Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC- gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC- delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.     

Role of Galphaq and phospholipase C-beta2 in human platelets activation by thrombin receptors PAR1 and PAR4: studies in human platelets deficient in Galphaq and phospholipase C-beta2

Thrombin responses in human platelets are mediated by the protease-activated receptors (PAR), PAR1 and PAR4. The signalling pathways mediating PAR activation have not been fully delineated for human platelets. We assessed cytoplasmic Ca2+ mobilization in response to activation with thrombin and PAR1 (SFLLRN) and PAR4 (GYPGKF) peptides in two patients whose platelets were deficient in two major signalling proteins, Galphaq or phospholipase (PLC)-beta2. In normal platelets, thrombin induced a biphasic Ca2+ response with a rapid rise to a peak followed by a sustained elevation in Ca2+. The peak Ca2+ rise was impaired in both patients at lower thrombin concentrations. At higher concentrations, it was decreased in PLC-beta2-deficient platelets; the sustained Ca2+ elevation observed in normal and Galphaq-deficient platelets was reduced in PLC-beta2-deficient platelets. The response to SFLLRN was decreased in both patients at lower concentrations. The peak Ca2+ in response to GYPGKF was reduced in both patients; the sustained Ca2+ increase was markedly decreased in PLC-beta2-deficient platelets. These studies provide evidence that, in human platelets, both Galphaq and PLC-beta2 play a major role in responses to PAR1 and PAR4 activation, and that PLC-beta2 is required for the sustained Ca2+ rise upon thrombin activation.     

Selective interaction of the C2 domains of phospholipase C-β1 and -β2 with activated Gαq subunits: An alternative function for C2-signaling modules

Phospholipase C (PLC)-beta1 and PLC-beta2 are regulated by the Gq family of heterotrimeric G proteins and contain C2 domains. These domains are Ca2+-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-beta1 and PLC-beta2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS])-bound] Galphaq subunits. The C2 domain of PLC-beta1 effectively suppressed the activation of the intact isozyme by Galphaq(GTP[gammaS]), indicating that the C2-Galphaq interaction may be physiologically relevant. C2 affinity for Galphaq(GTP[gammaS]) was reduced when Galphaq was deactivated to the GDP-bound state. Binding to activated Galphai1 subunits or to Gbetagamma subunits was not detected. Also, Galphaq(GTP[gammaS]) failed to associate with the C2 domain of PLC-delta, an isozyme that is not activated by Galphaq. These results indicate that the C2 domains of PLC-beta1 and PLC-beta2 provide a surface to which Galphaq subunits can dock, leading to activation of the native protein.     

Novel splice variants of type I pituitary adenylate cyclase-activating polypeptide receptor in frog exhibit altered adenylate cyclase stimulation and differential relative abundance

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts its various effects through activation of two types of G protein-coupled receptors, a receptor with high affinity for PACAP named PAC1-R and two receptors exhibiting similar affinity for both PACAP and vasoactive intestinal polypeptide named VPAC1-R and VPAC2-R. Here, we report the characterization of PAC1-R and novel splice variants in the frog Rana ridibunda. The frog PAC1-R has 78% homology with human PAC1-R and is highly expressed in the central nervous system. Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized. PAC1-R25 carries a 25-amino acid insertion that matches the hop cassette of the mammalian receptor, whereas PAC1-R41 carries a cassette with no homology to any mammalian PAC1-R variant. A third splice variant of PAC1-R, exhibiting a completely different intracellular C-terminal domain, named PAC1-Rmc has also been identified. Determination of cAMP formation in cells transfected with the cloned receptors showed that PACAP activated PAC1-R, PAC1-R25, and PAC1-R41 with similar potency. In contrast, PACAP failed to stimulate adenylate cyclase in cells transfected with PAC1-Rmc. Fusion of PAC1-R or PAC1-Rmc with the green fluorescent protein revealed that both receptors are expressed and targeted to the plasma membrane in transfected cells. The different PAC1-R variants are highly expressed in the frog brain and spinal cord and to a lesser extent in peripheral tissues, where only certain isoforms could be detected. The present data indicate that in frog, PACAP may act through different PAC1-R splice variants that differ in their G(s) protein coupling and their abundance in various tissues.     

Differential effects of melatonin and its downstream effector PKCalpha on subcellular localization of RGS proteins

Regulators of G protein signaling (RGS) are proteins that bind specifically to activated Galpha subunits of heterotrimeric G proteins to terminate signaling by both Galpha and Gbetagamma subunits. Signal-induced RGS redistribution may affect their activity in G protein-mediated signaling. We have previously shown that melatonin and the cell permeable cGMP analog 8-bromo cGMP, which lead to protein kinase C (PKC) activation, enhanced cytoplasmic distribution of RGS10 and RGS2 in prostate carcinoma PC3-AR cells. In the present study, we transfected PC3-AR cells with myc-tagged Galphai/Galphaq specific RGS proteins RGS2, RGS4 and RGS10 and examined the effects of melatonin, 8-bromo cGMP and PKC inhibitors on their nuclear-cytoplasmic partitioning. RGS10 and RGS2 were predominantly localized in the nucleus and perinuclear regions whereas RGS4 was mostly cytoplasmic in the PC3-AR cells. Melatonin and the cell permeable cGMP analog 8-bromo cGMP, previously found to activate PKCalpha in the PC3-AR cells, enhanced cytoplasmic localization of RGS10 and RGS2 but induced nuclear accumulation of RGS4. The isozyme specific PKC inhibitor GO6976 (PKCalpha and PKCbeta1) but not hispidin (PKCbeta) negated the effects of melatonin on RGS10, RGS2 and RGS4 localization. These findings indicate that PKCalpha, a downstream effector of the melatonin receptor, differentially affects nuclear/cytoplasmic localization of both Galphai and Galphaq specific RGS proteins. These observations provide further insight into melatonin's ability to fine-tune multiple membrane G-proteins signaling in cells.     

Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.

A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.