Collections > Electronic Theses and Dissertations > Clathrin-dependent Internalization of Protease-activated Receptor-1: Regulation by Ubiquitination and Epsin

Thrombin, the main effector protease of the coagulation cascade elicits its cellular effects via activation of a family of G-protein coupled receptors (GPCRs) called proteaseactivated receptors (PARs). PAR1, the prototype of this family, is expressed in cells in and around blood vessels and mediates thrombin elicited cellular responses that are essential for hemostasis and thrombosis, embryonic development, and cancer progression. Thrombin binds to and cleaves the amino-terminal exodomain of PAR1, thus exposing a newly formed amino-terminus that binds intramolecularly to the body of the receptor and causes transmembrane signaling. Due to the irreversible, proteolytic mechanism of PAR1 activation, processes that contribute to signal termination, including desensitization, internalization and downregulation, are critically important for thrombin-elicited cellular responses. PAR1 displays two modes of receptor trafficking. Constitutive PAR1 internalization is mediated by the adaptor protein complex-2 (AP2), but is independent of arrestins, and is important for resensitization of thrombin signaling. Activated PAR1 internalizes by a clathrin- and dynamin-dependent pathway that is independent of arrestins and AP2 and is then sorted directly to lysosomes for degradation, a process that is important for signal termination. Studies of mammalian and yeast GPCRs have demonstrated a role for ubiquitination in trafficking of GPCRs. A major focus of this dissertation was to understand the role of ubiquitination in PAR1 trafficking. We found that PAR1 is basally ubiquitinated and undergoes agonist-induced deubiquitination. Ubiquitination of PAR1 negatively regulates constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 ubiquitination appears to inhibit binding of AP2 to PAR1 and therefore helps to retain PAR1 at the cell surface. The clathrin adaptor protein epsin binds to activated and ubiquitinated PAR1 to mediate receptor internalization. A deubiquitinated form of the receptor is sorted to lysosomes by a ubiquitin-independent mechanism that requires sorting nexin-1. These studies reveal a novel function for ubiquitination in regulation of GPCR trafficking.