in Sst2 localization during the pheromone response are controlled by phosphorylation, removing Sst2 from regions of barrier proteins, known as septins. Furthermore, our data suggests that this phosphorylation event in turn changes the localization of the MAPK. We show that the formin Bni1 is the pheromone responsive formin and is required for endocytic rates to be maintained during chemotropic growth. Finally, we provide evidence that the defects observed in endocytosis may be due to the improper localization of Gα bound MAPK. These results provide insight into previously unknown regulatory roles of the RGS during the yeast pheromone response.

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