P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms.

Adenosine 5'-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein-coupled receptors P2Y(1) and P2Y(12). Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y(1)- and P2Y(12)-mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein-coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y(12), but not P2Y(1), desensitization is mediated by GRKs. In contrast, desensitization of P2Y(1), but not P2Y(12), is largely dependent on protein kinase C activity. This study is the first to show that both P2Y(1) and P2Y(12) desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered.

[1]  H. Holmsen,et al.  ADP-induced refractory state of platelets in vitro. II. Functional and ultra studies on gel filtered platelets. , 2009, Scandinavian journal of haematology.

[2]  H. Holmsen,et al.  ADP-induced refractory state of platelets in vitro. I. Methodological studies on aggregation in platelet rich plasma. , 2009 .

[3]  H. Holmsen,et al.  Platelet disaggregation and refractoriness to ADP. , 2009, Acta medica Scandinavica. Supplementum.

[4]  J. Benovic,et al.  Bimodal Regulation of the Human H1 Histamine Receptor by G Protein-coupled Receptor Kinase 2* , 2005, Journal of Biological Chemistry.

[5]  R. Challiss,et al.  Non-visual GRKs: are we seeing the whole picture? , 2003, Trends in pharmacological sciences.

[6]  T. K. Harden,et al.  Purification and functional reconstitution of the human P2Y12 receptor. , 2003, Molecular pharmacology.

[7]  S. Kunapuli,et al.  ADP receptors--targets for developing antithrombotic agents. , 2003, Current pharmaceutical design.

[8]  M. Salter,et al.  Differential Frequency Dependence of P2Y1- and P2Y2- Mediated Ca 2+ Signaling in Astrocytes , 2003, The Journal of Neuroscience.

[9]  J. Benovic,et al.  G Protein-coupled Receptor Kinase 2/Gαq/11Interaction: A NOVEL SURFACE ON A REGULATOR OF G PROTEIN SIGNALING HOMOLOGY DOMAIN FOR BINDING Gα SUBUNITS , 2003 .

[10]  J. Stankova,et al.  Agonist-induced Internalization of the Platelet-activating Factor Receptor Is Dependent on Arrestins but Independent of G-protein Activation , 2002, The Journal of Biological Chemistry.

[11]  T. Saito,et al.  Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor. , 2001, Molecular pharmacology.

[12]  S. Mundell,et al.  Rapid Agonist-induced Desensitization and Internalization of the A2B Adenosine Receptor Is Mediated by a Serine Residue Close to the COOH Terminus* , 2001, The Journal of Biological Chemistry.

[13]  C. Gachet ADP Receptors of Platelets and their Inhibition , 2001, Thrombosis and Haemostasis.

[14]  S. Ferguson,et al.  Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. , 2001, Pharmacological reviews.

[15]  David Julius,et al.  Identification of the platelet ADP receptor targeted by antithrombotic drugs , 2001, Nature.

[16]  Carol L. Williams,et al.  P2Y2 Purinergic and M3 Muscarinic Acetylcholine Receptors Activate Different Phospholipase C-β Isoforms That Are Uniquely Susceptible to Protein Kinase C-dependent Phosphorylation and Inactivation* , 2000, The Journal of Biological Chemistry.

[17]  J. Cazenave,et al.  Desensitization of the Platelet Aggregation Response to ADP: Differential Down-regulation of the P2Y1 and P2cyc Receptors , 2000, Thrombosis and Haemostasis.

[18]  S. Mundell,et al.  Selective Regulation of Endogenous G Protein-coupled Receptors by Arrestins in HEK293 Cells* , 2000, The Journal of Biological Chemistry.

[19]  A. Schonbrunn,et al.  Protein Kinase C Activation Stimulates the Phosphorylation and Internalization of the sst2A Somatostatin Receptor* , 2000, The Journal of Biological Chemistry.

[20]  Patricia D. Christie,et al.  Targeted disruption of cd39/ATP diphosphohydrolase results in disordered hemostasis and thromboregulation , 1999, Nature Medicine.

[21]  B. J. Knoll,et al.  Partial agonists and G protein-coupled receptor desensitization. , 1999, Trends in pharmacological sciences.

[22]  J. Benovic,et al.  Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor. , 1999, Molecular endocrinology.

[23]  L. L. Lin,et al.  Protein kinase C epsilon-dependent pathway of extracellular signal-regulated protein kinase activation by P2Y1 and P2Y2 purinoceptors that activate cytosolic phospholipase A2 in endothelial cells. , 1999, European journal of pharmacology.

[24]  P. Devreotes,et al.  Desensitization of G-protein-coupled Receptors , 1999, The Journal of Biological Chemistry.

[25]  T. K. Harden,et al.  Agonist action of adenosine triphosphates at the human P2Y1 receptor. , 1998, Molecular pharmacology.

[26]  S. Mundell,et al.  A dominant negative mutant of the G protein-coupled receptor kinase 2 selectively attenuates adenosine A2 receptor desensitization. , 1997, Molecular pharmacology.

[27]  P. Mannucci,et al.  Deficiency of (33P)2MeS-ADP Binding Sites on Platelets with Secretion Defect, Normal Granule Stores and Normal Thromboxane A2 Production , 1997, Thrombosis and Haemostasis.

[28]  S. Coughlin,et al.  Role of the Thrombin Receptor's Cytoplasmic Tail in Intracellular Trafficking , 1996, The Journal of Biological Chemistry.

[29]  J. Benovic,et al.  Differentially regulated expression of the G-protein-coupled receptor kinases, betaARK and GRK6, during myelomonocytic cell development in vitro. , 1996, Blood.

[30]  J. Cazenave,et al.  Cloning and sequencing of a human cDNA encoding endothelial P2Y1 purinoceptor. , 1996, Gene.

[31]  T. K. Harden,et al.  Second messenger cascade specificity and pharmacological selectivity of the human P2Y1‐purinoceptor , 1996, British journal of pharmacology.

[32]  A. Strosberg,et al.  New molecular and structural determinants involved in beta 2-adrenergic receptor desensitization and sequestration. Delineation using chimeric beta 3/beta 2-adrenergic receptors. , 1996, The Journal of biological chemistry.

[33]  E. Barnard,et al.  Cloning and chromosomal localization of the human P2Y1 purinoceptor. , 1996, Biochemical and biophysical research communications.

[34]  S. Watson,et al.  Regulation of cytosolic calcium by collagen in single human platelets , 1995, British journal of pharmacology.

[35]  R. Clark,et al.  cAMP-dependent protein kinase and protein kinase C consensus site mutations of the beta-adrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase. , 1994, The Journal of biological chemistry.

[36]  J. Benovic,et al.  A beta-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the beta 2-adrenergic receptor. , 1994, The Journal of biological chemistry.

[37]  G. Burnstock,et al.  Cloning and functional expression of a brain G‐protein‐coupled ATP receptor , 1993, FEBS letters.

[38]  M. Heath,et al.  ADP induces desensitisation of equine platelet aggregation responses: studies using ADP beta S, a stable analogue of ADP. , 1993, Research in veterinary science.

[39]  J. Faure,et al.  Immunopurification of an S-antigen-like protein from human platelets. , 1991, Thrombosis research.

[40]  M. Caron,et al.  Turning off the signal: desensitization of β‐adrenergic receptor function , 1990, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[41]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[42]  S. Hourani,et al.  PARTIAL AGONIST BEHAVIOUR OF ADENOSINE 5′O‐(2‐THIODIPHOSPHATE) ON HUMAN PLATELETS , 1981, British journal of pharmacology.

[43]  S. Niewiarowski,et al.  Proceedings: Effect of ADP and thrombin on fibrin retraction induced by human platelets and fibroblasts. , 1975, Thrombosis et diathesis haemorrhagica.

[44]  M. Etherington,et al.  Refractory state of platelet aggregation with major operations. , 1971, Lancet.

[45]  J. Mustard,et al.  Influence of Apyrase on Stability of Suspensions of Washed Rabbit Platelets , 1971, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[46]  J. Benovic,et al.  G protein-coupled receptor Kinase 2/G alpha q/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding G alpha subunits. , 2003, The Journal of biological chemistry.

[47]  M. Salter,et al.  Differential Frequency Dependence of P 2 Y 1-and P 2 Y 2-Mediated Ca 2 Signaling in Astrocytes , 2003 .

[48]  J. Benovic,et al.  The role of receptor kinases and arrestins in G protein-coupled receptor regulation. , 1998, Annual review of pharmacology and toxicology.

[49]  M. Mancilla,et al.  Properties of two apyrases from Solanum tuberosum , 1982 .

[50]  H. Holmsen,et al.  ADP-Induced Refractory State of Platelets in Vitro , 1975, Thrombosis and Haemostasis.