Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0155339 (Brown)
 12,436 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, we have reported that the isolated guanine nucleotide-binding regulatory protein, Gh, couples to the alpha 1-adrenergic receptor (Im, M.-J., and Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951 and Im, M.-J., Riek, R.P., and Graham, R. M. (1990) J. Biol. Chem. 265, 18952-18960) and has a molecular mass of approximately 74 kDa, and the approximately 50-kDa protein which is copurified probably regulates guanine nucleotide binding of the 74-kDa GTP-binding protein. In this paper, we describe the role of purified Gh in the regulation of phospholipase C in the reconstitution system. The stimulation of phospholipase C activity by Gh effectively occurred at a low calcium concentration (less than or equal to 2 microM), but the phospholipase C (PLC) itself required at least 50-100 times more calcium to become fully activated. The characteristic nature of phospholipase C stimulation by Gh is its response to the calcium concentration. Thus, the enzyme activity changes in narrow submicromolar ranges and reaches maximal stimulation, but it does not extend to the levels above those stimulated by calcium alone. The calcium concentrations for the maximal stimulation of phospholipase C activity were 10-20 microM with phospholipid vesicles and 100-200 microM with detergent solution. These calcium concentrations were further decreased when Gh and phospholipase C were co-reconstituted into the phospholipid vesicles or in the detergent solution. The maximal stimulations of the PLC activity were reached at less than 5 microM calcium in both the vesicles and the detergent solution. The changes of calcium concentration for the activation of PLC are quite different from those obtained by reconstituting PLC-beta 1 with Gq-like G-proteins (Smarcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991) Science 251, 804-807 and Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991) Nature 350, 516-518). The phospholipase C activity was stimulated in a Gh concentration-dependent manner in the presence of GTP gamma S. The phospholipase C activity was activated by Gh alpha in the presence of aluminum fluoride, but not by Gh beta. Furthermore, a Gh.PLC complex can be induced by incubation with aluminum fluoride in a detergent solution and partially purified without the dissociation of related proteins. Thus, our reconstitution studies show that the pattern of stimulation of PLC by AIF-4-activated Gh in the ternary complex is similar to the stimulation of PLC activated by Gh in both detergent solution and phospholipid vesicles.
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PMID:Characterization of a phospholipase C activity regulated by the purified Gh in reconstitution systems. 157 27

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp. 212 Dec 70

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.
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PMID:The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system. 215 36

Although many functions of phagocytes are known to be regulated by guanosine triphosphate (GTP)-binding proteins, phagocytosis itself has not been considered one of these. However, previous studies have examined only unstimulated neutrophil phagocytosis. Motivated by our previous work, which showed that stimulated neutrophil phagocytosis is regulated by GTP-binding proteins (H. D. Gresham, M. G. Peters, and E. J. Brown. 1986. J. Cell Biol. 103:215a), we have examined the effect of pertussis toxin (PT) on monocyte receptor-mediated phagocytosis. PT inhibited unstimulated and fibronectin-stimulated IgG-mediated phagocytosis and also inhibited C3b-mediated phagocytosis stimulated by fibronectin or phorbol dibutyrate. Cholera toxin (CT) had no effect on unstimulated or stimulated phagocytosis mediated by IgG or C3b. PT inhibition of phagocytosis was not mediated via increases in cellular cAMP levels or by inhibition of the respiratory burst. Inhibition of phagocytosis did not result from decreased numbers of plasma membrane opsonin receptors nor decreased ability to bind opsonized targets. Although phorbol ester-stimulated phagocytosis was inhibited by PT, ligand-independent internalization of CR1 stimulated by phorbol dibutyrate proceeded normally in PT-intoxicated cells. We conclude that a PT-sensitive GTP-binding protein does regulate phagocytic function in monocytes. This protein operates on a molecular mechanism specific to the process of ingestion in both unstimulated monocytes and in cells stimulated to increase phagocytosis. Because unstimulated neutrophil phagocytosis is unaffected by PT or CT, and stimulated neutrophil phagocytosis is inhibited by both PT and CT, our data also demonstrate that monocytes and neutrophils have distinct mechanisms for regulation of phagocytic function.
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PMID:Molecular regulation of phagocyte function. Evidence for involvement of a guanosine triphosphate-binding protein in opsonin-mediated phagocytosis by monocytes. 311 17

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.
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PMID:Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go. 312 Jun 96

Members of the Rho subfamily of Ras-related GTP-binding proteins play important roles in the organization of the actin cytoskeleton and in the regulation of cell growth. We have shown previously that the dbl oncogene product, which represents a prototype for a family of growth regulatory proteins, activates Rho subfamily GTP-binding proteins by catalyzing the dissociation of GDP from their nucleotide binding site. In the present study, we demonstrate that the acidic phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), provides an alternative mechanism for the activation of Cdc42Hs. Among a variety of lipids tested, only PIP2 was able to stimulate GDP release from Cdc42Hs in a dose-dependent manner, with a half-maximum effect at approximately 50 microM. Unlike the Dbl oncoprotein, which requires the presence of (free) guanine nucleotide in the medium to replace the GDP bound to Cdc42Hs, PIP2 stimulates GDP release from Cdc42Hs in the absence of free guanine nucleotide. PIP2, when incorporated into phosphatidylcholine carrier vesicles, binds tightly to the guanine nucleotide-depleted form of Cdc42Hs and weakly to the GDP-bound form of the GTP-binding protein but does not bind to GTP-bound Cdc42Hs, similar to what was observed for the Dbl oncoprotein. However, mutational analysis of Cdc42Hs indicates that the site that is essential for the functional interaction between PIP2 and Cdc42Hs is distinct from the Dbl-binding site and is located at the positively charged carboxyl-terminal end of the GTP-binding protein. The GDP-releasing activity of PIP2 is highly effective toward Cdc42Hs and Rho (and is similar to the reported effects of PIP2 on Arf (Terui, T., Kahn, R. A., and Randazzo, P. A., (1994) J. Biol. Chem. 269, 28130-28135)), is less effective with Rac, and is not observed with Ras, Rap1a, or Ran. The ability of PIP2 to activate Cdc42Hs (or Rho) and Arf provides a possible point of convergence for the biological pathways regulated by these different GTP-binding proteins and may be related to the synergism observed between Arf and Rho-subtype proteins in the stimulation of phospholipase D activity (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950).
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PMID:Phosphatidylinositol 4,5-bisphosphate provides an alternative to guanine nucleotide exchange factors by stimulating the dissociation of GDP from Cdc42Hs. 879 10