Cannabinoid Receptor Agonist Efficacy for Stimulating [35S]GTPgamma S Binding to Rat Cerebellar Membranes Correlates with Agonist-induced Decreases in GDP Affinity*

Christopher S. Breivogel, Dana E. Selley, and Steven R. ChildersDagger

From the Department of Physiology and Pharmacology, Center for the Neurobiological Investigation of Drug Abuse, and Center for Investigative Neuroscience, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The relationship between GDP and cannabinoid-stimulated [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgamma S) binding was investigated in rat cerebellar membranes. Kinetic analyses showed that [35S]GTPgamma S 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]GTPgamma S occurred with two rates (t1/2 = 7 and 170 min), and WIN 55212-2 increased the proportion of sites exhibiting the faster rate. Without GDP, [35S]GTPgamma S bound to membranes with high and low affinity, and WIN 55212-2 had no effect. With 30 µM GDP, [35S]GTPgamma S bound to low and intermediate affinity sites, and WIN 55212-2 induced high affinity [35S]GTPgamma S binding without affecting low affinity sites. GDP competed for high affinity [35S]GTPgamma S 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]GTPgamma S 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 Ki values that were proportional to agonist Emax values, indicating that agonist efficacy is determined by displacement of GDP from G-proteins.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cannabinoid receptors mediate the actions of Delta 9-tetrahydrocannabinol (Delta 9-THC)1 and other cannabimimetic ligands (1). To date, two types of cannabinoid receptors have been discovered, CB1 (2, 3) and CB2 (4). A splice variant of CB1, termed CB1A, has also been reported (5). Apart from a recent report of CB2 in mouse cerebellum (6), CB1 has been the only cannabinoid receptor found in brain. All cannabinoid receptors discovered to date belong to the superfamily of G-protein-coupled receptors (3, 4); their effectors include inhibition of adenylyl cyclase (7, 8), inhibition of calcium influx (9), and activation of inwardly rectifying potassium channels (10, 11). The physiological actions of cannabinoid ligands have been shown to be mediated through the activation of pertussis toxin-sensitive G-proteins (Gialpha and Goalpha subtypes) (7, 12), although some effects have been implicated via Gsalpha as well (13, 14).

G-proteins are heterotrimeric proteins that transduce the agonist binding signal from G-protein-coupled receptors to effectors (15, 16). Upon activation by an agonist-occupied receptor, the alpha  subunit of a G-protein (Galpha ) releases bound GDP, binds a molecule of GTP, and dissociates from the G-protein beta gamma subunit complex. Both Galpha and beta gamma subunits act upon effectors until Galpha cleaves the bound GTP to GDP by its intrinsic GTPase activity, and Galpha re-associates with a beta gamma dimer (15, 16). 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 (17-19). 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 (20). 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 (21) and may be the most relevant step in measuring agonist efficacy at G-protein-coupled receptors (22).

Agonist-stimulated binding of the hydrolysis-resistant GTP analog, [35S]GTPgamma S, to G-protein alpha  subunits measures receptor activation of G-proteins in purified and reconstituted systems (23), native cell membrane preparations (24), and brain sections (25). The present study focuses on three aspects of the role of GDP in the agonist-stimulated [35S]GTPgamma S binding assay. First, GDP has been shown to decrease basal [35S]GTPgamma S 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]GTPgamma S binding has been demonstrated (24, 26-28). Second, GDP has been reported to modulate the kinetics of [35S]GTPgamma S binding. The presence of micromolar concentrations of GDP was shown to decrease the magnitude and rate of [35S]GTPgamma S binding to purified and reconstituted G-proteins (23). However, early reports of [35S]GTPgamma S binding to purified G-protein Gialpha (29) and Goalpha (30) 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]GTPgamma S binding (31). Therefore, a problem frequently noted for [35S]GTPgamma S 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]GTPgamma S binding. In the adenosine A1 receptor system, a full agonist was shown to be maximally effective for the stimulation of [35S]GTPgamma S binding at a higher concentration of GDP than a partial agonist (32). Similar results were found in the mu opioid system, where increasing the concentration of GDP increased relative efficacy differences among agonists (33). In order to determine whether GDP plays similar roles in modulating cannabinoid agonist efficacy, it is necessary to compare [35S]GTPgamma S binding stimulated by agonists of different efficacies. Previous studies which showed that Delta 9-THC (34, 35), CP 55940 (36), and anandamide (37-39) 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 (40) and cannabinoid-activated G-proteins (25) compared with other G-protein-coupled receptors in brain. These experiments provide evidence that cannabinoid agonist-stimulated [35S]GTPgamma S 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Male Sprague-Dawley rats were purchased from Zivic Miller (Zelienople, PA). [35S]GTPgamma S (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). Delta 9-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 GTPgamma S were purchased from Boehringer Mannheim. All other reagent grade chemicals were obtained from Sigma or Fisher.

Membrane Preparations-- 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 (41). For assays including anandamide, thawed membranes were pretreated with 50 µM phenylmethylsulfonyl 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 (42) before addition to assay tubes.

[35S]GTPgamma S Binding-- Assays were performed as described previously (41). 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]GTPgamma S in a final volume of 1 ml, and nonspecific binding was determined with 30 µM unlabeled GTPgamma S. 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 (41). 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 GTPgamma S at various times; reactions were terminated as above.

Data Analysis-- 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 (43).
<UP>B = B<SUB>final</SUB> × </UP>(1−e<SUP>−kt</SUP>) (Eq. 1)
where B is the amount of [35S]GTPgamma S bound at time t; Bfinal is the maximum amount of ligand bound under steady-state conditions, and k is the apparent association rate constant (kobs). Dissociation parameters were determined by fitting for biphasic bimolecular dissociation as shown in Equation 2 (43).
<UP>B = B<SUB>01</SUB> × </UP>e<SUP>−k1t</SUP>+<UP>B<SUB>02</SUB></UP>×e<SUP>−k2t</SUP> (Eq. 2)
where B is the amount of [35S]GTPgamma S bound at time t; B01 and B02 are the amounts of ligand bound to rapidly and slowly dissociating sites at time 0, and k1 and k2 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 (kobs or k-1). EC50 and Emax values for each agonist were determined by fitting concentration-effect curves to Equation 3.
E=<FR><NU>[<UP>L</UP>]×E<SUB><UP>max</UP></SUB></NU><DE>[<UP>L</UP>]+<UP>EC</UP><SUB><UP>50</UP></SUB></DE></FR> (Eq. 3)
where E is amount of [35S]GTPgamma S bound at receptor ligand concentration [L]; Emax is the amount of [35S]GTPgamma S bound at maximally effective concentrations of receptor ligand, and EC50 is the concentration of receptor ligand producing half-maximal [35S]GTPgamma S binding. IC50 and Imax values for GDP competition curves were determined by fitting the biphasic Equation 4.
I=<FR><NU>[I]×I<SUB><UP>max</UP>(<UP>H0</UP></SUB></NU><DE>[I]+<UP>IC</UP><SUB><UP>50</UP>(<UP>H</UP>)</SUB></DE></FR>+<FR><NU>[I]×I<SUB><UP>max</UP>(<UP>L</UP>)</SUB></NU><DE>[I]+<UP>IC</UP><SUB><UP>50</UP>(<UP>L</UP>)</SUB></DE></FR> (Eq. 4)
where I is the amount of [35S]GTPgamma S binding inhibited at GDP concentration [I]; Imax(H) and Imax(L) are the maximum amounts of [35S]GTPgamma S 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]GTPgamma S binding from each site, respectively. Ki values were estimated by the Cheng-Prusoff equation (44). [35S]GTPgamma S saturation binding was analyzed using EBDA and LIGAND (45) to determine apparent high and low affinity Bmax and KD 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Effects of GDP and Cannabinoid Agonist on the Kinetics of [35S]GTPgamma S Binding-- The association and dissociation rates of [35S]GTPgamma S 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. 1A shows association of [35S]GTPgamma S binding; Table I provides maximal binding and t1/2 values of association under these conditions. [35S]GTPgamma S 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]GTPgamma S binding reached maximum values within 1 and 2 h, respectively, and actually decreased slightly between 2 and 4 h. Maximal [35S]GTPgamma S 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]GTPgamma S binding by agonist was increased by increasing concentrations of GDP, up to a maximum of 125% at 30 µM GDP (Table I).


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Fig. 1.   Effects of GDP and WIN 55212-2 on the association and dissociation of [35S]GTPgamma S binding in rat cerebellar membranes. For association assays (A), membranes were incubated at 30 °C for various times with 0.05 nM [35S]GTPgamma S in the presence of various concentrations of added GDP and in the presence and absence of 3 µM WIN 55212-2. B depicts dissociation assays that were conducted by incubating membranes for 1-2 h under the same conditions used for the association assays before the addition of 30 µM unlabeled GTPgamma S at various times. Data in B are expressed on a logarithmic scale as percent of steady-state [35S]GTPgamma S binding.

                              
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Table I
Association and dissociation of [35S]GTPgamma S binding
Kinetics of [35S]GTPgamma S 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]GTPgamma S-binding sites that exhibited a rapid dissociation rate (t1/2 = 6.8 min) versus a slow dissociation rate (t1/2 = 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]GTPgamma S association, as determined by the apparent t1/2 values (Table I). As the concentration of added GDP was increased from 0 to 30 µM, t1/2 values of basal [35S]GTPgamma S 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 the t1/2 of association in the absence of GDP but significantly decreased the t1/2 from 101 to 72 min at 30 µM GDP.

Data for dissociation of [35S]GTPgamma S binding are shown in Fig. 1B 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. 1A). In contrast to previous reports of irreversible binding of [35S]GTPgamma S to purified G-proteins in the presence of millimolar concentrations of Mg2+ (29, 30), [35S]GTPgamma S dissociated with both a rapid (t1/2 of 6.8 min) and a slow (t1/2 of 170 min) dissociation rate from cerebellar membranes. The biphasic nature of [35S]GTPgamma S dissociation is shown by the logarithmic plot of the data in Fig. 1B. Nonlinear regression analysis of these data determined that neither GDP nor agonist affected the t1/2 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]GTPgamma S-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]GTPgamma S binding at 30 µM GDP. Unlike the effects of WIN 55212-2 on [35S]GTPgamma S 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]GTPgamma S 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]GTPgamma S 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]GTPgamma S binding reaches steady-state levels within 2 h and is readily dissociable.


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Fig. 2.   Effects of GDP on net WIN 55212-2-stimulated [35S]GTPgamma S binding to rat cerebellar membranes. Net agonist-stimulated [35S]GTPgamma S binding was determined from the data shown in Fig. 1 by subtracting basal [35S]GTPgamma S binding values from values obtained in the presence of WIN 55212-2 at each time point.

Effects of GDP and Cannabinoid Agonist on Steady-state [35S]GTPgamma S Binding Parameters-- To characterize [35S]GTPgamma S-binding sites and the effect of agonist on these sites, GTPgamma S 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]GTPgamma S binding was biphasic, displaying both high (apparent KD = 2.7 nM) and low (apparent KD = 800 nM) affinity sites (Table II). Addition of WIN 55212-2 had no effect on the apparent KD or Bmax of either site in the absence of GDP (Fig. 3A). In the presence of 30 µM GDP alone (Fig. 3B), [35S]GTPgamma S binding was best fit to sites with intermediate (apparent KD = 14 nM) and low affinity; apparent Bmax values were decreased by 70-80% compared with those in the absence of added GDP. Addition of agonist with 30 µM GDP produced [35S]GTPgamma S binding with high (apparent KD = 4 nM) and low affinity sites (Fig. 3B). The apparent KD and Bmax values of the low affinity sites were not significantly affected by agonist. The apparent KD of the agonist-induced high affinity (4 nM) site was significantly lower than the apparent KD of the intermediate affinity (14 nM) site of basal [35S]GTPgamma S binding (p = 0.010); however, there was no significant different between the Bmax values of these sites. Whereas there was no net agonist-stimulated [35S]GTPgamma S binding in the absence of added GDP (Fig. 3A), net WIN 55212-2-stimulated [35S]GTPgamma S binding in the presence of GDP was monophasic with an apparent high affinity KD value of 2.7 nM (Fig. 3B, inset, and Table II), similar to previous results with mu and delta opioid agonists (19, 27, 33, 46).


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Fig. 3.   Effect of GDP and WIN 55212-2 on [35S]GTPgamma S-binding sites in rat cerebellar membranes. Representative biphasic Scatchard plots of [35S]GTPgamma S binding with and without 3 µM WIN 55212-2 in the absence (A) and presence (B) of 30 µM GDP. Saturation binding was accomplished by incubating membranes at 30 °C for 2 h with 0.05 nM [35S]GTPgamma S plus 0.5 nM to 10 µM unlabeled GTPgamma S. B, inset, Scatchard plot of net agonist-stimulated [35S]GTPgamma S binding determined by subtracting basal [35S]GTPgamma S binding from that obtained in the presence of WIN 55212-2 at each concentration of GTPgamma S. Data shown are representative of three experiments that gave similar results; mean apparent KD and Bmax values are given in Table II.

                              
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Table II
[35S]GTPgamma S binding parameters in rat cerebellar membranes
Apparent Bmax and KD values were determined using 0.05 nM [35S]GTPgamma S plus 0.5 nM to 10 µM unlabeled GTPgamma S in the absence and presence of 3 µM WIN 55212-2, to determine basal and agonist-stimulated binding, respectively. (H) designates KD and Bmax 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]GTPgamma S binding, determined by subtracting basal from WIN 55212-2-stimulated [35S]GTPgamma S binding at each concentration of GTPgamma S, 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 Galpha for [35S]GTPgamma S, agonists have been reported to reduce the affinity of Galpha for GDP (15, 16, 23). To explore this possibility, cerebellar membranes were incubated with [35S]GTPgamma S and 0.3 nM to 1000 µM GDP in the presence and absence of WIN 55212-2 (Fig. 4). Since the standard concentration of [35S]GTPgamma S used (0.05 nM) results in low occupancy of high affinity [35S]GTPgamma S-binding sites (0.5-2%), these assays were also conducted using two higher concentrations of [35S]GTPgamma S (0.2 and 1 nM) to produce approximately 7 and 25% occupancy of the high affinity sites. Nevertheless, at any of these concentrations of [35S]GTPgamma S, high affinity [35S]GTPgamma S binding would predominate, since 1 nM [35S]GTPgamma S would occupy less than 0.15% of the low affinity sites. Thus, the GDP-binding sites investigated under these conditions represented only sites that bound [35S]GTPgamma S with high affinity.


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Fig. 4.   Effect of WIN 55212-2 on competition binding of [35S]GTPgamma S and GDP in rat cerebellar membranes. Membranes were incubated with 0.05, 0.20, or 1.0 nM [35S]GTPgamma S plus 0.3 nM to 1 mM GDP in the presence and absence of 3 µM WIN 55212-2. B, data from A re-plotted on a logarithmic scale y axis to show the effect of WIN 55212-2 at low levels of [35S]GTPgamma S binding. GDP Ki and Imax values are provided in Table III.

The results showed that as cannabinoid agonist increased the apparent affinity of a fraction of [35S]GTPgamma S-binding sites (Fig. 3), it also decreased the affinity of a fraction of GDP-binding sites (Fig. 4). The effect of agonist on GDP affinity in Fig. 4A is best observed for the upper set of curves (1 nM [35S]GTPgamma S), where significant increases in binding by WIN 55212-2 were not observed until GDP concentrations exceeded 0.1 µM. To show that this effect of agonist was also observed at lower concentrations of [35S]GTPgamma S (0.05 and 0.20 nM), these data were re-plotted in a logarithmic fashion (Fig. 4B). When plotted in this manner, it is clear that WIN 55212-2 had no effect on [35S]GTPgamma S binding at low (<0.1 µM) concentrations of GDP but increased binding in the presence of micromolar concentrations (1-100 µM) of GDP, i.e. WIN 55212-2 shifted the lower affinity component of the GDP competition curve to the right. Nonlinear regression analysis showed that GDP inhibited basal [35S]GTPgamma S binding in a biphasic manner, with high affinity Ki values of 20-30 nM and intermediate affinity Ki values of 800-1000 nM, regardless of the [35S]GTPgamma S concentration used (Table III). In the presence of WIN 55212-2, GDP competed for [35S]GTPgamma S binding with high affinity (Ki of 30-40 nM) and low affinity (Ki of 7000 nM). These data show that high affinity Ki values for GDP in the presence of WIN 55212-2 were indistinguishable from those measured under basal conditions but that the agonist-induced low affinity Ki value was 8-fold lower than the intermediate affinity component observed under basal conditions. Although high affinity GDP sites represented approximately 60% of the total high affinity basal [35S]GTPgamma S-binding sites, variability in the Imax calculations prevented any definitive determination of agonist-induced changes in the proportion of high affinity GDP-binding sites.

                              
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Table III
Parameters of GDP competition for high affinity [35S]GTPgamma S binding in rat cerebellar membranes
Imax and IC50 values were determined by displacement of 0.05, 0.2, or 1.0 nM [35S]GTPgamma S by 0.3 nM to 1 mM GDP in the presence and absence of 3 µM WIN 55212-2. (H), (I), and (L) designate Ki and Imax values for high, intermediate, and low affinity binding sites, respectively. Ki values were calculated from IC50 values as described under "Experimental Procedures."

Relationship between GDP and Cannabinoid Agonist Efficacy-- Previous studies have demonstrated differences in cannabinoid agonist efficacies by different methods. Since G-protein activation is the first step in the signal transduction cascade of G-protein-coupled receptors, it was of interest to measure cannabinoid efficacy by agonist-stimulated [35S]GTPgamma S binding. Several cannabinoid ligands with different structural bases were selected including the following: Delta 9-THC, the primary psychoactive constituent of marijuana; WIN 55212-2, a synthetic aminoalkylindole agonist; levonantradol, a potent Delta 9-THC analog; CP 55940, a synthetic bicyclic compound; anandamide and methanandamide, an endogenous cannabinoid agonist and its esterase-resistant analog; and SR141716A, the CB1-selective antagonist.

To establish the relative efficacies of these agonists for [35S]GTPgamma S binding, concentration-effect curves were generated in the presence of 30 µM GDP (Fig. 5). Some of the agonists exhibited shallow concentration-effect curves, indicating stimulation of [35S]GTPgamma S binding by more than one site (or affinity state of the receptor). Since this study focused on differences in the maximal stimulation of [35S]GTPgamma S binding by each agonist, concentration-effect curves were analyzed for EC50 and Emax values monophasically. Full biphasic analysis of agonist stimulation of [35S]GTPgamma S binding will be conducted in a future study. Potencies (EC50 values) varied widely for these compounds. CP 55940 displayed the greatest potency with an EC50 of 6.6 ± 0.5 nM; levonantradol was next at 9.0 ± 0.4 nM, followed by Delta 9-THC at 87 ± 42 nM, WIN 55212-2 at 160 ± 38 nM, and methanandamide and anandamide at 320 ± 26 and 390 ± 96 nM, respectively (Fig. 5). As previously shown for receptor binding (39), pretreatment of the membranes with the irreversible esterase inhibitor PMSF greatly increased the potency of anandamide, since without PMSF pretreatment, anandamide stimulated [35S]GTPgamma S binding with an EC50 of 1750 ± 570 nM (data not shown). In contrast, none of the potencies or efficacies of the other agonists, including methanandamide, were significantly affected by PMSF pretreatment (data not shown).


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Fig. 5.   Concentration-effect curves of cannabinoid ligands in stimulating [35S]GTPgamma S binding to rat cerebellar membranes. Membranes were incubated with 0.05 nM [35S]GTPgamma S, 30 µM GDP, and various concentrations of each ligand. Data are expressed as the percent of [35S]GTPgamma S binding obtained in the presence of a maximally effective concentration of levonantradol (1 µM), which was 190 ± 45 fmol/mg. Emax values for each agonist are provided in Table IV.

Concentration-effect analysis revealed that these ligands produced a wide range of efficacies for G-protein activation in the [35S]GTPgamma S binding assay (Fig. 5). WIN 55212-2 and levonantradol displayed the highest efficacies, and these two ligands were designated as full agonists. For this reason, results for other ligands were normalized to the amount of net agonist-stimulated [35S]GTPgamma S binding obtained with a maximally effective concentration of levonantradol (1 µM), which was defined as 100% within each experiment (Fig. 5). Likewise, Emax values obtained by nonlinear regression analysis for each agonist were normalized to the Emax value obtained with levonantradol (Table IV). Whereas the Emax value of WIN 55212-2-stimulated [35S]GTPgamma S binding (106 ± 2%) was not significantly different from that of levonantradol, CP 55940 acted as a high efficacy partial agonist stimulating 81 ± 2% as much as levonantradol. Anandamide and methanandamide each produced Emax values of approximately 70% (70 ± 6 and 68 ± 2%, respectively) of levonantradol. In agreement with previous results (34, 35), Delta 9-THC stimulated only 21 ± 0.7% as much as levonantradol, confirming that this ligand exhibits weak partial agonist activity. Finally, SR141716A failed to stimulate [35S]GTPgamma S binding at any concentration, indicating that this ligand is a pure antagonist with zero efficacy. However, SR141716A slightly but consistently inhibited basal [35S]GTPgamma S binding at the highest concentration (10 µM) used (Fig. 5).

                              
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Table IV
Stimulation of [35S]GTPgamma S binding Emax and GDP Ki values induced by cannabinoid agonists
Emax values were obtained by nonlinear regression analysis of the stimulation of [35S]GTPgamma S binding by various concentrations of each agonist, shown in Fig. 5, except the Emax value reported for 0.1 µM WIN 55212-2, which was determined by the amount of [35S]GTPgamma S binding obtained with that concentration of the agonist. Data are expressed as mean ± S.E. of percent of the Emax value obtained with levonantradol for each experiment. GDP Ki values were calculated from IC50 values determined by competition of 0.05 nM [35S]GTPgamma S with 0.3 nM to 1 mM GDP in the presence and absence of 3 µM WIN 55212-2, shown in Fig. 6. ND, not determined. Emax values that are not marked with the same letter are significantly different from each other by the Tukey-Kramer test at p < 0.05. 

In other receptor systems, increasing the GDP concentration was reported to increase differences between full and partial agonists for stimulating [35S]GTPgamma S binding (32, 33). Therefore, the relationship between GDP and cannabinoid agonist efficacy was directly explored using a few representative cannabinoid ligands. To determine whether the affinity of the low affinity GDP-binding site was related to the efficacy of the agonist, GDP competition curves were generated with 0.05 nM [35S]GTPgamma S in the presence and absence of maximally effective concentrations of these ligands. As described above (Table III), GDP displaced [35S]GTPgamma S binding with both high and intermediate affinity or high and low affinity in the absence or presence of agonist, respectively. Full biphasic analysis (Table IV) indicated that the agonists had no significant effect on the Ki values of high affinity GDP binding, which were all between 20 and 47 nM. In the absence of agonist, the low affinity GDP-binding sites had an intermediate Ki value of 1.1 µM; in the presence of agonist, the affinity of this low affinity GDP site depended on the agonist. Delta 9-THC had no statistically significant effect on the low affinity GDP Ki value (1.3 µM), whereas the full agonists WIN 55212-2 and levonantradol produced low affinity GDP Ki values of 8 µM, and the partial agonist methanandamide produced a Ki value of 6.6 µM. Addition of a submaximally effective concentration of WIN 55212-2 (0.1 µM), which stimulated 44% of maximal [35S]GTPgamma S binding values, produced an intermediate low affinity GDP Ki value of 4.2 µM (Table IV).

The finding that these agonists decreased GDP affinity in proportion to their efficacies predicts that saturating concentrations of full agonists will be maximally effective for the stimulation of [35S]GTPgamma S binding at higher concentrations of GDP than saturating concentrations of partial agonists. This relationship is depicted in Fig. 6A where net agonist-stimulated [35S]GTPgamma S binding is plotted as a function of the concentration of added GDP. For each agonist assayed, net agonist-stimulated [35S]GTPgamma S binding increased with increasing GDP concentrations until maximum net-stimulated binding was achieved. The GDP concentration that produced maximal net-stimulated binding depended on the efficacy of the agonist. Maximal net agonist-stimulated [35S]GTPgamma S binding was observed with Delta 9-THC at approximately 0.1-0.2 µM GDP, with methanandamide at approximately 1 µM GDP, and with WIN 55212-2 at 2-3 µM GDP. SR141716A failed to significantly stimulate [35S]GTPgamma S binding at any GDP concentration, and results for 1 µM levonantradol were similar to those obtained with 10 µM WIN 55212-2 (data not shown). When the data were plotted as percent stimulation by each agonist as a function of GDP concentration (Fig. 6B), differences in percent stimulation among the agonists increased as the concentration of GDP was increased to an optimum level of approximately 100 µM. Thus, at 0.1 µM GDP there was little difference between the full and partial agonists, at 1 µM GDP there was a significant difference between Delta 9-THC and all of the higher efficacy agonists, and at 30 µM GDP the efficacies of the high efficacy partial agonists CP 55940, anandamide, and methanandamide were different from the full agonists WIN 55212-2 and levonantradol (data for representative ligands are shown in Fig. 6B). CP 55940 was different from the anandamide compounds only at 100 µM GDP (data not shown). The antagonist SR141716A was different from all of the agonists at every GDP concentration assayed with this ligand (1-100 µM).


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Fig. 6.   Effect of GDP on the relative efficacies of cannabinoid agonists in stimulating [35S]GTPgamma S binding to rat cerebellar membranes. Membranes were incubated with 0.05 nM [35S]GTPgamma S plus 0.3 nM to 100 µM GDP, in the presence and absence of maximally effective concentrations of each agonist as determined by data shown in Fig. 5. Net agonist-stimulated [35S]GTPgamma S binding values were determined by subtracting values obtained in the absence from those obtained in the presence of WIN 55212-2, and percent stimulation values were determined by dividing net agonist-stimulated [35S]GTPgamma S binding values by basal binding values at each concentration of GDP. Data are expressed as a percentage of the maximum values obtained with levonantradol, which were 466 ± 28 fmol/mg of net agonist-stimulated binding and 399 ± 43% stimulation.

Fig. 7 shows the correlation between agonist efficacy for the stimulation of [35S]GTPgamma S binding (expressed as a percent of levonantradol Emax) and agonist-induced GDP low affinity Ki values (from Table IV). These data show that agonists of high efficacy produced higher low affinity GDP Ki values than agonists of lower efficacy. The correlation between these two parameters was highly significant (r = 0.979, analysis of variance, p = 0.0007). In contrast, there was no significant correlation (r = 0.333, p = 0.519) between Emax values and high affinity GDP Ki values obtained in the presence of each agonist (data not shown).


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Fig. 7.   Correlation between relative agonist efficacy (Emax) in stimulating [35S]GTPgamma S binding and agonist-induced low affinity GDP Ki values. Data for relative agonist efficacy (agonist Emax values) and low affinity GDP binding Ki values were obtained from Table IV.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

This study characterized several aspects of the role of GDP in the activation of G-proteins by cannabinoid receptors in brain membranes. In kinetics studies, [35S]GTPgamma S binding to cerebellar membranes reached an apparent steady state and was readily dissociable. Moreover, cannabinoid agonist increased the rate of both [35S]GTPgamma S association and dissociation. Addition of GDP decreased the rate and magnitude of [35S]GTPgamma S association, consistent with the competitive binding of [35S]GTPgamma S and GDP as previously shown in purified G-proteins (43).

It was significant that agonist-stimulated [35S]GTPgamma S binding was shown to be dissociable under these assay conditions, which include 3 mM Mg2+, and that both GDP and agonist increased the rate of [35S]GTPgamma S dissociation, just as muscarinic agonists and guanine nucleotides increased the dissociation of [35S]GTPgamma S from native cardiac membranes (47). The findings of reversible [35S]GTPgamma S binding in membranes seem to contradict earlier studies where [35S]GTPgamma S binding to purified Goalpha and Gialpha was virtually irreversible in the presence of millimolar concentrations of Mg2+ (29, 30). This discrepancy might be explained by the lower ratio of G-protein beta gamma to Galpha subunits in purified systems compared with those that may be present in native membranes (29, 30, 48, 49). [35S]GTPgamma S binding to purified Goalpha and Gialpha exhibits both rapid and slow dissociation rates, and the ratio of slowly to rapidly dissociating sites is proportional to the concentration of Mg2+ (50). beta gamma subunits increase the dissociation of [35S]GTPgamma S from Galpha , but this effect is inhibited by Mg2+, which inhibits beta gamma coupling to Galpha (30, 50). The present study found that upon addition of excess GTPgamma S, cannabinoid agonist increased the ratio of rapidly to slowly dissociating [35S]GTPgamma S-binding sites by the same degree (62-86%) regardless of the concentration of GDP. It is possible that the agonist-induced increase in rapidly dissociating [35S]GTPgamma S binding was the result of the liberation of large amounts of beta gamma by the agonist-accelerated binding of the unlabeled GTPgamma S to Galpha subunits. The finding that GDP produced a slight increase in the ratio of rapidly dissociating [35S]GTPgamma S-binding sites is consistent with the fact that GDP increases the ratio of low affinity to high affinity [35S]GTPgamma S-binding sites (Fig. 3), which would be expected to display different dissociation rates.

In cerebellar membranes, basal and cannabinoid-stimulated [35S]GTPgamma S binding appeared to follow the characteristics of bimolecular reactions, allowing the data to be analyzed in the manner of traditional radioligand binding. However, any study involving the binding of guanine nucleotide analogs to G-proteins must consider the presence of pre-bound GDP. It has been shown that GDP remains bound to Galpha in high molar ratios even after purification of Galpha subunits (43). Thus, all parameters of [35S]GTPgamma S binding to native cell membranes must be considered "apparent" in the presence or absence of added GDP. The present study has also demonstrated that occupancy of 2% of high affinity [35S]GTPgamma S-binding sites using 0.05 nM [35S]GTPgamma S accurately assesses high affinity [35S]GTPgamma S-binding sites, since concentrations of [35S]GTPgamma S that occupied up to 25% of high affinity sites yielded identical results with respect to the effects of GDP and agonist (Fig. 4).

Concentration-effect curves comparing the relative efficacies of several cannabinoid agonists determined that WIN 55212-2 and levonantradol produced the highest Emax values for the stimulation of [35S]GTPgamma S binding and are therefore referred to as full agonists. CP 55940 was a high efficacy partial agonist, confirming the results of a previous study (36). Anandamide and methanandamide both acted as partial agonists, in agreement with previous studies demonstrating partial agonism for the inhibition of Ca2+ currents (37) and adenylyl cyclase activity (38, 39). As previously shown (34, 35), Delta 9-THC acted as a weak partial agonist, stimulating only 20% of the [35S]GTPgamma S binding of the full agonists. SR141716A appeared to be a neutral antagonist, although the decreased [35S]GTPgamma S binding at 10 µM SR141716A seemed to agree with other recent reports of inverse agonism by SR141716A (51-53). However, since SR141716A has a KD of 0.3 nM in brain membranes (41), it is unlikely that inhibition of [35S]GTPgamma S binding was a CB1 receptor-mediated effect. If some CB2 receptors are present in cerebellum (6), then the 700 nM Ki of SR141716A at CB2 receptors (54) makes it possible that this inhibitory effect was mediated by CB2 receptors.

In agreement with results obtained in the mu opioid system, increasing the concentration of GDP between 0.1 and 100 µM increased efficacy differences among agonists (33). Significant stimulation of [35S]GTPgamma S binding was observed only in this range of GDP concentrations. The slight and variable stimulation of [35S]GTPgamma S binding observed in the absence (or at nanomolar concentrations) of added GDP may have been due to agonist-induced release of pre-bound GDP on the G-proteins (43).

A question that can be addressed by these data is which change in G-protein affinity is mediating agonist efficacy, i.e. is an increase in GTP(gamma S) affinity the fundamental mechanism or is the agonist-induced increase in apparent GTP(gamma S) affinity caused by a decrease in GDP affinity? These data suggest that the agonist-induced increases in the apparent affinity of [35S]GTPgamma S were due to decreases in the affinity of GDP, since these changes were observed only in the presence of added GDP (Fig. 3 and Table II). Moreover, the agonist-induced [35S]GTPgamma S affinities (measured with 30 µM GDP) and GDP affinities were reciprocal, high affinity for [35S]GTPgamma S and low affinity for GDP (Fig. 8). The two affinity states for [35S]GTPgamma S (measured with 30 µM GDP) and GDP observed in the basal state may also be reciprocal; the low affinity [35S]GTPgamma S-binding sites may correspond to the high affinity GDP-binding sites, and basal binding also exhibited intermediate affinities for both ligands (Fig. 3, lower panel, and Fig. 4; and Tables II and III; and Fig. 8). The affinities of [35S]GTPgamma S for the three sites observed in the presence of 30 µM GDP can actually be predicted based on the observed affinities for GDP and previous reports of the actual affinity of purified Galpha subunits for [35S]GTPgamma S. This prediction was made on the basis that the presence of a binding competitor will decrease the apparent affinity of a radioligand by an amount proportional to the ratio of the competitor's concentration and inhibition constant, according to a rearrangement of the Cheng-Prusoff equation: KD ratio = ([C]/Ki) + 1, where KD ratio is the ratio of the apparent KD and the actual KD for [35S]GTPgamma S in the presence and absence of GDP, respectively; [C] is the concentration of GDP, and Ki is the inhibition constant for GDP at each binding site. Therefore, 30 µM GDP would shift the apparent [35S]GTPgamma S affinity at each GDP-binding site (Ki values of 30, 1000, and 7000 nM; see Table III) by approximately 1000-, 30-, and 5-fold, respectively. The apparent high affinity KD value of [35S]GTPgamma S binding in the absence of added GDP was 3 nM, but this value is probably higher than the actual KD of G-proteins for [35S]GTPgamma S due to the presence of pre-bound GDP on Galpha (43). The actual KD value was probably less than 1 nM, as previously reported for purified Goalpha (30), Gialpha (29), and Gsalpha (48). If the affinity of [35S]GTPgamma S at membrane G-proteins was 0.5 nM, for example, the apparent affinities of these sites for [35S]GTPgamma S in the presence of 30 µM GDP would be 500, 15, and 2.5 nM, which are almost identical to the apparent KD values measured in the present study, 540-980, 14, and 4 nM (Table II). Thus, it appears that the three apparent affinity states of G-proteins for [35S]GTPgamma S can be explained in terms of three affinity states for GDP. If all of the observed changes in [35S]GTPgamma S binding affinity in the presence of GDP and/or agonist can be explained in terms of competition by GDP with three different affinities, then the apparent increases in [35S]GTPgamma S binding affinity that were induced by agonist were due to agonist-induced decreases in G-protein affinity for GDP. This is in agreement with studies of purified Go indicating that the primary mechanism of agonist activation is an increase in the dissociation rate and a decrease in the association rate of GDP (23). Moreover, the correlation between agonist-induced Ki values and agonist Emax values (Fig. 7 and Table IV) indicates that the maximal ability of each agonist to stimulate [35S]GTPgamma S binding is dependent on the degree of GDP release induced by each agonist. This model is in agreement with a previous study where mu opioid agonists were observed to induce high affinity states for [35S]GTPgamma S that were proportional to their efficacy for stimulating [35S]GTPgamma S binding in concentration-effect curves (33).


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Fig. 8.   Proposed reciprocity of guanine nucleotide-binding sites. The binding sites for [35S]GTPgamma S (when measured in the presence of 30 µM GDP) and GDP that appear only in the presence of agonist display affinities that are reciprocal, with [35S]GTPgamma S exhibiting high apparent affinity and GDP exhibiting low affinity. The remaining binding sites that appear in the presence of agonist, which exhibit low apparent affinity for [35S]GTPgamma S and high affinity for GDP, appear to be present also in the absence of agonist, under basal conditions. The binding sites for each ligand that are apparent only in the absence of agonist both display intermediate affinities. Since the apparent affinities of [35S]GTPgamma S in the presence of GDP appear to depend on the affinity for GDP, it suggests the possibility that each ligand binds to the same three sites with reciprocal apparent affinities.

This model of agonist-induced GDP release also explains the requirement for micromolar concentrations of GDP to observe significant agonist effects in the [35S]GTPgamma S binding assay. Addition of GDP has widely been observed to decrease basal [35S]GTPgamma S binding more than agonist-stimulated binding (Figs. 3 and 4), and the reason is now clear: G-proteins exhibit a lower affinity for GDP in the presence of agonist (30 nM and 8 µM) than under basal conditions (30 nM and 1 µM). GDP is only effective in the micromolar range because it must compete at the intermediate and low affinity GDP-binding sites with [35S]GTPgamma S, which exhibits nanomolar affinities for these sites. Thus, in the absence of added GDP, [35S]GTPgamma S binds to G-proteins regardless of the presence of agonist because there is insufficient GDP to result in significant re-association to either 1 or 8 µM affinity sites. In the presence of agonist, GDP competes with [35S]GTPgamma S significantly better at unactivated G-proteins than at agonist-activated G-proteins due to these affinity differences, resulting in greater decreases in basal than agonist-stimulated [35S]GTPgamma S binding.

These data do not provide direct evidence concerning the source of the agonist-induced [35S]GTPgamma S-binding sites. However, it is clear that the agonist increases the apparent affinity of [35S]GTPgamma S binding between 3- and 200-fold or more, depending on whether the high affinity (4 nM) sites were derived from sites that displayed intermediate (14 nM) or low affinity (800 nM) or for [35S]GTPgamma S binding under basal conditions. Moreover, this study presents no direct evidence for the identity of the two basal binding sites observed for each ligand. It may be that the basal intermediate affinity binding sites and high affinity GDP-/low affinity [35S]GTPgamma S-binding sites represent receptor-coupled and non-coupled G-proteins, respectively, a concept that is currently being investigated by further studies. However, a large portion of the total low affinity [35S]GTPgamma S-binding sites may be non-G-protein sites such as tubulin, guanylyl cyclase, or other nucleotide triphosphatases (55).

Previous reports of the mechanisms of receptor activation of purified G-proteins have found that agonists induced G-proteins to release GDP, allowing GTP or [35S]GTPgamma S to bind to G-protein alpha  subunits (15, 16, 23). Studies with adenosine receptors in membranes showed that the magnitude of agonist-induced release of [3H]GDP from membranes corresponded to agonist efficacy (32). In the current study, experiments measuring the effect of different cannabinoid agonists on GDP binding affinities indicated that the mechanism of agonist efficacy is the magnitude of the decrease in G-protein affinity for GDP. These results explain why increases in GDP concentration magnified differences in agonist efficacy in both the present study and in the mu opioid system (33). Thus, the results of the present study appear to generalize to G-protein-coupled receptors based on similarities to previously published results from both purified and native membrane systems. It appears that the agonist-induced low affinity state of the G-protein for GDP is necessary and sufficient to explain agonist-induced stimulation of [35S]GTPgamma S binding by G-protein-coupled receptors.

    FOOTNOTES

* These studies were supported by Public Health Service Grants DA-06784 and DA-07246 from the National Institute on Drug Abuse.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-3791; Fax: 336-716-0237; E-mail: childers{at}wfubmc.edu.

1 The abbreviations used are: Delta 9-THC, Delta 9-tetrahydrocannabinol; [35S]GTPgamma S, [35S]guanosine-5'-O-(3-thiotriphosphate); PMSF, phenylmethylsulfonyl fluoride.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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