Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Pharmacology and Toxicology

Supervisor

Dr. Peter Chidiac

Abstract

Signal transduction via heterotrimeric G proteins in response to transmembrane G protein-coupled receptors plays a central aspect in how cells integrate extracellular stimuli and produce biological responses. In addition to receptor-mediated activation of heterotrimeric G proteins, during the last few decades, accessory proteins have been found to regulate G protein activity via different mechanisms. Several proteins have been identified that contain multiple G protein regulatory domains. Using various molecular and biochemical approaches, we have characterized the effects of two such proteins, G18 and RGS14, on G protein activity. Both proteins contain a second G protein binding domain in addition to the GoLoco domain, which primarily acts as a guanine nucleotide dissociation inhibitor (GDI) on Gi/o proteins. Our results identified that the N-terminal region of G18 is a novel G protein-interacting domain, which may have distinct regulatory effects within the Gi/o subfamily and could potentially play a role in differentiating signals between these related G proteins. In addition, we characterized the tissue and cellular distribution of G18. We found that G18 is expressed in primary isolated rat aortic smooth muscle cells and endothelial cells. A protein-protein interaction assay indicated that G18 is able to directly interact with RGS5, an RGS protein that is also highly expressed in vascular tissue. This interaction results in an increase in RGS5 GTPase accelerating protein (GAP) activity with little or no effect on G18 activity. In Chapter 4 of the thesis, we identified a novel GAP enhancing domain located at the Ras-binding (RB) region of RGS14. This enhancement may be due to the intramolecular interaction between the RB domain and the RGS domain. Furthermore, this interaction may also result in an inhibitory effect on the GDI activity of the RGS14 GoLoco motif. Overall, my work suggests that GoLoco motif containing proteins G18 and RGS14 are organizers of G protein signaling that also modulate RGS protein function.

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