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
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INTRODUCTION |
Cannabinoid receptors mediate the actions of
9-tetrahydrocannabinol
(
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
(Gi
and Go
subtypes) (7, 12), although
some effects have been implicated via Gs
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
subunit of a
G-protein (G
) releases bound GDP, binds a molecule of GTP, and
dissociates from the G-protein 
subunit complex. Both G
and

subunits act upon effectors until G
cleaves the bound GTP to
GDP by its intrinsic GTPase activity, and G
re-associates with a

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]GTP
S, to G-protein
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]GTP
S binding assay. First, GDP
has been shown to decrease basal [35S]GTP
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]GTP
S binding has been
demonstrated (24, 26-28). Second, GDP has been reported to modulate
the kinetics of [35S]GTP
S binding. The presence of
micromolar concentrations of GDP was shown to decrease the magnitude
and rate of [35S]GTP
S binding to purified and
reconstituted G-proteins (23). However, early reports of
[35S]GTP
S binding to purified G-protein
Gi
(29) and Go
(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]GTP
S binding (31).
Therefore, a problem frequently noted for [35S]GTP
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]GTP
S
binding. In the adenosine A1 receptor system, a full agonist was shown
to be maximally effective for the stimulation of
[35S]GTP
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]GTP
S
binding stimulated by agonists of different efficacies. Previous
studies which showed that
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]GTP
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.
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EXPERIMENTAL PROCEDURES |
Materials--
Male Sprague-Dawley rats were purchased from
Zivic Miller (Zelienople, PA). [35S]GTP
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).
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
GTP
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]GTP
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]GTP
S in a final volume of 1 ml, and nonspecific
binding was determined with 30 µM unlabeled GTP
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 GTP
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).
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(Eq. 1)
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where B is the amount of [35S]GTP
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).
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(Eq. 2)
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where B is the amount of [35S]GTP
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.
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(Eq. 3)
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where E is amount of [35S]GTP
S bound
at receptor ligand concentration [L]; Emax is
the amount of [35S]GTP
S bound at maximally effective
concentrations of receptor ligand, and EC50 is the
concentration of receptor ligand producing half-maximal
[35S]GTP
S binding. IC50 and
Imax values for GDP competition curves were
determined by fitting the biphasic Equation 4.
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(Eq. 4)
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where I is the amount of [35S]GTP
S
binding inhibited at GDP concentration [I];
Imax(H) and Imax(L) are
the maximum amounts of [35S]GTP
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]GTP
S binding from each site, respectively.
Ki values were estimated by the Cheng-Prusoff
equation (44). [35S]GTP
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.
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RESULTS |
Effects of GDP and Cannabinoid Agonist on the Kinetics of
[35S]GTP
S Binding--
The association and
dissociation rates of [35S]GTP
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]GTP
S binding; Table
I provides maximal binding and
t1/2 values of association under these conditions.
[35S]GTP
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]GTP
S binding
reached maximum values within 1 and 2 h, respectively, and
actually decreased slightly between 2 and 4 h. Maximal
[35S]GTP
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]GTP
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]GTP S binding in
rat cerebellar membranes. For association assays (A),
membranes were incubated at 30 °C for various times with 0.05 nM [35S]GTP 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 GTP S at various times. Data in
B are expressed on a logarithmic scale as percent of
steady-state [35S]GTP S binding.
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Table I
Association and dissociation of [35S]GTP S binding
Kinetics of [35S]GTP 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]GTP 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.
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Both GDP and agonist significantly affected the rate of
[35S]GTP
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]GTP
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]GTP
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]GTP
S to
purified G-proteins in the presence of millimolar concentrations of
Mg2+ (29, 30), [35S]GTP
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]GTP
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]GTP
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]GTP
S binding at 30 µM
GDP. Unlike the effects of WIN 55212-2 on [35S]GTP
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]GTP
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]GTP
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]GTP
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]GTP S binding to rat cerebellar membranes.
Net agonist-stimulated [35S]GTP S binding was
determined from the data shown in Fig. 1 by subtracting basal
[35S]GTP S binding values from values obtained in the
presence of WIN 55212-2 at each time point.
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Effects of GDP and Cannabinoid Agonist on Steady-state
[35S]GTP
S Binding Parameters--
To characterize
[35S]GTP
S-binding sites and the effect of agonist
on these sites, GTP
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]GTP
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]GTP
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]GTP
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]GTP
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]GTP
S binding in the
absence of added GDP (Fig. 3A), net WIN 55212-2-stimulated
[35S]GTP
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]GTP S-binding sites in rat cerebellar
membranes. Representative biphasic Scatchard plots of
[35S]GTP 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]GTP S plus 0.5 nM to 10 µM unlabeled GTP S. B, inset,
Scatchard plot of net agonist-stimulated [35S]GTP S
binding determined by subtracting basal [35S]GTP S
binding from that obtained in the presence of WIN 55212-2 at each
concentration of GTP 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]GTP S binding parameters in rat cerebellar membranes
Apparent Bmax and KD values were
determined using 0.05 nM [35S]GTP S plus 0.5 nM to 10 µM unlabeled GTP 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]GTP S binding, determined by
subtracting basal from WIN 55212-2-stimulated [35S]GTP S
binding at each concentration of GTP S, was not detectable (N/A, not
applicable) in the absence of GDP and was monophasic and high affinity
in the presence of GDP.
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In addition to increasing the apparent affinity of G
for
[35S]GTP
S, agonists have been reported to reduce the
affinity of G
for GDP (15, 16, 23). To explore this possibility,
cerebellar membranes were incubated with [35S]GTP
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]GTP
S used (0.05 nM) results in low occupancy of high affinity [35S]GTP
S-binding sites (0.5-2%), these assays were
also conducted using two higher concentrations of
[35S]GTP
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]GTP
S, high affinity [35S]GTP
S
binding would predominate, since 1 nM
[35S]GTP
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]GTP
S
with high affinity.

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Fig. 4.
Effect of WIN 55212-2 on competition binding
of [35S]GTP S and GDP in rat cerebellar membranes.
Membranes were incubated with 0.05, 0.20, or 1.0 nM
[35S]GTP 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]GTP S binding. GDP Ki and
Imax values are provided in Table III.
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The results showed that as cannabinoid agonist increased the apparent
affinity of a fraction of [35S]GTP
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]GTP
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]GTP
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]GTP
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]GTP
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]GTP
S concentration used (Table
III). In the presence of WIN 55212-2, GDP
competed for [35S]GTP
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]GTP
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]GTP S binding in rat cerebellar membranes
Imax and IC50 values were determined by
displacement of 0.05, 0.2, or 1.0 nM [35S]GTP 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."
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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]GTP
S binding.
Several cannabinoid ligands with different structural bases were
selected including the following:
9-THC, the primary
psychoactive constituent of marijuana; WIN 55212-2, a synthetic
aminoalkylindole agonist; levonantradol, a potent
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]GTP
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]GTP
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]GTP
S binding by each
agonist, concentration-effect curves were analyzed for EC50
and Emax values monophasically. Full biphasic analysis of agonist stimulation of [35S]GTP
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
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]GTP
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]GTP S binding to rat
cerebellar membranes. Membranes were incubated with 0.05 nM [35S]GTP S, 30 µM GDP, and
various concentrations of each ligand. Data are expressed as the
percent of [35S]GTP 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.
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Concentration-effect analysis revealed that these ligands produced a
wide range of efficacies for G-protein activation in the
[35S]GTP
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]GTP
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]GTP
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),
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]GTP
S binding at any concentration, indicating
that this ligand is a pure antagonist with zero efficacy. However,
SR141716A slightly but consistently inhibited basal
[35S]GTP
S binding at the highest concentration (10 µM) used (Fig. 5).
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Table IV
Stimulation of [35S]GTP S binding Emax and GDP
Ki values induced by cannabinoid agonists
Emax values were obtained by nonlinear regression
analysis of the stimulation of [35S]GTP 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]GTP 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]GTP 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.
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In other receptor systems, increasing the GDP concentration was
reported to increase differences between full and partial agonists for
stimulating [35S]GTP
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]GTP
S in the presence and
absence of maximally effective concentrations of these ligands. As
described above (Table III), GDP displaced [35S]GTP
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.
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]GTP
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]GTP
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]GTP
S binding is plotted as a
function of the concentration of added GDP. For each agonist assayed,
net agonist-stimulated [35S]GTP
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]GTP
S binding was observed
with
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]GTP
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
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]GTP S binding
to rat cerebellar membranes. Membranes were incubated with 0.05 nM [35S]GTP 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]GTP 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]GTP 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.
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Fig. 7 shows the correlation between
agonist efficacy for the stimulation of [35S]GTP
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]GTP 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.
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DISCUSSION |
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]GTP
S binding to cerebellar
membranes reached an apparent steady state and was readily dissociable.
Moreover, cannabinoid agonist increased the rate of both
[35S]GTP
S association and dissociation. Addition of
GDP decreased the rate and magnitude of [35S]GTP
S
association, consistent with the competitive binding of [35S]GTP
S and GDP as previously shown in purified
G-proteins (43).
It was significant that agonist-stimulated [35S]GTP
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]GTP
S dissociation,
just as muscarinic agonists and guanine nucleotides increased the
dissociation of [35S]GTP
S from native cardiac
membranes (47). The findings of reversible [35S]GTP
S
binding in membranes seem to contradict earlier studies where
[35S]GTP
S binding to purified Go
and
Gi
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 
to G
subunits in purified systems compared with those that may be
present in native membranes (29, 30, 48, 49).
[35S]GTP
S binding to purified Go
and
Gi
exhibits both rapid and slow dissociation rates, and
the ratio of slowly to rapidly dissociating sites is proportional to
the concentration of Mg2+ (50). 
subunits increase
the dissociation of [35S]GTP
S from G
, but this
effect is inhibited by Mg2+, which inhibits 
coupling
to G
(30, 50). The present study found that upon addition of excess
GTP
S, cannabinoid agonist increased the ratio of rapidly to slowly
dissociating [35S]GTP
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]GTP
S binding was the result of the liberation of
large amounts of 
by the agonist-accelerated binding of the
unlabeled GTP
S to G
subunits. The finding that GDP produced a
slight increase in the ratio of rapidly dissociating
[35S]GTP
S-binding sites is consistent with the fact
that GDP increases the ratio of low affinity to high affinity
[35S]GTP
S-binding sites (Fig. 3), which would be
expected to display different dissociation rates.
In cerebellar membranes, basal and cannabinoid-stimulated
[35S]GTP
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 G
in high molar ratios even after purification of
G
subunits (43). Thus, all parameters of [35S]GTP
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]GTP
S-binding sites using 0.05 nM
[35S]GTP
S accurately assesses high affinity
[35S]GTP
S-binding sites, since concentrations of
[35S]GTP
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]GTP
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),
9-THC
acted as a weak partial agonist, stimulating only 20% of the
[35S]GTP
S binding of the full agonists. SR141716A
appeared to be a neutral antagonist, although the decreased
[35S]GTP
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]GTP
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]GTP
S binding was observed only in this range of
GDP concentrations. The slight and variable stimulation of
[35S]GTP
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(
S) affinity the fundamental mechanism or is the
agonist-induced increase in apparent GTP(
S) affinity caused by a
decrease in GDP affinity? These data suggest that the agonist-induced increases in the apparent affinity of [35S]GTP
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]GTP
S affinities
(measured with 30 µM GDP) and GDP affinities were
reciprocal, high affinity for [35S]GTP
S and low
affinity for GDP (Fig. 8). The two
affinity states for [35S]GTP
S (measured with 30 µM GDP) and GDP observed in the basal state may also be
reciprocal; the low affinity [35S]GTP
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]GTP
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 G
subunits for
[35S]GTP
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]GTP
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]GTP
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]GTP
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]GTP
S due
to the presence of pre-bound GDP on G
(43). The actual
KD value was probably less than 1 nM, as
previously reported for purified Go
(30),
Gi
(29), and Gs
(48). If the affinity of
[35S]GTP
S at membrane G-proteins was 0.5 nM, for example, the apparent affinities of these sites for
[35S]GTP
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]GTP
S can be explained in terms of three affinity
states for GDP. If all of the observed changes in
[35S]GTP
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]GTP
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]GTP
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]GTP
S that were
proportional to their efficacy for stimulating [35S]GTP
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]GTP 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]GTP 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]GTP 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]GTP 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.
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This model of agonist-induced GDP release also explains the requirement
for micromolar concentrations of GDP to observe significant agonist
effects in the [35S]GTP
S binding assay. Addition of
GDP has widely been observed to decrease basal
[35S]GTP
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]GTP
S, which exhibits
nanomolar affinities for these sites. Thus, in the absence of added
GDP, [35S]GTP
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]GTP
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]GTP
S binding.
These data do not provide direct evidence concerning the source of the
agonist-induced [35S]GTP
S-binding sites. However, it
is clear that the agonist increases the apparent affinity of
[35S]GTP
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]GTP
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]GTP
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]GTP
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]GTP
S to bind to G-protein
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]GTP
S binding by G-protein-coupled receptors.