We investigated the role of Akt-1, one of the major downstream effectors of phosphoinositide 3-kinase (PI3K), in platelet function using mice in which the gene for Akt-1 had been inactivated. Using ex vivo techniques, we showed that Akt-1-deficient mice exhibited impaired platelet aggregation and spreading in response to various agonists. These differences were most apparent in platelets activated with low concentrations of thrombin. Although Akt-1 is not the predominant Akt isoform in mouse platelets, its absence diminished the amount of total phospho-Akt and inhibited increases in intracellular Ca 2؉ concentration in response to thrombin. Moreover, thrombin-induced platelet ␣-granule release as well as release of adenosine triphosphate from dense granules was also defective in Akt-1-null platelets. Although the absence of Akt-1 did not influence expression of the major platelet receptors for thrombin and collagen, fibrinogen binding in response to these agonists was significantly reduced. As a consequence of impaired ␣ IIb  3 activation and platelet aggregation, Akt-1 null mice showed significantly longer bleeding times than wild-type mice. IntroductionUnder normal conditions, platelets circulate freely in the blood without interacting with each other or the vessel wall. On vascular injury, subendothelial matrix proteins, including collagens, or soluble agonists trigger platelet activation. The hallmark of platelet activation is the transformation of the major platelet glycoprotein, ␣ IIb  3 , from its resting to active state, which serves as a fibrinogen receptor, thereby mediating platelet aggregation. One of the most potent platelet agonists, thrombin, acts via a dual system of G protein-coupled protease-activated receptors, PAR3 and PAR4. Both of these receptors are required for optimal thrombin-induced aggregation and secretion. 1 The majority of platelet agonists, including thrombin and collagen, activate phosphoinositide 3-kinase (PI3K) in platelets. Inhibitors of PI3K (wortmannin and LY294002) block fibrinogen binding and platelet aggregation induced by thrombin and collagen, indicating a role for PI3K in ␣ IIb  3 activation. 2 Platelets contain 2 major forms of PI3K, p85/p110 PI3K, composed of a p110 catalytic and p85 regulatory subunit and PI3K␥, composed of a p110␥ catalytic and p101 regulatory subunit. Both forms of PI3K are involved in the inside-out signaling that activates ␣ IIb  3 and induces platelet aggregation. 2 Recent studies demonstrated that a deficiency in the p85␣ regulatory subunit in mice leads to a significant reduction of collagen-induced platelet aggregation, particularly at low doses of stimulus. . 3 The absence of PI3K␥ activity, indicated by the lack of Akt phosphorylation, leads to impaired platelet aggregation in response to adenosine diphosphate (ADP) and protects against thrombosis. 4 PI3Ks generate phosphoinositide products that target the Tec family tyrosine kinases, serine/threonine protein kinases such as Akt, guanosine diphosphate/guanosine triphosphate exchange f...
Sphingosylphosphorylcholine (SPC) is a bioactive lipid that acts as an intracellular and extracellular signalling molecule in numerous biological processes. Many of the cellular actions of SPC are believed to be mediated by the activation of unidentified G-protein-coupled receptors. Here we show that SPC is a high-affinity ligand for an orphan receptor, ovarian cancer G-protein-coupled receptor 1 (OGR1). In OGR1-transfected cells, SPC binds to OGR1 with high affinity (Kd = 33.3 nM) and high specificity and transiently increases intracellular calcium. The specific binding of SPC to OGR1 also activates p42/44 mitogen-activated protein kinases (MAP kinases) and inhibits cell proliferation. In addition, SPC causes internalization of OGR1 in a structurally specific manner.
Abstract-Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca 2ϩ cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca 2ϩ but no significant difference between Ht31-expressing and control cells in the rate of Ca Key Words: A-kinase anchoring proteins Ⅲ protein kinase A Ⅲ cardiac myocyte Ⅲ -adrenergic receptor Ⅲ contractility S timulation of the -adrenergic signaling pathway in cardiac myocytes results in activation of cAMPdependent protein kinase A (PKA) and phosphorylation of several PKA substrates. These include the sarcolemmal L-type Ca 2ϩ channel, the ryanodine receptor (RyR) and phospholamban (PLB) of the sarcoplasmic reticulum (SR), and the myofibrillar proteins troponin I (TnI) and myosin binding protein C (MBP-C). 1-5 Phosphorylation of these substrates acts in concert to generate both enhanced contractility and accelerated relaxation in response to -adrenergic stimulation. Although PKA has broad substrate specificity, it can be highly selective in a physiological setting, because different stimuli are capable of eliciting phosphorylation of specific subsets of targets. 6 The high level of specificity may be explained, in part, by targeting of PKA to distinct subcellular locations via interaction with A-kinase anchoring proteins (AKAPs). 7 Intracellular gradients of cAMP within the cytosol may also contribute to PKA specificity. 8 PKA holoenzyme contains 2 regulatory (R) and 2 catalytic (C) subunits. The 2 isoforms of PKA, types I and II, are distinguished by the R isoform. Most RII is targeted to specific subcellular sites by interaction with AKAPs. 7,9 Targeting of RI by AKAPs has also been recently reported. 10 Members of the AKAP family are functionally similar; they bind RII dimers and, by means of a targeting domain, anchor PKA II holoenzyme to specific subcellular locations. However, the subcellular ...
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