![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 55, Issue 3, 473-480, March 1999
Sanofi Recherche, Montpellier cedex 04, France
 |
Summary |
|---|
 Top
 Summary
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The peripheral cannabinoid receptor (CB2) is a G protein-coupled receptor that is both positively and negatively coupled to the mitogen-activated protein kinase (MAPK) and cAMP pathways, respectively, through a Bordetella pertussis toxin-sensitive G protein. CB2 receptor-transfected Chinese hamster ovary cells exhibit high constitutive activity blocked by the CB2-selective ligand, SR 144528, working as an inverse agonist. We showed here that in addition to the inhibition of autoactivated CB2 in this model, we found that SR 144528 inhibited the MAPK activation induced by Gi-dependent receptors such as receptor-tyrosine kinase (insulin, insulin-like growth factor 1) or G protein-coupled receptors (lysophosphatidic acid), but not by Gi-independent receptors such as the fibroblast growth factor receptor. We showed that this SR 144528 inhibitory effect on Gi-dependent receptors was mediated by a direct Gi protein inhibition through CB2 receptors. Indeed, we found that through binding to the CB2 receptors, SR 144528 blocked the direct activation of the Gi protein by mastoparan analog in Chinese hamster ovary CB2 cell membranes. Furthermore, we described that sustained treatment with SR 144528 induced an up-regulation of the cellular Gi protein level as shown in Western blotting as well as in confocal microscopic experiments. This up-regulation occurred with a concomitant loss of SR 144528 ability to inhibit the insulin or lysophosphatidic acid-induced MAPK activation. This inverse agonist-induced modulation of the Gi strongly suggests that the modulated protein is functionally associated with the complex SR 144528/CB2 receptors, and that the Gi level may account for the heterologous desensitization phenomena.
| |
Introduction |
|---|
|
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
To
date, two cannabinoid receptors have been characterized: 1) the central
cannabinoid receptor (CB1) primarily expressed in central nervous
system tissues (Matsuda et al., 1990
; Herkenham et al., 1991;
Matsuda et al., 1993) and 2) the peripheral cannabinoid receptor (CB2)
expressed in the immune system but not in the brain (Munro et al.,
1993; Galiegue et al., 1995). The CB1 is a prime target for the
psychoactive effect of cannabinoids, whereas cannabinoid-induced immunomodulation is predominantly CB2-mediated. Both CB1 and CB2 receptors belong to the G protein-coupled receptor (GPCR) superfamily, and their stimulation by cannabinoid agonists induced several biological responses among which are the inhibition of adenylyl cyclase
(Howlett, 1985; Felder et al., 1995), the activation of the MAPKs
(Bouaboula et al., 1995b, 1996), and the in vitro induction of the
immediate-early gene Krox 24 (Bouaboula et al., 1995a, 1996), an effect
also observed in vivo (Mailleux et al., 1994; Glass and Dragunow,
1995). All of these actions appear to be exerted through one or more
members of the Bordetella pertussis toxin (PTX)-sensitive
Gi family of GTP-binding regulatory proteins such as Gi and Go. Although
natural (
9-tetrahydrocannabinol) or synthetic
(CP-55,940, WIN 55212-2) cannabinoid ligands are not selective for CB1
and CB2 receptors, selective antagonists have recently been developed
specifically targeting either CB1 receptor (SR 141716; Rinaldi-Carmona
et al., 1994, 1996) or CB2 receptor (SR 144528; Rinaldi-Carmona et al.,
1998).
The classical model of GPCR action implies that the binding of an
agonist to a receptor is essential for the receptor activation and the
transduction of the biological signal across the plasma membrane.
However, several studies have revealed that receptor activation may
occur spontaneously without any agonist binding. This discovery led to
a reclassification of antagonists as neutral antagonists or inverse
agonists. Although neutral antagonist blocks agonistic actions being
ineffective on the receptor-constitutive activity, inverse
agonists not only block the action of agonist, but also suppress
constitutive activity (Costa and Herz, 1989; Lefkowitz et al., 1993;
Chidiac et al., 1994; Bond et al., 1995). Inverse agonism was first
described as a property of 
-carbolines at the 
-aminobutyric acid
receptor (Braestrup et al., 1982), and is now identified for numerous
GPCRs (Kenakin, 1996).
In this context, we recently demonstrated: 1) an agonist-independent
activity for the CB1 receptor when expressed in mammalian cells after
transfection and 2) that the CB1-selective antagonist SR141716 not only
blocks the actions of cannabinoid agonists but also suppresses the
constitutive activity of the receptor, indicating that SR141716 acts as
an inverse agonist (Bouaboula et al., 1997). Furthermore, we revealed
for the first time a novel and provocative property for this inverse
agonist. We indeed demonstrated that SR141716 not only inhibits
autoactivated CB1 but also switches off, through CB1, the activation
induced by other and unrelated Gi-dependent
receptors such as insulin or insulin-like growth factor 1 (IGF1)
receptors (Bouaboula et al., 1997). We hypothesized that SR141716 could
promote or stabilize CB1-coupled Gi complex, making this protein unavailable for further coupling. According to this
model, designated a "three-state receptor model", inverse agonist
converted the autoactivated receptor to a suppresser receptor acting in
trans by sequestration of the Gi protein, which
remains inactive. It is assumed that the receptor is in equilibrium
among three conformations: R°, R+, and
R
. In the R° state, the receptor is uncoupled
from the G protein, whereas both forms R+
and R are able to bind to the G protein.
R+/G represents the active positive conformation
and leads to the classical signal transduction induced by agonists. In
contrast, the R/G form does not likely induce
any signal transduction, but by capturing the G protein, prevents the
activation of nonrelated GPCRs localized in its vicinity. Therefore,
R/G was termed the active negative state.
Interestingly, this three-state model fits with the cubic ternary
complex model elaborated from thermodynamic calculations predicting the
existence of an inactive receptor-G protein complex that is consistent
with our biological data (Kenakin, 1996; Weiss et al., 1996a,b).
In the present work, we investigated whether this property could be extended to another couple receptor/inverse agonist model. We used the novel CB2-selective inverse agonist SR 144528 and the human CB2 stably transfected into Chinese hamster ovary (CHO) cells. We confirmed that inverse agonist may inhibit transduction pathways through a PTX-sensitive Gi protein of other receptors such as receptor-tyrosine kinases (RTKs) and GPCR. In addition, using this property as a starting point, we further demonstrated that inverse agonist induces through CB2 a modulation of the Gi to which it is coupled. These data provide new insights into the relationship between inverse agonist, GPCR, and its cognate G protein.
| |
Materials and Methods |
|---|
|
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Reagents.
[35S]GTPS (1250 Ci/mmol) was purchased from DuPont-NEN (Paris, France).
[32P]ATP (3000 Ci/mmol) was purchased from
Amersham (Les Ulis, France). Bovine myelin basic protein,
9-tetrahydrocannabinol
(9-THC), and WIN 55212.2 PTX were purchased
from Sigma Chemical Co. (Saint-Quentin-Fallavier, France). SR 144528 [N-(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]
hepta-2-yl]-5(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) and CP-55,940 were synthesized at the Chemistry Department of Sanofi
(Montpellier, France). Phospho-MAPK rabbit polyclonal antibodies were
purchased from Biolabs (Hertfordshire, England). Mastoparan analog (Mas-7) was purchased from Calbiochem (Meudon, France). GDP and
GTPS were purchased from Boehringer Mannheim (Meylan, France).
Cholera toxin (CTX) was obtained from Calbiochem. Anti-rabbit antibody,
Gi
3,
Gi1-2,
Gi3-0 and G were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Stable Cell Lines and Culture Conditions.
For stable
expression of the human CB2, the CHO dihydrofolate reductase negative
cell line was transfected using a modified calcium phosphate
precipitation method (Graham and Eb, 1973) with plasmid p1211
coding for human CB2, and was selected for dihydrofolate reductase
expression as described previously (Bouaboula et al., 1996). CHO
wild-type (CHO-wt) cells were routinely grown as monolayers at 37°C
in a humidified atmosphere containing 5% CO2 in
-modified Eagle's medium (Gibco-BRL) supplemented with 5% dialyzed
fetal calf serum (FCS), 40 µg/ml L-proline, 1 mM sodium
pyruvate, 60 µg/ml tylocine, and 20 µg/ml gentamycin.
Preparation of Cellular Membranes.
Cells grown to confluence
were collected by scraping and spun at 200g for 10 min at
4°C. Crude membranes were prepared by homogenization of cells in 5 mM
Tris-HCl (pH 7.5) and centrifugation at 1000g for 5 min. The
supernatant was centrifuged at 40,000g for 40 min at 4°C,
the pellet was resuspended in a buffer consisting of 50 mM Tris-HCl, pH
7.5, 5 mM MgCl2, 1 mM EDTA, and stored at 
80°C until use.
[35S]GTPS Binding.
The
[35S]GTPS binding was measured as described
by Selley et al. (1996). Briefly, membranes from CHO-CB2 or CHO-wt
cells (30 µg protein) were incubated with various drugs for 60 min at
30°C in assay buffer (50 mM Tris-HCl, pH 7.4; 3 mM
MgCl2; 0.2 mM EGTA; 100 mM NaCl; 0.1% BSA) in
the presence of 0.1 nM [35S]GTPS and 50 µM
GDP, in a final volume of 200 µl. The reaction was carried out in
96-well microtitration plates (Multiscreen FB Glass Fiber, Millipore
Corp., Bedford, MA). Nonspecific binding was measured in the
presence of 10 µM unlabeled GTPS. The reaction was terminated by
rapid filtration, the microfiltration plates washed five times
with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4), and bound
radioactivity was determined.
CTX-Catalyzed ADP Ribosylation. 25 µg of CHO-CB2 membranes were treated with dithiothreitol-activated CTX (5 µg) in reaction mixture (100 µl) containing 3 mM [32P]-NAD+ (5 µCi/tube), 3 mM MgCl2, 1 mM ATP, 10 mM thymidine, 0.2% bovine serum albumin, and 0.1 M potassium phosphate. After incubation for 60 min at 30°C, the reactions were terminated by addition of 20 mM HEPES/NaOH pH 7.4 (4°C), and centrifuged at 12,000g for 10 min at 4°C. The membrane pellets were then dissolved in Laemmli's buffer and resolved in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was dried and analyzed by autoradiography. The spot signals in the autoradiography were scanned, and the data was expressed in histogram representation.
MAPK Assay.
MAPK activity was measured as described
previously (Bouaboula et al., 1995b). Briefly, cells grown to 80%
confluence in 24-well plates were placed in medium containing 0.5% FCS
for 24 h (0% FCS when 9-THC was used)
before assay. After treatment, cells were washed twice in buffer A (50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM Na4 P2O7, 50 mM NaF, 1 mM EDTA,
20 mM glycerophosphate, 1 mM EGTA, 2 mM
Na3VO4) and lysed for 15 min in 100 µl of buffer A containing 1% (v/v) Triton X-100, 100 U/ml
aprotinin, 20 µM leupeptin, 0.2 mg/ml phenylmethylsulfonyl
fluoride and 2 mM dithiothreitol. Solubilized cell extracts were
centrifuged at 14,000g for 15 min and 18 µl of
supernatants (20 µg of proteins) were analyzed for MAPK activity. The
protein contents in the supernatants were determined using a micro BCA
protein assay kit (Pierce Chemical Co., Rockford, IL). The
phosphorylation of MAPK-specific peptide substrate was carried out at
30°C for 30 min (linear assay conditions) with [32P]ATP by using the Biotrack p42/p44 MAPK
enzyme system (Amersham).
Western Blot Analysis.
After treatment, cells were
phosphate-buffered saline-washed and directly lysed in Laemmli's
loading buffer containing 6 M urea (Laemmli, 1970). Fifty-µg proteins
were run on 4 to 20% gradient polyacrylamide gel before being blotted
onto nitrocellulose filters. Nonspecific antibodies-binding was
prevented by incubating filters in 10% dried milk powder in
Tris-buffered saline (TBS) buffer (10 mM Tris-HCl pH 7.6, 150 mM
NaCl, and 0.05% Tween 20). Blots were incubated with the
anti-phospho-p42 MAPK for 3 h in TBS 1% dried milk. After
extensive washes with TBS, the blots were subsequently incubated for
1 h at room temperature with a peroxidase-labeled anti-IgG
antibody. After washing, immunostained phospho-MAPKs were visualized
using an enhanced chemiluminescence detection system (Amersham).
Immunofluorescence Analysis of Gi.
Immunocytochemical fluorescence labeling of Gi
was analyzed by confocal laser microscopy. CHO-CB2 cells were grown on
12-mm glass coverslips (Prolabo, Paris, France) and treated with
cannabinoid ligands for various periods. Washed cells were incubated
with anti-Gi3 polyclonal
antibody for 30 min at 4°C, washed, and incubated with 1/200 dilution
of CY3-conjugated anti-rabbit IgG (Sigma Immunochemicals) for
another 30 min at 4°C. After washing, coverslips were inverted and
mounted on glass microscope slides using glycerol mountant containing
the antibleaching reagent DABCO at 50 ng/ml (Sigma). Fluorescence
analyses were performed using a confocal microscope (LSM410; Zeiss,
Oberkochen, Germany).
| |
Results |
|---|
|
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
SR 144528 Acted as an Inverse Agonist on Autoactivated CB2
Receptor.
The biological properties of the CB2-selective
antagonist SR 144528 were investigated in CHO cells stably transfected
with the human CB2 cDNA (CHO-CB2 cells). Many receptors that couple to
heterotrimeric guanine-nucleotide binding proteins (G proteins) have
been shown to mediate rapid activation of the MAPK (Van Biesen et al.,
1996), a response that is also observed with CB2 (Bouaboula et al.,
1996). Stimulation of CB2 by the synthetic cannabinoid agonist
CP-55,940 resulted in a dose-dependent activation of MAPKs with an
EC50 of 8 nM (Fig.
1A), an effect that was completely prevented in CHO-CB2 cells after treatment with SR 144528 (not shown).
Comparison of CHO-CB2 and CHO-wt cells showed an enhanced basal MAPK
activity in CHO-CB2 cells (not shown); this activity was reduced in a
dose-dependent manner by SR 144528 with an IC50 of 18 nM (Fig. 1B). Neither the CP-55,940 ability to stimulate MAPK
activity nor that of SR 144528 to inhibit this activity are measurable
in parental CHO cells (not shown). On the other hand, the natural
cannabinoid ligand 9-THC, which acted as a CB2
neutral antagonist (Bayewitch et al., 1996), was unable to modulate
MAPK either positively or negatively in these studies. As expected for
a competitive interaction, 9-THC was able to
block both the stimulating effect of CP 55,940 (Fig. 1A, inset) and the
inhibitory effect of SR 144528 (Fig. 1B, inset), thus ruling out the
possibility that the constitutive activity of the receptor was due to a
putative endogenous cannabinoid ligand carried over from cell culture.
|
S binding. The comparison of basal
[35S]GTPS binding in CHO-wt and CHO-CB2
cellular membranes showed an enhanced G protein activity in membranes
from CHO-CB2 cells (not shown). The treatment with CP-55,940 induced a
stimulation of [35S]GTPS binding to the
CHO-CB2 but not to the CHO-wt cellular membranes in a dose-dependent
manner with a ED50 of 5 ± 1.5 nM (Table
1). In contrast, as shown in Table 1, the
basal [35S]GTPS binding was reduced in a
dose-dependent manner by SR 144528 with an IC50
of 3.1 ± 1.2 nM. SR 144528 treatment had no effect in membranes
from nontransfected parental CHO-wt cells (not shown).
|
(43-45 Kda) but also that of
Gi (40-41 Kda) when the assay was performed
in the absence of guanine nucleotides and when the
Gi-coupled GPCR is stimulated by its specific
agonist (Milligan et al., 1991). In the absence of exogenously added
nucleotides, CTX catalyzed the labeling of a 45-Kda protein that
corresponds to the stimulatory Gs, and of
another substrate of 40-41 Kda that is attributed to
Gi because, when a similar experiment was carried out with membrane prepared from cells previously treated with
PTX, CTX could no longer label the 40-41 Kda band (data not shown). As
illustrated in Fig. 2, the quantification
of the Gi [32P]ADP
ribosylation showed that the CP-55,940 treatment of CHO-CB2 cellular
membranes markedly enhanced the Gi labeling,
whereas SR 144528 provoked a strong inhibition of
Gi labeling. In addition, neither CP-55,940
nor SR 144528 treatment affected the [32P]ADP
ribosylation of the Gs (data not shown).
|
protein and MAPK, and that these
spontaneous activities can be drastically attenuated by addition of SR
144528, which acted as an inverse agonist.
SR 144528 Inhibits Gi Activity in CHO-CB2 Cells.
We have recently demonstrated that an inverse agonist could inactivate
MAPK activation induced by other receptors (Bouaboula et al., 1997). We
examined whether such a property could also be observed in our present
model. To question this, MAPK was stimulated with one of three
different ligands such as insulin, basic fibroblast growth factor
(FGF-b), or lysophosphatidic acid (LPA), in the presence or absence of
SR 144528. MAPK activation in response to insulin or LPA exposure was
prevented by SR 144528 in CHO-CB2 cells. In contrast, MAPK stimulated
by FGF-b was not affected by SR 144528 treatment. These results,
obtained by the detection of the active p42-isoform of MAPK proteins in
Western blot experiments (Fig. 3), were
confirmed by the measure of MAPK activities in cell lysates as
described in the Fig. 1 legend (data not shown). Furthermore, SR 144528 had no effect on LPA- or insulin-stimulated MAPKs in CHO-wt cells (not
shown), establishing that the above effects did require the interaction
of SR 144528 with CB2 receptors.
|
). We indeed observed that the MAPK
activation induced by insulin, IGF-1, or LPA was markedly inhibited by
PTX, whereas induction of MAPK by FGF-b was unaffected by PTX (data not
shown). Because the Gi protein is the key element coincident with both RTK and GPCR and that can be affected by SR
144528, one may assume that the binding of SR 144528 to CB2 interfered
with Gi activity. To test this possibility, the
effect of SR 144528 on Gi or MAPK activation
induced by an analog of mastoparan was studied. Mastoparan is a direct
activator of G protein with a reported selectivity for
Gi/Go (Gil et al., 1991). Mas-7 is an analog exhibiting a 5-fold greater potency than does mastoparan (Leyte et al., 1992).
We measured the effect of SR 144528 on Gi
activation induced by Mas-7 in cellular membranes from CHO-CB2 cells by
the use of [35S]GTPS binding. Figure
4 clearly indicates that a 3 µM Mas-7 treatment stimulated [35S]GTPS binding to
CHO-CB2 cell membranes. SR 144528 was able to induce a marked
inhibition of constitutive as well as Mas-7-induced [35S]GTPS binding to CHO-CB2 cell membranes.
This effect was not observed in CHO-wt cell membranes, indicating that
the SR 144528-induced inhibition is not a nonspecific direct
interaction with the Gi protein, but a
receptor-mediated response. These results suggest that the inhibition
of the Gi protein might be the key feature that
controls signal inhibition.
|
Sustained Treatment of the CB2 with Inverse Agonist Causes a
Cellular Gi Protein Up-Regulation.
It is tempting to
hypothesize that Gi would be physically linked to
CB2, making this protein unavailable for further coupling to GPCR or
RTK. If so, we speculated that prolonged exposure to SR 144528 may
provoke a cellular response that affects the cellular level of the
Gi protein to which the receptor is coupled. The analysis of cellular content of the Gi subunit in
CHO-wt or CHO-CB2, measured immunologically by Western blot analysis,
revealed the presence of the following G protein subunits: G
(i0/1, i1/2, i3) and G. As shown in Fig.
5A, a 24-h treatment of CHO-CB2 cells
with the agonist CP-55,940 significantly decreased the abundance of the
Gi protein. In striking contrast, similar
treatment with SR 144528 induced a completely opposite scenario by
up-regulating the Gi protein levels (i3,
i0, i1/2, and ). Interestingly, when SR 144528-treated cells
were washed and further exposed for another 5-h time period to
CP-55,940, the SR 144528-induced enhancement is shown to be
reversible (Fig. 5B). Neither CP-55,940 nor SR 144528 affected
Gs. On the other hand, treatment of parental nontransfected CHO cells with either CP-55,940 or SR 144528 had no
effect on levels of Gi (results not shown).
|
|
|
| |
Discussion |
|---|
|
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
SR 144528 is an Inverse Agonist.
In the first part of this
paper, we demonstrated that the CB2-selective antagonist SR 144528 functions as an inverse agonist for the autoactivated CB2. These
conclusions emerged from observations on two independent biological
responses. First, we showed that the basal level of MAPK activity was
enhanced in CHO cells after transfection of CB2 receptors, and that
this increase was reversed by treatment with SR 144528. Second, we
showed that SR 144528 inhibited the basal level of G protein activity.
This was demonstrated by [35S]GTPS binding
to G proteins that are directly regulated by the receptor. We showed
that membranes derived from CHO-CB2 displayed a basal binding activity
stimulated markedly by CP-55,940 but inhibited by SR 144528 in a
dose-dependent manner. We also used an indirect test in which the
Gi protein (PTX-sensitive) becomes substrate of
CTX-catalyzed ADP ribosylation only when it is activated by a receptor
(Milligan et al., 1991; Mullaney et al., 1996). This assay has
previously been used to examine the coupling of the delta opioid
receptor to Gi-like protein in NG 108-15 (Roerig et al., 1992). In membranes of CB2 cells, in the absence of receptor ligand, in addition to the expected [32P]ADP
ribosylation of Gs, CTX was able to
catalyze the [32P]ADP ribosylation of a 40-Kda
Gi. The treatment of CHO-CB2 cell membranes
with CP-55,940 markedly enhanced the Gi
labeling, whereas the [32P]ADP ribosylation of
the Gs isoforms was unaffected. Conversely, addition of SR 144528 provoked a strong inhibition of
Gi labeling with no effect on the
radioactivity incorporation into the other bands (data not shown).
9-THC acts
as a neutral CB2 antagonist in our assay, such a possibility could be
ruled out. Together, these results support the notion that the observed
effects of the CB2 antagonist SR 144528 reflect the direct consequences
of its binding to unoccupied receptors and the notion that it acts as
an inverse agonist with high intrinsic activity.
SR 144528-Induced Heterologous Desensitization.
We
demonstrated here that the inverse agonist SR 144528 not only inhibits
autoactivated CB2, but also switches off MAPK activation from RTKs,
including insulin and IGF1 receptors or the GPCR LPA receptor. By
contrast, SR 144528 did not affect MAPK activation induced by FGF-b in
CHO-CB2 cells. These effects were CB2-mediated because they were not
observed in the parental CHO cells. These results indicated that SR
144528 binding to CB2 induced biological responses that negatively
interfered with particular RTK or GPCR pathways. We have already
described this novel property for the central cannabinoid receptor
inverse agonist, SR 141716 (Bouaboula et al., 1997). The present
results extended this property to another couple inverse
agonist/receptor model.
). Both receptor types used
many of the same intermediates and the upstream point at which all
these mitogenic signals, originating from either insulin, IGF1, or LPA
and CB2 receptors, converge is very likely the Gi
protein. The following observations argue for the
Gi protein being this key element: 1) in
CHO-transfected cells, we showed that CB2, insulin, IGF1, and LPA
shared a PTX-sensitive signaling pathway, 2) FGF-b-stimulated MAPK
phosphorylation, which is not Gi-mediated, is not
affected by the presence of the inverse agonist, 3) SR 144528 bound to CB2 receptor prevents the activation of both Gi
protein and MAPKs induced by Mas-7, which acts directly at the
Gi level, and 4) the effects of SR 144528 were
abolished in parallel to those of the agonist when the ability of the
receptor to couple to G protein was lost by ADP ribosylation of the
Gi after treatment by PTX (data not shown).
A Gi-dependent cross-talk between
cannabinoid receptors and other receptors was shown for CB1 and
dopamine D2 (receptors in striatal neuron by analyzing the other
cannabinoid cellular response, Gi-dependent cAMP
production (Glass and Felder, 1997).
SR 144528 Induces Gi Protein Modulation.
How did
SR144528 mediate Gi protein inhibition?
Inverse agonist may induce or stabilize a CB2/Gi
protein complex that remains inactive. Initial experiments where we
attempted to visualize the CB2/Gi complex by
immunoprecipitation and Western blotting were unsuccessful very likely
due to the high detergent concentration needed for the receptor
solubilization, which could be incompatible with maintaining a
putative CB2/Gi complex interaction. We
therefore addressed this question from another point of view. As
prolonged cell exposure to a GPCR agonist can result in a
down-regulation of cellular levels of the G protein to which the
receptor is normally coupled (Milligan, 1993), we reasoned that, as a
corollary, if indeed CB2 stably interacts with Gi
in the presence of SR 144528, then sustained treatment with inverse
agonist is expected to affect the cellular level of
Gi protein.
subunit of Gi1,
Gi2, and
Gi3 without altering
levels of Gs, an effect that was observed
together with a redistribution of Gi proteins
from the cytosol to the plasma membrane. Remarkably, we showed
that chronic exposure to CP-55,940 produces a time-dependent down-regulation of the same G-protein subunits. This effect indicated that SR 144528 enhanced the G-protein subclasses, which are precisely those coupled to the receptor in the presence of the agonist. This
point was further supported by the ability of CP-55,940 to reduce the
same G-protein subunit levels that have been increased by SR 144528. Although it has not been formally demonstrated, from these results it
is reasonable to surmise that the binding of SR 144528 to the receptor
promotes or stabilizes the SR 144528/CB2/Gi complex that controls signal transduction. A predicted result from this
model is that, if the Gi stoichiometry increases
when compared to the receptor CB2 load, then the capacity of SR 144528 to inhibit RTK or GPCR signaling should be lost. This is in agreement with our observations of the Gi protein being
up-regulated by the inverse agonist.
The Gs protein down-regulation by an agonist
treatment has already been reported. For instance, sustained treatment
of NG 108-15 cells transfected to express the human
2-adrenoreceptor with isoprenaline resulted in a decrease in
membrane-associated levels of the subunit of the
Gs protein that interacts with the receptor (Kim
and Milligan, 1994). However, sustained treatment with inverse agonist
failed to cause any alteration in levels of this
Gs protein in this model (MacEwan and Milligan,
1996), indicating that the property of SR 144528 we described here may not be common for all inverse agonists.
Predictive Model for Receptor Activation of G Proteins. The "two-state model" currently in use suggests that the mode of action of inverse agonists can be explained by a higher affinity of these compounds for the inactive uncoupled form of the receptor. Binding of a compound preferentially to this form leads to a reduction in constitutive activity by shifting the equilibrium from the active form to the inactive uncoupled form. Our results cannot be adequately interpreted in this theoretical framework.
An agonist-bound receptor activates an appropriate G protein that promotes dissociation of GDP. Although the interactions between receptor and G protein are poorly understood, both mutagenesis and biochemical experiments with a variety of GPCR suggest that, first of all, the receptor activation by ligand binding causes a change in the relative orientations of the transmembrane helices 3 and 6. This modification then affects the conformation of the G protein-interacting intracellular loop of the receptor and thus uncovers previously masked G protein-binding sites (Wess, 1997). Secondly, as GDP is buried within
the G protein, the receptor-G protein interaction must, in addition,
promote changes in interdomain interactions necessarily. Thus, to
activate the G protein, the receptor had to deliver two pieces of
information-one for the formation of R/G complex and the other to
induce the exchange of bound GDP to GTP on heterotrimeric G proteins,
resulting in the dissociation of the G protein into active G-GTP and
G subunits.
Although the physical process involved in the stabilization and
inactivation of the complex remains undefined, we suggest that inverse
agonist may trigger only the first part of this activation process.
Another alternative would be the involvement of the recently described
regulators of G protein signaling or RGS proteins (De Vries et al.,
1996; Druey et al., 1996). Given the evidence that RGS proteins are
negative regulators of G protein signaling that act at the level of the
dissociated G subunit or above, one may hypothesize that RGS
proteins would be recruited in the presence of an inverse agonist, thus
acting as inhibitors of GDP dissociation and blocking G protein
activation. This possibility could be addressed by analyzing whether
RGS interacts with Gi3 in the presence of SR
144528. The detailed mechanisms responsible for inverse agonist-induced G protein inhibition are the basis of ongoing studies.
The data provided herein demonstrate that inverse agonist occupation of
CB2 receptor can selectively regulate both the activity and the
cellular level of Gi. The inverse agonist-induced
modulation of the Gi strongly suggested that the
modulated protein must be physically associated with the complex SR
144528/CB2, which accounts for the heterologous desensitization
phenomena. The results presented in this study demonstrate a complex
pattern of cellular G protein regulation after inverse agonist that may
be as complex as that associated with the agonist-induced activation of
the receptor.
| |
Footnotes |
|---|
Received August 17, 1998; Accepted December 15, 1998
Send reprint requests to: Dr. Pierre Casellas, Sanofi Recherche, 371 rue du Prof. Joseph Blayac, 34184 Montpellier cedex 04, France. E-mail: pierre.casellas{at}sanofi.com
| |
Abbreviations |
|---|
CB1, central cannabinoid receptor;
CB2, peripheral cannabinoid receptor;
CHO, Chinese hamster ovary;
CHO-wt, CHO wild type;
FGF-b, basic fibroblast growth factor;
GPCR, G
protein-coupled receptor;
IGF1, insulin-like growth factor 1;
LPA, lysophosphatidic acid;
MAPKs, mitogen-activated protein kinases;
Mas-7, mastoparan analog;
PTX, Bordetella pertussis toxin;
CTX, cholera toxin;
RTK, receptor-tyrosine kinase, 9-THC,
delta 9 tetrahydrocannabinol;
FCS, fetal calf serum;
TBS, Tris-buffered
saline.
| |
References |
|---|
|
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
)-Delta9-tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated inhibition of adenylyl cyclase.
J Biol Chem
271:
9902-9905This article has been cited by other articles:
|
|
 |
|
  F. Montecucco, F. Burger, F. Mach, and S. Steffens CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined intracellular signaling pathways Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1145 - H1155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Lunn, J. S. Fine, A. Rojas-Triana, J. V. Jackson, X. Fan, T. T. Kung, W. Gonsiorek, M. A. Schwarz, B. Lavey, J. A. Kozlowski, et al. A Novel Cannabinoid Peripheral Cannabinoid Receptor-Selective Inverse Agonist Blocks Leukocyte Recruitment in Vivo J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 780 - 788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. K. Rao, W. Zhang, and N. E. Kaminski Cannabinoid receptor-mediated regulation of intracellular calcium by {Delta}9-tetrahydrocannabinol in resting T cells J. Leukoc. Biol., May 1, 2004; 75(5): 884 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kenakin Efficacy as a Vector: the Relative Prevalence and Paucity of Inverse Agonism Mol. Pharmacol., January 1, 2004; 65(1): 2 - 11. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Wenzel-Seifert and R. Seifert Molecular Analysis of beta 2-Adrenoceptor Coupling to Gs-, Gi-, and Gq-Proteins Mol. Pharmacol., November 1, 2000; 58(5): 954 - 966. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. W. Klein, B. Lane, C. A. Newton, and H. Friedman The Cannabinoid System and Cytokine Network Experimental Biology and Medicine, October 1, 2000; 225(1): 1 - 8. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. X. Zhu, S. Sharma, M. Stolina, B. Gardner, M. D. Roth, D. P. Tashkin, and S. M. Dubinett {Delta}-9-Tetrahydrocannabinol Inhibits Antitumor Immunity by a CB2 Receptor-Mediated, Cytokine-Dependent Pathway J. Immunol., July 1, 2000; 165(1): 373 - 380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Chen, J. Way, S. Armour, C. Watson, K. Queen, C. K. Jayawickreme, W.-J. Chen, and T. Kenakin Use of Constitutive G Protein-Coupled Receptor Activity for Drug Discovery Mol. Pharmacol., January 1, 2000; 57(1): 125 - 134. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. Vasquez and D. L. Lewis The CB1 Cannabinoid Receptor Can Sequester G-Proteins, Making Them Unavailable to Couple to Other Receptors J. Neurosci., November 1, 1999; 19(21): 9271 - 9280. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bouaboula, D. Dussossoy, and P. Casellas Regulation of Peripheral Cannabinoid Receptor CB2 Phosphorylation by the Inverse Agonist SR 144528. IMPLICATIONS FOR RECEPTOR BIOLOGICAL RESPONSES J. Biol. Chem., July 16, 1999; 274(29): 20397 - 20405. [Abstract] [Full Text] [PDF] < |
