Phosphatidylinositol 3,4,5-Trisphosphate-dependent Stimulation of Phospholipase C-γ2 Is an Early Key Event in FcγRIIA-mediated Activation of Human Platelets

In pancreatic islets the activation of phospholipase C (PLC) by the muscarinic receptor agonist carbamyolcholine (carbachol) results in the hydrolysis of both phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) and phosphatidylinositol (PtdIns). Here we tested the hypothesis that PtdIns hydrolysis is mediated by PLC␥1, which is known to be regulated by activation of tyrosine kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than that of PtdInsP 2 to the tyrosine kinase inhibitor, genistein. Conversely, the tyrosine phosphatase inhibitor, vanadate, alone promoted PtdIns hydrolysis and acted non-additively with carbachol. Vanadate did not stimulate PtdInsP 2 breakdown. Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine phosphorylation of several islet proteins, although not of PLC␥1 itself. Two structurally unrelated inhibitors of PtdIns 3-kinase, wortmannin and LY294002, more effectively attenuated the hyrolysis of PtdIns compared with PtdInsP 2 . Adenovirally mediated overexpression of PLC␥1 significantly increased carbachol-stimulated PtdIns hydrolysis without affecting that of PtdInsP 2 . Conversely overexpression of PLC␤1 up-regulated the PtdInsP 2 , but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP 2 are independently regulated in pancreatic islets and that PLC␥1 selectively mediates the breakdown of PtdIns. The activation mechanism of PLC␥ involves tyrosine phosphorylation (but not of PLC␥ directly) and PtdIns 3-kinase. Our findings point to a novel bifurcation of signaling pathways downstream of muscarinic receptors and suggest that hydrolysis of PtdIns and PtdInsP 2 might serve different physiological ends.Many receptor tyrosine kinases and GPCRs 1 couple to PLC that catalyzes the cleavage of PtdInsP 2 and resultant generation of the two second messengers Ins(1,4,5)P 3 , which mobilizes Ca 2ϩ from intracellular stores, and DAG, which activates protein kinase C (1-4). These pathways are well described in the regulation of insulin secretion following occupation of m3 and m1 muscarinic receptors on the surface of pancreatic ␤Ϫcells (5-8). However we have shown that in addition to the classical pathway of PtdInsP 2 hydrolysis, exposure of pancreatic islets to the muscarinic receptor agonist carbachol also promotes the hydrolysis of PtdIns (9, 10). The latter predominates quantitatively over, and is a precursor of, PtdInsP 2 . Although hydrolysis of either phosphoinositide species results in generation of DAG, only PtdInsP 2 gives rise to a Ca 2ϩ signal via Ins(1,4,5)P 3 production. Therefore the two hydrolytic events may have different functional consequences. The inositol phosphate, corresponding to Ins(1,4,5)P 3 , that is derived from PtdIns, is Ins(1)P 1 . This has no biological function but can be used to quantify PtdIns hydrolysis, at least under the conditions verified in our previous studies (9).The PLC family is comprised of four major groups (PLCs ␤, ␥, ␦, and ⑀). Members of the PLC␤ family are activated by Gproteins in response to o...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Phospholipase C-γ Mediates the Hydrolysis of Phosphatidylinositol, but Not of Phosphatidylinositol 4,5-Bisphoshate, in Carbamylcholine-stimulated Islets of Langerhans

Mitchell

Kelly

Blewitt

et al. 2001

“…FcR␥ and Fc␥RIIA receptor signaling is initiated by Src kinase-dependent tyrosine phosphorylation of the receptors' ITAM motif, leading to the recruitment of p72 syk , PI 3-kinase, and the adaptor proteins (LAT, SLP-76, and vav), which ultimately promote the activation of phospholipase C␥2 (PLC␥2). The subsequent hydrolysis of phosphatidylinositol 4,5-diphosphate, leading to IP 3 generation and intracellular calcium release is a critical event for efficient platelet activation (19,20).…”

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confidence: 99%

Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization

Mangin

Yao

Goncalves

et al. 2003

105

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca 2؉ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP 3 ) and that treating platelets with the IP 3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca 2؉ flux and platelet shape change. Normal shape change was observed in G␣ q ؊/؊ mouse platelets, excluding a role for PLC␤ isoforms in this process. However, decreased shape change and Ca 2؉ mobilization were observed in mice lacking PLC␥2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca 2؉ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca 2؉ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-Fc␥RIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcR␥ chain. Taken together, these studies define an important role for PLC␥2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.

“…Both phosphatidylinositol 3-kinase (PI3-kinase) and phosphatidylinositol 4-kinase (PI4-kinase) and their products are present in all eukaryotic organisms and tissues that have been investigated, including platelets (12,13). PI3-kinase and PI4-kinase have been shown to be regulated upstream by small GTP-binding proteins in platelets and other cells (14,15).…”

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Phosphoinositides Are Required for Store-mediated Calcium Entry in Human Platelets

Rosado

Sage

2000

We have recently observed that small GTP-binding proteins are important for mediation of store-mediated Ca 2؉ entry in human platelets through the reorganization of the actin cytoskeleton. Because it has been shown in platelets and other cells that small GTP-binding proteins regulate the activity of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, whose products, phosphoinositides, play a key role in the reorganization of the actin cytoskeleton, we have investigated the role of these lipid kinases in store-mediated Ca 2؉ entry. Treatment of platelets with LY294002, an inhibitor of phosphatidylinositol 3-and phosphatidylinositol 4-kinases, resulted in a concentration-dependent inhibition of Ca 2؉ entry stimulated by thapsigargin or the physiological agonist, thrombin. In addition, wortmannin, another inhibitor of these kinases, which is structurally unrelated to LY294002, significantly reduced storemediated Ca 2؉ entry. The inhibitory effect of LY294002 was not mediated either by blockage of Ca 2؉ channels or by modification of membrane potential. LY294002 inhibited actin polymerization stimulated by thrombin or thapsigargin. These results indicate that both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase are required for activation of store-mediated Ca 2؉ entry in human platelets and that the mechanism could involve the reorganization of the actin cytoskeleton.Store-mediated Ca 2ϩ entry (SMCE) 1 is a mechanism present in many cell types; however, the intracellular processes underlying SMCE remain unclear (1). Several hypotheses have considered both direct and indirect coupling mechanisms (2). Indirect coupling assumes the existence of a diffusible messenger generated by the intracellular Ca 2ϩ stores; in contrast, direct coupling models propose a physical interaction between the endoplasmic reticulum (ER) and the plasma membrane (PM; Ref. 2). Recently a new model for SMCE has been proposed in several different cell types. This involves a physical but reversible interaction between the ER and the PM that may require translocation of portions of the ER toward the PM and mechanical support provided by the actin cytoskeleton (3, 4).