Cannabinoid Receptor Agonist Efficacy for Stimulating [35S]GTPγS Binding to Rat Cerebellar Membranes Correlates with Agonist-induced Decreases in GDP Affinity
1998; Elsevier BV; Volume: 273; Issue: 27 Linguagem: Inglês
10.1074/jbc.273.27.16865
ISSN1083-351X
AutoresC. S. Breivogel, Dana E. Selley, Steven R. Childers,
Tópico(s)Neurotransmitter Receptor Influence on Behavior
ResumoThe relationship between GDP and cannabinoid-stimulated [35S]guanosine-5′-O-(3-thiotriphosphate) ([35S]GTPγS) binding was investigated in rat cerebellar membranes. Kinetic analyses showed that [35S]GTPγS binding reached steady-state levels and that the association rate was increased by the agonist WIN 55212-2 proportional to the concentration of GDP. Dissociation of [35S]GTPγS occurred with two rates (t½ = 7 and 170 min), and WIN 55212-2 increased the proportion of sites exhibiting the faster rate. Without GDP, [35S]GTPγS bound to membranes with high and low affinity, and WIN 55212-2 had no effect. With 30 μm GDP, [35S]GTPγS bound to low and intermediate affinity sites, and WIN 55212-2 induced high affinity [35S]GTPγS binding without affecting low affinity sites. GDP competed for high affinity [35S]GTPγS binding with high and intermediate affinity in the absence of WIN 55212-2 and with high and low affinity in the presence of WIN 55212-2. Cannabinoid ligands displayed differential abilities to maximally stimulate [35S]GTPγS binding in the presence of GDP. Efficacy differences among ligands increased with increasing GDP concentrations. GDP competition curves revealed that agonists induced low affinity GDP K i values that were proportional to agonist E max values, indicating that agonist efficacy is determined by displacement of GDP from G-proteins. The relationship between GDP and cannabinoid-stimulated [35S]guanosine-5′-O-(3-thiotriphosphate) ([35S]GTPγS) binding was investigated in rat cerebellar membranes. Kinetic analyses showed that [35S]GTPγS binding reached steady-state levels and that the association rate was increased by the agonist WIN 55212-2 proportional to the concentration of GDP. Dissociation of [35S]GTPγS occurred with two rates (t½ = 7 and 170 min), and WIN 55212-2 increased the proportion of sites exhibiting the faster rate. Without GDP, [35S]GTPγS bound to membranes with high and low affinity, and WIN 55212-2 had no effect. With 30 μm GDP, [35S]GTPγS bound to low and intermediate affinity sites, and WIN 55212-2 induced high affinity [35S]GTPγS binding without affecting low affinity sites. GDP competed for high affinity [35S]GTPγS binding with high and intermediate affinity in the absence of WIN 55212-2 and with high and low affinity in the presence of WIN 55212-2. Cannabinoid ligands displayed differential abilities to maximally stimulate [35S]GTPγS binding in the presence of GDP. Efficacy differences among ligands increased with increasing GDP concentrations. GDP competition curves revealed that agonists induced low affinity GDP K i values that were proportional to agonist E max values, indicating that agonist efficacy is determined by displacement of GDP from G-proteins. Cannabinoid receptors mediate the actions of Δ9-tetrahydrocannabinol (Δ9-THC) 1The abbreviations used are: Δ9-THC, Δ9-tetrahydrocannabinol; [35S]GTPγS, [35S]guanosine-5′-O-(3-thiotriphosphate); PMSF, phenylmethylsulfonyl fluoride. and other cannabimimetic ligands (1Compton D.R. Rice K.C. DeCosta B.R. Razdan R.K. Melvin L.S. Johnson M.R. Martin B.R. J. Pharmacol. Exp. Ther. 1993; 265: 218-226PubMed Google Scholar). To date, two types of cannabinoid receptors have been discovered, CB1 (2Devane W.A. Dysarz F.A.I. Johnson M.R. Melvin L.S. Howlett A.C. Mol. Pharmacol. 1988; 34: 605-613PubMed Google Scholar, 3Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.L. Bonner T.I. Nature. 1990; 346: 561-564Crossref PubMed Scopus (4315) Google Scholar) and CB2 (4Munro S. Thomas K.L. Abu-Shaar M. Nature. 1993; 365: 61-65Crossref PubMed Scopus (4269) Google Scholar). A splice variant of CB1, termed CB1A, has also been reported (5Shire D. Carillon C. Kaghad M. Calandra B. Rinaldi-Carmona M. Le Fur G. 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Both Gα and βγ subunits act upon effectors until Gα cleaves the bound GTP to GDP by its intrinsic GTPase activity, and Gα re-associates with a βγ dimer (15Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (5031) Google Scholar, 16Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar). The cycle is then complete, and the heterotrimeric G-protein is able to be activated again. Receptors act catalytically, as one receptor can activate multiple G-proteins (17Asano T. Ross E.M. Biochemistry. 1984; 23: 5467-5471Crossref PubMed Scopus (31) Google Scholar, 18Gierschik P. Moghtader R. Straub C. Dieterich K. Jakobs K.H. Eur. J. Biochem. 1991; 197: 725-732Crossref PubMed Scopus (86) Google Scholar, 19Sim L.J. Selley D.E. Xiao R. Childers S.R. Eur. J. Pharmacol. 1996; 307: 95-107Crossref Scopus (140) Google Scholar). The activation and dissociation of the G-protein subunits occur very rapidly and thus do not appear to be rate-limiting steps in the signal transduction cascade (20Neubig R.R. Sklar L.A. Mol. Pharmacol. 1993; 43: 734-740PubMed Google Scholar). However, since the actions of G-protein-coupled receptors are mediated strictly via the activation of G-proteins, this step plays a key role in determining overall agonist efficacy (21Kenakin T. Pharmacologic Analysis of Drug-Receptor Interaction. Raven Press, Ltd., New York1993: 249-277Google Scholar) and may be the most relevant step in measuring agonist efficacy at G-protein-coupled receptors (22Keen M. Trends Pharmacol. Sci. 1991; 12: 371-374Abstract Full Text PDF PubMed Scopus (20) Google Scholar). Agonist-stimulated binding of the hydrolysis-resistant GTP analog, [35S]GTPγS, to G-protein α subunits measures receptor activation of G-proteins in purified and reconstituted systems (23Florio V.A. Sternweis P.C. J. Biol. Chem. 1989; 264: 3909-3915Abstract Full Text PDF PubMed Google Scholar), native cell membrane preparations (24Hilf G. Gierschik P. Jakobs K.H. Eur. J. Biochem. 1989; 186: 725-731Crossref PubMed Scopus (167) Google Scholar), and brain sections (25Sim L.J. Selley D.E. Childers S.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7242-7246Crossref PubMed Scopus (427) Google Scholar). The present study focuses on three aspects of the role of GDP in the agonist-stimulated [35S]GTPγS binding assay. First, GDP has been shown to decrease basal [35S]GTPγS binding and allow detection of agonist stimulation. The requirement for micromolar concentrations of GDP to observe agonist effects in native membrane preparations has been reported consistently in every system for which agonist-stimulated [35S]GTPγS binding has been demonstrated (24Hilf G. Gierschik P. Jakobs K.H. Eur. J. Biochem. 1989; 186: 725-731Crossref PubMed Scopus (167) Google Scholar, 26Lorenzen A. Fuss M. Vogt H. Schwabe U. Mol. Pharmacol. 1993; 44: 115-123PubMed Google Scholar, 27Traynor J.R. Nahorski S.R. Mol. Pharmacol. 1995; 47: 848-854PubMed Google Scholar, 28Selley D.E. Stark S. Sim L.J. Childers S.R. Life Sci. 1996; 59: 659-668Crossref PubMed Scopus (97) Google Scholar). Second, GDP has been reported to modulate the kinetics of [35S]GTPγS binding. The presence of micromolar concentrations of GDP was shown to decrease the magnitude and rate of [35S]GTPγS binding to purified and reconstituted G-proteins (23Florio V.A. Sternweis P.C. J. Biol. Chem. 1989; 264: 3909-3915Abstract Full Text PDF PubMed Google Scholar). However, early reports of [35S]GTPγS binding to purified G-protein Giα (29Bokoch G.M. Katada T. Northrup J.K. Ui M. Gilman A.G. J. Biol. Chem. 1984; 259: 3560-3567Abstract Full Text PDF PubMed Google Scholar) and Goα (30Sternweis P.C. Robishaw J.D. J. Biol. Chem. 1984; 259: 13806-13813Abstract Full Text PDF PubMed Google Scholar) subunits concluded that this binding is essentially irreversible in the presence of millimolar concentrations of Mg2+, which is also required for agonist stimulation of [35S]GTPγS binding (31Sim L.J. Selley D.E. Childers S.R. Misthra R. Baker G.B. Boulton A.A. Neuromethods: G Protein Methods and Protocols. Humana Press Inc., Totowa, NJ1997: 1-27Google Scholar). Therefore, a problem frequently noted for [35S]GTPγS binding is that it is performed under non-equilibrium conditions, thus complicating interpretation of the results. Finally, GDP has been shown to play an important role in determining agonist efficacy for the stimulation of [35S]GTPγS binding. In the adenosine A1 receptor system, a full agonist was shown to be maximally effective for the stimulation of [35S]GTPγS binding at a higher concentration of GDP than a partial agonist (32Lorenzen A. Guerra L. Vogt H. Schwabe U. Mol. Pharmacol. 1996; 49: 915-926PubMed Google Scholar). Similar results were found in the mu opioid system, where increasing the concentration of GDP increased relative efficacy differences among agonists (33Selley D.E. Sim L.J. Xiao R. Liu Q. Childers S.R. Mol. Pharmacol. 1997; 51: 87-96Crossref PubMed Scopus (193) Google Scholar). In order to determine whether GDP plays similar roles in modulating cannabinoid agonist efficacy, it is necessary to compare [35S]GTPγS binding stimulated by agonists of different efficacies. Previous studies which showed that Δ9-THC (34Sim L.J. Hampson R.E. Deadwyler S.A. Childers S.R. J. Neurosci. 1996; 16: 8057-8066Crossref PubMed Google Scholar, 35Burkey T.H. Quock R.M. Consroe P. Roeske W.R. Yamamura H.I. Eur. J. Pharmacol. 1997; 323: R3-R4Crossref PubMed Scopus (59) Google Scholar), CP 55940 (36Shen M. Piser T.M. Seybold V.S. Thayer S.A. J. Neurosci. 1996; 16: 4322-4334Crossref PubMed Google Scholar), and anandamide (37Mackie K. Devane W.A. Hille B. Mol. Pharmacol. 1993; 44: 498-503PubMed Google Scholar, 38Vogel Z. Barg J. Levy R. Saya D. Heldman E. J. Neurochem. 1993; 61: 352-355Crossref PubMed Scopus (285) Google Scholar, 39Childers S.R. Sexton T. Roy M.B. Biochem. Pharmacol. 1994; 47: 711-715Crossref PubMed Scopus (162) Google Scholar) are each partial agonists provide an effective starting point to examine this question. The present study explores these three aspects of GDP modulation of G-protein activation by cannabinoid agonists. The cannabinoid system is ideal for the study of G-protein activation in brain membranes, due to the very high levels of cannabinoid receptors (40Herkenham M. Lynn A.B. Little M.D. Johnson M.R. Melvin L.S. De Costa B.R. Rice K.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1932-1936Crossref PubMed Scopus (1993) Google Scholar) and cannabinoid-activated G-proteins (25Sim L.J. Selley D.E. Childers S.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7242-7246Crossref PubMed Scopus (427) Google Scholar) compared with other G-protein-coupled receptors in brain. These experiments provide evidence that cannabinoid agonist-stimulated [35S]GTPγS binding is dependent on the agonist-induced decrease in G-protein affinity for GDP and that cannabinoid agonist efficacy for G-protein activation is determined by the magnitude of this decrease in affinity. Male Sprague-Dawley rats were purchased from Zivic Miller (Zelienople, PA). [35S]GTPγS (1250 Ci/mmol), and ReflectionsTM film were obtained from NEN Life Science Products. Anandamide, (R)-(+)-methanandamide and WIN 55212-2 were purchased from Research Biochemicals International (Natick, MA). CP 55940 and levonantradol were obtained from Pfizer, Inc. (Groton, CT). Δ9-THC was provided by NIDA/Research Triangle Institute (Research Triangle Park, NC). SR141716A was a generous gift from Dr. Francis Barth at Sanofi Recherché(Montpellier, France). Guanosine diphosphate (GDP) and unlabeled GTPγS were purchased from Boehringer Mannheim. All other reagent grade chemicals were obtained from Sigma or Fisher. Rat cerebellar membranes were prepared in membrane buffer (50 mm Tris-HCl, 3 mm MgCl2, 0.2 mm EGTA, pH 7.4) and stored at −80 °C as described previously (41Breivogel C.S. Sim L.J. Childers S.R. J. Pharmacol. Exp. Ther. 1997; 282: 1632-1642PubMed Google Scholar). For assays including anandamide, thawed membranes were pretreated with 50 μmphenylmethylsulfonyl fluoride (PMSF) followed by centrifugation and homogenization of the pellet. All preparations were preincubated for 10 min at 30 °C with 0.004 units/ml adenosine deaminase (Sigma) and assayed for protein content (42Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222164) Google Scholar) before addition to assay tubes. Assays were performed as described previously (41Breivogel C.S. Sim L.J. Childers S.R. J. Pharmacol. Exp. Ther. 1997; 282: 1632-1642PubMed Google Scholar). Unless otherwise specified, 4–15 μg of cerebellar membrane protein were incubated for 2 h at 30 °C in membrane buffer containing 0.1% (w/v) bovine serum albumin, 100 mm NaCl, 30 μm GDP, and 0.05 nm[35S]GTPγS in a final volume of 1 ml, and nonspecific binding was determined with 30 μm unlabeled GTPγS. For association assays, membranes were added to assay tubes on ice, and assay tubes were transferred to a 30 °C water bath at various times. Reactions were terminated in all tubes simultaneously by rapid filtration as described previously (41Breivogel C.S. Sim L.J. Childers S.R. J. Pharmacol. Exp. Ther. 1997; 282: 1632-1642PubMed Google Scholar). For dissociation assays, assay tubes were allowed to associate for 1 h (0 and 0.1 μm GDP) or 2 h (3 and 30 μm GDP) before the addition of 30 μm unlabeled GTPγS at various times; reactions were terminated as above. Unless otherwise indicated, binding parameters were determined by nonlinear regression analysis using JMP for Macintosh (SAS Institute, Cary, NC). Association parameters were fitted to Equation 1 (43Ferguson K.M. Higashijima T. Smigel M.D. Gilman A.G. J. Biol. Chem. 1986; 261: 7393-7399Abstract Full Text PDF PubMed Google Scholar). B=Bfinal×(1−e−kt)Equation 1 where B is the amount of [35S]GTPγS bound at time t; Bfinal is the maximum amount of ligand bound under steady-state conditions, and k is the apparent association rate constant (k obs). Dissociation parameters were determined by fitting for biphasic bimolecular dissociation as shown in Equation 2 (43Ferguson K.M. Higashijima T. Smigel M.D. Gilman A.G. J. Biol. Chem. 1986; 261: 7393-7399Abstract Full Text PDF PubMed Google Scholar). B=B01×e−k1t+B02×e−k2tEquation 2 where B is the amount of [35S]GTPγS bound at time t; B01 and B02 are the amounts of ligand bound to rapidly and slowly dissociating sites at time 0, andk 1 and k 2 are the dissociation rate constants (k −1) for the rapidly and slowly dissociating sites, respectively. Half-times for each site were calculated by dividing −ln(0.5) by the respective rate constants (k obs or k −1). EC50 and E max values for each agonist were determined by fitting concentration-effect curves to Equation 3. E=[L]×Emax[L]+EC50Equation 3 where E is amount of [35S]GTPγS bound at receptor ligand concentration [L]; E max is the amount of [35S]GTPγS bound at maximally effective concentrations of receptor ligand, and EC50 is the concentration of receptor ligand producing half-maximal [35S]GTPγS binding. IC50 andI max values for GDP competition curves were determined by fitting the biphasic Equation 4. I=[I]×Imax(H0[I]+IC50(H)+[I]×Imax(L)[I]+IC50(L)Equation 4 where I is the amount of [35S]GTPγS binding inhibited at GDP concentration [I];I max(H) and I max(L) are the maximum amounts of [35S]GTPγS inhibited from either the high or low affinity sites, respectively, and IC50(H)and IC50(L) are the concentrations of GDP that inhibit half of the [35S]GTPγS binding from each site, respectively.K i values were estimated by the Cheng-Prusoff equation (44Cheng Y.-C. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-4002Crossref PubMed Scopus (12526) Google Scholar). [35S]GTPγS saturation binding was analyzed using EBDA and LIGAND (45Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (8014) Google Scholar) to determine apparent high and low affinity B max and K D values. Significant differences (p < 0.05) among values were determined using JMP to perform a two-tailed Tukey-Kramer HSD test for multiple comparisons or a two-tailed Student's t test to compare two values. Unless otherwise indicated, all data presented are mean ± S.E. of three or more determinations from assays that were each performed in triplicate. The association and dissociation rates of [35S]GTPγS binding were investigated in rat cerebellar membranes using different concentrations of GDP, in the presence and absence of a maximally effective concentration of the cannabinoid agonist WIN 55212-2. Fig. 1 A shows association of [35S]GTPγS binding; TableI provides maximal binding andt½ values of association under these conditions. [35S]GTPγS binding to cerebellar membranes reached steady state at a rate that was dependent on the concentration of GDP. At 0 and 0.1 μm GDP, [35S]GTPγS binding reached maximum values within 1 and 2 h, respectively, and actually decreased slightly between 2 and 4 h. Maximal [35S]GTPγS binding, both in the presence and absence of agonist, was decreased by increasing concentrations of GDP. Stimulation by WIN 55212-2 could only be observed with micromolar concentrations of GDP, and the percent stimulation of [35S]GTPγS binding by agonist was increased by increasing concentrations of GDP, up to a maximum of 125% at 30 μm GDP (Table I).Table IAssociation and dissociation of [35S]GTPγS binding[GDP]Maximum bindingBasal+ 3 μm WIN 55212–2μmpmol/mgAssociation 01.502 ± 0.032 a1.524 ± 0.029 a 0.10.997 ± 0.069 b1.080 ± 0.048 b 30.571 ± 0.023 c0.865 ± 0.021 caSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest. 300.246 ± 0.014 d0.548 ± 0.035 daSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest.Apparentt½min 08.5 ± 0.2 a8.4 ± 0.4 a 0.122 ± 0.3 ab19 ± 0.5 a 360 ± 6.8 b45 ± 3.4 ab 30101 ± 16 c72 ± 7.4 baSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest.% fast-dissociating sitesDissociation 014 ± 1.0 a25 ± 0.5aSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest. 0.123 ± 2.1 ab38 ± 5.0aSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest. 322 ± 2.8 ab41 ± 6.9aSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest. 3027 ± 1.8 b44 ± 2.7aSignificant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest.Kinetics of [35S]GTPγS binding to cerebellar membranes were determined in the absence (basal) and presence of WIN 55212–2 at different concentrations of GDP, as shown in Fig. 1. Kinetic values were obtained by nonlinear fitting of the data, as described under "Experimental Procedures." "% of fast dissociating sites" is the percentage of [35S]GTPγS-binding sites that exhibited a rapid dissociation rate (t½ = 6.8 min)versus a slow dissociation rate (t½ = 170 min). Letters indicate a significant effect of GDP; values within a column designated with different letters are significantly different (p < 0.05) by the Tukey-Kramer test.a Significant effects of WIN 55212–2 at a given GDP concentration are p < 0.05 by Student's ttest. Open table in a new tab Kinetics of [35S]GTPγS binding to cerebellar membranes were determined in the absence (basal) and presence of WIN 55212–2 at different concentrations of GDP, as shown in Fig. 1. Kinetic values were obtained by nonlinear fitting of the data, as described under "Experimental Procedures." "% of fast dissociating sites" is the percentage of [35S]GTPγS-binding sites that exhibited a rapid dissociation rate (t½ = 6.8 min)versus a slow dissociation rate (t½ = 170 min). Letters indicate a significant effect of GDP; values within a column designated with different letters are significantly different (p < 0.05) by the Tukey-Kramer test. Both GDP and agonist significantly affected the rate of [35S]GTPγS association, as determined by the apparentt½ values (Table I). As the concentration of added GDP was increased from 0 to 30 μm,t½ values of basal [35S]GTPγS association were increased from 8.5 to 101 min. The effect of agonist was increased by GDP; addition of WIN 55212-2 had no effect on thet½ of association in the absence of GDP but significantly decreased the t½ from 101 to 72 min at 30 μm GDP. Data for dissociation of [35S]GTPγS binding are shown in Fig. 1 B as percent of steady-state binding values obtained in the presence or absence of agonist at each concentration of GDP. Actual binding values at time 0 were very similar to those obtained under the same conditions at 2 h in the association assays (Fig. 1 A). In contrast to previous reports of irreversible binding of [35S]GTPγS to purified G-proteins in the presence of millimolar concentrations of Mg2+ (29Bokoch G.M. Katada T. Northrup J.K. Ui M. Gilman A.G. J. Biol. Chem. 1984; 259: 3560-3567Abstract Full Text PDF PubMed Google Scholar, 30Sternweis P.C. Robishaw J.D. J. Biol. Chem. 1984; 259: 13806-13813Abstract Full Text PDF PubMed Google Scholar), [35S]GTPγS dissociated with both a rapid (t½ of 6.8 min) and a slow (t½ of 170 min) dissociation rate from cerebellar membranes. The biphasic nature of [35S]GTPγS dissociation is shown by the logarithmic plot of the data in Fig. 1 B. Nonlinear regression analysis of these data determined that neither GDP nor agonist affected the t½ values of either rate, but both increased the fraction of sites that displayed rapid dissociation. In the absence of GDP and agonist, only 14% of the [35S]GTPγS-binding sites exhibited the rapid dissociation rate (Table I). Increasing the concentration of GDP alone increased the fraction of rapidly dissociating binding sites to 27% of total [35S]GTPγS binding at 30 μmGDP. Unlike the effects of WIN 55212-2 on [35S]GTPγS association, WIN 55212-2 significantly affected dissociation regardless of the concentration of GDP, increasing the fraction of rapidly dissociating sites to 25% in the absence of GDP up to 44% with 30 μm GDP. Moreover, although there was a significant increase in the dissociation by 30 μm GDP in the absence of agonist, the effect of GDP in the presence of WIN 55212-2 did not reach statistical significance. Net agonist-stimulated [35S]GTPγS binding kinetics are shown in Fig. 2. These curves were obtained by subtracting basal binding values from the values obtained in the presence of WIN 55212-2 at each respective time point and GDP concentration. Since there was significant stimulation by WIN 55212-2 only at micromolar GDP concentrations, net agonist-stimulated [35S]GTPγS association and dissociation are shown for 3 and 30 μm GDP. In Fig. 2, it can be seen that net WIN 55212-2-stimulated [35S]GTPγS binding reaches steady-state levels within 2 h and is readily dissociable. To characterize [35S]GTPγS-binding sites and the effect of agonist on these sites, GTPγS saturation experiments were performed after 2-h incubations in the presence and absence of a maximally effective concentration of WIN 55212-2 and 30 μm GDP (Fig. 3). In the absence of GDP, [35S]GTPγS binding was biphasic, displaying both high (apparent K D = 2.7 nm) and low (apparentK D = 800 nm) affinity sites (TableII). Addition of WIN 55212-2 had no effect on the apparent K D orB max of either site in the absence of GDP (Fig. 3 A). In the presence of 30 μm GDP alone (Fig. 3 B), [35S]GTPγS binding was best fit to sites with intermediate (apparent K D = 14 nm) and low affinity; apparent B maxvalues were decreased by 70–80% compared with those in the absence of added GDP. Addition of agonist with 30 μm GDP produced [35S]GTPγS binding with high (apparentK D = 4 nm) and low affinity sites (Fig. 3 B). The apparent K D andB max values of the low affinity sites were not significantly affected by agonist. The apparent K Dof the agonist-induced high affinity (4 nm) site was significantly lower than the apparent K D of the intermediate affinity (14 nm) site of basal [35S]GTPγS binding (p = 0.010); however, there was no significant different between theB max values of these sites. Whereas there was no net agonist-stimulated [35S]GTPγS binding in the absence of added GDP (Fig. 3 A), net WIN 55212-2-stimulated [35S]GTPγS binding in the presence of GDP was monophasic with an apparent high affinity K D value of 2.7 nm (Fig. 3 B, inset, and Table II), similar to previous results with mu and delta opioid agonists (19Sim L.J. Selley D.E. Xiao R. Childers S.R. Eur. J. Pharmacol. 1996; 307: 95-107Crossref Scopus (140) Google Scholar, 27Traynor J.R. Nahorski S.R. Mol. Pharmacol. 1995; 47: 848-854PubMed Google Scholar,33Selley D.E. Sim L.J. Xiao R. Liu Q. Childers S.R. Mol. Pharmacol. 1997; 51: 87-96Crossref PubMed Scopus (193) Google Scholar, 46Breivogel C.S. Selley D.E. Childers S.R. J. Neurochem. 1997; 68: 1462-1472Crossref PubMed Scopus (58) Google Scholar).Table II[35S]GTPγS binding parameters in rat cerebellar membranesNo GDPBasal+ 3 μm WIN 55212–2NetK D(H)(nm)2.7 ± 1.13.0 ± 1.1N/AB max(H)(pmol/mg)110 ± 33120 ± 33N/AK D(L) (nm)750 ± 110920 ± 180N/AB max(L) (pmol/mg)1060 ± 2201140 ± 430N/A30 μm GDPK D(I/H)(nm)14 ± 2.24.0 ± 0.7aBy Student's t test, p < 0.05 for WIN 55212–2 versus basal.2.7 ± 0.3B max(I/H) (pmol/mg)15 ± 3.823 ± 3.812.6 ± 1.1K D(L)(nm)540 ± 104980 ± 270N/AB max(L)(pmol/mg)220 ± 56350 ± 68N/AApparent B max and K D values were determined using 0.05 nm [35S]GTPγS plus 0.5 nm to 10 μm unlabeled GTPγS in the absence and presence of 3 μm WIN 55212–2, to determine basal and agonist-stimulated binding, respectively. (H) designatesK D and B max values for high affinity binding sites, (L) indicates low affinity sites, and (I) indicates intermediate affinity sites. Assays were conducted in the absence and presence of 30 μm added GDP. Net agonist-stimulated [35S]GTPγS binding, determined by subtracting basal from WIN 55212–2-stimulated [35S]GTPγS binding at each concentration of GTPγS, was not detectable (N/A, not applicable) in the absence of GDP and was monophasic and high affinity in the presence of GDP.a By Student's t test, p < 0.05 for WIN 55212–2 versus basal. Open table in a new tab Apparent B max and K D values were determined using 0.05 nm [35S]GTPγS plus 0.5 nm to 10 μm unlabeled GTPγS in the absence and presence of 3 μm WIN 55212–2, to determine basal and agonist-stimulated binding, respectively. (H) designatesK D and B max values for high affinity binding sites, (L) indicates low affinity sites, and (I) indicates intermediate affinity sites. Assays were conducted in the absence and presence of 30 μm added GDP. Net agonist-stimulated [35S]GTPγS binding, determined by subtracting basal from WIN 55212–2-stimulated [35S]GTPγS binding at each concentration of GTPγS, was not detectable (N/A, not applicable) in the absence of GDP and was monophasic and high affinity in the presence of GDP. In addition to increasing the apparent affinity of Gα for [35S]GTPγS, agonists have been reported to reduce the affinity of Gα for GDP (15Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (5031) Google Scholar, 16Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 23Florio V.A. Sternweis P.C. J. Biol. Chem. 1989; 264: 3909-3915Abstract Full Text PDF PubMed Google Scholar). To explore this possibility, cerebellar membranes were incubated with [35S]GTPγS and 0.3 nm to 1000 μm GDP in the pr
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