Characterization of protease-activated receptor-4 trafficking and heterodimerization in modulating receptor signaling

G protein-coupled receptors (GPCRs) are transmembrane proteins that allow cells to respond to extracellular stimuli. GPCR activation occurs when a ligand binds to the extracellular portion of the receptor. The ligand-bound receptor undergoes a conformational change that allows the intracellular domain of the receptor to engage and activate signaling effectors. The protease-activated receptors (PARs) are a family of GPCRs that are activated by proteases such as thrombin. Unlike traditional GPCRs, which can return to their inactive state after signaling, PARs are irreversibly activated. Therefore, the eventual fate of an activated PAR is degradation. The internalization and endocytic sorting of PARs play key roles in their signal regulation.Trafficking has been well-characterized for PAR1, the prototypical thrombin receptor. PAR4 was the last of the four thrombin receptor to be discovered, and has in large part been relatively under-studied. Recent studies have shown that PAR4 plays distinct roles that differ from those of PAR1, and as such may represent a potential drug target. Thus, understanding the mechanisms that regulate PAR4 signal regulation is important.In this dissertation, I characterized the role that trafficking and heterodimerization play in regulating PAR4 signaling. I found that adaptor- protein complex-2 (AP-2) is a key mediator of PAR4 agonist-induced internalization, and that AP-2 binds to intracellular loop 3 (ICL3) of PAR4. Disruption of PAR4 internalization resulted in enhanced and prolonged ERK1/2 activation, suggesting that endocytosis may mediate PAR4 signal attenuation.I also characterized the role of heterodimerization with the puringeric receptor P2Y12 in modulating PAR4 signaling. Previous studies had demonstrated that PAR4 and P2Y12 coordinate β-arrestin-dependent Akt activation. My work strongly suggests that PAR4 and P2Y12 physically associate basally and co-internalize in response to activation of PAR4. I also demonstrate that β-arrestin is recruited to the co-internalized PAR4-P2Y12 complex. In contrast to the effect on ERK1/2 activation, disruption of PAR4 internalization diminished Akt activation. Taken together, the studies summarized in this dissertation highlight the importance of PAR4 internalization in modulating activity of functionally and spatially distinct signaling pathways