Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of (cid:2) -arrestin

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4)hasbeenshowntodimerizewiththepurinergicreceptor P2Y12 to coordinate (cid:2) -arrestin–mediated Akt signaling, an important mediator of integrin activation. However, the mech-anismbywhichthePAR4-P2Y12dimercontrols (cid:2) -arrestin–de-pendent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for (cid:2) arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation incellsexpressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer.

[1]  J. Trejo,et al.  Challenges and Opportunities in Protease-Activated Receptor Drug Development. , 2017, Annual review of pharmacology and toxicology.

[2]  J. Guay,et al.  Blockade of protease-activated receptor-4 (PAR4) provides robust antithrombotic activity with low bleeding , 2017, Science Translational Medicine.

[3]  Thomas H. Smith,et al.  Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins* , 2016, The Journal of Biological Chemistry.

[4]  P. Gurbel,et al.  Vorapaxar in the secondary prevention of atherothrombosis , 2015, Expert review of cardiovascular therapy.

[5]  M. Cattaneo,et al.  P2Y12 receptors: structure and function , 2015, Journal of thrombosis and haemostasis : JTH.

[6]  Aasma A Khan,et al.  The Physical Association of the P2Y12 Receptor with PAR4 Regulates Arrestin-Mediated Akt Activation , 2014, Molecular Pharmacology.

[7]  A. Arachiche,et al.  Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin* , 2013, The Journal of Biological Chemistry.

[8]  Thomas H. Smith,et al.  Cofactoring and Dimerization of Proteinase-Activated Receptors , 2013, Pharmacological Reviews.

[9]  J. Trejo,et al.  Transactivation of the PAR1-PAR2 Heterodimer by Thrombin Elicits β-Arrestin-mediated Endosomal Signaling* , 2013, The Journal of Biological Chemistry.

[10]  N. Hay,et al.  ADP-Stimulated Activation of Akt During Integrin Outside-In Signaling Promotes Platelet Spreading by Inhibiting Glycogen Synthase Kinase-3&bgr; , 2012, Arteriosclerosis, thrombosis, and vascular biology.

[11]  S. Mundell,et al.  Arrestin Scaffolds NHERF1 to the P2Y12 Receptor to Regulate Receptor Internalization* , 2012, The Journal of Biological Chemistry.

[12]  Michael R Dores,et al.  ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting , 2012, The Journal of cell biology.

[13]  Marc P. Bonaca,et al.  Vorapaxar in the secondary prevention of atherothrombotic events. , 2012, The New England journal of medicine.

[14]  D. Woulfe,et al.  Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets* , 2010, The Journal of Biological Chemistry.

[15]  Meryem Köse,et al.  High-affinity, non-nucleotide-derived competitive antagonists of platelet P2Y12 receptors. , 2009, Journal of medicinal chemistry.

[16]  Pascual Ferrara,et al.  The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Eric Reiter,et al.  GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling , 2006, Trends in Endocrinology & Metabolism.

[18]  D. Siderovski,et al.  Clathrin Adaptor AP2 Regulates Thrombin Receptor Constitutive Internalization and Endothelial Cell Resensitization , 2006, Molecular and Cellular Biology.

[19]  C. Derian,et al.  Blocking the Protease-Activated Receptor 1-4 Heterodimer in Platelet-Mediated Thrombosis , 2006, Circulation.

[20]  S. Marley,et al.  Targeting primary human leukaemia cells with RNA interference: Bcr‐Abl targeting inhibits myeloid progenitor self‐renewal in chronic myeloid leukaemia cells , 2005, British journal of haematology.

[21]  R. Kriz,et al.  N‐linked glycosylation of platelet P2Y12 ADP receptor is essential for signal transduction but not for ligand binding or cell surface expression , 2004, FEBS letters.

[22]  Michel Bouvier,et al.  Heterodimerization of V1a and V2 vasopressin receptors determines the interaction with beta-arrestin and their trafficking patterns. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Kuliopulos,et al.  Protease-activated receptor-4 uses dual prolines and an anionic retention motif for thrombin recognition and cleavage. , 2003, The Biochemical journal.

[24]  M. Freund,et al.  Differential Involvement of the P2Y1 and P2Y12 Receptors in Platelet Procoagulant Activity , 2003, Arteriosclerosis, thrombosis, and vascular biology.

[25]  P. Conley,et al.  P2Y12 regulates platelet adhesion/activation, thrombus growth, and thrombus stability in injured arteries. , 2003, The Journal of clinical investigation.

[26]  P. Nurden,et al.  Role of ADP Receptor P2Y12 in Platelet Adhesion and Thrombus Formation in Flowing Blood , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[27]  T. Kohout,et al.  β-Arrestins Regulate Protease-activated Receptor-1 Desensitization but Not Internalization or Down-regulation* , 2002, The Journal of Biological Chemistry.

[28]  S. Coughlin,et al.  Protease-activated Receptors 1 and 4 Are Shut Off with Distinct Kinetics after Activation by Thrombin* , 2000, The Journal of Biological Chemistry.

[29]  S. Coughlin,et al.  Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. , 1999, The Journal of clinical investigation.

[30]  Robert V Farese,et al.  A dual thrombin receptor system for platelet activation , 1998, Nature.

[31]  Scott R. Presnell,et al.  Cloning and characterization of human protease-activated receptor 4. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Stephen S. G. Ferguson,et al.  Synergistic Regulation of β2-Adrenergic Receptor Sequestration: Intracellular Complement of β-Adrenergic Receptor Kinase and β-Arrestin Determine Kinetics of Internalization , 1997 .

[33]  V. Wheaton,et al.  Domains specifying thrombin–receptor interaction , 1991, Nature.

[34]  V. Wheaton,et al.  Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation , 1991, Cell.

[35]  R. Abagyan,et al.  A novel approach to quantify G-protein-coupled receptor dimerization equilibrium using bioluminescence resonance energy transfer. , 2013, Methods in molecular biology.