Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.
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PMID:Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. 133 52

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.
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PMID:G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma. 142 82

Hydrolysis by phospholipase C (PLC) of phosphatidylinositol 4,5-bisphosphate is a key mechanism by which many extracellular signalling molecules regulate functions of their target cells. At least eight distinct isozymes of PLC are recognized in mammalian cells. Receptor-controlled PLC is often regulated by G proteins, which can be modified by pertussis toxin in some cells but not in others. In the latter cells, PLC-beta 1, but not PLC-gamma 1 or PLC-delta 1, may be activated by members of the alpha q-subfamily of the G protein alpha-subunits. An unidentified PLC in soluble fractions of cultured human HL-60 granulocytes is specifically stimulated by G protein beta gamma subunits purified from retina and brain. Identification of a second PLC-beta complementary DNA (PLC-beta 2) in an HL-60 cell cDNA library prompted us to investigate the effect of purified G protein beta gamma subunits on the activities of PLC-beta 1 and PLC-beta 2 transiently expressed in cultured mammalian cells. We report here that PLC-beta 1 and PLC-beta 2 were stimulated by free beta gamma subunits and that PLC-beta 2 was the most sensitive to beta gamma stimulation. Thus stimulation of PLC by beta gamma subunits is isozyme-selective and PLC-beta 2 is a prime target of beta gamma stimulation. Activation of PLC-beta 2 by beta gamma subunits may be an important mechanism by which pertussis toxin-sensitive G proteins stimulate PLC.
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PMID:Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. 146 33

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).
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PMID:Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. 146 34

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.
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PMID:Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa. 151 Oct 86

The 36-kDa beta 1, 35-kDa beta 2, and 6.5-kDa gamma 2 subunits of the heterotrimeric guanine nucleotide-binding proteins have been overexpressed in Sf9 cells using a baculovirus expression system. The gamma 2 subunit expressed in Sf9 cells incorporated label derived from [3H]mevalonate and is therefore likely to be isoprenylated, as is its mammalian counterpart. Extracts of Sf9 cells doubly infected with viruses encoding a beta subunit and viruses encoding a gamma subunit are active in promoting the pertussis toxin-catalyzed ADP-ribosylation of a G protein alpha subunit. However, extracts from Sf9 cells singly infected with viruses encoding either a beta or gamma subunit are not active in this assay. Results demonstrate utility of the insect/baculovirus system for expressing G protein beta gamma subunits of defined composition.
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PMID:Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system. 161 13

cDNAs corresponding to a previously uncharacterized phospholipase C were isolated from an HL-60 cell cDNA library. The cDNAs encodes a putative polypeptide of 1181 amino acids with a calculated molecular mass of 133,700 daltons. Comparison of the amino acid sequence of the predicted protein with those of five mammalian phospholipase C isoforms (PLC-beta 1, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) revealed that the new enzyme is most closely related to PLC-beta 1 with an overall amino acid sequence identity of 48%. Thus, the new phospholipase C was named PLC-beta 2. The least similarity between PLC-beta 1 and PLC-beta 2 is apparent in the carboxyl-terminal 450 amino acids. Both PLC-beta 1 and PLC-beta 2 were purified from extracts of HeLa cells that had been transfected with vaccinia virus containing the corresponding cDNAs. Like other mammalian PLC isoforms, including PLC-beta 1, the catalytic activity of PLC-beta 2 was entirely dependent on Ca2+, and PLC-beta 2 preferred phosphatidyl-inositol 4,5-bisphosphate to phosphatidylinositol as substrate. Recently, the alpha subunit of the pertussis toxin-insensitive G-protein alpha q has been shown to activate PLC-beta 1 but not PLC-gamma 1 and PLC-delta 1. When alpha q purified from bovine brain was reconstituted with PLC-beta 1 or PLC-beta 2, no stimulation of PLC-beta 2 was observed in the presence of either AlF4- or guanosine 5-O-(3-thiotriphosphate) (GTP gamma S), whereas PLC-beta 1 activity was enhanced markedly in the presence of AlF4- and less markedly but significantly in the presence of GTP gamma S. These results suggest that the receptor-dependent stimulation of PLC-beta 1 and that of PLC-beta 2 may require different G-protein alpha subunits. (see also accompanying article (Lee, C. H., Park, D., Wu, D., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044-16047).
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PMID:Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta 2. 164 92

Plasma membranes from bovine liver contain a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity that is activated by guanine nucleotides. The G-proteins involved retained their ability to activate bovine brain PLC-beta 1 in a guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent manner following extraction from the membranes with cholate and reconstitution with phospholipids. This reconstitution assay was used to purify the G-proteins by chromatography on heparin-Sepharose, DEAE-Sephacel, octyl-Sepharose, hydroxylapatite, Mono Q, and Sephacryl S-300 gel filtration. Gel electrophoresis showed that two alpha-subunits with molecular mass of 42 and 43 kDa were isolated to a high degree of purity, together with a beta-subunit. Neither alpha-subunit was a substrate for pertussis toxin-catalyzed ADP-ribosylation. Gel filtration of the final activity indicated an apparent molecular mass of 95 kDa, suggesting the presence of an alpha beta gamma heterotrimer. Immunological data revealed that the 42- and 43-kDa proteins were related to alpha-subunits of the Gq class recently purified from brain (Pang, I.-H., and Sternweis, P. C. (1990) J. Biol. Chem. 265, 18707-18712) and identified by molecular cloning (Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117). The activation of PLC-beta 1 by the purified G-protein preparation was specific for nonhydrolyzable guanine nucleotides, the efficacy decreasing in order GTP gamma S greater than guanylimidodiphosphate greater than guanylyl(beta,gamma-methylene)-diphosphonate. Half-maximal activation required 4 microM GTP gamma S suggesting that the affinity of the G-proteins for GTP analogues is low. The GTP gamma S-dependent activation of PLC-beta 1 required millimolar Mg2+ and was inhibited by guanosine 5'-O-(2-thiodiphosphate) and by excess beta gamma-subunits. Aluminum fluoride also activated PLC-beta 1 in the presence of the G-proteins. The G-proteins were inactive toward PLC-gamma 1 or PLC-delta 1. In summary, these findings identify two G-protein activators of PLC-beta 1 that have the properties of heterotrimeric G-proteins and are members of the Gq class.
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PMID:Purification and characterization of two G-proteins that activate the beta 1 isozyme of phosphoinositide-specific phospholipase C. Identification as members of the Gq class. 165 41

Changes in the beta-adrenergic receptor-G protein-adenylate cyclase complex were investigated in an experimental canine model of low-output heart failure produced by chronic rapid ventricular pacing. The contractile response occurring after exposure to the beta-adrenergic agonist dobutamine, measured as peak left ventricular + dP/dt, was decreased after 3 weeks of pacing. To further characterize the diminished functional responsiveness to beta-adrenergic receptor stimulation, beta-adrenergic receptor-adenylate cyclase coupling was investigated using membranes prepared from both control and paced animals. The density of beta-adrenergic receptors was decreased by 40% with a selective downregulation of the beta 1-subtype. The affinity of the receptor for the antagonist radioligand [125I]iodocyanopindolol remained unchanged. A defect in coupling was suggested by a decreased ability of isoproterenol, fluoride, and forskolin to stimulate adenylate cyclase in membranes prepared from failing hearts. Determination of the levels of Gi alpha (the alpha-subunit of Gi) by immunoblotting and pertussis toxin labeling revealed modest increases of approximately 30%. Furthermore, Mn2+ and purified Gs failed to stimulate adenylate cyclase in membranes prepared from failing hearts, indicating an impairment in the catalytic moiety of adenylate cyclase itself or in the ability of adenylate cyclase to couple to Gs. In contrast, complementation assay did not reveal differences in the functional activity of Gs alpha (the alpha-subunit of Gs). Taken together, these data demonstrate a selective decrease in the beta 1-subtype of adrenergic receptors and an increase in a 40-kd G1-like protein in the failing heart. Similar changes have been described in human idiopathic dilated cardiomyopathy. In addition to these changes, we identified a possible defect at the level of the catalytic subunit of adenylate cyclase.
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PMID:Beta-adrenergic receptor-G protein-adenylate cyclase complex in experimental canine congestive heart failure produced by rapid ventricular pacing. 165 3

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.
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PMID:Phospholipase C-beta 1 is regulated by a pertussis toxin-insensitive G-protein. 166 86


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