title: Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins.
abstract: Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation.
authors.affiliation: From the Biomedical Sciences Graduate Program and Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Smith
authors.affiliation: 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: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Julia G
authors.initials: JG
authors.lastname: Li
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California 92093, Department of Biology, Hofstra University, Hempstead, New York 11549, and.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Dores
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44016.
authors.forename: Marvin T
authors.initials: MT
authors.lastname: Nieman
authors.affiliation: 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: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D021381:Protein Transport / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins / Q000235:genetics / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; D018179:Receptors, Thrombin; D000071557:beta-Arrestins; D051057:Proto-Oncogene Proteins c-akt; C112908:protease-activated receptor 4
keywords: Akt; G protein-coupled receptor (GPCR); adaptor protein complex-2; arrestin; clathrin; extracellular-signal-regulated kinase (ERK); lysosome; megakaryocyte; thrombin