title: Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.
abstract: Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.
authors.affiliation: Biomedical Sciences Graduate Program, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Isabel
authors.initials: I
authors.lastname: Canto
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D033963:Adaptor Protein Complex 3 / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D009154:Mutation; D021381:Protein Transport / Q000502:physiology; D059748:Proteolysis; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D044463:Receptor, PAR-1
keywords: 7-Helix Receptor; Adaptor Proteins; Lysosomes; Protein Palmitoylation; Receptor Endocytosis; Thrombin; Trafficking