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Distinct Roles for Protein Kinase C Isoforms in Regulating Platelet Purinergic Receptor Function

Department of Pharmacology, School of Medical Sciences, University Walk, Bristol, United Kingdom (S.J.M., M.L.J., A.R.H., J.F.B., A.W.P.); Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Bristol, United Kingdom (S.M.B.); and Portola Pharmaceuticals Inc., South San Francisco, California (P.B.C.)

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y₁ and P2Y₁₂. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y₁ is regulated by protein kinase C (PKC) and P2Y₁₂ by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKC and PKC dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y₁ receptor signaling and trafficking, although only PKC was capable of regulating P2Y₁₂, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y₁ and P2Y₁₂ receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y₁₂ function, whereas both novel and classic isoforms regulate P2Y₁ activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y₁ receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y₁₂ receptor is unaffected. Both P2Y₁ and P2Y₁₂ receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y₁₂, whereas both novel and classic isoforms regulate P2Y₁ internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.

Address correspondence to: Dr. Alastair W. Poole, Department of Pharmacology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK. E-mail: a.poole{at}bris.ac.uk

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