title: Adaptor protein complex-2 (AP-2) and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals.
abstract: Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Buxin
authors.initials: B
authors.lastname: Chen
authors.forename: Michael R
authors.initials: MR
authors.lastname: Dores
authors.forename: Neil
authors.initials: N
authors.lastname: Grimsey
authors.forename: Isabel
authors.initials: I
authors.lastname: Canto
authors.forename: Breann L
authors.initials: BL
authors.lastname: Barker
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D021382:Protein Sorting Signals; D021381:Protein Transport; D044463:Receptor, PAR-1 / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D033962:Adaptor Protein Complex 2; D033942:Adaptor Proteins, Vesicular Transport; D021382:Protein Sorting Signals; D044463:Receptor, PAR-1; C114154:epsin