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. IntroductionPlatelets are activated by a variety of extracellular stimuli and are an essential component of the normal response to vascular injury. Central among these stimuli is adenosine 5Ј-diphosphate (ADP), which induces multiple platelet responses and potentiates platelet aggregation to other agonists (for reviews, see Gachet 1 and Kunapuli et al 2 ). Indeed, since it was recognized 40 years ago, ADP has been regarded as a central mediator of hemostasis and thrombosis by providing a positive feedback mechanism for the activation of platelets by multiple agonists. For example, both thrombin and collagen promote ADP release from platelet-dense granules; ADP subsequently acts on purinergic receptors to reinforce platelet aggregation responses 1,2 and thrombus formation.ADP acts on 2 G protein-coupled receptors (GPCRs), P2Y 1 and P2Y 12 . 1,2 The P2Y 1 purinergic receptor was the first of the ADP receptors to be cloned. [3][4][5] This receptor is widely expressed throughout the body and couples to G q , leading to the activation of phospholipase C␤, a subsequent increase in cytosolic calcium, and the activation of protein kinase C (PKC). The P2Y 12 receptor was only recently identified 6 and is the target of the clinically effective antithrombotic drugs clopidogrel and ticlopidine. P2Y 12 couples through G i to the inhibition of adenylyl cyclase and the activation of PI3-kinase. On the basis of pharmacologic and genetic studies, it is now accepted that P2Y 1 is required for platelet activation by ADP, whereas P2Y 12 is important in synergizing with P2Y 1 or other G q -coupled receptors to induce platelet activation by ADP and other agonists playing a major role in stabilizing platelet thrombi in vivo.Given the established crucial role of ADP in platelet activation, it is likely that the responsiveness of platelets to ADP is tightly regulated, and knowledge of the mechanisms responsible for t...

The ability of two opioid agonists, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) and morphine, to induce -opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.