(Received for publication, March 25, 1997, and in revised form, June 19, 1997)
From the In the present study, we showed that Chinese
hamster ovary (CHO) cells transfected with human central cannabinoid
receptor (CB1) exhibit high constitutive activity at both levels of
mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These
activities could be blocked by the CB1-selective ligand, SR 141716A,
that functions as an inverse agonist. Moreover, binding studies showed
that guanine nucleotides decreased the binding of the agonist
CP-55,940, an effect usually observed with agonists, whereas it
enhanced the binding of SR 141716A, a property of inverse agonists.
Unexpectedly, we found that CB1-mediated effects of SR 141716A included
inhibition of MAPK activation by pertussis toxin-sensitive
receptor-tyrosine kinase such as insulin or insulin-like growth factor
1 receptors but not by pertussis toxin-insensitive receptor-tyrosine
kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein.
Furthermore, SR 141716A inhibited guanosine
5 The mechanism of action of CB1 has been associated with several biological responses including
inhibition of adenylyl cyclase (AC) and modulation of ion channels (10,
11). More recently, we showed that treatment by cannabinoid agonist of
CHO cells expressing CB1 or U373 MG cells induced activation of both
mitogen-activated protein kinases (MAPKs) and immediate-early gene
Krox 24, also known as NGFI-A, zif/268, egr-1,
and TIS 8 (12, 13). Cannabinoid-induced Krox 24 expression was also described in vivo (14, 15). These
cannabinoid effects were mediated by a pertussis toxin (PTX)-sensitive
guanine nucleotide-binding protein (Gi protein) and
prevented by the potent CB1-selective antagonist SR 141716A (12, 13,
16, 17).
In the last few years, a new class of antagonist molecules designated
as inverse agonists has been identified (18-21). The first to be
characterized were the In the present study, we explored in detail the pharmacological and
biological properties of the CB1-selective antagonist SR 141716A, using
CHO cells transfected with human CB1. We here demonstrated that SR
141716A functions not only as an antagonist of cannabinoid-mediated
effects but also as an inverse agonist. We next provided evidence for a
coupling between CB1 and receptor-tyrosine kinase (RTK) transduction
pathways, involving a PTX-sensitive Gi protein. These
results suggest that biological functions of inverse agonists have been
underestimated and enable us to conceive a new ligand/receptor
interaction model.
[3H]CP-55,940, [3H]SR
141716A, and [35S]GTP A synthetic oligonucleotide
containing six cAMP-response elements (CREs) was obtained as described
(24). This CRE cassette was placed upstream from the herpes simplex
virus-thymidine kinase (tk) promoter and cloned into the pSE1 plasmid
(25) in place of the SV40 early enhancer-promoter. The interleukin-2
coding sequence was then replaced with luciferase, and the resulting CRE-luciferase reporter construct was called p661.
For stable
expression of the CRE-luciferase gene, the CHO dihydrofolate reductase
negative cell line was co-transfected using a modified calcium
phosphate precipitation method (26) with the p661 plasmid mixed with a
tk-neo plasmid. Selection was achieved by culturing cells for 12 days
in CHO wild-type (CHO-WT) cells were routinely grown as monolayers at
37 °C in a humidified atmosphere containing 5% CO2 in
Cells grown to confluence in 24-well plates
were washed and incubated for 30 min in serum-free medium containing
Ro-20-1724 (0.1 mM) and isobutylmethylxanthine (0.1 mM). Cells were either pretreated or not with cannabinoid
receptor ligands for 5 min before a 20-min stimulation with forskolin
(3 µM). The reaction was stopped by the addition of 50 mM Tris-HCl, 4 mM EDTA. The determination of
cAMP levels was performed by a radioimmunoassay (Amersham Corp.)
according to the manufacturer's instructions. Each value is the mean
of triplicate determinations ± S.E.
Cells were seeded at 104
cells/well in white 96-well microplates for luminescence, in MEM medium
supplemented with 5% FCS. Twenty-four hours later, cells were
pretreated for 5 min with various concentrations of cannabinoid
receptor ligands before stimulation with 1 µM forskolin.
Four hours after cell stimulation, cells were washed twice with
phosphate-buffered saline and lysed by the addition of cell culture
lysis buffer from the Luciferase Assay System (Promega,
Charbonnières, France). Measurement of light emission was
determined after the addition of reconstituted luciferase assay reagent
following the supplier's instructions. Luminescence was detected by a
CCD camera (MTP Reader, Hamamatsu Photonics, Hamamatsu, Japan). The
quantitation of light emission was made by accumulation of photon
counting, and mean values from triplicate determinations were expressed
as percentage of values from forskolin-stimulated cells.
Cells grown to confluence were
collected by scraping and spun at 200 × g 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 1,000 × g for 5 min. The supernatant was centrifuged at 40,000 × g for 40 min at 4 °C, and 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
The
[35S]GTP MAPK activity was measured as
described previously (12). Briefly, cells grown to 80% confluence in
6-well plates were placed in medium containing 0.5% FCS for 24 h
before assay. After treatment, cells were washed twice in buffer A (50 mM Hepes, pH 7.4, 150 mM NaCl, 10 mM Na4P2O7, 50 mM NaF, 1 mM EDTA, 20 mM
glycerophosphate, 1 mM EGTA, 2 mM
Na3VO4) and lysed for 15 min in buffer A
containing 1% (v/v) Triton X-100, 100 units/ml aprotinin, 20 µM leupeptin, and 0.2 mg/ml phenylmethylsulfonyl
fluoride. Solubilized cell extracts were centrifuged at 14,000 × g for 15 min, and supernatants (100 µg of proteins) were
incubated for 3 h with the agarose-coupled anti-p44 and anti-p42
antibodies. The protein contents in the supernatants were determined
using micro BCA protein assay kit (Pierce). Immune complexes were
assayed for phosphorylation of MBP as a substrate. Reaction was
initiated by the addition of buffer containing 150 µg/ml MBP, 10 mM magnesium acetate, 1 mM dithiothreitol, and
5 µM [ Phosphorylation of p42 and p44 MAPKs was
determined by the electrophoretic mobility shift assay (EMSA) as
described (12). Briefly, quiescent cells were incubated in 6-well
plates for 10 min at 37 °C in the presence of various compounds.
Cells were further washed once in ice-cold buffer containing 50 mM Hepes, pH 7.4, 0.2 mM sodium orthovanadate
and then directly lysed in Laemmli's loading buffer containing 6 M urea. Proteins were heated for 10 min at 95 °C and
separated by SDS-PAGE on 11% acrylamide gel. Following gel transfer
onto nitrocellulose filters in 25 mM Tris, 0.19 M glycine, 20% methanol, membranes were blotted in TN
buffer (20 mM Tris, pH 7.5, 120 mM NaCl)
supplemented with 10% dried milk powder. Blots were then incubated
with anti-p42 and anti-p44 antibodies (0.25 µg/ml) in blotting TN
solution containing 2% dried milk powder for 3 h at room
temperature. After extensive washing in TN buffer containing 0.1%
Tween 20, a peroxidase-labeled anti-rabbit IgG antibody (Sigma) was
added for 45 min at room temperature. After 5 washes, immunostained
MAPKs were visualized using the enhanced chemiluminescence detection
system according to the supplier's instructions (Amersham Corp.) and
subjected to autoradiography.
Quiescent cells were incubated at
37 °C for 10 min in the presence of various compounds and lysed in
the Laemmli's buffer, and proteins were analyzed for MAPK activity
using an in-gel kinase assay as already described (28). Samples were
subjected to SDS-PAGE in 10% polyacrylamide gel containing 0.2 mg/ml
MBP. The gel was incubated once in buffer A (50 mM
Tris-HCl, pH 8, 0.5 mM The biological properties of the CB1 selective antagonist
SR 141716A were studied in CHO cells stably expressing the human CB1
(CHO-CB1 cells) in which we measured MAPK activity. MAPK activity was
assayed toward MBP as substrate, after specific immunoprecipitation of
p42 and p44 MAPK isoforms. We showed that stimulation of CB1 by the
synthetic cannabinoid agonist CP-55,940 resulted in a strong activation
of MAPKs which is in agreement with our previously described results
(12). This effect was not observed in parental cells (CHO-WT) (Fig.
1A) and was completely
prevented in CHO-CB1 cells by treatment with SR 141716A (data not
shown). Interestingly, a comparison of CHO-CB1 and CHO-WT cells showed
an enhanced basal MAPK activity in CHO-CB1 cells (Fig. 1B).
Furthermore, this basal activity was reduced in a
dose-dependent manner by SR 141716A with an
IC50 of 5 nM (Fig. 1B). A
concentration of 30 nM SR 141716A reduced the basal level
of MAPKs in CHO-CB1 cells to that observed in CHO-WT cells, whereas SR
141716A treatment had no effect on CHO-WT cells. Pretreatment of
CHO-CB1 cells by PTX induced an inhibition of MAPK basal level, whereas
it had no significant effect in CHO-WT cells. When CHO-CB1 cells were
co-treated with PTX and SR 141716A, no additive inhibitory effects were
observed (data not shown). This suggested that the enhanced MAPK
activation was related to a CB1-coupled PTX-sensitive Gi
protein. Activation of MAPKs has been described to lead to the
appearance of slower migrating forms in SDS-PAGE, resulting from the
phosphorylation on specific threonine and tyrosine residues of p42 and
p44 MAPK isoforms (29). In CHO-CB1 cells, the presence of
phosphorylated proteins, which was markedly enhanced by treatment with
CP-55,940, was clearly apparent in the untreated cells (Fig.
2A). Treatment with SR 141716A
abolished the CP-55,940-induced forms and also the constitutively
activated forms. Similar results were obtained in an in-gel kinase
assay performed with proteins extracted from CHO-CB1 cells and run in
SDS-PAGE. Intact MBP protein, trapped in the polyacrylamide gel, was
efficiently phosphorylated by p42/44 MAPKs from unstimulated cells or
cells treated with CP-55,940 (Fig. 2B). Again, the intensity
of MAPKs was decreased by treatment with SR 141716A in cells stimulated
with CP-55,940 as well as in unstimulated cells. In a control
experiment, we did not observe any modulation of p42/p44 MAPKs by SR
141716A in either Western blot or in-gel kinase assays on CHO-WT cells
(data not shown). These results indicate that the enhanced basal MAPK
activity in CHO cells expressing CB1 is related to the autoactivation
of CB1 receptors and that it is specifically decreased by SR 141716A which thereby acts as an inverse agonist.
We
examined whether SR 141716A inverse agonist activity was also
manifested by other cellular responses. Previous studies have
established that cannabinoids alter cAMP production through the
GTP-binding protein Gi (10, 30). The increase of
intracellular cAMP levels leads to the activation of protein kinase A. The catalytic subunit of the activated protein kinase A is translocated
to the nucleus where it phosphorylates the cAMP response element
binding protein which thereafter binds to cAMP response elements
(CREs), leading to an increase in gene transcription (31). To monitor cAMP metabolism, we generated a cAMP-responsive reporter construct by
linking CRE sequences to the luciferase gene, and we stably transfected
this chimeric gene in CHO-WT cells (CHO-CRE). As expected, forskolin,
by elevating intracellular cAMP, strongly stimulated luciferase gene
expression. The CHO-CRE cell line was used as recipient to stably
transfect human CB1, and transformants were denoted as CHO-CRE-CB1
cells. We further analyzed the effects of cannabinoid receptor ligands
on forskolin-induced CRE-luciferase response in CHO-CRE-CB1 cells.
CP-55,940, WIN 55212-2, and
Table I.
Effect of SR 141716A and CP-55,940 on forskolin-stimulated cAMP
production
Sanofi,
Sanofi,
-0-(thiotriphosphate) uptake induced by CP-55,940 or
Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to
the inhibition of autoactivated CB1, SR 141716A can deliver a
biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses.
By contrast, SR 141716A had no effect on MAPK activation by insulin or
IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of
a direct interaction of SR 141716A with the Gi protein.
This supports the notion that the Gi protein may act as a
negative intracellular signaling cross-talk molecule. From these
original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.
9-tetrahydrocannabinol
(
9-THC),1 the
main active principle of marijuana, began to be described only a few
years ago. It is now known that
9-THC, and other potent
synthetic cannabinoid receptor agonists as well as anandamide, the
putative endogenous ligand (1), bind to specific cannabinoid receptors.
The central cannabinoid receptor (CB1) was cloned from both rat and
human (2, 3) and was shown to be expressed primarily in brain tissue
(4, 5). Although to a lower extent, CB1 mRNA has also been found in
testis (3), spleen (6), and leukocytes (7). A second cannabinoid
receptor (CB2) has been cloned and recently characterized. CB2 is
expressed in macrophages from the marginal zone of spleen, in B
lymphocytes, and NK cells but not in brain tissues (8, 9). Both CB1 and
CB2 receptors belong to the G protein-coupled receptor (GPCR)
superfamily.
-carbolines acting toward the ionotropic
-aminobutyric acid receptor (22). These molecules contrast with
classical antagonists in that they exhibit a biological activity by
blocking the signal transduction mediated by constitutively activated
receptors. Most of the other inverse agonist molecules identified so
far are ligands for receptors of the GPCR superfamily (23).
Reagents
S (1250 Ci/mmol) were purchased
from DuPont NEN (Paris, France). [
32P]ATP (3000 Ci/mmol) was from Amersham Corp. (Les Ullis, France). Phorbol
12-myristate 13-acetate, forskolin, isobutylmethylxanthine, bovine
myelin basic protein (MBP),
9-tetrahydrocannabinol
(
9-THC), WIN 55212-2, and pertussis toxin (PTX) were
purchased from Sigma (Saint-Quentin-Fallavier, France). Wortmannin was
from Biomol (Plymouth Meeting, PA). SR 141716A
(N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide) and CP-55,940 were synthesized at the Chemistry Department of Sanofi
(Montpellier, France). Anti-p44 (C-16, anti-ERK-1) and anti-p42 (C-14,
anti-ERK-2) rabbit polyclonal antibodies were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA). Mastoparan analog (Mas-7) was from
Calbiochem (Meudon, France). G418 was from Life Technologies, Inc.
(Cergy Pontoise, France); Ro 20-1724 was from Research Biochemicals
International (Illkirch, France). GDP and GTP
S were purchased from
Boehringer Mannheim (Meylan, France).
-essential medium (Life Technologies, Inc.) supplemented with
10% fetal calf serum (FCS) and 300 µg/ml G418. Individual clones
were tested for an inducible expression of luciferase following
stimulation with 1 µM forskolin as described below. The
selected clones were subcloned by limiting dilution, and the clone
Z661.11.9, exhibiting an induction rate of 80-100-fold upon
stimulation with forskolin, was chosen for further studies. For stable
expression of CB1, Z661.11.9 cells were transfected as indicated above
with plasmid p1211 coding for human CB1 and selected for dihydrofolate
reductase expression as described previously (12). The CHO-CRE-CB1 cell
line that exhibited the highest response to
9-THC was
chosen for further analyses.
-essential medium supplemented with 5% FCS, 60 µg/ml tylocine,
and 20 µg/ml gentamycin. CHO cells stably transformed with CB1
(CHO-CB1) or CB1 plus CRE luciferase (CHO-CRE-CB1) were grown in
MEM (Life Technologies, Inc.) supplemented with 5% dialyzed FCS, 40 µg/ml L-proline, 1 mM sodium pyruvate, 60 µg/ml tylocine, and 20 µg/ml gentamycin.
80 °C until use. To determine the effect of GTP
S on displacement of [3H]CP-55,940 or [3H]SR
141716A, membranes (50 µg of protein) were incubated at 30 °C for
1 h in binding buffer (50 mM Tris-HCl, pH 7.7) with
0.25 nM [3H]CP-55,940 or 0.8 nM
[3H]SR 141716A and increasing concentrations of unlabeled
SR 141716A or CP-55,940, respectively, in the presence or absence of
100 µM GTP
S. Effects of GTP
S were also studied by
incubating membranes at 37 °C for 30 min with 0.25 nM
[3H]CP-55,940 or 0.8 nM [3H]SR
141716A and increasing concentrations of GTP
S. A rapid filtration technique using Whatman GF/B filters (pretreated with 0.5% (w/v) polyethyleneimine) and a 48-well filtration apparatus (Brandel) was
used to harvest and rinse labeled membranes with cold buffer containing
0.05 M Tris-HCl, pH 7.7, and 0.25% bovine serum albumin. Filter-bound radioactivity was counted with 4 ml of biofluor liquid scintillator. Nonspecific binding was determined in the presence of 1 µM CP-55,940.
S Binding
S binding was measured as described by Selley
et al. (27). Briefly, membranes from CHO-CB1 cells (30 µg
of 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% bovine serum albumin) in the presence of 0.1 nM
[35S]GTP
S 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).
Nonspecific binding was measured in the presence of 10 µM
unlabeled GTP
S. The reaction was terminated by rapid filtration,
washing 5 times with ice-cold wash buffer (50 mM Tris-HCl,
pH 7.4), and bound radioactivity was determined.
-32P]ATP. The phosphorylation
reaction was performed for 30 min at 30 °C (linear assay condition)
and was stopped by spotting on Whatman P-81 filter papers which were
then dropped into 0.1% (v/v) orthophosphoric acid. Papers were washed
in this solution, rinsed with ethanol, and air-dried, and radioactivity
incorporated in MBP was measured by liquid scintillation counting.
-mercaptoethanol) containing 20%
methanol for 20 min, once in buffer A for 30 min, twice in buffer A
containing 6 M guanidine-HCl for 30 min, twice in buffer A
containing 0.04% Tween 20 at 4 °C for 16 h, and once in buffer
A containing 100 µM Na3VO4, 2 mM MgCl2, 50 µM ATP, and 50 µCi
of [
32P]ATP for 2 h at 30 °C. The reaction was
stopped by intensive washing of the gel in 5% trichloroacetic acid
solution. The gel was dried and subjected to autoradiography.
Effect of SR 141716A on MAPK Basal Activity in CHO-CB1
Cells
Fig. 1.
Effect of cannabinoid receptor ligands on
MAPK activity in CHO-WT and CHO-CB1 cells. Growth-arrested CHO-WT
cells () or CHO-CB1 cells (
) were treated with increasing
concentrations of cannabinoid receptor ligands for 10 min. The
activities of p42/p44 MAPK immunoprecipitates were measured by using
MBP as a substrate as described under "Materials and Methods." Data
points are mean values ± S.D. of duplicate samples, and
experiments were repeated five times. A, dose-response
effects of CP-55,940 on MAPK activity. B, dose-response
effects of the CB1-selective antagonist SR 141716A on basal MAPK
activity in CHO-CB1 and CHO-WT cells. MAPK activities in CHO-WT and
CHO-CB1 cells were normalized by their protein contents; the MAPK
activity in CHO-CB1 cells was taken as 100%.
[View Larger Version of this Image (20K GIF file)]
Fig. 2.
Effect of SR 141716A on basal MAPK activity
in CHO-CB1 cells. Growth-arrested CHO-CB1 cells were treated or
not for 10 min with cannabinoid receptor ligands. Lane 1,
untreated cells, or cells treated with the following: lane
2, 100 nM SR 141716A; lane 3, 10 nM CP-55,940; lane 4, 10 nM
CP-55,940 plus 100 nM SR 141716A. A, Western
blot of p42/p44 MAPK in the protein extracts. B, in-gel
kinase assay. Protein extracts were run in SDS-PAGE gel containing MBP.
Proteins renatured in the gel were assayed for kinase activities.
[View Larger Version of this Image (53K GIF file)]
9-THC decreased
forskolin-induced luminescence in a dose-dependent manner,
with IC50 values of 0.16 ± 0.023, 17.95 ± 3.15, and 2.25 ± 0.024 nM, respectively (Fig.
3A). The higher potency of
CP-55,940 compared with that of the agonists
9-THC or
WIN 55212-2 to inhibit AC is consistent with their respective ability
to displace [3H]CP-55,940 binding (17). Interestingly, SR
141716A not only reversed the inhibition induced by 10 nM
CP-55,940 in a dose-dependent manner but dramatically
enhanced forskolin-induced CRE-luciferase gene expression by 5-fold
above the control level. On its own, SR 141716A produced similar
effects at a 20-fold lower concentration (Fig. 3B). No
stimulation of luciferase by SR 141716A was observed in the absence of
forskolin, excluding a direct stimulation of GTP-binding protein
Gs by SR 141716A (data not shown). The effects of SR
141716A per se on CRE-luciferase are consistent with the blockage of AC inhibition mediated by autoactivated CB1 receptors. As
an additional control, we observed that when experiments were carried
out with CHO cells transfected with CRE-luciferase but lacking CB1
(CHO-CRE cells), neither CP-55,940 nor SR 141716A affected the level of
luciferase induced by forskolin (data not shown). These results were
confirmed by direct measurement of cAMP levels (Table
I), which showed that SR 141716A
treatment induced a marked increase in forskolin-stimulated cAMP
production in a dose-dependent manner in CHO-CB1 cells.
Under the same conditions, CP-55,940 inhibited the forskolin-induced
cAMP production in CHO-CB1 cells.
Fig. 3.
Effect of cannabinoid receptor ligands on
CRE-luciferase in CHO-CRE-CB1 cells. Growth-arrested CHO cells
stably expressing CRE-luciferase and CB1 genes (CHO-CRE-CB1) were
pretreated for 5 min with increasing concentrations of cannabinoid
receptor ligands before addition of 1 µM forskolin. After
4 h of stimulation, cell extracts were prepared, and luciferase
activities were measured by photon counting accumulation. Results are
mean values of triplicate determinations ± S.E. and were expressed as
percentage of values from cells stimulated with forskolin alone.
A, dose-response effects of cannabinoid receptor agonists
CP-55,940 (),
9-THC (
), and WIN 55212-2 (
).
B, dose-response effects of SR 141716A (
) or SR 141716A
in the presence of 10 nM CP-55,940 (
).
[View Larger Version of this Image (24K GIF file)]
Cannabinoid receptor ligands
(M)
cAMP
SR 141716A
CP-55,940
pmol/well
%
pmol/well
%
0 (control)
41.8
± 4
100 ± 9.5
41.8 ± 4
100 ± 9.5
10
10
42.75 ± 3.2
102.2 ± 7.6
37.2
± 1.7
88.9 ± 4
10
9
50.25 ± 3.2
120.2
± 7.6
20.95 ± 2
50.1 ± 4.7
10
8
72
± 10
172.2 ± 23
9.61 ± 2
22.9 ± 4.7
10
7
73.5 ± 4
175.8 ± 9.5
7.2
± 3
17.2 ± 7.1
10
6
69.5 ± 2.8
166.2
± 6.6
NDa
ND
a
ND, not determined.
It has often been
observed that guanidylic nucleotide analogs decrease the binding of
agonists while increasing the binding of inverse agonists (32). To
determine whether SR 141716A behaved as an inverse agonist, we first
compared the capacity of the unlabeled ligands SR 141716A and CP-55,940
to displace the binding of either [3H]CP-55,940 or
[3H]SR 141716A on CHO-CB1 membranes, in the presence of
GTPS. As shown in Fig. 4A,
the addition of 100 µM GTP
S shifted the displacement curve of [3H]CP-55,940 by SR 141716A to lower
concentrations (IC50 = 22.77 ± 6.6 nM;
plus GTP
S, IC50 = 4.2 ± 1.4 nM). Fig.
4B indicates a shift of the displacement curves of
[3H]SR 141716A by CP-55,940 to higher concentrations
(IC50 = 10.37 ± 3.6 nM; plus GTP
S,
IC50 = 25.73 ± 5.5 nM). Furthermore, we observed that GTP
S enhanced the binding of [3H]SR
141716A, while decreasing the binding of [3H]CP-55,940
(Fig. 4C). This pharmacology agrees with what has already
been described for agonists and inverse agonists.
Effect of SR 141716A on Receptor-Tyrosine Kinase-mediated MAPK Activation in CHO-CB1 Cells
The above results provide strong evidence that autoactivation of CB1 is shut off by SR 141716A treatment. We next asked the following question: does SR 141716A-mediated CB1 inactivation affect other signal pathways?
MAPKs can be activated in response to both GPCR or receptor-tyrosine
kinase (RTK) stimulation. Insulin receptors belong to the RTK family,
and their stimulation by insulin has been shown to activate MAPKs in
CHO cells (33). When CHO-CB1 or CHO-WT cells were stimulated with 1 µg/ml insulin, a time-dependent activation of MAPKs
(evaluated by phosphorylation of MBP) was observed (Fig. 5). The maximum level of induction was
obtained after a 10-min stimulation and then declined with time.
Surprisingly, 100 nM SR 141716A completely inhibited
insulin-activated MAPK in CHO-CB1 cells (Fig. 5A). On the
other hand, SR 141716A had no effect on insulin-stimulated MAPKs in
CHO-WT cells (Fig. 5D), establishing that the above effects
required the interaction of SR 141716A with CB1 receptors. These
results were confirmed by Western blot analysis of p42/44-kDa MAPK
proteins (Fig. 6A) as well as
by a MAPK renaturation assay (Fig. 6B).
We next wondered whether this inhibition could be extended to other RTKs. MAPK phosphorylation in response to 10 ng/ml insulin-like growth factor 1 (IGF1) exposure followed a similar time course and was also prevented by SR 141716A in CHO-CB1 but not in CHO-WT (Fig. 5, B and E). Both Western blot analysis and MAPK renaturation assay also confirmed these results (Fig. 6). In contrast, MAPK stimulated by 10 ng/ml basic fibroblast growth factor (FGF-b) was not affected by SR 141716A treatment (Fig. 5, C and F). It is noticeable that the level of MAPK activation induced by FGF-b was much higher than that elicited by insulin or IGF-1. However, this difference could not account for the lack of effect of SR 141716A. Indeed, even when using low concentrations of FGF-b (0.1 ng/ml), for which a 3-fold lower MAPK activation was observed, SR 141716A still did not inhibit this response (data not shown).
Transduction Pathway Elicited by CB1, Insulin, and IGF1 ReceptorsSince MAPK activation by CB1, insulin, and IGF1
receptors could be blocked by SR 141716A, an attractive hypothesis was
that they shared a common transduction pathway. Several authors have reported that the PTX-sensitive activation of MAPK by GPCR required the
activation of a phosphatidylinositol 3-kinase (PI-3K) upstream to the
MAPKs (34). Treatment of CHO-CB1 cells with the PI-3K inhibitor
wortmannin resulted in a significant inhibition of both CP-55,940 and
insulin-mediated MAPK activation, with IC50 values of
26 ± 4 and 38 ± 7 nM, respectively (Fig.
7). Similar results were obtained with
IGF1 (data not shown). On the contrary, wortmannin did not affect the
FGF-b-mediated effect (Fig. 7). These data show that stimulations of
MAPKs by the Gi-coupled CB1 receptor and the insulin
receptor are equally sensitive to wortmannin.
We next wondered whether the same molecular entities were committed in the transduction of CB1 and insulin signals. To address this question, we analyzed their costimulatory effects. The results are summarized in Table II. At suboptimal concentrations of both CP-55,940 and insulin, additive effects on MAPK activation were observed. In contrast, when saturating concentrations of 10 nM CP-55,940 were used, no additional effect with insulin was noted in costimulatory experiments. On the other hand, the effects of CP-55,940 and FGF-b were additive independently of their concentrations (Table II). These results are in agreement with a common transduction pathway for cannabinoid- and insulin-mediated MAPK activation and suggest the involvement of the same limiting factor for optimal activation.
|
It has already been shown that the Gi
protein, which is involved in the CB1 response, could also be required
for MAPK activation induced by insulin and IGF1 but not by FGF-b (35).
Our results shown on Fig. 8 indicate
similar results. The MAPK activation induced by insulin or IGF-1 was
markedly inhibited by PTX, whereas induction of MAPK by FGF-b was
unaffected by PTX. Since the Gi component is one of the
early known steps in signaling, we hypothesized that the binding of SR
141716A to CB1 interfered with Gi activity. To test this
hypothesis, we examined the effect of SR 141716A on MAPK activation
induced by an analog of mastoparan. Mastoparan is a direct activator of
G protein with a reported selectivity for Gi/Go
(36), and Mas-7 is an analog exhibiting a 5-fold greater potency than
mastoparan (37). Exposure of CHO-CB1 or CHO-WT cells to 3 µM Mas-7 for 10 min resulted in a marked increase in MAPK
activity (Fig. 9A). In
agreement with a Gi coupling, this increase was completely
prevented by pretreatment with PTX. Interestingly, 100 nM
SR 141716A completely inhibited the Mas-7-induced MAPK activation in
CHO-CB1 cells but not in CHO-WT cells. We next examined whether SR
141716A could act directly at the Gi level. This was investigated by measuring the binding of [35S]GTPS to
G protein. Indeed, this binding is regulated by the receptor and
provides direct information about the interaction between receptors and
G protein activation (38, 39). Fig. 9B clearly indicates
that 20 nM CP-55,940 and 30 µM Mas-7
treatment stimulated [35S] GTP
S binding to the
CHO-CB1 cell membranes. Conversely, SR 141716A was able to induce a
marked inhibition of constitutive as well as CP-55,940- and
Mas-7-induced [35S]GTP
S binding to CHO-CB1 cell
membranes. This effect was not observed in CHO-WT cell membranes,
indicating that the SR 141716A-induced inhibition is not a nonspecific
direct interaction with the Gi protein but a
receptor-mediated response.
In the first part of this
paper, we demonstrated that the CB1-selective antagonist SR 141716A
functions as an inverse agonist for the autoactivated CB1. This was
concluded from major observations on two independent signaling
pathways, G-mediated MAPK and Gi
-mediated AC
responses. First, we showed that the basal level of MAPK activity was
enhanced in CHO cells following transfection of CB1 receptors, and this
increase was reversed by treatment with SR 141716A. These results,
observed by measuring the phosphorylation of MBP substrate in p42/p44
immunoprecipitates, were confirmed by EMSAs and in-gel kinase assays.
Second, we showed that SR 141716A prevented the inhibition of AC
mediated by autoactivated CB1 receptors. This was demonstrated by
direct measurement of cAMP and by the use of CHO-CB1 cells transfected
with a reporter construct containing CREs linked to a luciferase
gene.
We observed that GTPS decreased the binding of CP-55,940 and
enhanced the binding of SR 141716A, properties usually described for
agonists and inverse agonists, respectively (32). Moreover, it is
noticeable that GTP
S was more potent at blocking
[3H]CP-55,940 than at potentiating [3H]SR
141716A binding. Assuming that the proportion of sites for agonists
that are inhibited by guanine nucleotides is an indication of the
receptor status in a precoupled autoactivated form, this correlated
well with 1) the marked autoactivation of MAPK in CHO-CB1 cells and 2)
the degree of negative and positive effects of SR 141716A on MAPK and
AC activities, respectively.
An often raised alternative interpretation of the inhibition of autoactivated receptor by inverse antagonists could be a blockage of endogenous agonists present in culture medium or produced by the cells. We ruled out such a possibility for the following reasons. First, the human astrocytoma cell line U373-MG, which expresses CB1, was not affected in its cAMP metabolism when exposed to culture supernatants from CHO-CB1 cells, whereas in these cells AC is exquisitely sensitive to minute amounts of cannabinoid receptor agonist (data not shown). Second, the dose-response curves for SR 141716A modulation of MAPK and AC activities showed an EC50 in the nM range, which fits with the binding property of [3H]SR 141716A (16, 17, 40). A significantly higher EC50 would be expected if endogenous agonist molecules were acting in competition with SR 141716A. Indeed, we observed that the presence of as low as 10 nM CP-55,940 decreased the potency of SR 141716A on cAMP metabolism by 20-fold (Fig. 3B). Together, these results support the notion that the observed effects of the CB1 antagonist SR 141716A reflect the direct consequences of its binding to unoccupied receptors and the notion that it acts as an inverse agonist.
The properties of autoactivated receptor have already been described in
heterologous expression systems for a variety of GPCRs including
dopamine receptors D1A and D1B, 5HT2C receptor,
opioid receptor, and
-adrenergic receptor (20, 39, 41, 42). Our
results provide an additional example of such autoactivated receptors.
One of the most important and provocative findings of this paper, developed in its second part, describes a novel property for the inverse agonist SR 141716A. This concerns the link between CB1 and some growth factor receptors belonging to the RTK family. CHO cells naturally express RTKs such as those for insulin, IGF-1, and FGF. The specific stimulation of these receptors by their natural ligands has been shown to lead to MAPK activation (35). We demonstrated here that the inverse agonist not only inhibits autoactivated CB1 but also switches off MAPK activation from some RTKs, including insulin and IGF1 receptors. This was shown by measuring phosphorylation of MBP in MAPK immunoprecipitates, by EMSA, and by in-gel kinase assay. On the contrary, SR 141716A did not affect MAPK activation induced by FGF-b in CHO-CB1 cells. These effects were related to CB1 since they were not observed in CHO-WT cells. We concluded from these results that the binding of SR 141716A to CB1 induced biological responses that negatively interfered with particular RTK pathways. Moreover, exposure of cells to a saturating concentration of SR 141716A, followed by its removal by washing, did not prevent MAPK stimulation by CP-55,940 or insulin, indicating that the effect of SR 141716A was reversible and that the binding of SR 141716A to CB1 was required to mediate its inhibitory effect.
MAPKs represent a point of convergence of mitogenic signals emerging
from several distinct types of GPCRs and RTKs, suggesting the
integration of redundant information. Although these interactions remain poorly understood, recent findings support such a network, which
may function as a mechanism of enhancement or counter-regulation. For
instance, tyrosine phosphorylation of the 2-adrenergic receptor by
insulin has been shown to produce supersensitization of adrenergic signaling (43), whereas protein kinase A activation by
-adrenergic receptor leads to an attenuation of insulin and EGF-stimulated MAPK
activity (44). More recently, lysophosphatidic acid (LPA) and thrombin,
two GPCR ligands, were shown to mediate cell proliferation through
ligand-independent induction of tyrosine phosphorylation of the EGF
receptor belonging to RTK (45). For the first time, we demonstrate an
original situation where an inverse agonist specific for GPCR may
induce an RTK inhibition through an intracellular signal cross-talk.
Although the mechanism by which SR 141716A counteracts insulin or IGF
receptors remains to be elucidated, several points can be raised. It
has been suggested that the responses mediated by RTKs segregate into
two groups, one PTX-sensitive, resembling the Gi-coupled
LPA receptor, and one PTX-insensitive (35). Unlike the FGF receptor
pathway, the insulin or IGF-1 signal can be blocked by either PTX
treatment or wortmannin, an inhibitor of PI-3K, which is an early
intermediate of G
-mediated MAPK signaling pathway (34). This
latter pathway has been implicated in MAPK activation mediated by
classical Gi-coupled receptors such as CB1. The mechanism
whereby insulin or IGF1 receptors can induce the generation of free
G
subunits is unclear, although indirect evidence suggests a
direct protein-protein interaction between receptor and G protein (46).
It has been reported that insulin attenuates the sensitivity of a
40-kDa Gi-like protein toward PTX in hepatocyte membranes
(47, 48). Jo et al. (49) reported that the insulin receptor
is directly associated with two proteins of 40 and 67 kDa that bind
GTP. Recently, peptides derived from the insulin receptor
subunit
have been shown to directly activate Gi in phospholipid
vesicles (50). Whatever the molecular mechanism, the physiological
importance of the Gi protein in insulin receptor signaling
is supported by recent results from Moxham and Malbon (51) who
established that Gi
2 deficiency in liver and
adipose tissues of transgenic mice impaired glucose tolerance and
induced insulin resistance.
However, it is noteworthy that the RTK segregation as described above cannot be considered as a stringent rule. Indeed in some cells, FGF receptors have been reported to be PTX-sensitive (52). Conversely, in other cases, PTX-sensitive G proteins are not involved in MAPK activation by insulin or IGF-1 (32, 53). More recently, Harada et al. (54) have demonstrated that insulin exhibits multiple signal transduction pathways that vary from cell to cell. For instance, they showed that, in the mouse myeloid cells 32D, insulin-stimulated MAP kinase did not involve PI 3-kinase activation (54), which differs from our observation.
Using CHO-transfected cells, we showed that CB1, insulin, and IGF shared a PTX-sensitive signaling pathway. We have shown here that when CHO-CB1 cells were co-stimulated with saturating concentrations of insulin and CP-55,940, the MAPK response was not cumulative, suggesting that a pool of common proteins or key enzymes is used downstream from both CB1 and the insulin receptor. Mitogenic signals originating from insulin, IGF1, and CB1 receptors converge at a point upstream of p21ras, probably represented by the Gi protein. We thus hypothesized that Gi represents the limiting factor for optimal stimulation and that the binding of an inverse agonist to CB1 may inactivate the Gi protein. The two following observations argue in favor of this: (i) SR 141716A bound to CB1 receptor prevents the activation of MAPKs induced by Mas-7, which acts directly at the Gi level; (ii) the effects of SR 141716A 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 by PTX.
The effect of SR 141716A on PTX-sensitive RTK was shown in CHO-CB1 cells, but it is likely to be a general phenomenon since we could extend our observations to Rat-1 fibroblast cells (data not shown). These cells naturally express insulin, IGF1, FGFb, EGF, as well as LPA receptors. When transfected with CB1, SR 141716A blocked the PTX-sensitive-MAPK activation induced by insulin, IGF1, or LPA, whereas the PTX-insensitive MAPK activation induced by EGF or FGF-b was not altered by SR 141716A. Thus, the SR 141716A·CB1 complex, acting as a reversible negative dominant of the Gi, can be considered as a potent and valuable tool to study the signal transduction pathway involving Gi.
Predictive Model of Receptor Activation of G ProteinsIt is
tempting to hypothesize that sequestration of CB1-coupled
Gi protein makes this protein unavailable for coupling to insulin or IGF1 receptors. According to this model, which we designate a "three-state receptor model" as illustrated in Fig.
10, SR 141716A converted the CB1
autoactivated receptor to a suppressor receptor acting in
trans by sequestration of the Gi protein, which
very likely remains inactive under a GDP-bound form. One possibility could be that the SR 141716A·CB1·Gi complex blocks the
GDP-GTP exchange. We assume that the receptor could be in equilibrium between three conformations: R°, R+, R. In
the R° state, the receptor is uncoupled to the G protein, whereas the
two forms R+ and R
are able to bind 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 induce signal
transduction, but by capturing the G protein, this state prevents the
activation of nonrelated G protein-coupled receptors localized in its
vicinity. We therefore term R
/G the active negative
state. To activate the G protein the receptor should deliver two
simultaneous pieces of information, one for the formation of R·G
complex and the other to catalyze guanine nucleotide exchange by
dramatically increasing the GDP dissociation rate. A study of
structure-activity by site-directed mutagenesis of both R and G
components would help to elucidate the molecular nature of these two
distinct signals involved in G activation.
Such a model is not consistent with the theoretical framework of the "two-state model" currently in use. This latter model postulates that an antagonist with negative intrinsic activity stabilizes the inactivated form of the receptor that is uncoupled to the G protein. One of the main arguments for this is based on the increase in affinity of inverse agonists in the presence of guanine nucleotides, which are assumed to increase the level of uncoupled receptors. Thus, antagonists with negative activity and guanine nucleotides are both believed to promote or stabilize the inactivated form of the receptor that is uncoupled to the G protein. This is one possible interpretation for these effects, but several observations make that interpretation questionable.
We show here that the addition of guanine nucleotide is indeed
accompanied by a decrease in agonist binding and a reciprocal enhancement in antagonist binding. However, these effects do not occur
at equivalent concentrations of nucleotides, as opposed to what could
be expected. In our case, the concentration of GTPS needed to
produce a half-maximal effect on SR 141716A binding is 20 times greater
than that required to produce a half-maximal effect on CP-55,940
binding (Fig. 4C). Similar observations have already been
described for the 5-HT2C receptor (32, 55).
Thus, nucleotide-induced changes in binding do not necessarily indicate
a dissociation of receptor-G protein complexes. Indeed, guanylate
imidodiphosphate has been shown to reduce agonist binding to
solubilized cardiac muscarinic acetylcholine receptors under conditions
that did not prevent G protein co-immunoprecipitation (56). As already
raised by Chidiac (57), this indicates that there is no obvious
relationship between inverse agonist nucleotide-induced changes in
binding and the stability of receptor-G protein complexes. On the
contrary, we think from these observations, together with the results
presented here, that the inverse agonist promotes or stabilizes an
active negative state R/G. The enhancement of both
binding and affinity of the inverse agonist, which occurred only at
very high nucleotide concentrations, requires further studies to be
correctly interpreted.
Interestingly the three-state model that we propose fits with the cubic ternary complex model elaborated from thermodynamic calculations that predict the existence of an inactive receptor-G protein complex that is consistent with our biological data (23, 58, 59).
One important question that arises from a stoichiometric point of view is how the SR 141716A·CB1 complexes can alter most of the cellular Gi responses, as manifested here by the inhibition of Mas-7-mediated Gi activation. Two possibilities may account for such an observation as follows: 1) although G protein/receptor-coupled systems often seem to have much more G proteins than receptors (60), in our case the very high CB1 expression level per cell (around 2 pmol/mg proteins) could be in excess when compared with the Gi protein level; 2) as proposed by Schlegel et al. (61) and by Jahangeer and Rodbell (62) in the GDP-bound state, G proteins are likely complexed as multimeric structures, estimated as dodecamers. During the activation process, activated monomers are released from multimers. In that context, one may envision that one SR 141716A molecule, which inhibits G protein activation, prevents the dynamic progression of several G proteins. A precise measurement of the CB1/Gi stoichiometry may favor one of these alternatives; in addition, it would provide an interesting opportunity for validating the existence of such a G protein multimeric structure.
Concluding RemarksIf the above-mentioned hypothesis is valid, then the full range of properties and therapeutic potential of inverse agonists remain to be explored. Moreover, the interpretation of biological effects observed in the presence of any antagonists has to be re-examined since some of them may be related to negative effects rather than zero efficacy.
Similar observations remain to be extended to cells naturally expressing CB1 receptors to assess their physiological relevance. However, one can anticipate that such observations may be difficult to obtain due to compartmentalization. Indeed, it is very likely that, in intact cells, some sets of receptors, G proteins, and effectors may be organized into separated microdomains and do not have direct access to other sets. In contrast, transfected cells may not faithfully mirror the organization of wild-type cells, because overexpression may saturate normal compartments and artificially introduce signaling components into abnormal sites. In our case, CHO cells that expressed a high level of CB1 per cell may account for the observed exacerbated trans-signal responses.
Additionally, our observations were made on an autoactivated receptor. We do not know yet whether they are also valid on receptors not constitutively activated and therefore in normal physiology. However, before this important point has been clarified, the new interaction described here deserves further exploration for therapeutic strategies. Indeed, our results could be important in pathologies where point mutations (63, 64) as well as overexpression of receptors (23, 20) are known to promote such autoactivated receptors and are often associated with human diseases (65, 66). Tumors expressing high levels of autoactivated receptors are an obvious example in which inverse agonists would offer a considerable therapeutic benefit by trans-inactivation of a variety of growth factors involved in cell proliferation.