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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoinositide phospholipase C (PLC) activity extracted from bovine liver plasma membranes with sodium cholate was stimulated by GTP gamma S-activated G alpha q/G alpha 11, whereas the enzyme from liver cytosol was not. The membrane-associated PLC was subjected to chromatography on heparin-Sepharose, Q Sepharose, and S300HR, enabling the isolation of the G-protein stimulated activity and its resolution from PLC-gamma and PLC-delta. Following gel filtration, two proteins of 150 and 140 kDa were found to correspond to the activatable enzyme. These proteins were identified immunologically as members of the PLC-beta family and were completely resolved by chromatography on TSK Phenyl 5PW. The 150-kDa enzyme was markedly responsive to GTP gamma S-activated alpha-subunits of G alpha q/G alpha 11 or to purified Gq/G11 in the presence of GTP gamma S. The response of this PLC was of much greater magnitude than that of the 140-kDa enzyme. The partially purified 150-kDa enzyme showed specificity for PtdIns(4,5)P2 and PtdIns4P as compared to PtdIns and had an absolute dependence upon Ca2+. These characteristics were similar to those of the brain PLC-beta 1. The immunological and biochemical properties of the 150-kDa membrane-associated enzyme are consistent with its being the PLC-beta isozyme that is involved in receptor-G-protein-mediated generation of inositol 1,4,5-triphosphate in liver.
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PMID:Identification in bovine liver plasma membranes of a Gq-activatable phosphoinositide phospholipase C. 132 Sep 35

The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.
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PMID:Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes. 132 89

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.
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PMID:Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells. 132 59

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family. 133 87

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are integral to the signal transduction pathways that mediate the cell's response to many hormones, neuromodulators, and a variety of other ligands. While many signaling processes are guanine nucleotide dependent, the precise coupling between a variety of receptors, G proteins, and effectors remains obscure. We found that the family of genes that encode the alpha subunits of heterotrimeric G proteins is much larger than had previously been supposed. These novel alpha subunits could account for some of the diverse activities attributed to G proteins. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha q and G alpha 11, that are 88% identical. They lack the site that is ordinarily modified by pertussis toxin and their sequences vary from the canonical Gly-Ala-Gly-Glu-Ser (GAGES) amino acid sequence found in most other G protein alpha subunits. Multiple mRNAs as large as 7.5 kilobases hybridize to G alpha q specific probes and are expressed at various levels in many different tissues. G alpha 11 is encoded by a single 4.0-kilobase message which is expressed ubiquitously. Amino acid sequence comparisons suggest that G alpha q and G alpha 11 represent a third class of alpha subunits. A member of this class was found in Drosophila melanogaster. This alpha subunit, DG alpha q, is 76% identical to G alpha q. The presence of the Gq class in both vertebrates and invertebrates points to a role that is central to signal transduction in multicellular organisms. We suggest that these alpha subunits may be involved in pertussis toxin-insensitive pathways coupled to phospholipase C.
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PMID:G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. 212 49

Soluble and membrane phosphoinositide-specific phospholipases obtained separately from dispersed circular and longitudinal intestinal muscle cells were characterized for substrate specificity and G protein dependence using selective antibodies to various isoforms of phospholipase C (PLC) and G protein subunits. Western blot analysis disclosed the presence of the main PLC isozymes, PLC-gamma 1, PLC-delta 1, and PLC-beta 1. Soluble PLC from circular and longitudinal muscle was stimulated by guanosine 5'-O-(3-thiophosphate) and inhibited by PLC-beta 1 antibody (80-90%) and PLC-beta 3 antibody (approximately 25%) but not by G protein antibodies. Membrane PLC from circular and longitudinal muscle was stimulated by cholecystokinin octapeptide (CCK-8) and inhibited selectively by PLC-beta 1 antibody (85%), PLC-beta 3 antibody (15%), and G alpha q/11 antibody (90%). CCK-8-induced contraction in permeabilized circular muscle cells was also selectively inhibited by PLC-beta 1 antibody (76%), PLC-beta 3 antibody (24%), and G alpha q/11 antibody (86%). The combined effects of PLC-beta 1 and PLC-beta 3 antibodies on PLC activity and muscle contraction were additive, causing complete inhibition. Soluble and membrane PLC from circular and longitudinal muscle were immunologically similar but functionally different. The enzymes from circular muscle preferentially hydrolyzed endogenous and exogenous phosphatidylinositol 4,5-biphosphate (PIP2), confirming previous findings of preferential hydrolysis of PIP2 in dispersed intestinal circular muscle cells.
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PMID:Functional characterization of phosphoinositide-specific phospholipase C-beta 1 and -beta 3 in intestinal smooth muscle. 748 67

The fission yeast Schizosaccharomyces pombe has proven useful for studying molecular interactions between a range of signal transduction components. We now report the first co-expression of a mammalian seven-transmembrane receptor and G-protein components in S. pombe. We selected the human neurokinin NK2 receptor together with its G-protein-signalling partner Gq for this study. Yeast membrane fractions showed high levels of NK2 receptor-binding activity (1159 +/- 534 (n = 3) fmol/mg protein) although initial experiments with intact cells revealed an absence of receptors at the cell surface. Using a construct comprising the NK2 coding sequence fused with the signal sequence from an endogenous phosphatase (phoI), we detected approximately 400 NK2 receptors/cell in unbroken yeast. Successful co-expression of the NK2 receptor with the G-protein subunits G alpha q, beta 1 or beta 2 and gamma 3 failed to modulate agonist binding, suggesting the absence of functional interaction between these components. As an alternative test of G alpha q function, we next expressed its downstream effector target phospholipase C-beta 1 (PLC beta 1) in S. pombe. Although PLC beta 1 undergoes powerful in vitro activation by G alpha q derived from baculovirus-infected Sf9 cells and mammalian cells, G alpha q expressed in S. pombe is totally ineffective. Similar results were also achieved with the G-protein subunit G alpha 16. Together, these data suggest that seven-transmembrane receptors can be expressed in S. pombe at high levels and directed to the cell surface although their interaction with co-expressed G-proteins in undetectable. Production of inactive G alpha-chains in S. pombe may account for these observations.
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PMID:Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeast Schizosaccharomyces pombe. 749 95

The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that different receptors which mediate similar biochemical responses may utilize distinct mechanisms to activate G-proteins. Differences among the signaling pathways triggered by chemoattractant factor receptors suggest an opportunity for pharmacologic modifications of the inflammatory response.
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PMID:Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins. 749 83

m3-Muscarinic cholinergic receptor (m3-AChR) and alpha 1-adrenergic receptor (alpha 1-AR) stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis (by a PIP2-specific phospholipase C, PLC) in rat parotid gland membranes is mediated via activation of alpha subunits of the Gq/11 family of G-proteins. This study examines m3-AChR and alpha 1-AR stimulation of PIP2 hydrolysis in membranes isolated from parotid glands of old (24 months) and young (3 months) rats (old and young rat membranes). Old rat membranes exhibited reduced stimulation of PIP2 hydrolysis in response to the addition of guanosine-5'-O-(3-thiotrisphosphate) (GTP gamma S) alone or GTP gamma S plus either carbachol (m3-AChR agonist) or epinephrine (alpha 1-AR agonist). This reduction in receptor-stimulated PIP2 hydrolysis was not due to a decrease in PLC activity per se since cholate-solubilized PLC activity was similar in old and young rat membranes. Additionally, these membranes exhibited comparable, immunologically detectable, levels of PLC beta 3, G alpha q/11, and G beta. In the presence of 10 microM AlCl3 and 10 mM NaF, stimulation of PIP2 hydrolysis in both old and young rat membranes was similar. Preincubation of membranes from old rats with GTP gamma S induced a time-dependent increase in the rate of PIP2 hydrolysis and, with 20 min preincubation, the rates of hydrolysis in old and young rat membranes were not statistically different. In aggregate, these data indicate that there is a defect in the activation of G alpha q/11 in parotid gland membranes from old rats.
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PMID:Decreased m3-muscarinic and alpha 1-adrenergic receptor stimulation of PIP2 hydrolysis in parotid gland membranes from aged rats: defect in activation of G alpha q/11. 757 3

The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the alpha subunits of at least two guanine-nucleotide-binding (G) proteins, G alpha i2 and G alpha q. Activation of cPLA2 is inhibited by pertussis toxin and G alpha i2 mutants. In addition, activation of phospholipase C via G alpha q results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C, both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of G alpha i2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative G alpha i2 mutant, [G203T]G alpha i2, in which Gly203 is mutated to Thr. [G203T]G alpha i2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]G alpha i2 was specific to G alpha i2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]G alpha i2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on G alpha i2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61 +/- 9% (n = 5) in [G203T]G alpha i2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]G alpha i2. Thus, [G203T]G alpha i2. specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]G alpha i2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is G alpha i2-dependent. The possibility is discussed that G alpha i2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.
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PMID:The guanine-nucleotide-binding protein subunit G alpha i2 is involved in calcium activation of phospholipase A2. Effects of the dominant negative G alpha i2 mutant, [G203T]G alpha i2, on activation of phospholipase A2 in Chinese hamster ovary cells. 760 Oct 96


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