UC San Diego

G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequent downstream signaling. Protease-activated receptor-1 (PAR1) is a member of a family of GPCRs that are proteolytically activated. PAR1 is a receptor for the coagulant protease thrombin, and is capable of coupling to multiple G-protein subtypes to elicit various cellular responses; however, the mechanisms that regulate this selectivity are not well understood. Palmitoylation is a post-translational modification that many GPCRs possess, and is defined as the addition of palmitate, a 16 carbon fatty acid, to a cysteine residue via a thioester linkage. Many GPCRs are palmitoylated on their C-terminal tails, but the role of this modification differs based on the GPCR being examined. In this dissertation, I examined the role of palmitoylation in PAR1 signaling and trafficking. I defined the sites of PAR1 palmitoylation to occur on conserved C-tail cysteine residues C387 and C388. I discovered that palmitoylation is important for other PAR1 post-translational modifications, specifically phosphorylation and ubiquitination. I also show that palmitoylation of PAR1 regulates the accessibility of a nearby tyrosine-based sorting motif to the adaptor-protein complex-2 (AP-2) and -3 (AP-3), which controls receptor internalization and degradation. Additionally, palmitoylation appears to be important for the regulation of selective PAR1-induced signaling pathways such as G[alpha]₁₂/₁₃ -induced RhoA activation, G[alpha]i coupling , and thrombin-stimulated p38 MAPK signaling pathways. However, thrombin-induced G[alpha]q-mediated phosphoinositide hydrolysis and ERK1/2 activation are unperturbed in the absence of PAR1 palmitoylation. Taken together, the studies summarized in this dissertation highlight the relevance of palmitoylation for PAR1 function, and suggest that palmitoylation governs a C-tail conformation that is important for accessibility of other proteins such as ligases, kinases, adaptor proteins, and G -proteins, ultimately regulating PAR1 signaling and trafficking