title: Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins.
abstract: Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms.
authors.affiliation: From the Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Grimsey
authors.affiliation: From the Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Luisa J
authors.initials: LJ
authors.lastname: Coronel
authors.affiliation: From the Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Isabel Canto
authors.initials: IC
authors.lastname: Cordova
authors.affiliation: From the Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093 joanntrejo@ucsd.edu.
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D001343:Autophagy; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D015723:Gene Library; D006367:HeLa Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
chemical_list: D050556:Fatty Acid-Binding Proteins; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; D043562:Receptors, G-Protein-Coupled; D013917:Thrombin; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C493410:FABP4 protein, human
keywords: G protein-coupled receptor; autophagy; endothelial cell; lysosome; thrombin; trafficking