title: Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1.
abstract: Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.
authors.affiliation: Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.
authors.forename: Breann L
authors.initials: BL
authors.lastname: Wolfe
authors.forename: Adriano
authors.initials: A
authors.lastname: Marchese
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000502:physiology*; D034281:Dynamins / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D021381:Protein Transport; D051381:Rats; D044463:Receptor, PAR-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; D002966:Clathrin; D044463:Receptor, PAR-1; D025801:Ubiquitin; D034281:Dynamins