Abstract
Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y1 and P2Y12 purinoceptors. Recently, we demonstrated that P2Y1 and P2Y12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y1 and P2Y12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y1 or P2Y12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.
Highlights
Adenosine diphosphate (ADP) plays a key role in platelet function
In order to study the role of ADP ribosylation factor 6 (ARF6) in either P2Y1 or P2Y12 purinoceptor function and circumvent the inherent methodological problems that this presents in platelets, we initially examined these receptors in a cell line system
Western blotting confirmed that both ARF1-dominant-negative mutant (DNM) and ARF6-DNM are expressed after transient transfection (70–90% cells transfected at 4–5 fold over endogenous ARF6 levels; data not shown)
Summary
Adenosine diphosphate (ADP) plays a key role in platelet function. Following its secretion from platelet-dense granules ADP amplifies the platelet responses induced by other platelet agonists and stabilizes platelet aggregates. ADP activates two platelet G protein-coupled receptors (GPCRs), P2Y1 and P2Y12, which couple respectively to Gq and Gi-mediated pathways, and synergise to induce full platelet aggregation responses to ADP [1,2]. Interaction of ADP with P2Y1 leads to mobilization of intracellular calcium and activation of Rho kinase, resulting in platelet shape change and an initial wave of rapidly reversible aggregation. ADP stimulation of P2Y12 is associated with adenylyl cyclase (AC) inhibition and PI3-kinase activation, resulting in sustained aggregation in synergy with the P2Y1 receptor. Activation of both receptors is required for a full aggregation response to ADP [3]
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