Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA encoding the human alpha 2-C-10-adrenergic receptor was transfected into Rat-1 fibroblasts by CaPO4 precipitation, and clones expressing the receptor were isolated and expanded. One clone (1C) expressing high levels of the receptor was studied in order to determine the contacts between this receptor and guanine nucleotide-binding proteins (G proteins) mediating second messenger signaling. The alpha 2-adrenergic agonist UK 14304 stimulated high affinity GTPase activity in membranes from these cells. Incubation of these membranes with Protein A-purified fractions from an antiserum able to identify the carboxyl-terminal decapeptide common to Gi1 alpha and Gi2 alpha was partially able to prevent agonist stimulation of high affinity GTPase activity. Similar results were produced with an antiserum that identifies the carboxyl-terminal decapeptide of Gi3 alpha. In contrast, equivalent fractions of antisera that identify the carboxyl-terminal decapeptides of Go alpha and Gs alpha did not inhibit receptor stimulation of high affinity GTPase activity. Coincubation of the membranes from the cells with Protein A-purified fractions from the anti-Gi1 alpha + Gi2 alpha antiserum and the anti-Gi3 alpha antiserum produced greater inhibition of UK14304-stimulated GTPase activity than did either of the two antisera in isolation. These data show direct interaction of the human alpha 2-C10-adrenergic receptor, when expressed in this clone of Rat-1 fibroblasts, with multiple pertussis toxin-sensitive G proteins and demonstrate that a single receptor has the physical capacity to interact functionally with more than a single pertussis toxin-sensitive G protein in a native membrane. Furthermore, because the two antisera were able to inhibit receptor stimulation of high affinity GTPase activity to similar degrees, the G protein pools identified by these antisera must contribute similar amounts of the total receptor activation of pertussis toxin-sensitive G proteins in these cells.
Mol Pharmacol 1991 Nov
PMID:Molecular interaction of the human alpha 2-C10-adrenergic receptor, when expressed in Rat-1 fibroblasts, with multiple pertussis toxin-sensitive guanine nucleotide-binding proteins: studies with site-directed antisera. 165 1

In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.
Mol Endocrinol 1991 Jul
PMID:Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells. 165 34

Cholera toxin treatment (up to 1 microgram/ml, 16 h) of neuroblastoma x glioma hybrid NG108-15 cells produced a decrease of some 35% in both delta opioid receptor-mediated stimulation of high-affinity GTPase activity and inhibition of forskolin-amplified adenylate cyclase. Coincident with these decreases was a down-regulation of some 35% in the delta opioid receptor population. A similar pattern of a decrease in signalling capacity was noted for the alpha 2B-adrenergic receptor in these cells after cholera toxin treatment. Half-maximal effects of cholera toxin on all of the parameters assayed were noted at concentrations between 2 and 5 ng/ml. Neither levels of Gi2, as assessed by immunoblotting with specific antisera, nor the intrinsic activity of the alpha subunit of the guanine-nucleotide-binding protein which acts as the inhibitory G-protein of the adenylate cyclase in these cells, as assessed by guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p)-mediated inhibition of adenylate cyclase, was lowered by cholera toxin treatment. Furthermore, levels of another pertussis toxin-sensitive G-protein (Go) expressed by these cells was also not lowered by cholera toxin treatment. However, as previously noted in other cells [Milligan, Unson & Wakelam (1989) Biochem. J. 262, 643-649], marked down-regulation of the alpha subunit of the stimulatory G-protein (Gs) of the adenylate cyclase cascade was observed in response to cholera toxin treatment. Previous studies [Klee, Milligan, Simonds & Tocque (1985) Mol. Aspects Cell Regul. 4, 117-129] have shown that cholera toxin treatment can result in a decrease in the maximal effectiveness of agonists which function to inhibit adenylate cyclase. These data have been used as evidence to suggest a functional interaction between Gs and 'Gi'. The results provided herein demonstrate that such effects of the toxin can be explained adequately by a decrease in the number of receptors that function to produce inhibition of adenylate cyclase.
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PMID:Cholera toxin impairment of opioid-mediated inhibition of adenylate cyclase in neuroblastoma x glioma hybrid cells is due to a toxin-induced decrease in opioid receptor levels. 167 34

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.
Mol Cell Biol 1990 Jun
PMID:Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation. 169 62

Guanine nucleotide-binding proteins (G proteins) transduce signals from agonist- and light-sensitive receptors. In the visual excitation system, the photon receptor rhodopsin is coupled to the G protein Gt (transducin). Gt is composed of alpha, beta, and gamma subunits; the alpha subunit binds guanine nucleotide, whereas the beta and gamma subunits, which are tightly associated, appear to facilitate interaction of alpha with receptor and pertussis toxin-catalyzed ADP-ribosylation of alpha. To study the function of transducin, monoclonal antibodies were developed against the purified protein. Monoclonal antibody 2H3 reacted with Gt gamma but not G gamma from bovine brain or rabbit liver. In the absence of photolyzed rhodopsin, both intact 2H3 and Fab fragments of 2H3 were able to inhibit completely, in a concentration-dependent manner, ADP-ribosylation of transducin by pertussis toxin 2H3 had no effect on ADP-ribosylation in the presence of photolyzed rhodopsin. The GTPase activity of transducin, which is dependent on rhodopsin, was inhibited only 50% by 2H3. These data are consistent with the hypotheses that an epitope recognized by 2H3 may be important in the formation of the alpha beta gamma complex or that interaction of 2H3 with gamma may alter conformation of the latter and, thereby, inhibit complex formation. Further, reactions of gamma with 2H3 appear to be prevented by interaction with rhodopsin, suggesting that its interaction either shields or alters the epitope recognized by 2H3.
Mol Pharmacol 1990 Jun
PMID:Immunological characterization of guanine nucleotide-binding proteins: effects of a monoclonal antibody against the gamma subunit of transducin on guanine nucleotide-binding protein-receptor interactions. 169 60

The peptide hormones bradykinin and kallidin (Lys-bradykinin), as well as their analogues [des-Arg9]-bradykinin, a selective B1 agonist, [des-Arg9,Leu8]-bradykinin, a selective B1 antagonist, and [Thi5,8,D-Phe7]-bradykinin and D-Arg0-[Hyp3,D-Phe7]-bradykinin, two selective B2 antagonists, induced rapid histamine release from purified rat peritoneal mast cells. In contrast, the N-terminal fragment bradykinin-(1-5) was inactive. These peptides also activate the GTPase activity of GTP-binding proteins (G proteins) (Go/Gi) purified from calf brain, with an order of potency identical to that observed on mast cells, [Thi5,8,D-Phe7]-bradykinin much greater than kallidin greater than bradykinin greater than D-Arg0-[Hyp3,D-Phe7]-bradykinin greater than [des-Arg9]-bradykinin greater than [des-Arg9,Leu8]-bradykinin greater than bradykinin-(1-5). This correlation suggested that G proteins are the targets of kinins in mast cells. Accordingly, the concomitant increase in inositol trisphosphates and release of histamine elicited by kinins were inhibited by pertussis toxin pretreatment of mast cells. The inhibitory effect of benzalkonium chloride showed that the G proteins involved belong to the Gi type. GTPase activity was measured in the supernatant of homogenized mast cells but not in the membranous fraction. This activity was stimulated by kinins and by the venom peptide mastoparan. The potency of peptides was similar to that observed with purified bovine G proteins. Sodium dodecyl sulfate-gel electrophoresis of mast cell supernatant revealed pertussis toxin-induced ADP-ribosylation of two proteins, in the Mr 41,000 and 40,000 range, i.e., similar to purified alpha-subunits of Gi1 and Gi2 or Gi3 subtypes. The data support the proposal that bradykinin and analogues act like mastoparan, substance P, and compound 48/80, interacting first with sialic acid residues of the cell surface and then with Gi-like proteins, inducing phospholipase C activation and intracellular calcium mobilization.
Mol Pharmacol 1990 Dec
PMID:Activation of Gi-like proteins, a receptor-independent effect of kinins in mast cells. 170 Dec 14

Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.
Mol Cell Biol 1992 Jan
PMID:Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes. 172 98

We show by nuclear magnetic resonance studies that, following GTP hydrolysis during phage T4 sheath contraction, GDP remains bound to the sheath protein (gp18), whereas orthophosphate is released. gp18 in the contracted state has GTPase activity and can hydrolyse exogenous GTP; the reaction is calcium-dependent and displays high substrate specificity. The process comprises two steps: (1) displacement of GDP from gp18 by exogenous GTP, and (2) GTP hydrolysis proper. The first step appears to be rate-limiting and to be accelerated when the nucleotide-protein interaction is mechanically disrupted by sonication.
J Mol Biol 1992 Jan 05
PMID:GTPase activity of bacteriophage T4 sheath protein. 173 Oct 71

Regulation of GTP and GDP binding and GTPase activity of cardiac sarcolemmal guanine nucleotide-binding proteins was investigated. In purified sarcolemmal membranes, carbachol and a variety of other muscarinic receptor (MR) agonists induced increases in [3H]GTP, [gamma-32P]GTP, and [3H]GDP binding to relatively high affinity sites. Carbachol-dependent GTP and GDP binding changes were maximal within 5 sec at 30 degrees and thereafter remained at steady state. Carbachol increased GTP binding to two sites with apparent Kapp values of 50 nM and 250 nM and GDP binding to a single site with a Kapp of 100 nM. N-Ethylmaleimide attenuated carbachol-dependent GDP and GTP binding, tentatively identifying the binding sites as Gi and/or Go. Further studies showed that [3H]GDP and [3H]GTP bound to Gi/Go in the presence of carbachol rapidly exchanged with GTP and GDP in the medium. In membranes preincubated with carbachol and [gamma-32P]GTP or carbachol and [3H]GDP, postaddition of atropine resulted in complete hydrolysis of [gamma-32P]GTP bound to Gi/Go, to unlabeled GDP and 32Pi, by GTPase, within 10 sec, whereas [3H]GDP remained bound. This study also showed that bound [3H]GDP did not exchange with GDP or GTP in the absence of an MR agonist. Under identical conditions, atropine reversed adenylate cyclase (AC) inhibition by carbachol and GTP or GDP in 5-10 sec. MR agonists appear to increase the rate of dissociation of GDP from Gi/Go, which results in rapid GTP turnover on these sites by a combination of GTPase and GDP/GTP exchange reactions. Furthermore, MR-Gi/Go may be tightly coupled during AC inhibition, so that GTP hydrolysis as well as MR-Gi/Go uncoupling may be required to reverse AC inhibition.
Mol Pharmacol 1992 Jan
PMID:Regulation of GDP and GTP binding in cardiac sarcolemma by muscarinic receptor agonists. 173 18

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.
Mol Cell Biol 1992 Feb
PMID:A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity. 173 35


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