N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)- 4-methyl-1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS 3.28(192) is crucial for its inverse agonism at the cannabinoid CB1 receptor

Article abstract of DOI:10.1124/mol.62.6.1274

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)- 4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca²⁺ current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-

Full text of DOI:10.1124/mol.62.6.1274

0026-895X/02/6206-1274–1287$7.00
MOLECULAR PHARMACOLOGY
Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 62:1274–1287, 2002
Vol. 62, No. 6
1915/1023870
Printed in U.S.A.
N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide (SR141716A) Interaction
with LYS 3.28(192) Is Crucial for Its Inverse Agonism at the
Cannabinoid CB1 Receptor
DOW P. HURST, DIANE L. LYNCH, JUDY BARNETT-NORRIS, STEPHEN M. HYATT, HERBERT H. SELTZMAN,
MIAO ZHONG, ZHAO-HUI SONG, JINGJIANG NIE, DEBORAH LEWIS, and PATRICIA H. REGGIO
Department of Chemistry, Kennesaw State University, Kennesaw, Georgia (D.P.H., D.L.L., J.B.-N., P.H.R.); RTI International, Research Triangle
Park, North Carolina (S.M.H., H.H.S.); Pharmacology and Toxicology Department, University of Louisville, Louisville, Kentucky (M.Z., Z.-H.S.);
and Pharmacology and Toxicology Department, Medical College of Georgia, Augusta, Georgia (J.N., D.L.)
Received June 10, 2002; accepted August 23, 2002
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4- SR141716A only in R. To test this hypothesis, a “mutant thermody-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3- namic cycle” was constructed that combined the evaluation of
carboxamide (SR141716A) competitively antagonizes the Ca2ϩ SR141716A affinity at WT CB1 and K3.28A with an evaluation of the
current effect of the cannabinoid (CB) agonist (R)-(ϩ)-[2,3-dihydro-5- wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-
methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6- chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-
yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an in- methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential
verse agonist by producing opposite current effects when applied at C3. Binding affinities suggested that K3.28 is involved in a strong
alone. In contrast, in neurons expressing CB1 with a K3A mutation interaction with SR141716A in WT CB1, but does not interact with
at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antag- VCHSR. Thermodynamic cycle calculations indicated that a direct
onizes the effects of WIN55212-2, but behaves as a neutral antago- interaction occurs between the C3 substituent of SR141716A and
nist by producing no current effects itself. Receptor modeling studies K3.28 in WT CB1. Consistent with these results, VCHSR acted as
suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes a neutral antagonist at WT CB1. These results support the hypoth-
transmembrane helix 6 in its inactive conformation via aromatic esis that hydrogen bonding of the SR141716A C3 substituent with
stacking with F3.36/W6.48. In this binding site, SR141716A would K3.28 is responsible for its higher affinity for the inactive R state,
exhibit higher affinity for CB1 R due to a hydrogen bond between the leading to its inverse agonism.
SR141716A C3 substituent and K3.28(192), a residue available to
To date, two subtypes of the cannabinoid receptor, CB1 enhancement of inwardly rectifying potassium currents is
(Gerard et al., 1991) and CB2 (Munro et al., 1993), have been
identified. These receptors belong to the rhodopsin family of
G protein-coupled receptors. CB1 and CB2 receptor agonists
inhibit forskolin-stimulated adenylyl cyclase by activation of
a pertussis toxin-sensitive G protein (Felder et al., 1995). In
heterologous cells, CB1 but not CB2 receptors inhibit N-, P-,
and Q-type calcium channels and activate inwardly rectify-
ing potassium channels (Felder et al., 1995; Mackie et al.,
1995; Pan et al., 1996). Inhibition of calcium channels and
pertussis toxin-sensitive, but independent of cAMP inhibi-
tion, suggestive of a direct G protein mechanism (Mackie et
al., 1995).
The CB1 antagonist SR141716A (1) displays nanomolar
CB1 affinity, but very low affinity for CB2. SR141716A an-
tagonizes the pharmacological and behavioral effects pro-
duced by CB1 agonists after intraperitoneal or oral adminis-
tration (Rinaldi-Carmona et al., 1994).
SR141716A (1) has been shown to act as a competitive
antagonist and inverse agonist in host cells transfected with
exogenous CB1 receptor, as well as in biological preparations
RTI International is a trademark of Research Triangle Institute.
ABBREVIATIONS: SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; WT, wild-
type; CB, cannabinoid; VCHSR, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole; WIN, WIN55212-2
[(R)-(ϩ)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone]; R, inactive state; hCB,
human cannabinoid; TMH, transmembrane helix; Rho, rhodopsin; R*, active state; CM, conformational memory; CG, conjugate gradient; SCG,
superior cervical ganglion; MEP, molecular electrostatic potential; GPCR, G protein-coupled receptor; MC, monochlorophenyl; DC, dichlorophe-
nyl.
1274

SR141716A Interaction with K3.28 Crucial for Inverse Agonism
1275
endogenously expressing CB1. Bouaboula et al. (1997) re- onist at WT CB1 in this same assay. This result lends further
ported that Chinese hamster ovary cells transfected with
hCB1 receptor exhibit high constitutive activity at both lev-
els of mitogen-activated protein kinase and adenylyl cyclase.
Guanine nucleotides enhanced the binding of SR141716A, a
property of inverse agonists. Lewis and coworkers (Pan et al.,
1998) demonstrated constitutive activity of CB1 receptors in
inhibiting Ca2ϩ currents that was not due to endogenous
agonist. These investigators reported that SR141716A antag-
onized the Ca2ϩ current inhibition induced by the cannabi-
noid agonist WIN55212-2, in neurons heterologously ex-
pressing either rat or hCB1 receptors. Furthermore, when
applied alone, SR141716A increased the Ca2ϩ current, with
an EC50 value of 32 nM, via a pertussis toxin-sensitive path-
way, indicating that SR141716A can act as an inverse ago-
nist by reversal of tonic CB1 receptor activity (Fig. 1). Me-
schler et al. (2000) demonstrated that constitutive activity is
demonstrable in neuronal cells that endogenously express
CB1 (N18TG2 cells) and that SR141716A acts as a competi-
tive antagonist and reduces basal activity in the manner of
an inverse agonist in these cells.
support to the hypothesis that a K3.28(192) direct interaction
with the C3 substituent of SR141716A is crucial for the inverse
agonist activity of SR141716A at CB1.
In some experiments, SR141716A has been found to be
more potent in blocking the actions of CB1 agonists than in
eliciting inverse responses by itself. For example, in their
study that focused upon rat brain membrane and brain sec-
tions, Sim-Selley et al. (2001) suggested that SR141716A
may bind to two sites on the cannabinoid receptor, a high-
affinity site at which it exerts its competitive antagonism and
a lower affinity site at which it exerts its inverse agonism.
The present study was prompted by results reported by
Pan et al. (1998). These investigators found that Ca2ϩ cur-
rent was tonically inhibited in neurons expressing a mutant
CB1 K3.28(192)A receptor. Surprisingly, SR141716A had no
effect on the Ca2ϩ current in these neurons, but SR141716A
could still antagonize the effect of WIN55212-2. The authors
concluded that the K3.28(192) site is critical for the inverse
agonist activity of SR141716A and that SR141716A seemed
to become a neutral antagonist at the K3.28(192)A mutant
receptor. Prompted by these intriguing SR141716A/
K3.28(192)A results, we assessed through modeling studies,
the proximity of K3.28(192) to SR141716A in CB1. Our mod-
eling suggested that SR141716A has equivalent aromatic
stacking interactions in both the inactive and active states of
CB1, but only in the inactive state of the CB1 receptor can
Fig. 1. A, WIN decreased and SR increased the Ca2ϩ current in a neuron
SR141716A (via its carboxamide oxygen) hydrogen bond with expressing human CB1 receptors after cDNA microinjection. The double-
K3.28(192).
pulse protocol was used to elicit control (⅜) and facilitated (●) Ca2ϩ
currents, and current amplitudes were plotted over the time course of the
The work presented herein was designed to test the hypoth-
esis that a direct interaction between K3.28(192) and the C3
substituent region of SR141716A is responsible for the inverse
agonist activity of SR141716A at CB1. An SR141716A analog,
5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichloro-
phenyl)-4-methyl-1H-pyrazole (VCHSR; 2) was designed and
synthesized in which the C3 substituent carboxamide trans-
geometry was preserved, but in which all hydrogen bonding
capability was removed. A mutant cycle was constructed in
which the binding affinities of SR141716A and VCHSR at both
CB1 WT and K3.28(192)A receptors were analyzed. Results of
this study suggest that K3.28 is a direct interaction site for the
C3 substituent of SR141716A in CB1. Furthermore, consistent
with the neutral antagonism displayed by SR141716A at CB1
K3.28(192)A in a Ca2ϩ current assay, VCHSR, which lacks the
experiment. Application of 1 ␮M WIN55,212-2 decreased the Ca2ϩ cur-
rent, which slowly recovered to a greater amplitude. A subsequent appli-
cation of 1 ␮M SR141716A rapidly increased the Ca2ϩ current. Inset,
superimposed current traces recorded at the beginning of the experiment
(control), in the presence of 1 ␮M WIN55,212-2, and in the presence of 1
␮M SR141716A. B, summary of the changes in Ca2ϩ current amplitudes
in the presence of 1 ␮M WIN55,212-2 (negative axis) or 1 ␮M SR141716A
(positive axis) in neurons microinjected with hCB1 or mutant
K3.28(192)A receptor cDNA is shown. In neurons micro-injected with
hCB1 receptor cDNA (hCB1), WIN55,212-2 inhibited the Ca2ϩ current
and 1 ␮M SR141716A increased the Ca2ϩ current. In neurons microin-
jected with mutant K3.28(192)A receptor cDNA (K192A),
1 ␮M
WIN55,212-2 inhibited the Ca2ϩ current, but 1 ␮M SR141716A did not
increase the Ca2ϩ current. In neurons microinjected with hCB1 cDNA
and recorded in the absence of Ca2ϩ, using Ba2ϩ as the charge carrier and
BAPTA to chelate intracellular Ca2ϩ (hCB1 Bapta), SR141716A in-
creased the current. SR141716A also increased the current in neurons
microinjected with hCB1 cDNA and preincubated with 300 nM anand-
amide (hCB1 Anand). This figure is reprinted with permission from Pan
ability to interact with K3.28(192), behaved as a neutral antag- et al. (1998).

1276
Hurst et al.
gences included the absence of helix kinking proline residues in TMH
1 and TMH 5, the lack of a GG motif in TMH 2, as well as the
presence of extra flexibility in TMH 6.
Materials and Methods
Molecular Modeling
Because TMH 6 figures prominently in the R-to-R* transition (i.e.,
activation), we have studied the conformations accessible to TMH 6
in CB1 and CB2 using conformational memories (CMs) (Barnett-
Norris et al., 2002a). These studies revealed that TMH 6 in CB1 has
high flexibility due to the small size of residue 6.49 (a Gly) immedi-
ately preceding Pro 6.50. Two families of conformers were identified
by CM for TMH 6 in CB1. Cluster 1 showed a pronounced proline
kink (40 members of 100, 71.2° average kink angle). Cluster 2 con-
A recent crystal structure of SR141716A confirms that the carbox-
amide of the C3 substituent is in a trans-geometry (C. George,
personal communication). Complete conformational analyses of
SR141716A and VCHSR were performed using the semiempirical
method, AM1 within the Spartan molecular modeling program
(Wavefunction, Inc., Irvine, CA). AM1 six-fold Conformer Searches
were performed for the rotatable bonds in SR141716A (Fig. 2; C3
substituent: C3-C1Ј and N2Ј-N3Ј; C5 substituent: C5-C1ٞ; N-1 sub-
stituent: N1-C1Љ) and in VCHSR (C3 substituent: C3-C1Ј and C2Ј- tained helices with less pronounced kinks (51 members of 100, 30.1°
C3Ј; C-5 substituent: C5-C1ٞ; N-1 Substituent: N1-C1Љ). The energy average kink angle). A conformer from the more kinked CM family of
separation between a smaller set of conformers was recalculated
using an ab initio Hartree Fock calculation at the 6-31G* level as
encoded in Jaguar (Schro¨dinger, Inc., Portland, OR).
CB1 TMH 6 (cluster 1) was used in our model of the R state of CB1.
This conformer was selected (Pro kink angle of 53.1°) so that
R3.50(214) and D6.30(338) could form a salt bridge at the intracel-
lular ends of TMHs 3 and 6 in the CB1 TMH bundle. An analogous
salt bridge has been shown to be an important stabilizer of the
inactive state of the ␤2-adrenergic receptor (Ballesteros et al., 2001)
and to be present in Rho (Palczewski et al., 2000).
Receptor Model Construction
Amino Acid Numbering System. In the discussion of receptor
residues that follows, the amino acid numbering scheme proposed by
Ballesteros and Weinstein (1995) is used. In this numbering system,
the most highly conserved residue in each transmembrane helix
(TMH) is assigned a locant of 0.50. This number is preceded by the
TMH number and may be followed in parentheses by the sequence
number. All other residues in a TMH are numbered relative to this
residue. In this numbering system, for example, the most highly
conserved residue in TMH 2 of the CB1 receptor is D2.50(163). The
residue that immediately precedes it is A2.49(162).
Model of R Form of CB1. A model of the R form of CB1 was
created using the 2.8-Å crystal structure of bovine rhodopsin (Rho)
(Palczewski et al., 2000). First, the sequence of the human CB1
receptor (Gerard et al., 1991) was aligned with the sequence of
bovine Rho using the same highly conserved residues as alignment
guides that were used initially to generate our first model of CB1
(Bramblett et al., 1995). TMH 5 in CB1 lacks the highly conserved
proline in TMH 5 of Rho. The sequence of CB1 in the TMH 5 region
was aligned with that of Rho as described previously using its hy-
drophobicity profile (Bramblett et al., 1995). Helix ends for CB1 were
chosen in analogy with those of Rho (Palczewski et al., 2000); TMH
1: N1.28(112)-R1.61(145); TMH 2: R2.37(150)-H2.68(181); TMH 3:
S3.21(185)-R3.56(220); TMH 4: T4.38(229)-C4.66(257); TMH 5:
H5.34(270)-K5.64(300); TMH 6:R6.28(336)-K6.62(370); TMH 7:
K7.32(376)-S7.57(401); and intracellular extension of TMH 7:
D7.59(403)-C7.71(415). With the exception of TMH 1, these helix
ends were found to be within one turn of the helix ends originally
calculated by us and reported in Bramblett et al. (1995). Changes to
the general Rho structure that were necessitated by sequence diver-
Model of R* Form of CB1. An R* CB1 model was created by
modification of our rhodopsin-based model of the R form of CB1. This
R* model construction was guided by the biophysical literature on
the R-to-R* transition in Rho and the ␤2-adrenergic receptor. This
literature has indicated that for the ␤2-adrenergic receptor, a salt
bridge between R3.50 and E6.30 at its intracellular end stabilizes
this receptor in its inactive state (Ballesteros et al., 2001). Biophys-
ical studies of Rho and/or the ␤2-adrenergic receptor have indicated
that rotation of both TMH 3 and 6, as well as a conformational
change in TMH 6 occurs upon activation (Farrens et al., 1996; Lin
and Sakmar, 1996; Javitch et al., 1997; Jensen et al., 2001). Jensen
et al. (2001) recently demonstrated through fluorescence studies in
the ␤2-adrenergic receptor that P6.50 in the highly conserved CWXP
motif of TMH 6 can act as a flexible hinge that mediates the transi-
tion from R to R*. In the R state, these investigators proposed that
TMH 6 is kinked at P6.50 such that its intracellular end is nearly
perpendicular to the membrane and close to the intracellular end of
TMH 3. The transition to the R* state is accomplished by the
straightening of TMH 6 such that the intracellular part of TMH 6
moves away from the receptor core and upwards toward the lipid
bilayer (Jensen et al., 2001). All of these experimental findings were
used to create the R* model of CB1 described herein.
Our conformational memories study of CB1 TMH 6 revealed two
distinct conformational families for TMH 6 that differed in the de-
gree of kinking at CWGP (Barnett-Norris et al., 2002a). These con-
formationally distinct TMH 6s were used to create the R and R*
states depicted herein. In the R* bundle, a TMH 6 conformer from
Fig. 2. Structures of SR141716A (1)
and VCHSR (2).

SR141716A Interaction with K3.28 Crucial for Inverse Agonism
1277
the second major conformational family (less kinked: 21.8° kink
angle) identified by CM (Barnett-Norris et al., 2002a) was substi- amide, to the corresponding stabilized phosphorus ylide, and treat-
tuted for the TMH 6 conformer used in the inactive model of CB1. ment with cyclohexanecarboxaldehyde afforded the putative olefin 2.
Deprotonation of this phosphonium salt with lithium diisopropyl-
This conformer was chosen so that the salt bridge in the inactive The trans-geometry of the olefin was proven by an NMR shift reagent
state between R3.50(214) and D6.30(338) would be broken due to the experiment that resolved the overlapping vinyl resonances to reveal
movement of the intracellular end of TMH 6 away from that of TMH
3 and out into lipid (Ballesteros et al., 2001). In addition, the R* CB1
bundle was created by rotating TMH 3 so that residue 3.41 changes
environments (Lin and Sakmar, 1996). This was accomplished by a
20° counterclockwise (extracellular view) rotation of TMH 3 from its
orientation in the R bundle. TMH 6 was also rotated (counterclock-
wise from extracellular view) so that Cys 6.47 became accessible
from inside the binding site crevice (Javitch et al., 1997).
Preparation of Helices. Each helix of the model was capped as
the acetamide at its N terminus and as the N-methyl amide at its C
terminus. Ionizable residues in the first turn of either end of the
helix were neutralized, as were any lipid-facing charged residues.
Ionizable residues were considered charged if they appeared any-
where else in the helix.
their 16-Hz coupling constant, indicative of trans (E)-geometry.
Ligand Binding Assay
Materials. Dulbecco’s modified Eagle’s medium, fetal bovine se-
rum, penicillin/streptomycin, L-glutamine, trypsin, and geneticin
were purchased from BioWhittaker (Walkersville, MD). Enzymes
and reagents used for recombinant DNA experiments were pur-
chased from Invitrogen (Carlsbad, CA) or Promega (Madison, WI).
Adenovirus-transformed 293 cells were obtained from American
Type Culture Collection (Rockville, MD). Glass tubes used for dilut-
ing cannabinoid drugs and for ligand binding assays were silanized
through exposure to dichlorodimethylsilane (Sigma-Aldrich, St.
Louis, MO) vapor while under vacuum for 3 h. [3H]SR141716A was
purchased from Amersham Biosciences (Piscataway, NJ).
Ligand-Receptor Complex. Each ligand was docked in the ar-
omatic, residue-rich TMH 3-4-5-6 region of the CB1 R or R* TMH
bundle using interactive computer graphics. The energy of the li-
gand/CB1 R or R* TMH bundle complex was minimized using the
AMBER* united atom force field in Macromodel 6.5 (Schro¨dinger
Inc.). A distance-dependent dielectric, 8.0 Å, extended nonbonded
cutoff (updated every 10 steps), 20.0-Å electrostatic cutoff, and 4.0-Å
hydrogen bond cutoff were used. The first stage of the calculation
consisted of 2000 steps of Polak-Ribier conjugate gradient (CG) min-
imization in which a force constant of 225 kJ/mol was used on the
helix backbone atoms to hold the TMH backbones fixed, while per-
mitting the side chains to relax. The second stage of the calculation
consisted of 100 steps of CG in which the force constant on the helix
backbone atoms was reduced to 50 kJ/mol to allow the helix back-
bones to adjust. Stages 1 and 2 were repeated with the number of CG
steps in stage two incremented from 100 to 500 steps until a gradient
of 0.001 kJ/(mol ⅐ Å2) was reached.
Assessment of Aromatic Stacking Interactions. Burley and
Petsko (1985) have reported that aromatic-aromatic (␲-␲) stacking
interactions in proteins operate at distances (d) of 4.5 to 7.0 Å
between ring centroids. The angle (␣) between normal vectors of
interacting aromatic rings typically is between 30° and 90°, produc-
ing a “tilted-T” or “edge-to-face” arrangement of interacting rings.
Hunter et al. (1991) have reported that ␲-␲ parallel stacking inter-
actions (␣ Ͻ 30°) between phenylalanine residues in proteins are
favorable if the rings are offset from each other. Residues and/or
ligand regions were designated herein as participating in an aro-
matic stacking interaction if they had centroid-to-centroid distances
between 4.5 and 7.0 Å. These interactions were further classified as
tilted-T arrangements if 30° Յ ␣ Յ 90° and as parallel arrangements
for ␣ Ͻ 30°. Parallel arrangements were considered favorable only if
the interacting rings were offset from each other (Hunter et al.,
1991). All measurements were made using Macromodel 6.5 (Schro¨-
dinger Inc.).
Expression and Mutagenesis of CB1 Cannabinoid Receptor
Gene. A 1.5-kilobase SstI/XbaI fragment of the human CB1 gene
containing the entire coding region was subcloned into expression
vector pRC/CMV (Invitrogen) to construct the expression plasmid
pHCB1-RC/CMV (Song and Bonner, 1996). A lysine-to-alanine mu-
tation at the position 192 of the CB1 cannabinoid receptor was made
by site-directed mutagenesis (Song and Bonner, 1996).
Cell Transfection and Culture. Expression plasmids contain-
ing wild-type and mutant cannabinoid receptors were purified with
QIAGEN plasmid kit (QIAGEN, Chatsworth, CA) and then trans-
fected into human embryonic kidney 293 cells using the calcium
phosphate precipitation method (Chen and Okayama, 1987). Trans-
fected cells were selected in culture medium containing 500 ␮g/ml
geneticin, and cell lines stably expressing wild-type and mutant
cannabinoid receptors were established according to a method estab-
lished previously (Chen and Okayama, 1987). Cells were grown as
monolayers in Dulbecco’s modified Eagle’s medium containing 10%
fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100
␮g/ml streptomycin in a humidified atmosphere consisting of 5% CO2
and 95% air at 37°C.
Ligand Binding Assay. For membrane preparations, cells were
washed twice with cold phosphate-buffered saline and scraped off the
tissue culture plates. Subsequently, cells were homogenized in bind-
ing buffer (50 mM Tris-HCl, 5 mM MgCl2, and 2.5 mM EDTA, pH
7.4) with a Tissumizer (Tekmar-Dohrmann, Mason, OH). The ho-
mogenate was centrifuged at 32,000g for 20 min at 4°C. The pellet
was resuspended in binding buffer and stored at Ϫ80°C. Protein
concentrations were determined by the use of a bicinchoninic acid
protein reagent kit (Pierce Chemical, Rockford, IL).
For binding assays, cannabinoid ligand dilutions were made in
binding buffer containing 25 mg/ml bovine serum albumin and then
added to assay tubes. [3H]SR141716A was used as a labeled ligand
for ligand binding assays. Binding assays were performed in 0.5 ml
of binding buffer containing 5 mg/ml bovine serum albumin for 60
min at 30°C. Free and bound radioligands were separated by rapid
filtration through polyethylenimine-treated GF/B filters (Whatman,
Synthesis
The conformationally constrained compound VCHSR (2), a vinyl- Maidstone, UK) that had been soaked in cold wash buffer (50 mM
cyclohexyl analog of SR141716, was synthesized by Wittig olefina- Tris-HCl, pH 7.4, and 1 mg/ml bovine serum albumin). The filters
tion. The route started with the reported pyrazole ester ethyl 1-(2,4-
were washed three times with 3 ml of cold wash buffer. The bound
dichlorophenyl)-5-(4-chlorophenyl)-4-methylpyrazole-3-carboxylate [3H]SR141716A was determined by liquid scintillation counting after
(Barth et al., 1995), an intermediate in the synthesis of SR141716, overnight equilibration of the filter in 5 ml of scintillation cocktail
which was reduced with lithium aluminum hydride to the alcohol (Hydrofluor; National Diagnostics, Manville, NJ). The assays were
[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol-3- performed in duplicate, and the results represent the combined data
yl]methanol. The latter was converted with CBr4 and triphenylphos- from three independent experiments.
phine to the benzylic bromide 3-(bromomethyl)-5-(4-chlorophenyl)-1-
(2,4-dichlorophenyl)-4-methyl-1H-pyrazole and then to the
phosphonium salt, 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-
Data Analysis. Data from ligand binding assays were analyzed,
and curves were generated with use of the Prism program (Graph-
Pad Software, San Diego, CA). IC50 values were determined through
methyl-1H-pyrazol-3-yl-methyl-triphenylphosphonium bromide nonlinear regression analysis performed with the Prism program. Kd
with triphenylphosphine. and Bmax values were estimated from competition binding experi-

1278
Hurst et al.
ments with the following equations: Kd ϭ IC50 Ϫ L and Bmax
ϭ
marker. After an overnight incubation, Ca2ϩ currents from injected
(B0IC50)/L, where L is the concentration of free radioligand and B0 is neurons were recorded at room temperature (24–26°C) with an Axo-
specifically bound radioligand (DeBlasi et al., 1989). The Ki values patch 200A patch-clamp amplifier (Axon Instruments, Union City,
were calculated based on the Cheng-Prusoff equation: Ki ϭ IC50/(1 ϩ CA). The cell membrane capacitance and series resistance were
L/Kd) (Cheng and Prusoff, 1973).
electronically compensated to Ͼ80%. Whole-cell currents were low
pass-filtered at 5 kHz using the Bessel filter of the clamp amplifier.
Voltage-clamp protocols were generated with a Power Macintosh
8600 computer (Apple Computer, Cupertino, CA) equipped with a
PCI-16 Host Interface card connected to an ITC-16 Data Acquisition
Interface (InstruTECH Corporation, Port Washington, NY) using
Pulse Control 5.0 XOPs (Richard J. Bookman, Jack D. Herrington,
and Kenneth R. Newton, University of Miami, Miami, FL) with Igor
software (WaveMetrics, Lake Oswego, OR). Ca2ϩ currents were elic-
ited by voltage steps from a holding potential of Ϫ80 mV and digi-
tized at 180 ␮s/point. A double pulse protocol consisting of two 25-ms
steps to ϩ5 mV was used to elicit Ca2ϩ currents. The first step to ϩ5
mV was followed by a 50-ms step to ϩ 80 mV to reverse G protein-
dependent inhibition of the Ca2ϩ current. Current amplitudes were
measured isochronally 10 ms after the voltage step to ϩ5mV and
current traces show the current elicited by the first voltage step to
ϩ5 mV.
Mutant Cycle Calculations
To assess whether a direct interaction occurs between K3.28(192)
and the C3 substituent of SR141716A, we performed a mutant cycle
analysis using Kd and Ki data for WT CB1/SR141716A, WT CB1/
VCHSR, K3.28(192)A/SR141716A, and K3.28(192)A/VCHSR dis-
placement of [3H]SR141716A. Mutant cycles have commonly been
used in the literature to analyze whether indirect or direct interac-
tions occur between certain amino acid residues in a protein (Faiman
and Horovitz, 1996) and also between an amino acid residue and a
ligand (Ambrosio et al., 2000). In the mutant cycle, two nonidentical
perturbations (WT CB13K3.28(192)A and SR141716A3VCHSR)
are applied to the system. As described by Ambrosio et al. (2000), the
effects on the binding energy produced by the two perturbations
should obey the principle of free energy conservation and allow us to
consider the thermodynamic cycle below:
Ca2ϩ currents were isolated with an external solution that con-
tained 140 mM tetraethylammonium methanesulfonate, 10 mM
HEPES, 15 mM glucose, 10 mM CaCl2, and 0.0001 mM tetrodotoxin,
pH 7.4 (adjusted with methanesulfonic acid). The intracellular solu-
tion contained 120 mM N-methyl-D-glucamine, 20 mM tetraethylam-
monium chloride, 10 mM HEPES, 11 mM EGTA, 1 mM CaCl2, 4 mM
MgATP, 0.1 mM Na2GTP, and 14 mM phosphocreatine, pH 7.2
(adjusted with methanesulfonic acid). Stock solutions of 10 nM
WIN55,212-2 mesylate (Sigma/RBI, Natick, MA) and 10 mM VCHSR
were prepared in dimethyl sulfoxide. On the day of the experiment,
stock solutions were diluted to 1 ␮M in external solution and briefly
sonicated to facilitate dispersion. WIN55,212-2 and VCHSR were
applied by a fast-switching device (SF-77B Perfusion Fast-Step;
Warner Instrument, Hamden, CT).
Herein, ⌬Gs indicate free energy changes measured in binding ex-
periments using the equation
⌬G ϭ ϪRTln(1/Kd)
(1)
and ⌬⌬Gs depict alchemical free energy changes that are computed
from the ⌬Gs. These ⌬⌬Gs were calculated by taking the difference
between final and initial states
⌬⌬G ϭ ⌬Gf Ϫ ⌬Gl
(2)
The overall free energy change for the transition from the unchanged
state (WT CB1/SR141716A) to the final state (CB1 K3.28(192)A/
VCHSR) should not be path-dependent (see Table 2 for a diagram of
the thermodynamic cycle discussed here).
Results
⌬⌬G(T) ϭ ⌬⌬G(1) ϩ ⌬⌬G(2/1) ϭ ⌬⌬G(2) ϩ ⌬⌬G(1/2)
(3)
Chemistry
In the thermodynamic cycle described above, each pair of opposite
paths indicates a change that is applied either before [ ⌬⌬G(1) or
⌬⌬G(2)] and when [⌬⌬G(1/2) or ⌬⌬G(2/1)] the second change is also
present. From eq. 3, it follows that the differences between these
paths should be equal; i.e.,
X-ray crystal structure analysis of SR141716A has re-
vealed that the carboxamide in the C3 substituent of
SR141716A is in a trans-geometry (C. George, personal com-
munication). VCHSR (2) was designed to mimic this carbox-
amide trans-geometry in the C3 substituent of SR141716A,
but to lack hydrogen bonding potential in this substituent
region. NMR spectroscopy was used to confirm the trans
geometry of 2. Compound 2 was further characterized by thin
layer chromatography, gas chromatography, high-pressure
liquid chromatography, and high-resolution mass spectrom-
etry. The high-resolution mass spectral data confirm the
empirical formula for 2 as C24H23N2Cl3: Calculated m/z ϭ
444.0927; observed high-resolution mass spectra, 444.0927.
Further details of the synthesis and characterization of 2 will
be published elsewhere.
⌬⌬G(1/2) Ϫ ⌬⌬G(1) ϭ ⌬⌬G(2/1) Ϫ ⌬⌬G(2)
(4)
If we let ␦G1,2 equal this constant difference, it follows from eqs. 3
and 4 that
⌬⌬G(T) ϭ ⌬⌬G(1) ϩ ⌬⌬G(2) ϩ ␦ G1,2
(5)
Herein, ␦G1,2 is the coupling free energy between the effects of the
two perturbations. When ␦G1,2 ϭ 0, ⌬⌬G(T) ϭ ⌬⌬G(1) ϩ ⌬⌬G(2), the
two effects on binding energy are totally additive and are therefore
acting independent of each other. When ␦G1,2
0, interaction be-
tween the two perturbations is implied, with the magnitude of the
␦G1,2 reflecting the extent to which the two effects are coupled.
Conformational Analysis Results
Ca2؉ Channel Assay
SR141716A Global Minimum Energy Conformer. The
global minimum energy conformer of SR141716A (Fig. 3, top,
left) has the carboxamide oxygen of the C3 substituent nearly
in plane with the pyrazole ring and pointing in the direction
of the C4 methyl group (O-C1Ј-C3-C4 ϭ 9.2°). The piperidine
ring is in a chair conformation with the nitrogen lone pair of
electrons pointing in the same direction as the carboxamide
hydrogen (LP-N3Ј-N2Ј-H ϭ 0.2°). The monochlorophenyl ring
Cannabinoid Receptor Expression and Electrophysiology.
Mammalian expression plasmids pCI (Promega), containing the hu-
man brain hCB1 cannabinoid receptor cDNA (from Dr. Tom I. Bon-
ner, Laboratory of Cell Biology, National Institute of Mental Health,
Bethesda, MD), were injected (100 ng/␮l) into nuclei of isolated rat
superior cervical ganglion (SCG) neurons as described previously
(Pan et al., 1998; Va´squez and Lewis, 1999). The pEGFP-N1plasmid
(10 ng/␮l) containing the coding sequence of enhanced green fluores-
cent protein (CLONTECH, Palo Alto, CA) was used as a coinjection is out of plane with the pyrazole ring (C4-C5-C1ٞ-C2ٞ ϭ

SR141716A Interaction with K3.28 Crucial for Inverse Agonism
1279
Ϫ46.2°), and the dichlorophenyl ring is also out of plane with enhanced negative potential region associated with the C3
the pyrazole ring (N2-N1-C1Љ-C2Љ ϭ Ϫ63.1°). In this position, substituent. Although AM1 calculations showed that this
the ortho-chloro is in the bottom face of the molecule (i.e., conformer was 4.83 kcal/mol higher in energy than the global
below the plane of the paper).
minimum, ab initio Hartree Fock 6-31G* calculations indi-
VCHSR Global Minimum Energy Conformer. Fig. 3, cated that the energy separation between these two conform-
top, right, illustrates the global minimum energy conformer ers was only 0.92 kcal/mol. For docking studies, we chose the
of VCHSR. This conformer of VCHSR differs from that of global minimum energy conformer of VCHSR and the mini-
SR141716A only in the orientation of its cyclohexyl ring mum energy conformer of SR141716A that matches this con-
compared with that of the piperidine ring in SR141716A. In formation of VCHSR. Figure 3 (bottom) illustrates the super-
the global minimum of VCHSR, the trans-ethylene group is position of these two conformers at their pyrazole rings. This
oriented such that the hydrogen attached to C1Ј is nearly in conformer of SR141716A was chosen because it produces the
the plane of the pyrazole ring, pointing toward the C4 methyl highest negative electrostatic potential in the C3 substituent
group (H-C1Ј-C3-C4 ϭ Ϫ0.4°) and the hydrogen attached to region, potentially enabling the C3 substituent to form the
C3Ј (cyclohexyl ring) points in the opposite direction from the strongest hydrogen bond possible with a hydrogen bond do-
C2Ј hydrogen (H-C3Ј-C2Ј-H ϭ Ϫ179.7°).
nor.
Conformer Selection for Docking. A molecular electro-
static potential (MEP) map calculated at the AM1 level (data
not shown) indicated that the piperidine nitrogen of
Receptor Docking Studies
One of the significant features of the CB1 R TMH bundle is
SR141716A generates an MEP minimum (i.e., negative po- a salt bridge between K3.28(192) and D6.58(366) (N-O dis-
tential region), second only to that generated by the carbox- tance ϭ 2.6 Å; N-H-O angle ϭ 159°). Unlike the intracellular
amide oxygen in SR141716A. AM1 conformational searches R3.50/E6.30 (or R3.50/D6.30) salt bridge shown to stabilize G
identified another minimum energy conformation of protein-coupled receptors (GPCRs) in their inactive states
SR141716A in which the piperidine nitrogen’s lone pair (Ballesteros et al., 2001), the extracellular K3.28/D6.58 salt
points in the same direction as the carboxamide oxygen (LP- bridge in CB1 (present only in the inactive state of CB1)
N3Ј-N2Ј-H ϭ 178.5°). An MEP of this conformer showed an seems to be important for positioning K3.28 for ligand inter-
action in the inactive state, rather than for stabilizing the
receptor in the inactive state. In fact, Pan et al. (1998) found
that WT CB1 and the CB1 K3.28(192)A mutant exhibit the
same level of constitutive activity. So, the absence of the
K3.28/D6.58 salt bridge does not lead to greater ease of
activation.
The K3.28/D6.58 salt bridge is made possible by two spe-
cial structural features of the CB1 receptor, its EC-2 loop and
the flexibility of TMH 6 in CB1. Despite the fact that the CB1
and CB2 receptors belong to the Rho family of GPCRs, there
are important differences between CB1/CB2 and Rho that
impact the ligand binding pocket in the TMH 3-4-5-6 region.
The CB1 and CB2 extracellular loop 2 (E-2 loops) are shorter
than that of Rho (CB1 15 residues in length; CB2 13 residues
in length; Rho 25 residues in length) and there is no corre-
sponding Cys residue in TMH 3 of CB1 or CB2 that would
cause the E-2 loop to dip down into the binding site crevice as
the E-2 disulfide bridge with Cys3.25(110) causes in Rho.
However, there is a Cys residue at the extracellular end of
TMH 4 and a Cys near the middle of the E-2 loop in the CB
receptors. Mutation results of these residues (C174 and
C179) in CB2 suggest that a disulfide bridge between these
two Cys residues may exist, but protein expression problems
have hampered attempts to prove the existence of this bridge
in CB1 (Shire et al., 1999). As the result of this important
difference between Rho and the CB receptors, the binding
site crevice around TMHs 3-4-5-6 is likely to be different with
the E-2 loop occupying less volume in the upper part of the
binding pocket than does the E-2 loop in Rho.
The different spatial requirements of the E-2 loop are
important because this difference permits the extracellular
end of TMH 6 to occupy a different position in the TMH
Fig. 3. Global minimum energy conformer of SR141716A (1) (top, left)
bundle than is seen in Rho. We have recently shown that the
small size of residue 6.49 in CB1 (a Gly) results in pro-
nounced flexibility of the CWXP motif in TMH 6 (Barnett-
Norris et al., 2002a). This motif has been suggested to func-
and global minimum energy conformer of VCHSR (2) (top, right) as
determined by the AM1 semiempirical method. Bottom, superposition at
their pyrazole rings of the global min of 2 (in yellow) and a higher
minimum energy conformation of 1 (⌬E ϭ 0.92 kcal/mol; ab initio Hartree
Fock 6-31G*; in pink). These are the conformations used for docking
studies (Figs. 4 and 5).
tion as
a flexible hinge, permitting agonist-promoted

1280
Hurst et al.
movement of the intracellular end of TMH 6 that occurs W5.43(279)/Y5.39(275)/W4.64(255)/F5.42(278). We found that
during activation (Jensen et al., 2001). In addition to permit- each ligand could insert itself into this aromatic residue-rich region
ting the intracellular end of TMH 6 to come close to the to become an integral part of an extended aromatic cluster.
intracellular end of TMH 3 in the inactive state of CB1, this
SR141716A/CB1 Complexes. SR was docked in the TMH
flexibility in CB1 TMH 6 permits the extracellular end of 3-4-5-6 region in a model of both the R and R* states of CB1.
TMH 6 to bend toward TMH 3, resulting in the formation of Figures 4A and 5A illustrate SR141716A docking results.
a salt bridge between D6.58 (near the extracellular end of
SR141716A/CB1 R Complex. In the inactive CB1 bundle,
TMH 6) and K3.28 in TMH 3. In the R* TMH bundle, the the carboxamide oxygen of SR141716A forms a hydrogen
K3.28(192) and D6.58(366) salt bridge is broken (N-O dis- bond with K3.28(192), which is part of the salt bridge with
tance ϭ 16.8 Å).
D6.58(366) (Fig. 4A). In this interaction, the nitrogen of
Both the CB1 inverse agonist SR141716A (SR) and its analog K3.28(192) is central and provides a hydrogen to an oxygen of
VCHSR are highly aromatic compounds. We hypothesized that D6.58(366) (N-O distance ϭ 2.6 Å; N-H-O angle ϭ 159°) and
aromatic stacking interactions might be important for the binding to the carboxamide oxygen of SR141716A (N-O distance ϭ
of these compounds at CB1. The CB1 TMH 3-4-5-6 region is rich in 2.7 Å; O-H-N angle ϭ 163°). The geometry of this interaction
aromatic residues that face into the ligand binding pocket, includ- mimics that of complex salt bridges in proteins. In their
ing F3.25(189), F3.36(200), W4.64(255), Y5.39(275), W5.43(279), statistical analysis of salt bridges in proteins, Musafia et al.
and W6.48(356). Shire et al. (1999) have shown in CB1/CB2 chi- (1995) documented that in 35.6% of the protein complex salt
mera studies that the TMH-4-E-2-TMH5 region of CB1 contains bridges analyzed that had the lysine nitrogen as the connect-
residues critical for the binding of SR141716A. In Monte Carlo/ ing position (central portion) of the salt bridge, a lysine N-H
Stochastic Dynamics Simulations of the inactive state of WT CB1, had an interaction with a single oxygen from each of two
McAllister et al. (2002) found a persistent aromatic stack between separate acidic residues (Fig. 3K in Musafia et al., 1995).
Y5.39(275) and W4.64(255) that seemed to be important for stabi- Figure 4A illustrates the geometry of this D6.58/K3.28/
lizing the positions of TMHs 4 and 5 in the TMH bundle on the SR141716A interaction in the R state.
extracellular side and
a
second aromatic stack between
Using the criteria described under Materials and Methods,
F3.36(200), W5.43(279), and W6.48(356) that seemed to be open we analyzed each minimized ligand/CB1 complex for the
for additional interaction with ligand. We therefore pursued the presence of aromatic stacking interactions. As described be-
aromatic residue-rich TMH 3-4-5-6 region as the binding site for low, W5.43(279) and F3.36(200) formed the closest aromatic
SR and VCHSR. The R bundle in the TMH 3-4-5-6 region is stacking interactions with SR141716A in CB1. In the
characterized by a W6.48(356)/W5.43(279)/F3.36(200) and a SR141716A/CB1 R complex, both the monochlorophenyl
Y5.39(275)/W4.64(255)/F5.42(278) aromatic cluster. In the R* (MC) and dichlorophenyl (DC) rings of SR were found to be
TMH bundle, an aromatic cluster exists between W6.48(356)/ involved in aromatic stacking interactions with W5.43(279)
Fig. 4. SR141716A in a minimum energy conformation (⌬E ϭ 0.92 kcal/mol) (A) and VCHSR in its global minimum energy conformation (B), each
docked in the computer TMH bundle model of the R state of CB1. The R state is characterized by a salt bridge between K3.28(192) and D6.58(366)
and two patches of aromatic residues that form clusters in the TMH 3-4-5-6 region of CB1, W5.43(279)/F3.36(200)/W6.48(356), and Y5.39(275)/
W4.64(255)/F5.42(278). Residues with which ligands have direct interactions are yellow. Residues that form part of the aromatic cluster complex with
ligand but that do not stack directly with ligand are green. Residues that have no direct or indirect interaction with ligand are cyan. In A, SR141716A
has a hydrogen bonding interaction with K3.28(192) in the salt bridge. Because SR141716A directly stacks with both F3.36(200) and W5.43(279) and
with Y5.39(275), the ligand joins the F3.36(200)/W5.43(279)/W6.48(356) and Y5.39(275)/W4.64(255)/F5.42(278) aromatic clusters into one large
extended cluster in the minimized complex. In B, VCHSR has aromatic stacking interactions with W5.43(279), F3.36(200) and Y5.39(275). In binding,
VCHSR bridges between the F3.36(200)/W5.43(279)/W6.48(356) and Y5.39(275)/W4.64(255)/F5.42(278) aromatic clusters and helps form one large
extended cluster in the minimized complex. However, VCHSR has no interaction with K3.28(192) due the lack of hydrogen bonding potential in the
C3 substituent region of VCHSR.

SR141716A Interaction with K3.28 Crucial for Inverse Agonism
1281
(MC d ϭ 4.8 Å, ␣ ϭ 50°; DC d ϭ 4.8 Å, ␣ ϭ 90°). In addition, in R*, breaking their salt bridge (O-N distance ϭ 16.8 Å). In R*,
the dichlorophenyl ring was found to have a stacking inter- K3.28 is no longer accessible in the TMH 3-5-6 region, but has
action with F3.36(200) (d ϭ 5.0 Å, ␣ ϭ 80°) and the mono- shifted toward the TMH 2-3-7 region, having rotated away from
chlorophenyl ring, an interaction with Y5.39(275) (MC d ϭ the SR141716A binding pocket in the R* state. D6.58(366) has
6.5 Å, ␣ ϭ 25°). Aromatic residues in the TMH 3-4-5-6 region rotated toward the TMH 5-6 interface and is raised higher
form two networks or clusters that are bridged by above the ligand binding pocket with the moderation of the
SR141716A. In one cluster, F3.36(200) directly stacks with TMH 6 proline kink angle. The carboxamide oxygen of
W6.48(356) (d ϭ 4.9 Å, ␣ ϭ 50°) and with W5.43(279) (d ϭ 5.9 SR141716A is now 11.4 Å from the nitrogen of the K3.28(192)
Å, ␣ ϭ 60°). In a second cluster, Y5.39(275) directly stacks side chain. As the result of these changes, K3.28(192) is no
with W4.64(255) (d ϭ 6.5 Å, ␣ ϭ 80°), which in turn stacks longer available for interaction with the C3 substituent of
with F5.42(278) (d ϭ 4.6 Å, ␣ ϭ 30°). Because SR141716A SR141716A. The F3.36(200)/W5.43(279)/W6.48(356) aromatic
directly stacks with both F3.36(200) and W5.43(279) and cluster also undergoes rearrangement, with F3.36(200) and
with Y5.39(275), the ligand joins the F3.36(200)/W5.43(279)/ W6.48(356) rotating away from each other in R*.
W6.48(356) and Y5.39(275)/W4.64(255)/F5.42(278) aromatic
clusters into one large extended cluster in the minimized interact with both phenyl rings (MC d ϭ 4.8 Å, ␣ ϭ 70°; DC
complex. d ϭ 4.9 Å, ␣ ϭ 80°) by tilted-T interactions that are charac-
In the ligand/receptor complex, W5.43(279) is positioned to
Importance of the F3.36/W6.48 Interaction to the In- terized by centroid-to-centroid distances at the low end of the
active State. In the minimized complex, SR141716A stabi- 4.5 to 7.0 Å range defined for a tilted-T aromatic stacking
lizes the inactive (bent) conformation of TMH 6 by its inter- interaction (see Assessment of Aromatic Stacking Interac-
action with F3.36/W6.48. Residues W6.48 [␹ ϭ gϩ (Ϫ60°)] tions under Materials and Methods; Hunter et al., 1991). The
1
and F3.36 [␹ ϭ trans (180°)] in the inactive bundle are monochlorophenyl ring also has an offset parallel stack with
1
engaged in a direct stacking interaction. Rotations of TMH 3 Y5.39(275) (MC d ϭ 5.7 Å, ␣ ϭ 0°). Aromatic residues in the
and 6 concomitant with activation move F3.36 and W6.48 TMH 3-4-5-6 region form a network or cluster with which
away from each other. We have recently proposed that in SR141716A interacts. W5.43(279) directly stacks with
CB1, ␹ of F3.36 must change from trans to gϩ during acti- Y5.39(275) (d ϭ 6.0 Å, ␣ ϭ 40°); Y5.39(275) stacks both with
1
vation (Barnett-Norris et al., 2002b). Spectroscopic studies W4.64(175) (d ϭ 5.5 Å, ␣ ϭ 80°) and F5.42 (d ϭ 5.7 Å, ␣ ϭ
(Lin and Sakmar, 1996) have indicated that W6.48 under- 30°); and W4.64(175) stacks with F5.42(275) (d ϭ 5.7 Å, ␣ ϭ
goes a conformational change when Rho is activated. Visiers 80°). In binding, SR141716A becomes part of an aromatic
et al. (2002) have proposed that a ␹ change in W6.48 from cluster that includes W5.43(279)/Y5.39(275)/W4.64(175)/
1
gϩ (Ϫ60°) to trans (180°) is part of the activation mechanism F5.42(278) in the minimized complex.
for the 5-hydroxytryptamine-2A receptor. In CM studies of
In evaluating the preference that SR141716A shows for R
5-hydroxytryptamine-2A TMH 6, these investigators found a versus R*, the overall extent of aromatic stacking (both direct
correlation between the proline kink angle of TMH 6 and the and indirect) created by ligand binding was assessed in each
W6.48 ␹ torsion angle. More kinked (inactive state) TMH 6 state. Results depicted herein in Figs. 4A and 5A show that
1
conformers were found to have a gϩ (Ϫ60°) ␹ torsion angle although the extent of aromatic stacking is similar in both
1
value for W6.48. Less kinked (active state) TMH 6 conform- the R and R* states, SR141716A will have a preference for
ers were found to have a trans (180°) ␹ torsion angle value the R state, because only in this state is a hydrogen bonding
1
for W6.48. In agreement with the results reported by Balles- interaction possible. Consequently, based on modeling re-
teros et al. (1998), CM studies of CB1 TMH 6 revealed that sults, SR141716A should compete with agonist, because it
the kink angle of TMH 6 clusters and the ␹ of W6.48 were has affinity for the R* state and behave as an inverse agonist,
1
highly correlated in this same way (unpublished observa- because it has higher affinity for the R state.
tions). Therefore, in our models, both W6.48 and F3.36 un-
VCHSR/CB1 Complexes. VCHSR was docked in the
dergo a change in their ␹ values from R to R*. ␹ in W6.48 TMH 3-4-5-6 region in a model of both the R and R* states of
1
1
changes from gϩ (Ϫ60°) to trans (180°) and ␹ of F3.36 CB1. Figures 4B and 5B illustrate VCHSR docking results.
1
changes from trans to gϩ. Because SR141716A in R stacks
VCHSR/CB1 R Complex. The interactions in which
directly with F3.36, which in turn, has a direct stacking VCHSR engages are very similar to those described above for
interaction with W6.48 in the CB1 inactive state, the binding SR141716A with one important exception (Fig. 4B). Because
of SR141716A to the R state of CB1 stabilizes this F3.36 (␹
the C3 substituent in VCHSR lacks hydrogen bonding poten-
1
ϭ trans)/W6.48 (␹1 ϭ gϩ) aromatic stacking interaction, pre- tial, it cannot form a hydrogen bonding interaction with
venting any changes in the ␹1 values of F3.36 and W6.48 and K3.28(192), despite its proximity to the K3.28(192)-
therefore stabilizing the bent (inactive state) conformation of D6.58(366) salt bridge. Similar to interactions found for the
TMH 6.
CB1/SR141716A complex, W5.43(279) and F3.36(200)
SR141716A/CB1 R* Complex. The conformational changes formed the closest aromatic stacking interactions with
that occur upon receptor activation result in rotations of TMHs VCHSR in the CB1 model. In the VCHSR/CB1 R complex,
3 and 6 as well as a change in the conformation of TMH 6 (by both the MC and DC rings of VCHSR were found to be
moderation of its proline kink angle) (Fig. 5A). As a result, the involved in aromatic stacking interactions with W5.43(279)
position and accessibility of residues in the TMH 3-4-5-6 region (MC d ϭ 4.7 Å, ␣ ϭ 50°; DC d ϭ 4.9 Å, ␣ ϭ 80°). In addition,
to SR141716A are altered. Figures 4 and 5 illustrate the differ- the monochloro ring was found to have a stacking interaction
ence between the TMH bundle conformation in R versus R*. It with Y5.39(275) (MC d ϭ 6.7 Å, ␣ ϭ 25°), and the dichloro-
is clear herein that activation has caused significant changes in phenyl ring was found to have a stacking interaction with
the relative position of certain TMH 3 and TMH 6 residues, F3.36(200) (d ϭ 5.0 Å, ␣ ϭ 80°). Aromatic residues in the
with K3.28(192) and D6.58(366) rotating away from each other TMH 3-4-5-6 region form a network with which VCHSR

1282
Hurst et al.
interacts. F3.36(200) directly stacks with W6.48(356) (d ϭ 4.8
In evaluating the preference that VCHSR will show for R
Å, ␣ ϭ 50°) as well as with W5.43(279) (d ϭ 5.8 Å, ␣ ϭ 60°), versus R*, the overall extent of aromatic stacking (both direct
and Y5.39(275) directly stacks with W4.64(255) (d ϭ 6.4 Å, and indirect) created by ligand binding was assessed in each
␣ ϭ 80°), which stacks with F5.42(278) (d ϭ 4.7 Å, ␣ ϭ 40°). In state. Results depicted here in Figs. 4B and 5B show that the
binding, VCHSR bridges between the F3.36(200)/W5.43(279)/ extent of aromatic stacking is similar in both the R and R*
W6.48(356) and Y5.39(275)/W4.64(255)/F5.42(278) aromatic states. As a result, VCHSR should have nearly equal affinity
clusters and helps form one large extended cluster in the for both the R and R* states, causing it to behave as a neutral
minimized complex.
antagonist.
VCHSR/CB1 R* Complex. As illustrated in Fig. 5B, in
CB1 R*, W5.43(279) is positioned to form an aromatic cluster
by inserting itself between the monochloro- and the dichlo-
Ligand Binding in hCB1 WT and K3.28(192)A
Scatchard analysis and ligand binding results for WT CB1
rophenyl ring of VCHSR. In so doing, W5.43(279) forms tilt- and the K3.28(192)A mutant cell lines are presented in Table
ed-T interactions with VCHSR that are characterized by 1. To test whether K3.28(192) is an interaction site for
centroid-to-centroid distances at the low end of the 4.5 to 7.0 SR141716A, we evaluated the binding of [3H]SR141716A in
Å range defined for a tilted-T aromatic stacking interaction CB1 WT and K3.28(192)A mutant cell lines (Fig. 6A). Results
(see Assessment of Aromatic Stacking Interactions under Ma- in Table 1 demonstrate the importance of K3.28(192) for
terials and Methods; Hunter et al., 1991) (MC d ϭ 4.9 Å, ␣ ϭ SR141716A binding at CB1. In WT CB1, the Kd value for
60°; DC d ϭ 4.7 Å, ␣ ϭ 80°). In addition, the dichloro ring of SR141716A was 2.3 Ϯ 1.1 nM, whereas in CB1 K3.28(192)A,
VCHSR has an off-set parallel aromatic stacking interaction the Kd value for SR141716A was 39.6 Ϯ 10.5 nM.
with W6.48(356) (DC d ϭ 6.5 Å, ␣ ϭ 30°). Aromatic residues
As a further test of our hypothesis that K3.28(192) is an
in the TMH 3-4-5-6 region form a network with which interaction site for SR141716A, VCHSR was used in compe-
VCHSR interacts. W5.43(279) has a stacking interaction tition binding assays (Fig. 6B). The Ki value for VCHSR
with Y5.39(275) (d ϭ 6.2 Å, ␣ ϭ 30°); Y5.39(275) has a binding in cloned human WT CB1 and CB1 K3.28(192)A cell
stacking interaction with both F5.42(278) (d ϭ 5.2 Å, ␣ ϭ 40°) lines versus [3H]SR141716A was 31.3 Ϯ 9.6 and 35.2 Ϯ 1.4
and W4.64(255) (d ϭ 5.6 Å, ␣ ϭ 70°); and W4.64(255) stacks nM, respectively. Figure 6C presents a comparison of Kd or Ki
with F5.42(278) (d ϭ 5.3 Å, ␣ ϭ 80°). In binding, VCHSR values for [3H]SR141716A binding. The bar graphs represent
becomes part of an F5.42(278)/W4.64(255)/Y5.39(275)/ the mean Ϯ S.E. of three independent experiments per-
W5.43(279)/W6.48(356) extended aromatic cluster in the formed in duplicate. It is clear herein that the Ki values for
minimized complex.
VCHSR binding to WT CB1 and to CB1 K3.28(192)A are
Fig. 5. SR141716A in a minimum energy conformation (⌬E ϭ 0.92 kcal/mol) (A) and VCHSR in its global minimum energy conformation (B), each
docked in the computer TMH bundle model of the R* state of CB1. In the R* state, the K3.28(192)-D6.58(366) salt bridge is broken due to rotations
of TMHs 3 and 6 and a conformational change in TMH 6. The aromatic cluster in this region has rearranged with F5.42(278)/W4.64(255)(175)/
Y5.39(275)/W5.43(279)/W6.48(356), forming a cluster. Residues with which ligands have direct interactions are yellow. Residues that form part of the
aromatic cluster complex with ligand but that do not stack directly with ligand are green. Residues that have no direct or indirect interaction with
ligand are cyan. In A, SR141716A interacts directly with W5.43(279) and Y5.39(275). In binding, SR141716A becomes part of an aromatic cluster that
includes W5.43(279)/Y5.39(275)/W4.64(175)/F5.42(278) in the minimized complex. In B, VCHSR interacts directly with W5.43(279) and W6.48(356).
In binding, VCHSR becomes part of an F5.42(278)/W4.64(255)/Y5.39(275)/W5.43(279)/W6.48(356) extended aromatic cluster in the minimized
complex.

SR141716A Interaction with K3.28 Crucial for Inverse Agonism
1283
comparable and that these Ki values, in turn, are comparable demonstration that this residue is also important for inverse
with the Kd value of SR146716A for CB1 K3.28(192)A, but agonist binding at CB1.
not to the Kd value for SR141716A binding to WT CB1.
Mutant Cycle
These results suggest that K3.28(192) is important to the
binding of SR141716A at WT CB1, but this residue is not
To evaluate whether a direct interaction takes place
important to the binding of VCHSR at WT CB1. Residue between the C3 substituent of SR141716A and K3.28(192)
K3.28(192) has previously been shown to be a very important in WT CB1, a mutant cycle was constructed herein. An
residue for agonist binding at CB1 (Song and Bonner, 1996; analog of SR141716A, VCHSR (2; Fig. 2), was designed and
Chin et al., 1998). The results reported herein are the first synthesized to be used in this study. As illustrated in Fig.
TABLE 1
Ligand binding profile and free energy data for WT and K3.28A CB1
Saturation experiments using [3H]SR141716A were performed on stably transfected human embryonic kidney 293 cells to evaluate binding affinities and relative levels of
receptor expression in the WT and mutant receptors. Inhibition constants were obtained from competition experiments using stably transfected human embryonic kidney293
cells. Data are the means Ϯ S.E. of three independent experiments each performed in duplicate. ⌬G values were calculated at 303°K using eq. 1. Statistics on free energy
data were computed using propagation of error formula (Mortimer, 1981).
SR141716A
VCHSR
Bmax
Kd
⌬G
Ki
⌬G
pmol/mg
nM
kJ/mol
nM
kJ/mol
WT CB1
K3.28A
1.45 Ϯ 0.41
1.61 Ϯ 0.31
2.3 Ϯ 1.1
39.6 Ϯ 10.5*
Ϫ50.11 Ϯ 1.20
Ϫ42.94 Ϯ 0.67
31.3 Ϯ 9.6
35.2 Ϯ 1.4
Ϫ43.53 Ϯ 0.77
Ϫ43.24 Ϯ 0.10
*, statistically significant difference from WT (p Ͻ 0.05).
Fig. 6. Competition of [3H]SR141716A binding in wild-type CB1 and CB1 K3.28(192)A receptors. Ligand binding assays were performed on
membranes prepared from human embryonic kidney 293 cells stably expressing wild-type CB1 or CB1 K3.28(192)A receptor. The competition by
SR141716A and VCHSR are shown in A and B, respectively. Points represent mean Ϯ S.E. of three independent experiments performed in duplicate.
Curves were generated as described under Materials and Methods. The comparison of Kd or Ki values for [3H]SR141716A binding is shown in C.
Graphs represent mean Ϯ S.E. of Ki or Kd values obtained from three independent experiments performed in duplicate.

1284
Hurst et al.
3, VCHSR (2) can mimic a minimum energy conformation the determination of whether deletions have occurred be-
of SR141716A, but unlike SR141716A, VCHSR has no tween two groups that interact indirectly or directly is the
hydrogen bonding capability in its C3 substituent region. effect produced by simultaneous deletion of both groups [i.e.,
Table 1 summarizes the values of ⌬G calculated for each K3.28(192)A/VCHSR]. If the modified groups do not interact
state in the thermodynamic cycle using eq. 1. Table 2 directly with each other in the WT state then the effect of the
summarizes the results obtained for the mutant cycle us- two simultaneous changes will be additive [i.e., ⌬⌬G(1) Ϸ
ing eqs. 2 to 5.
⌬⌬G(1/2), ⌬⌬G(2) Ϸ ⌬⌬G(2/1) and ␦G1,2 Ϸ 0]. This will be
In the mutant cycle, a set of complementary chemical reflected in a higher Ki value for the double deletion. If, on
groups was deleted from both ligand (SR141716A 3 VCHSR) the other hand, the two groups interact directly, then the
and receptor (WT CB1 3 K3.28(192)A). The resultant losses effect of the two simultaneous changes will be nonadditive
in binding energy for each of these deletions [⌬⌬G(1) for [i.e., ⌬⌬G(1/2) Ϸ ⌬⌬G(2/1) Ϸ 0 and ⌬⌬G(1) Ϸ ⌬⌬G(2) Ϸ
SR/WT CB1 3 SR/K3.28A and ⌬⌬G(2) for SR/WT CB1 3 Ϫ␦G1,2], and the Ki value for the double deletion will be
VCHSR/WT CB1, respectively] were found not to be statisti- comparable in value with the Ki values for each single dele-
cally different from each other (p ϭ 0.77, Student’s paired t tion. Ambrosio et al. (2000) suggest that such “clear-cut”
test; Table 2). However, even if the two deletions produce results may not be encountered frequently in proteins such
similar or identical losses in binding energy, this result is as receptors, in which binding and conformational change are
insufficient evidence to conclude that the two groups interact inextricably linked. However, these authors conclude that, in
directly with each other. The ligand may interact in one general, if the magnitude of the free energy of coupling
receptor region and the receptor residue, although remote (␦G1,2) is comparable (even if not necessarily identical) with
from the ligand, may be engaged in a network of interactions the magnitude of ⌬⌬G(1) or ⌬⌬G(2), it can be concluded that
that are crucial to the binding process. The decrease in bind- a direct interaction occurs. It is clear from Tables 1 and 2 that
ing energy due to deletion of ligand functionality may result such a direct interaction is the case herein, with the effect of
from a loss in binding energy, whereas the effects on the free the double change clearly being nonadditive (␦G1,2 0) and
energy due to receptor residue substitution may come from the effect of each single change being comparable [(⌬⌬G(1) ϭ
conformational contributions. These losses may have similar 7.17 Ϯ 1.37 kJ/mol) Ϸ (⌬⌬G(2) ϭ 6.58 Ϯ 1.43 kJ/mol; these
magnitudes, even if the deleted groups do not directly inter- values were shown not to be statistically different, p ϭ 0.77,
act with each other (Ambrosio et al., 2000). Therefore, key to using Student’s paired t test]. This result suggests, therefore,
that there is a direct interaction between the C3 substituent
of SR141716A and K3.28(192) in WT CB1. In addition, the
magnitude of the free energy of coupling (␦G1,2 ϭ Ϫ6.88 Ϯ
2.55 kJ/mol) is comparable with the free-energy change as-
sociated with a hydrogen bonding interaction.
TABLE 2
Thermodynamic cycle for removal of hydrogen bond potential at
residue 3.28 from CB1 receptor
Calcium Current Effects of SR141716A and VCHSR in
SCG Neurons
Figure 7 illustrates that in SCG neurons injected with
hCB1 receptor cRNA, VCHSR attenuated the inhibition of
the Ca2ϩ current by the cannabinoid agonist WIN55,212-2.
Figure 7A shows the Ca2ϩ current amplitude recorded over
time during a patch-clamp experiment from an SCG neuron
expressing the human CB1 cannabinoid receptor. Applica-
tion of 1 ␮M WIN (shaded column) rapidly inhibited the Ca2ϩ
current. The current slowly recovered during washout of
WIN55,212-2. Application of 1 ␮M VCHSR slightly increased
the Ca2ϩ current and attenuated the effect of WIN55,212-2.
Figure 7B shows superimposed Ca2ϩ current traces for the
same neuron shown in Fig. 7A in the absence (control) and
presence of WIN, VCHSR, and VCHSR ϩ WIN. Figure 7C
shows a graph of the percentage of change in Ca2ϩ current
amplitude in the presence of WIN, VCHSR, and VCHSR ϩ
WIN. VCHSR significantly attenuated the effect of
WIN55,212-2 (p Ͻ 0.05). The number of neurons tested is
indicated in each bar of Fig. 7C.
Free energy changes were computed as described under Materials and Methods
using the data summarized in Table 1. Standard errors were calculated using
propagation of error formula (Mortimer, 1981). Mutation free energy changes were
computed as ⌬⌬G
⌬GVCHSR/K3.28A
ϭ
⌬Gmut
Ϫ
⌬GWT
.
The fact that ⌬⌬G(1/2) (⌬GVCHSR/WT
3
)
Ϸ
⌬⌬G(2/1) (⌬GSR/K3.28A
3
⌬GVCHSR/K3.28A and
)
Ϸ
0
⌬⌬G(1)(⌬GSR/WT 3 ⌬GSR/K3.28A) Ϸ ⌬⌬G(2) (⌬GSR/WT 3 ⌬GVCHSR/WT) Ϸ Ϫ␦G1,2
suggests a strong direct interaction between and, therefore, lack of additivity of the
two deletions.
These results show that VCHSR behaved as a neutral
antagonist because 1 ␮M VCHSR significantly (n ϭ 6, p Ͻ
0.05) attenuated the WIN-induced inhibition of Ca2ϩ current,
but alone VCHSR produced a change in the Ca2ϩ current
that was not significantly different (p ϭ 0.259) from the
control current amplitude. Experiments performed with 10
␮M VCHSR (data not shown) were consistent with results
pictured in Fig. 7, indicating that VCHSR behaves as a
Mean
Free Energy
S.E.
kJ/mol
⌬⌬G(1/2)
⌬⌬G(2/1)
⌬⌬G(1)
⌬⌬G(2)
␦G1,2
0.29
Ϫ0.30
7.17*
6.58*
Ϫ6.88
0.78
0.68
1.37
1.43
2.55
*, values that were shown not to be statistically different from each other using a
Student’s paired t test (P ϭ 0.77).

SR141716A Interaction with K3.28 Crucial for Inverse Agonism
1285
neutral antagonist in SCG neurons expressing the human signaling pathway. The binding of a neutral/null antagonist
CB1 receptor.
is thought not to alter the equilibrium between R and R*
because the neutral antagonist has equal affinity for both
states.
Discussion
The central hypothesis tested in this article is that the
interaction between K3.28(192) and SR141716A in WT CB1,
an interaction that is possible only in the R state, causes
SR141716A to have higher affinity for the R state and con-
sequently behave as an inverse agonist. Pan et al. (1998)
reported that SR141716A antagonized the Ca2ϩ current in-
hibition induced by the cannabinoid agonist WIN55212-2 in
neurons heterologously expressing either rat or human CB1
receptors (Fig. 1). Furthermore, when applied alone,
SR141716A increased the Ca2ϩ current, indicating that
SR141716A can act as an inverse agonist by reversal of tonic
CB1 receptor activity.
The extended ternary complex model for GPCR activation
invokes the existence of two receptor conformational states, a
ground or inactive R state and an active R* state, which are
in equilibrium with each other (Leff, 1995). An agonist has
higher affinity for R* and agonist binding is thought to shift
the equilibrium toward R*, resulting in G protein activation
with an increase in GDP/GTP exchange. An inverse agonist
has higher affinity for R and its binding shifts the equilib-
rium toward R, resulting in a decrease in the activation of the
These investigators reported that the Ca2ϩ current was
also tonically inhibited in neurons expressing the mutant
CB1 K3.28(192)A receptor. Surprisingly, SR141716A had no
effect on the Ca2ϩ current in these neurons, but SR141716A
could still antagonize the effect of WIN55212-2 (Fig. 1). There
are two possible explanations for these results.
First, it is possible that the K3.28(192)A mutation results
in a receptor population 100% in the inactive state (R 4 R*).
In this case, SR141716A would have no effect when applied
alone because there is no R* population to convert back into
the inactive state. However, Pan et al. (1998) found that the
facilitation ratios for neurons expressing WT CB1 (1.41 Ϯ
0.09, n ϭ 4) and neurons expressing mutant K3.28(192)A
receptors (1.42 Ϯ 0.06, n ϭ 8) were both significantly differ-
ent (p Ͻ 0.001) from the Ca2ϩ current facilitation ratio in
uninjected neurons (1.17 Ϯ 0.01, n ϭ 11). The finding that
the K3.28(192)A mutant has the same facilitation ratio as
WT CB1 receptor suggests that both the mutant and WT CB1
can exist in a spontaneously active, G protein-coupled R*
state.
The second possible explanation (and the one offered by
Pan et al., 1998) is that the K3.28(192) site is critical for the
inverse agonist activity of SR141716A, and therefore
SR141716A becomes a neutral antagonist at the K3.28(192)A
mutant receptor. The modeling, mutation and Ca2ϩ current
assay results reported herein are clearly consistent with this
latter interpretation of the results depicted in Fig. 1 for WT
CB1 versus the K3.28(192)A mutant.
Central to the hypothesis tested herein is the determina-
tion of the interactions available to SR141716A in R and R*.
Modeling results reported herein suggest that the overall
extent of aromatic stacking (both direct and indirect) created
by SR141716A binding is equivalent in the R and R* states.
However, SR141716A has an additional interaction, a hydro-
gen bond with K3.28(192), only in the R state. Thus, the
collection of aromatic and hydrogen bonding interactions in
the R state suggest that SR141716A will have higher affinity
for the R (over the R*) state of CB1, rendering it an inverse
agonist.
It is important to emphasize that although SR141716A
displayed inverse agonism in the Ca2ϩ assay, it also compet-
itively antagonized the effect produced by WIN55,212-2 in
this assay (Pan et al., 1998). This effect is consistent with
modeling studies reported herein, which show that
SR141716A will have affinity for the R* state due to aromatic
Fig. 7. VCHSR attenuated the inhibition of the Ca2ϩ current by the
cannabinoid agonist WIN55,212-2. A, Ca2ϩ current amplitude recorded
over time during a patch-clamp experiment from a superior cervical
ganglion neuron expressing the human CB1 cannabinoid receptor. Ap-
plication of 1 ␮M WIN rapidly inhibited the Ca2ϩ current. The current
slowly recovered during washout of WIN55,212-2. Application of 1 ␮M
VCHSR slightly increased the Ca2ϩ current and attenuated the effect of
a subsequent application of WIN55,212–2. B, superimposed Ca2ϩ current
traces for the same neuron shown in A in the absence (control) and
presence of WIN, VCHSR, and VCHSR ϩ WIN. C, graph of the percent-
age of change in Ca2ϩ current amplitude in the presence of WIN, VCHSR,
and VCHSR ϩ WIN. VCHSR significantly attenuated the effect of
WIN55,212-2 (p Ͻ 0.05). However, VCHSR alone produced a change in
the Ca2ϩ current that was not significantly different (p ϭ 0.259) from the
control current amplitude. The number of neurons tested is indicated.

1286
Hurst et al.
stacking interactions (Fig. 5A). SR141716A should therefore receptors. Results presented by Bouaboula et al. (1997) sug-
be able to compete with an agonist for binding to the R* state. gest that the SR141716A⅐CB1 complex acts as a reversible
Modeling studies reported herein also show that because negative dominant of Gi. Therefore, it can be expected for the
VCHSR has no hydrogen bonding capability in its C3 sub- CB1/SR141716A system that the RϪ and Rϩ states will pre-
stituent, it can engage only in aromatic stacking interactions dominate and the inactive R0 state can be expected to have a
in both the R and R* states of WT CB1. Because the overall very low population. As a result, the system can be expected
extent of aromatic stacking (both direct and indirect) created to behave essentially as a two-state system.
by VCHSR binding is nearly equivalent in the R and R*
Va´squez and Lewis (1999) have further documented the
states, VCHSR will have equal affinity for both states and ability of WT CB1 receptors to sequester G proteins. These
would therefore be expected to behave as a neutral antago- investigators proposed that WT CB1 receptors exist predom-
nist at WT CB1. In fact, this is what was seen herein in Ca2ϩ inantly in either of two states, a G protein-coupled inactive
current assay results for VCHSR (Fig. 7). In SCG neurons, R-GGDP state or an active R*-GGTP state. It is possible that
expressing the human CB1 receptor, VCHSR behaved as a the inactive state represented herein in which SR141716A
neutral antagonist because 1 ␮M VCHSR significantly (n ϭ interacts with K3.28(192) and stabilizes the F3.36/W6.48
6, p Ͻ 0.05) attenuated CB agonist (WIN) induced inhibition interaction (Fig. 4A) may correspond to the CB1 conforma-
of Ca2ϩ current, but alone produced a change in the Ca2ϩ tion recognized by Gi/0 in its GGDP state. Experiments are
current that was not significantly different from the control currently underway to test this hypothesis.
current amplitude.
Acknowledgments
Consistent with the modeling results reported herein, the
mutant thermodynamic cycle results show that K3.28 is a
We thank Jannie Jones for technical assistance. This work was
direct interaction site with SR141716A. It is important to
note, however, that although the modeling results suggest
that it is the carboxamide oxygen of SR141716A that inter-
acts with K3.28(192), the mutant cycle calculations indicate
only that there is a direct interaction between K3.28(192)
and the C3 substituent of SR141716A. We are currently
engaged in a synthesis effort to develop analogs that may
help to identify the specific K3.28(192) hydrogen bonding site
within the C3 substituent of SR141716A.
Implications for Models of Inverse Agonism. The data
presented in this article are consistent with the most widely
discussed mechanism of inverse agonism in which the in-
verse agonist preferentially binds to the ground state (R) over
the R* state (Samama et al., 1994), thus suppressing agonist-
independent (constitutive) activation. An alternative mecha-
nism has been proposed for the ␮-opioid receptor in which the
inverse agonist binds preferentially to the uncoupled forms of
the receptor (R and R*), rather than to the receptor coupled
to G protein (R*G), suppressing constitutive activity (Costa
et al., 1992). For other GPCRs, inverse agonist preferential
binding to a particular conformational state (i.e., R, R*, R*G,
etc.) has not been demonstrated. In such cases, it has been
suggested that the inverse agonist binds to the receptor and
stabilizes the receptor in an inactive state that cannot acti-
vate G protein (McLoughlin and Strange, 2000).
We have shown herein that the inverse agonism of
SR141716A can be explained by a two-state model in which
SR141715A can interact with K3.28(192) only in the CB1
inactive state. A two-state representation for the CB1 recep-
tor in the presence of SR141716A is consistent with models
proposed by Bouaboula et al. (1997) and by Va´squez and
Lewis (1999). Based upon results which showed that the
interaction of SR141716A with the CB1 receptor can seques-
ter Gi proteins, preventing the signaling of other Gi-coupled
receptors, Bouaboula et al. (1997) proposed a three-state
model for GPCR activation in which agonists stabilize the Rϩ
form, inverse agonists stabilize the RϪ form, and antagonists
stabilize the inactive state, R0. The RϪ state herein is one in
which the inverse agonist converts a tonically active hCB1
receptor into an active negative state in which the receptor is
coupled to a GDP-bound G protein. Those G proteins trapped
by the inverse agonist would be unavailable to couple to other
supported by National Institute on Drug Abuse Grants DA03934 and
DA00489 (to P.H.R.), DA10350 (to D.L.L.), and DA11551 (to Z.H.S.).
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