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1.
Cholecystokinin induces the interaction of its receptor with a guanine nucleotide binding protein 总被引:1,自引:0,他引:1
To evaluate the relation between the pancreatic cholecystokinin (CCK) receptor and guanine nucleotide-binding protein(s) we studied the effects of nucleotides on 125I-CCK binding to pancreatic acinar plasma membranes, 125I-CCK binding to solubilized 125I-CCK receptors, and the stability of the solubilized 125I-CCK-receptor complex. In plasma membranes, guanine nucleotides both inhibited CCK binding and increased the dissociation of CCK from its receptor. The potency of the nucleotides studied was GTP gamma S = GMP-PNP greater than GTP much greater than ATP. When membranes were solubilized with digitonin, subsequent binding of CCK was insensitive to guanine nucleotides including GTP, GMP-PNP and GTP gamma S. However, if CCK binding occurred before solubilization of the membranes, guanine nucleotides increased dissociation at concentrations and with a specificity similar to that observed for effects on intact pancreatic membranes. It is concluded that guanine nucleotides act via a protein which is separable from the receptor to induce dissociation of bound CCK. Moreover, CCK binding induces an association in the plasma membrane of the CCK receptor with this guanine nucleotide binding protein. 相似文献
2.
Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles 总被引:11,自引:0,他引:11
Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides. 相似文献
3.
[3H]GTP [guanosine triphosphate] and [3H]GMP-PNP [guanosine 5'-(beta, 8-imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP-PNP is saturable, with respective KD values of 0.76 and 0.42 microM. The number of binding sites for GMP-PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 microM-ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP-PNP (at 0.2--1.0 microM), but no inhibition by other nucleotides at 100 microM concentrations. The bindings sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cyclase. 相似文献
4.
The binding of substance P (SP) to receptors in peripheral tissues as well as in the CNS is subject to regulation by guanine nucleotides. In this report, we provide direct evidence that this effect is mediated by a guanine nucleotide-binding regulatory protein (G-protein) that is required for high-affinity binding of SP to its receptor. Rat submaxillary gland membranes bind a conjugate of SP and 125I-labeled Bolton-Hunter reagent (125I-BHSP) with high affinity (KD = 1.2 +/- 0.4 X 10(-9) M) and sensitivity to guanine nucleotide inhibition. Treatment of the membranes with alkaline buffer (pH 11.5) causes a loss of the high-affinity, GTP-sensitive binding of 125I-BHSP and a parallel loss of [35S]guanosine 5'-(3-O-thio)triphosphate ([35S]GTP gamma S) binding activity. Addition of purified G-proteins from bovine brain to the alkaline-treated membranes restores high-affinity 125I-BHSP binding. Reconstitution is maximal when the G-proteins are incorporated into the alkaline-treated membranes at a 30-fold stoichiometric excess of GTP gamma S binding sites over SP binding sites. Both Go (a pertussis toxin-sensitive G-protein having a 39,000-dalton alpha-subunit) and Gi (the G-protein that mediates inhibition of adenylate cyclase) appear to be equally effective, whereas the isolated alpha-subunit of Go is without effect. The effects of added G-proteins are specifically reversed by guanine nucleotides over the same range of nucleotide concentrations that decreases high-affinity binding of 125I-BHSP to native membranes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
ATP and guanine nucleotide dependence of neutrophil activation. Evidence for the involvement of two distinct GTP-binding proteins 总被引:1,自引:0,他引:1
In phagocytes, activation of the respiratory burst by chemoattractants requires ATP and involves a pertussis toxin-sensitive G protein. ATP is also required for the response elicited in permeabilized neutrophils by nonhydrolyzable GTP analogs, indicating that at least one of the ATP-dependent steps lies downstream of the receptor-coupled G protein(s). A respiratory burst can also be produced in a reconstituted cell-free system by addition of arachidonic acid. Most investigators find this response to be independent of ATP, yet stimulated by GTP analogs, implying that the ATP-dependent steps observed in the unbroken cells must precede the guanine nucleotide-requiring event. To resolve this apparent discrepancy, we studied the ATP and guanine nucleotide dependence of the oxidative response elicited by arachidonic acid in electrically permeabilized human neutrophils. Two components of the response were apparent: one was ATP-dependent, the other ATP-independent. The ATP-dependent component was partially inhibited by staurosporine, suggesting involvement of protein kinase C. This kinase signals activation of the NADPH oxidase without intervening G proteins, since stimulation by phorbol ester was unaffected by guanosine 5'-(beta-thio)diphosphate (GDP beta S). Although nonhydrolyzable GTP analogs failed to stimulate the oxidase in the absence of ATP, the ATP-independent response stimulated by arachidonic acid was found to require GTP or one of its analogs and to be inhibited by GDP beta S. The relative potency of the guanine nucleotides to support the arachidonic acid response in the absence of ATP (5'-guanylyl imidodiphosphate (GMP-PNP) greater than or equal to guanosine 5'-(gamma-thio)triphosphate GTP gamma S) greater than or equal to (GTP) differed from their efficacy to stimulate the burst in the presence of ATP (GTP gamma S greater than GMP-PNP much greater than GTP). These observations suggest the involvement of two distinct GTP-binding proteins in oxidase activation: a receptor-coupled, heterotrimeric, pertussis toxin-sensitive G protein, and a second GTP-binding protein(s) located downstream of the ATP-requiring steps, which may lie in close proximity to the NADPH oxidase. This secondary GTP-binding protein could be part of the pathway activated by chemoattractants, but does not mediate stimulation via protein kinase C. Therefore multiple parallel routes may exist for activation of the NADPH oxidase. 相似文献
6.
Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells 总被引:16,自引:0,他引:16
The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion. 相似文献
7.
Muscarinic acetylcholine receptors (mAChR) purified from porcine atrium were reconstituted into lipid vesicles with GTP-binding regulatory proteins (G proteins, Gi, Go, or Gn) purified from porcine cerebrum. Apparent affinities of the reconstituted mAChR and G proteins for carbachol and GDP, respectively, were estimated from the effects of these ligands on the binding of [3H]-L-quinuclidinyl benzilate ([3H]QNB) to mAChR and [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) to G proteins in the presence of different concentrations of MgCl2. A total of 30-35% of reconstituted mAChRs exhibited low affinity for carbamylcholine, irrespective of the presence or absence of guanine nucleotides, and the remainder of the mAChRs showed high affinities for carbamylcholine in the absence of GTP or GDP and a low affinity in their presence. The affinity for carbamylcholine in the absence of guanine nucleotides, but not in their presence, increased with increases in MgCl2 concentration. Apparent Kd's for carbamylcholine were estimated to be approximately 100 microM in the presence of guanine nucleotides, 1.5 microM in the absence of guanine nucleotide and Mg2+ (< 0.1 microM), and 0.1 microM in the absence of guanine nucleotide and the presence of MgCl2 (10 mM). These results indicate that mAChRs may assume at least three different conformations that are characterized by different affinities for agonists. Furthermore, the data suggest that MgCl2 is not necessary for the formation of the mAChR-G protein complex, but can induce a conformational change in the complex. On the other hand, the presence of MgCl2 was necessary for carbamylcholine to influence the binding of guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins 总被引:30,自引:0,他引:30
The association of agonists with muscarinic receptors in membranes from bovine brain was affected only slightly by guanine nucleotides. However, solubilization of these membranes with deoxycholate and subsequent removal of detergent resulted in a preparation of receptors with increased affinity for agonists and a large increase in response to guanine nucleotides. Chromatography of deoxycholate extracts of membranes on DEAE-Sephacel resulted in the separation of receptors from 95% of the guanine nucleotide-binding activity. Guanine nucleotides had no effect on the binding of agonists to these resolved receptors. The effect of guanine nucleotides was restored after the addition of either of two purified guanine nucleotide-binding proteins from bovine brain. One of these proteins, presumably brain GI, is composed of subunits with the same molecular weights (alpha, 41,000; beta, 35,000; gamma, 11,000) and functions as the inhibitory guanine nucleotide-binding protein isolated from liver. The other protein, termed Go, is a novel guanine nucleotide-binding protein that possesses a similar subunit composition (alpha, 39,000; beta, 35,000; gamma, 11,000) but whose function is not yet known. Addition of either protein to the resolved receptor preparation increased agonist affinity by at least 10-20-fold, and low concentrations of guanine nucleotides specifically reversed this effect. Reconstitution of receptors with the resolved subunits of Go demonstrates that the beta subunit alone had no effect on agonist binding, but that this subunit does appear to enhance the effects observed with the alpha subunit alone. 相似文献
9.
A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go 总被引:3,自引:0,他引:3
R Munshi I H Pang P C Sternweis J Linden 《The Journal of biological chemistry》1991,266(33):22285-22289
A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins. 相似文献
10.
An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism. 总被引:5,自引:0,他引:5
S M Strittmatter D Valenzuela Y Sudo M E Linder M C Fishman 《The Journal of biological chemistry》1991,266(33):22465-22471
G protein-coupled membrane receptors activate G proteins by enhancing guanine nucleotide exchange. G0 is a major component of the growing regions (growth cones) of neurons. GAP-43 is a neuronal protein associated with the cytosolic face of the growth cone plasma membrane and stimulates binding of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to Go (Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. (1990) Nature 344, 836-841). Here we have examined the mechanism by which GAP-43 affects G0. Like G protein-coupled receptors, GAP-43 enhances GDP release from G0, increases the initial rate of GTP gamma S binding, and increases the GTPase activity of Go, all without altering the intrinsic kappa cat for the GTPase. Unlike the case for receptors, however, the GAP-43 effect is not blocked by pertussis toxin, nor affected by the presence or absence of beta gamma or of phospholipids. There is specificity to the interaction, in that GAP-43 increases GTP gamma S binding to recombinant alpha o and alpha i1, but not to recombinant alpha s. Thus, GAP-43 is a guanine nucleotide release protein with a novel mechanism of action, potentially controlling membrane-associated G proteins from within the cell. 相似文献
11.
Effect of Guanosine 5''-O-(3-Thiotriphosphate) and Calcium on γ-Aminobutyric AcidB Binding as a Function of Postnatal Development 总被引:1,自引:1,他引:0
We have examined the development of gamma-aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of approximately 50% with the GABAB ligand (-)-[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium- and guanosine triphosphate-binding protein (G protein)-regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTP gamma S was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptors affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTP gamma S may affect the GABAB receptor by different mechanisms. 相似文献
12.
Yung H. Wong Catherine D. Demoliou-Mason Eric A. Barnard 《Journal of neurochemistry》1989,52(4):0999-1009
Opioid receptors solubilized in Mg2+-digitonin (2%, wt/vol) from Mg2+-pretreated rat brain membranes maintain, in addition to high-affinity opioid agonist binding, the modulation by guanine nucleotides. One of the modes of expression of the latter property is an attenuation of agonist binding by guanine nucleotides in the presence of Na+. To investigate the molecular basis of this modulation and to identify the G protein(s) involved, the soluble receptors were [32P]ADP-ribosylated by means of Bordetella pertussis toxin and subjected to molecular size exclusion chromatography. In addition, soluble extracts were chromatographed on lectin and hydrophobic affinity columns. The binding of 35S- and 3H-labelled analogues of GTP was also monitored in the species separated. The oligomeric G protein-coupled opioid receptors and the guanine nucleotide/pertussis toxin-sensitive species showed similar chromatographic properties in all three systems. This indicates that the biochemically functional G protein-opioid receptor complex formed in Mg2+-pretreated membranes in the absence of an agonist is stable in digitonin solution and to chromatographic separation. Further analysis showed that the guanine nucleotide modulation of opioid receptors is via the pertussis toxin substrates with Mr of 41,000 and 39,000, which are identified as Gi and Go alpha subunits, respectively. 相似文献
13.
Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors 总被引:4,自引:0,他引:4
Investigation of the properties of the binding of the radiolabelled antagonists (125I)-iodohydroxybenzylpindolol, (125I)-iodopindolol, and (125I)-iodocyanopindolol to beta-adrenergic receptors of L6 myoblast membranes revealed that guanine nucleotides caused a 2 to 4.5 fold increase in the apparent affinity of these antagonists. No significant effects of GTP were observed on the density of binding sites determined with each radioligand. GTP, GDP, and GMPPNP were of similar high affinity in producing this effect, while GMP was much less potent, and ATP was without effect. Under similar assay conditions GTP reduced the apparent binding affinity of the agonist isoproterenol for the beta-adrenergic receptors of L6 cells. The results indicate that, contrary to previous observations, guanine nucleotides affect not only the interactions of agonists with beta-adrenergic receptors, but also the interaction of antagonists with these adenylate cyclase-linked receptors. 相似文献
14.
Rubin Maribel A. Medeiros Alan C. Rocha Paula C. B. Livi Carolina B. Ramirez Galo Souza Diogo O. 《Neurochemical research》1997,22(2):181-187
GMP-PNP, a non-hydrolyzable analog of GTP binds tightly to G-protein in the presence of Mg2+, so that the binding is stable even after exhaustive washings. This property was exploited to prepare membrane samples of rat brain where G-protein GTP-binding sites were saturated with GMP-PNP. Experiments carried out with these membranes showed that GTP, GMP-PNP, GDP-S and GMP (1 mM) inhibit the sodium-independent [3H]glutamate binding by 30–40% [F(4,40) = 5.9; p < .001], whereas only GMP-PNP activates adenylate cyclase activity [F(6,42) = 3.56; p < .01]. The inhibition of sodium-independent [3H]glutamate binding occurred in the absence of Mg2+. These findings suggest that guanine nucleotides may inhibit glutamate binding and activate adenylate cyclase through distinct mechanisms by acting on different sites. 相似文献
15.
Involvement of a pertussis toxin-sensitive G protein in the action of gastrin on gastric parietal cells 总被引:2,自引:0,他引:2
The mechanism whereby gastrin triggers phosphoinositide breakdown was investigated in an enriched preparation of isolated rabbit parietal cells (approx. 75%). In a permeabilized preparation of myo-[3H]inositol-labelled cells, GTP[S], a non-hydrolysable GTP analogue, enhanced [3H]inositol trisphosphate ([3H]InsP3 accumulation in a dose-dependent manner; submaximal concentrations of GTP[S] (less than 10 microM), potentiated gastrin-induced [3H]InsP3 release; preincubation for 5 min with GDP[S], a non-hydrolysable GDP analogue, dose-dependently reduced [3H]InsP3 accumulation stimulated by gastrin even in presence of GTP[S]. Exposure of intact parietal cells for 3 h to pertussis toxin (PTx) (200 ng/ml) led to a 15-50% reduction in gastrin-induced [14C]aminopyrine [(14C]AP) uptake (an index of in vitro acid secretion) and [3H]inositol phosphate ([3H]InsP) accumulation. A decrease in the accumulation of the different [3H]inositol phosphate occurred in gastrin-stimulated parietal cells treated with PTx. A rightward shift of gastrin dose-response curves in the presence of PTx was observed for [14C]AP uptake (EC50 values: 0.125 +/- 0.045 nM without PTx and 1.05 +/- 0.63 nM with PTx), for [3H]InsP accumulation (EC50 values: 0.16 +/- 0.08 nM without PTx and 1.56 +/- 0.58 nM with PTx) and [125I]gastrin binding (IC50 values: 0.247 +/- 0.03 nM without PTx and 2.38 +/- 0.56 nM with PTx). In contrast, cholera toxin (CTx) treatment (100 ng/ml) for 3 h was without effect on gastrin-induced [3H]InsP accumulation. CTx induced a pronounced potentiation of gastrin-stimulated [14C]AP uptake; this effect can be mimicked by IBMX (a phosphodiesterase inhibitor) and by forskolin (an activator of adenylyl cyclase). We conclude that: (i) one or more than one G protein appeared to be involved in gastrin receptor coupling to phospholipase C (PL-C); (ii) these G proteins are not substrates for CTx; (iii) one of these appeared to be a PTx-sensitive 'Gi-like' protein which could be involved in hormone-induced acid secretion, (iiii) the potentiating effect of CTx observed on AP uptake stimulated by gastrin suggests the existence of a cooperative effect between cAMP pathway (CTx) and the gastrin-induced phosphoinositide breakdown in acid secretory activity of parietal cells. 相似文献
16.
Kayvan Ariani Mark W. Hamblin Grace L. Tan Carol A. Stratford Dr. Roland D. Ciaranello 《Neurochemical research》1989,14(9):835-843
Several manipulations that affect G protein/receptor coupling also alter the binding of [125I]iodocyanopindolol ([125I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO4 in these assays results in a small but significant increase in the affinity of [125I]ICYP (fromK
D=0.046 nM toK
D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [125I]ICYP affinity (K
D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT1B sites labeled by [3H]dihydroergotamine ([3H]DE) also displays a small but significant reduction (from Ki=1.4 nM to Ki=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT1B specific binding of [125I]ICYP. The NEM induced reduction in [125I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [125I]ICYP to 5-HT1B sites is dependent on G proteins. The effects of Mg2+ ion, guanine nucleotides, NEM and G protein reconstitution on [125I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT1B sites. The relatively slight stabilization of [125I]ICYP and ±CYP binding conferred by G protein/5-HT1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT1B agoinists. 相似文献
17.
Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [3H]quinuclidinyl benzylate [( 3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor. 相似文献
18.
Somatostatin (SST) receptors activate potassium channels, stimulate protein phosphatases, inhibit adenylate cyclase and close calcium channels. These multiple effects are controlled by guanine nucleotide binding (G) proteins of the pertussis toxin-sensitive Gi and Go types. In the present study we have identified the G proteins coupling with brain SST receptors. To this end, brain SST receptors were solubilized in G-protein coupled form. Binding of the SST analogue MK 678 to the solubilized receptor was completely inhibited by guanosine 5'-O-thiotriphosphate (IC50 = 100 nM), reflecting decreased receptor affinity for agonist following uncoupling of the receptor and G protein(s). Antibodies raised against specific COOH-terminal peptides of the G proteins Gi(1-3), Go, and Gz were used to probe for SST receptor-G protein coupling in this system. Antibodies binding to the COOH-terminal regions of Gi1 and Gi2 (antibody AS) and Gi3 (antibody EC) inhibited binding of 125I-MK 678 (75 pM) by 57 +/- 4% and 48 +/- 5%, respectively. The effects of these antibodies were concentration-dependent and additive, such that in combination AS and EC completely inhibited binding. Antibodies binding to the COOH-terminal region of Go (GO) and Gz (QN) did not affect binding of 125I-MK 678, indicating that neither Go nor Gz are associated with the brain SST receptor. Prelabeling of the receptor with 125I-MK 678 prior to addition of antibody induced the formation of a "locked conformation" of the agonist-bound receptor-G protein complex which was insensitive to antibody. In conclusion, Gi1 and/or Gi2 and Gi3 are coupled in approximately equal proportions to the brain 125I-MK 678-binding SST receptor, accounting for all of the G protein coupling of this receptor. 相似文献
19.
Identification of an extracellular motif involved in the binding of guanine nucleotides by a glutamate receptor. 总被引:3,自引:1,他引:2
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Y Paas A Devillers-Thiry J P Changeux F Medevielle V I Teichberg 《The EMBO journal》1996,15(7):1548-1556
The chick cerebellar kainate (KA) binding protein (KBP), a member of the family of ionotropic glutamate receptors, harbours a glycine-rich (GxGxxG) motif known to be involved in the binding of ATP and GTP to kinases and G proteins respectively. Here, we report that guanine, but not adenine, nucleotides interact with KBP by inhibiting [3H]KA binding in a competitive-like manner, displaying IC50 values in the micromolar range. To locate the GTP binding site, KBP was photoaffinity labelled with [alpha-32P]GTP. The reaction was blocked by KA, glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione and antibodies raised against a peptide containing the glycine-rich motif. Site-directed mutagenesis of residues K72 and Y73 within the glycine-rich motif followed by the expression of the KBP mutants at the surface of HEK 293 cells showed a decrease in GTP binding affinity by factors of 10 and 100 respectively. The binding of [3H]KA to the K72A/T KBP mutants was not affected but binding to the Y73I KBP mutant was decreased by a factor of 10. Accordingly, we propose that the glycine-rich motif of KBP forms part of a guanine nucleotide binding site. We further suggest that the glycine-rich motif is the binding site at which guanine nucleotides inhibit the glutamate-mediated responses of various members of the subfamily of glutamate ionotropic receptors. 相似文献
20.
The bombesin receptor is coupled to a guanine nucleotide-binding protein which is insensitive to pertussis and cholera toxins 总被引:10,自引:0,他引:10
The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation. In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue. Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources. In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent. In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM. Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for [125I-Tyr4]bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein. Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number. Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation. Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release. Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular [Ca2+] produced by bombesin in these cells. In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases. 相似文献