Abstract
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 PKCα and PKCδ dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCδ 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.
Footnotes
-
A.R.H. was supported by an A.J. Clark Studentship from the British Pharmacological Society. J.F.B. is supported by a studentship from the British Heart Foundation (grant number FS/04/023). The work was supported by program and project grants from the British Heart Foundation (grant numbers PG/04/097/17620 and RG/05/015) and the Wellcome Trust (grant numbers 064785 and 069572). S.J.M. holds a British Heart Foundation Basic Science Lectureship.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; DNM, dominant-negative mutant; PMA, phorbol 12-myristate 13-acetate; ECL, enhanced chemiluminescence; A3P5P, adenosine-3′,5′-diphosphate; 2MeSADP, 2-methylthioadenosine 5′-diphosphate; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide); Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; PKC, protein kinase C; AM, Ro201724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; AR-C69931MX, 5′-adenylic acid, N-[2-(methylthio)-ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid]; ELISA, enzyme-linked immunosorbent assay.
- Received February 15, 2006.
- Accepted June 27, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|