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.

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y 1 and P2Y 12 , which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y 12 is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y 1 internalization is GRK-and arrestin-independent but requires protein kinase C. Importantly, the b 2 adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y 12 -containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the -opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [DAla 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K ϩ current in a concentrationdependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K ϩ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y 1 and P2Y 12 . We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y 1 is regulated by protein kinase C (PKC) and P2Y 12 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 P2Y 1 receptor signaling and trafficking, although only PKC␦ was capable of regulating P2Y 12 , 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 P2Y 1 and P2Y 12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y 12 function, whereas both novel and classic isoforms regulate P2Y 1 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 P2Y 1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y 12 receptor is unaffected. Both P2Y 1 and P2Y 12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y 12 , whereas both novel and classic isoforms regulate P2Y 1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.Activation of platelets occurs through a complex series of reactions in response to vessel injury and plays an essential role in thrombosis. One agonist, ADP, plays a central role in platelet activation by acting as a cofactor in the platelet responses to physiological agonists, including thromboxane A2, collagen, and thrombin. ADP activates two surface-expressed GPCRs, P2Y 1 and P2Y 12 (Kunapuli et al., 2003;Gachet, 2005). The combined stimulation of P2Y 1 receptor (coupled to G q and phospholipase C␤) and P2Y 12 receptor (negatively coupled to adenylyl cyclase through G i ) is necessary for the full platelet aggregation response to ADP, with platelet activation initiated by the P2Y 1 receptor and amplified by P2Y 12 (Gachet, 2005).The attenuation of receptor-stimulated signal output upon sustained or recurrent agonist stimulation, a process known as desensitization, is a crucial physiological mechanism of adaptation observed for many GPCRs. Because ADP plays a crucial role in platelet activation, it is likely that the responsiveness of P2Y 1 and P2Y 12 receptors is tightly regulated. We have shown that both P2Y 1 and P2Y 12 receptor responses desensitize in human platelets (Hardy et al., 2005), which may underlie the obse...

We investigated whether defects in the P2Y 12 ADP receptor gene (P2RY12) contribute to the bleeding tendency in 92 index cases enrolled in the European MCMDM-1VWD study. A heterozygous mutation, predicting a lysine to glutamate (K174E) substitution in P2Y 12 , was identified in one case with mild type 1 von Willebrand disease (VWD) and a VWF defect. Platelets from the index case and relatives carrying the K174E defect changed shape in response to ADP, but showed reduced and reversible aggregation in response to 10 M ADP, unlike the maximal, sustained aggregation observed in controls. The reduced response was associated with an approximate 50% reduction in binding of [ 3 H]2MeS-ADP to P2Y 12 , whereas binding to the P2Y 1 receptor was normal. IntroductionData from the European Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease (MCMDM-1VWD) study, and the Canadian Type 1 VWD Study, suggest locus heterogeneity contributes to the pathogenesis of type 1 VWD, though, as yet, no associated defect in platelet reactivity has been described. 1,2 Given the role of von Willebrand factor (VWF) in primary hemostasis, and the clinical similarities of patients with type 1 VWD and platelet-based bleeding disorders, the bleeding tendency in patients with type 1 VWD may be influenced by defects in the receptor or signaling proteins mediating platelet adhesion and aggregation.ADP is a feedback mediator of platelet aggregation, which elicits its response through the G protein-coupled receptors, P2Y 1 and P2Y 12 . Interaction of ADP with P2Y 1 leads to mobilization of intracellular calcium and activation of Rho kinase, resulting in platelet shape change and an initial wave of rapidly reversible aggregation. [3][4][5] In contrast, interaction with P2Y 12 is associated with adenylyl cyclase inhibition and PI3-kinase activation, resulting in sustained aggregation in synergy with the P2Y 1 receptor. 5 Both receptors are required for a full aggregation response to ADP. 6 We investigated the possible contribution of P2Y 12 gene (P2RY12) defects to the bleeding tendency in type 1 VWD patients recruited through the MCMDM-1VWD study. Methods Study design and evaluation of bleedingThe phenotypic and genotypic characteristics of the MCMDM-1VWD cohort have been described previously. 1 This study focused on those index cases having normal VWF multimer profiles, usually considered a criterion for type 1 VWD. This study has received local ethics committee approval at each of the centers that participated in the MCMDM-1VWD study, and informed consent was obtained in accordance with the Declaration of Helsinki. Genetic studiesP2RY12 coding sequences were amplified from genomic DNA and sequenced on an ABI 3730 DNA capillary sequencer. The P2RY12 520AϾG mutation was sought in other subjects by restriction analysis using BsmI. Platelet function studiesPlatelet aggregation and ATP secretion were assessed in platelet-rich plasma (PRP) using a dual Chronolog lumiaggregometer (Havertown, PA) as previously descr...

GABA B receptors (GABA B Rs) are involved in early events during neuronal development. The presence of GABA B Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GA-BA B R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA B1abcdf and GABA B2 subunits of the GABA B R. Using semiquantitative PCR analysis with GABA B R isoform-selective primers we found that the expression level of GABA B1abd was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA B2 isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA B1 and GABA B2 subunits are under independent control during oligodendroglial development. Activation of GABA B Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA B R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA B Rs in oligodendrocyte development. The presence of GABA B Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.

Background:The PDZ-binding motif of the P2Y 12 receptor regulates correct receptor traffic in human platelets. Results: The PDZ-binding protein NHERF1 binds to the P2Y 12 receptor to promote agonist-dependent internalization. Conclusion: Arrestin scaffolds NHERF1 to the P2Y 12 receptor to facilitate effective NHERF1-dependent receptor internalization. Significance: A novel model of arrestin-dependent GPCR internalization.