Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0017638 (glioma)
 30,880 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study was designed to investigate the relative ability of a series of cyclic opioid peptides to initiate the first activation steps following their binding of delta-opioid receptors. The extent of stimulation of low Km guanosine-triphosphatase (GTPase activity) and inhibition of hormonally-stimulated cAMP accumulation in the NG108-15 (neuroblastoma-glioma) hybrid cell line were determined and compared for six closely related peptides. In addition, their binding affinity was assessed by competition with 3H-[D-Pen2D-Pen5]-enkephalin (3H-DPDPE) in membranes from these cells. All peptides tested elicited comparable maximal effects for both functional responses. Different potencies in stimulating the low Km GTPase was observed at sub-maximal agonist concentrations, although the shallow dose-response behavior did not allow accurate determination of ED50s. Estimation of ED50s for inhibition of cAMP accumulation could be made by curve fitting and were similar for four of these peptides, while DCDPE and 3R-methylDCDPE, the highest affinity analogs, were considerably more potent. In general, the observed differences in hormonal activity somewhat parallel the rank order of binding affinities, but no strict relationship was found between receptor binding and activation.
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PMID:Assessment of delta-opioid receptor activation by a series of peptides in cultured cells. 132 97

Peptides derived from various regions of the alpha 2A-adrenergic receptor (alpha 2A-AR) were used to study receptor-G protein interactions. Binding of the partial agonist [125I]-p-iodoclonidine and the full agonist [3H]bromoxidine (UK14,304) to membrane preparations from human platelet was potently reduced by peptides (12-14 amino acids) from the second cytoplasmic loop (A) and the C-terminal side of the third cytoplasmic loop (Q). Binding of the antagonist [3H]yohimbine was significantly less affected. Five other peptides had no significant effects on ligand binding at concentrations less than 100 microM. The IC50 values for peptides A and Q were 7 and 27 microM for [125I]-p-iodoclonidine binding at the platelet alpha 2A receptor, 15 and 71 microM for the neuroblastoma-glioma (NG108-15) alpha 2B receptor, and greater than 300 microM for yohimbine binding at both alpha 2A and alpha 2B receptors. Competition studies demonstrate that at concentrations of 100 microM, peptides A and Q reduce the affinity of bromoxidine for the platelet alpha 2A-AR and this effect was abolished in the presence of guanine nucleotide. Alpha 2A-AR-stimulated GTPase activity in platelet membranes was inhibited by peptide Q with an IC50 of 16 microM but A was inactive. These data suggest that both the second cytoplasmic loop and the C-terminal part of the third cytoplasmic loop of the alpha 2A-AR are important in the interaction between the alpha 2-AR and Gi protein. Peptide Q appears to destabilize the high affinity state of the alpha 2-AR by binding directly to Gi thus preventing it from coupling to the receptor under both binding and GTPase assay conditions. The peptide from the second cytoplasmic loop (A) also reduces high affinity agonist binding in a G protein-dependent manner but its interaction with receptor and G protein is distinct in that it does not prevent activation of the G protein. These results provide new information about regions of the alpha 2-adrenergic receptor involved in G protein coupling and high affinity agonist binding.
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PMID:Two peptides from the alpha 2A-adrenergic receptor alter receptor G protein coupling by distinct mechanisms. 164 20

Ras (p21) proteins are involved in the control of cell growth and differentiation, but the mechanism by which they exert these effects is not yet known. Here we present evidence that c-Ha-ras (p21(Gly-12)) and its oncogenic mutant T24-ras (p21(Val-12)) selectively induce omega-conotoxin and dihydropyridine-sensitive Ca2+ currents within a few hours after introduction into the cytoplasm of neuroblastoma x glioma hybrid cells. Whereas control cells exhibited a mean Ca2+ current of 250 pA, it amounted to 730 pA in cells pretreated with ras protein. In cells loaded with p21(Gly-12), the effect occurred after 2 hours and was terminated after 8 hours. In contrast, introduction of p21(Val-12) resulted in a prolonged delay (6 hours) of the effect which lasted for more than 24 hours. When ras proteins were preactivated with the non-hydrolysable GTP analog GppNHp, the time courses of both p21(Gly-12) and p21(Val-12) effects were fast and sustained, suggesting that in intact cells (i) the GDP/GTP exchange is faster for p21(Gly-12) compared to p21(Val-12) and (ii) inactivation of p21(Gly-12) is mediated by GAP-induced GTPase activity. T-type Ca2+ currents and K+ currents were unaffected by ras proteins.
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PMID:Ras proteins activate calcium channels in neuronal cells. 165 68

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

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.
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PMID:Reconstitution of high-affinity opioid agonist binding in brain membranes. 184 95

Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.
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PMID:Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells. 215 84

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.
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PMID:Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2. 215 80

According to classical models of drug-receptor interactions, competitive antagonists share with agonists the ability to bind to a common site on the receptor molecule. However, they are different from agonists, as they cannot trigger the "stimulus" that leads to biological responses--i.e., they lack intrinsic activity. For those receptors whose signals are transduced to effector systems by GTP-binding regulatory proteins (G proteins), a mechanistic equivalent of such a stimulus is an increased ability of agonist-bound receptor to accelerate nucleotide exchange and thus GTPase activity on the G-protein molecule. Here we show that for a member of this family of receptors (delta opioid receptors in membranes of NG108-15 neuroblastoma-glioma cells), two types of competitive antagonists can be distinguished. One type has no intrinsic activity, since it neither stimulates nor inhibits the GTPase activity of G proteins and its apparent affinity for the receptor is not altered by pertussis toxin-mediated uncoupling of receptor and G protein. The second type, however, can inhibit GTPase and thus exhibits negative intrinsic activity; its affinity for receptors is increased following uncoupling from G proteins. The existence of antagonists with negative intrinsic activity may be a general feature of several classes of neurotransmitters or hormone receptors and calls for a reevaluation of biological effects produced by competitive antagonists.
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PMID:Antagonists with negative intrinsic activity at delta opioid receptors coupled to GTP-binding proteins. 255 39

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.
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PMID:Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells. 282 23

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.
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PMID:Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. 283 23


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