![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 291, Issue 2, 837-844, November 1999
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut (C.-N.C., J.W.M., D.A.K.); and Department of Chemistry, Clemson University, Clemson, South Carolina (J.W.H.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Two subtypes of the human cannabinoid receptor have been identified. The CB1 receptor is primarily distributed in the central nervous system, whereas the CB2 receptor is associated with peripheral tissue, including the spleen. These two subtypes are also distinguished by their ligand-binding profiles. The goal of this study was to identify critical residues in transmembrane region III (TM3) of the receptors that contribute to subtype specificity in ligand binding. For this purpose, a chimeric cannabinoid receptor [CB1/2(TM3)] was generated in which the TM3 of CB1 was replaced with the corresponding region of CB2. These receptors were stably expressed in Chinese hamster ovary cells for evaluation. The binding affinities of CB1/2(TM3) and the wild-type CB1 receptor to several prototype ligands were similar with one notable exception: the chimeric receptor exhibited a 4-fold enhancement in binding affinity to WIN 55,212-2 (Kd = 4.8 nM) relative to that observed with CB1 (Kd = 21.7 nM). Two additional aminoalkylindoles, JWH 015 and JWH 018, also bound the chimeric receptor (Ki = 1.0 µM and 1.4 nM, respectively) with higher affinity compared with the wild-type CB1 (Ki = 5.2 µM and 9.8 nM, respectively). Furthermore, the increase in binding affinities of the aminoalkylindoles were reflected in the EC50 values for the ligand-induced inhibition of intracellular cAMP levels mediated by the chimeric receptor. This pattern mirrors the selectivity of WIN 55,212-2 binding to CB2 compared with CB1. Site-specific mutagenesis of the most notable amino acid changes in the chimeric receptor, Gly195 to Ser and Ala198 to Met, revealed that the enhancement in WIN 55,212-2 binding is contributed to by the Ser but not by the Met residue. The data indicate that the amino acid differences in TM3 between CB1 and CB2 play a critical role in subtype selectivity for this class of compounds.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The
psychoactive component of marijuana,
9-tetrahydrocannabinol (THC), exerts many of
its effects through activation of specific G protein-coupled receptors.
Two subtypes of cannabinoid receptors, CB1 and CB2, have been
identified, and their cDNA has been cloned (Matsuda et al., 1990
;
Munro et al., 1993). Despite the high homology in their amino acid
sequence [44% sequence identity overall and 68% in the transmembrane
regions (TM)], they possess somewhat different pharmacological and
biochemical properties. The CB1 receptor is mainly distributed in the
central nervous system, and lesser amounts are found in peripheral
tissues (Gerard et al., 1991). The CB2 receptor was found to be
expressed predominantly in the immune system, including the marginal
zone of the spleen, and in macrophages, which may be associated with
the immunomodulation function of cannabinoids (Munro et al., 1993). The
differential tissue distribution of the two subtypes suggests that CB1
and CB2 play different roles in mediating cannabinoid-induced effects.
Biochemical characterization of the signal transduction of CB1 and CB2
expressed in cultured cells has suggested that the activation of both
receptors results in the inhibition of cAMP accumulation (Felder et
al., 1995; Slipetz et al., 1995). This coincides with the notion that
functional coupling to the inhibitory G protein
(Gi) is one of the major signaling pathways for
cannabinoid receptors (Howlett et al., 1986). CB1 has also been shown
to mediate the inhibition of N-type calcium channels in neuroblastoma
cells (Mackie and Hille, 1992), the inhibition of Q-type calcium
channels, and the activation of inwardly rectifying potassium channels
in AtT-20 cells (Mackie et al., 1995). These effects were not observed in AtT-20 cells transfected with CB2 (Felder et al., 1995). This suggests that CB1 and CB2 functionally couple to other effector systems
that are independent of Gi and are not shared
between the two subtypes.
To discriminate the physiological functions and cellular effects
mediated by either CB1 or CB2, cannabinoid ligands that can selectively
bind to one or the other of the subtypes have been developed. It was
found that cannabinol has a 10-fold higher binding affinity to CB2 than
to CB1 (Munro et al., 1993). (
)-9-THC, the
active component of Cannabis sativa, has been shown to act
as an agonist for CB1 but as a weak antagonist for CB2 (Bayewitch et
al., 1996). Derivatives of
8-THC-dimethylheptyl-8-THC
lacking a phenolic hydroxyl were found to give rise to high selectivity
for CB2 (Gareau et al., 1996; Huffman et al., 1996). It was also found
that the C3 substituents of classical and nonclassical cannabinoids
affected the selectivity for CB1 or CB2 (Lu et al., 1997). Selectivity
for CB2 has also been observed for WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)-
methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthalenyl)methanone monomethanesulfonate], the prototype of aminoalkyindoles
(AAIs), as well as other analogs in this series of cannabimimetic
ligands (Felder et al., 1995; Showalter et al., 1996). Anandamide, an endogenous ligand of the cannabinoid receptor isolated from porcine brain, has weak preference for CB1 over CB2. SR 141716A
[N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and SR 144528 are potent antagonists specific for CB1
and CB2, respectively (Rinaldi-Carmona et al., 1994, 1998). Comparisons
of pharmacological potency and binding affinities to CB1 and CB2
expressed in Chinese hamster ovary (CHO) cells have provided useful
information on cannabinoid ligands that can discriminate between
binding to CB1 or CB2 (Showalter et al., 1996). However, the molecular
basis for the selectivity is not fully understood.
One goal of our study was to understand the molecular interactions
between cannabinoid ligands and the receptor. A previous study on the
CB1 receptor indicated the importance of Lys192 in TM3 for the binding
of several cannabinoid agonists, including HU 210, CP 55,940 [()-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)cy-clohexanol], and anandamide but not for the binding of WIN 55,212-2 (Song and Bonner, 1996). Furthermore, evidence from our laboratory suggests that
a basic residue, in particular, at this position is essential to ensure
the high-affinity binding of CP 55,940 to the CB1 receptor (Chin et
al., 1998). To further explore the involvement of TM3 in ligand binding
and to delineate its role in subtype specificity, we examined the
selectivity displayed by several ligands for a chimeric mutant of CB1
in which the TM3 region was replaced by the corresponding region of
CB2. Remarkably, several AAI compounds, including WIN 55,212-2, showed
higher binding affinity for the chimeric receptor than for CB1. Our
data indicate that amino acid differences in this segment, particularly
Gly195 (CB1) versus Ser (CB2), contribute to the binding of AAIs and
play a role in subtype selectivity.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Reagents.
CP 55,940 was provided by Pfizer Inc. (Groton,
CT). WIN 55,212-2, (R)-methanandamide
[(R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide], and
CHO membrane expressing the CB2 receptor were purchased from Research
Biochemical Inc. (Natick, MA). SR 141716A and
[3H]SR141716A (19.4 Ci/mmol) were provided by the
National Institute on Drug Abuse (Bethesda, MD). JWH 015 and JWH 018 were provided by Dr. John Huffman of Clemson University (Bell et al.,
1991; Huffman et al., 1994). Indomethacin morpholinamide was purchased from Cayman Chemical (Ann Arbor, MI). [3H]CP 55,940 (165 Ci/mmol) and [3H]WIN 55,212-2 (43 Ci/mmol) were purchased
from DuPont-NEN (Boston, MA). G418 sulfate was obtained from Calbiochem
(La Jolla, CA). Protease inhibitor cocktail was obtained from Sigma
Chemical Co. (St. Louis, MO). The human CB1 cDNA was provided by Dr.
Marc Parmentier (University Libre de Bruxelles, Bruxelles, Belgium).
CHO-K1 cells were obtained from American Type Culture Collection
(Rockville, MD). Minimal essential medium (MEM) and fetal bovine serum
were purchased from Hyclone Laboratories, Inc. (Logan, UT).
Plasmid Construction and Stable Expression of Cannabinoid
Receptor in CHO Cells.
The human CB1 cDNA was subcloned into the
pAlterI vector for site-directed mutagenesis using the Alter Sites in
vitro mutagenesis system (Promega Corp., Madison, WI) as previously
described (Chin et al., 1998). Eight new restriction enzyme sites were
designed and introduced into the coding region of the CB1 receptor
without changing the amino acid sequence. These restriction enzyme
sites are unique in the coding region of CB1 to facilitate cassette mutagenesis. Two such sites, XbaI and
AgeI, were used in this study to generate the mutant CB1
receptor. Three pairs of oligonucleotides encoding the sequence of the
TM3 of the CB2 receptor were inserted between these two sites, which
resulted in the replacement of the TM3 of the CB1 receptor with that of
the CB2 receptor. The mutated CB1 gene was then subcloned into the
pcDNA3 vector (Invitrogen, San Diego, CA) for expression in mammalian
cells. CHO cells were maintained in MEM supplemented with 0.1 mM MEM
nonessential amino acid solution (Life Technologies, Inc.,
Gaithersburg, MD) and 5% fetal bovine serum under 5% CO2
at 37°C. Stably transfected cell lines expressing CB1 or the mutant
receptor were selected using reverse transcription-polymerase chain
reaction as previously described (Chin et al., 1998) and maintained in
the CHO cell growth medium with 0.5 mg/ml G418 sulfate. The single-site
mutations, G195S and A198M, were introduced into the CB1 coding region
using the QuikChange system (Stratagene, La Jolla, CA). These mutant receptors were evaluated after transient transfection of 293 cells with
calcium phosphate.
Ligand Binding Assay and cAMP Determination.
Membranes of
CHO or 293 cells were prepared as previously described (Abadji et al.,
1999). In brief, cells were grown to confluency and harvested by
scraping in PBS with a cell scraper. The cell suspension was
centrifuged at 1500g for 10 min at 4°C, and the pellet
was resuspended in TME buffer (25 mM Tris · HCl, pH 7.4, 5 mM
MgCl2, 1 mM EDTA) with 0.5% (v/v) protease inhibitor
cocktail containing 100 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 4 mM
transepoxysuccinyl-L-leucylamido(4-guanidino)butane,
2.2 mM leupeptin, 0.08 mM aprotinin, and 1.5 mM pepstatin A. The
resuspended cells were then subjected to nitrogen cavitation at 750 psi
for 5 min with a Parr Cell Disruption Bomb to rupture the cells. The fractionated cells were centrifuged at 1000g for 10 min
to remove cell debris and unbroken nuclei. The supernatant containing
cell membranes was stored at 70°C in aliquots of 1 to 2 mg
protein/ml until use in the radioligand-binding assay.
). The reactions were conducted in a total volume
of 200 µl in silanized glass tubes. Approximately 40 µg of membrane
protein was added to the reaction and incubated at 30°C for 1 h
with varying concentrations of radioligand and/or unlabeled ligand in
TME buffer containing 0.1% fatty acid-free BSA. The concentrations of
[3H]CP 55,940, [3H]WIN
55,212-2, or [3H]SR 141716A in saturation
binding studies ranged from 0.2 to 50 nM. Unlabeled ligands at a
concentration of 2 µM were used to determine the nonspecific binding.
In competition binding studies, the concentrations of compounds tested
ranged from 10
5 to 1011
M, and 1.5 nM [3H]CP 55,940 was used. The
reaction was terminated by the addition of 250 µl of cold TME
containing 5% BSA immediately preceding the separation of bound and
free radioligand via rapid filtration through GF/C filter paper
(Whatman Inc.) on a 24-manifold Brandel cell harvester (Gaithersburg,
MD). The filters were washed with 16 ml of cold TME buffer. Bound
radioactivity was determined by scintillation counting. Specific
binding accounted for >65% of the total binding at a
[3H]CP 55,940 concentration of 1.5 nM.
cAMP Determination.
The accumulation of cAMP was measured as
previously described (Chin et al., 1998). Forskolin was added at a
concentration of 1 µM to the reaction to stimulate adenylyl
cyclase and elevate the level of cAMP. The amount of cAMP was
determined using a [3H]cAMP assay system (Amersham,
Arlington Heights, IL).
Data Analysis.
Data obtained from binding assays and cAMP
assays were analyzed with the use of Prism (GraphPAD Software, San
Diego, CA). The Bmax and
Kd values of saturation binding assays were
determined using the template for fitting saturation binding curves to
a single binding site. For competition binding assays, IC50
values were determined by the nonlinear regression analysis of the
logarithm of ligand concentration versus percent displacement of
radioligand binding. These values were then converted to
Ki values according to the method of Cheng
and Prusoff (1973). The statistical significance of differences between
binding constants was determined with an unpaired t
test. Differences with values of P < .05 were
considered significant. Data obtained from cAMP assays were expressed
as the percentage of forskolin-stimulated cAMP accumulation as a function of the logarithm of ligand concentration. The EC50
values were determined by nonlinear regression analysis.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Evaluation of Effects of TM3 Replacement on Ligand Binding
Characteristics of CB1 Expressed in CHO Cells.
A chimeric mutant
of human CB1 was constructed in which the TM3 of CB1 was replaced with
the corresponding region of CB2, as shown in Fig.
1. This replacement gave rise to a mutant
CB1 receptor, CB1/2(TM3), containing six point mutations in the amino acid sequence of TM3 in CB1: F191L, L193I, G195S, A198M, S199T, and
F207L (Fig. 1).
|
). The chimeric
receptor CB1/2(TM3) exhibited binding of [3H]CP
55,940 with a Kd value of 10.7 ± 1.5 nM (Table 1). The affinity of CP 55,940 for the chimeric mutant was
similar to, although not quite as good as, the affinities for the
wild-type CB1 and CB2. The CHO cell lines stably transfected with the
wild-type or chimeric receptor tested in this study showed similar
levels of expression with Bmax values of
1124 ± 88 fmol/mg membrane protein for the wild-type CB1 and
1127 ± 60 fmol/mg membrane protein for the CB1/2(TM3) chimera.
The membrane preparation from CHO cells expressing CB2 showed a higher
expression level, with a Bmax value of
2960 ± 210 fmol/mg membrane protein.
|
|
). Saturation binding analysis of
[3H]SR141716A to CB1 and CB1/2(TM3) yielded
Kd values of 5.9 ± 0.7 and 11.8 ± 1.0 nM, respectively, showing that the mutation did not markedly
affect the binding affinity of this ligand to the receptor (Table 1).
(R)-Methanandamide is a chiral congener of anandamide that
displays higher affinity and better metabolic stability than anandamide
(Abadji et al., 1994). Like anandamide, (R)-methanandamide
(Fig. 2) possesses higher binding affinity to CB1 than to CB2
(Khanolkar et al., 1996). Our results showed that it binds to
CB1/2(TM3) with affinity similar to that of the wild-type CB1 (Table
1). The Ki value for the wild-type CB1 is comparable to that previously reported (Abadji et al., 1994; Khanolkar et al., 1996). Indomethacin morpholinamide is a selective ligand for
CB2 (Fig. 2), with a Ki value of 435 nM
(Gallant, 1996). Our results showed that this compound possesses very
low binding affinities to both the CB1 wild-type and mutant receptors
(Ki > 2 µM; Table 1).
Overall, little or no changes in the binding affinities of the
compounds tested above for CB1/2(TM3) were found relative to the
wild-type CB1. In no case was a pattern of subtype selectivity noted
for these compounds; thus, it appears that residues in TM3 specific to
CB2 are not critical to the selectivity of these compounds.
Selective Binding of AAIs to CB2 Is Reflected in Binding to
CB1/2(TM3) Chimera.
WIN 55,212-2 has been reported as a selective
ligand for CB2 with a 7- to 20-fold higher binding affinity than for
CB1 (Fig. 2; Felder et al., 1995; Slipetz et al., 1995; Showalter et
al., 1996). Selectivity of about 10-fold of WIN 55,212-2 for CB2 over CB1 was observed in our study (P < .05, unpaired
t test). As shown in Fig.
3, saturation binding analyses for
[3H]WIN 55,212-2 yielded a Kd
value of 21.7 ± 6.9 nM for CB1 and 2.3 ± 0.2 nM for CB2
(Fig. 3, top and middle; Table 1). The CB1/2(TM3) receptor bound
[3H]WIN 55,212-2 with a Kd
value of 4.8 ± 0.5 nM (Fig. 3, bottom; Table 1). Interestingly,
this indicates that the introduction of the TM3 of CB2 into CB1
resulted in a 4-fold enhancement in the binding affinity of WIN
55,212-2 to the mutant receptor relative to CB1. Therefore, unlike
other classes of cannabinoid compounds tested, WIN 55,212-2 is
significantly more selective for the mutant receptor than for CB1
(P < .05, unpaired t test). The
Bmax values obtained from these data are
1563 ± 231 fmol/mg for CB1, 2389 ± 60 fmol/mg for CB2, and
1510 ± 50 fmol/mg for CB1/2(TM3). These values are consistent
with the Bmax values obtained for these CHO
cell lines when assayed for [3H]CP 55,940 binding as
described above.
|
;
Wiley et al., 1998). However, a propyl side chain at this position (JWH 015) caused a significant loss of binding affinity for CB1 but retained
good binding affinity for CB2 (Showalter et al., 1996). Because JWH 015 and JWH 018 have been reported as selective ligands for CB2, although
to different degrees, we examined the binding activities of these two
AAI derivatives for CB1/2(TM3) relative to the wild-type CB1. As shown
in Fig. 4A, although JWH 015 bound to CB1
and CB1/2(TM3) with relatively low affinities, lower than previously
reported (Showalter et al., 1996), the Ki
values suggest a trend toward selectivity for the mutant receptor
(Table 1). JWH 018, on the other hand, displayed nearly a 7-fold
selectivity for CB1/2(TM3) over the wild-type CB1 (P < .05, unpaired t test), with Ki
values of 9.8 ± 2.8 nM for CB1, 3.1 ± 0.3 nM for CB2, and
1.4 ± 0.2 nM for CB1/2(TM3) (Fig. 4B; Table 1).
|
Efficacy of Inhibition of Forskolin-Stimulated cAMP Levels Mediated
by CB1/2(TM3) Chimera Reflect CB2 Selectivity for AAIs.
To examine
whether this mutant also changes the efficacy of CB1 to activate
Gi in response to the binding of ligands, cAMP assays were
performed as described in Materials and Methods.
Concentration-dependent inhibition of the level of forskolin-stimulated
cAMP by CP 55,940, WIN 55,212-2, and JWH 018 in CHO cells expressing
either the wild-type CB1 or the CB1/2(TM3) receptor are shown in Fig.
5.
|
|
Evaluation of Individual Amino Acid Changes Gly195 to Ser and Ala198 to Met within TM3 of CB1 for WIN 55,212-2 Binding. Assessment of the chemical nature of the six amino acids in TM3 that differ in CB1 and CB2 points to only a few candidates that are likely to contribute to the enhanced binding of the chimeric receptor with the AAIs. For example, L193I and S199T are conservative changes and retain the general characteristics of the amino acid side chain that was used. More notable are the changes of G195S and A195M; these changes provide an extra hydroxyl group (G195S) and a longer and more hydrophobic side chain (A198M). To examine the role of these two amino acid changes in WIN 55,212-2 binding, mutant CB1 receptors were generated in which only the Gly-to-Ser or the Ala-to-Met substitution was made. The resulting G195S and A198M receptors were separately evaluated after transient transfection of 293 cells. Membrane preparations of the wild-type CB1 receptor expressed in 293 cells versus CHO cells exhibit no differences in CP 55,940 binding (data not shown) or WIN 55,212-2 (Table 3). The ability of [3H]WIN 55,212-2 to bind to the A198M and G195S receptors was examined using saturation binding assays. Substitution of A198 in CB1 with Met produced no significant change in the binding isotherm relative to the wild-type receptor with a Kd value of 20.4 ± 5.1 nM and a Bmax value of 1949 ± 185 fmol/mg of protein (Table 3). In contrast, the G195S receptor bound [3H]WIN 55,212-2 with enhanced affinity relative to CB1 (P < .05, unpaired t test; Table 3). The Kd value of 5.6± 0.6 nM (Bmax = 1534 ± 56 fmol/mg protein) suggests that the Gly195-to-Ser exchange contributes substantially to the observed enhancement in WIN 55,212-2 binding by the CB1/2(TM3) chimera relative to the wild-type CB1 receptor.
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
In this study, we demonstrate that amino acid differences in the TM3 of CB1 and CB2 affect the binding profiles of some cannabinoid ligands. For most of the compounds, we found either no difference or only a small loss in binding affinity. Surprisingly, however, the exchange of TM3 resulted in a distinct enhancement in the binding affinity and efficacy of AAI analogs; this includes a 5-fold increase for WIN 55,212-2, a 5-fold increase for JWH 015, and a 7-fold increase for JWH 018 relative to binding to the wild-type CB1. This increase in binding affinity was also reflected in the EC50 values for the inhibition of intracellular levels of cAMP and further validates that the interactions of these ligands with the receptor were augmented in this mutant. This observation suggests that at least a part of the elements in CB2 responsible for the subtype selectivity of AAIs has been introduced into CB1 via the mutations that we generated.
A priori, sequence inspection of TM3 in CB1 and CB2 points to Lys192 in
CB1 (Lys109 in CB2) as the most likely potential contact point for
cannabinoid ligand/receptor interactions. The Lys residue stands out as
being the only charged residue in the TM regions that is not conserved
in the G protein-coupled receptor family yet is shared by both subtypes
of the cannabinoid receptor. However, previous work involving
single-site mutations of this residue has demonstrated that at least
for CB1, Lys192 is not critical for WIN 55,212-2 binding (Song and
Bonner, 1996; Chin et al., 1998). Furthermore, we have been able to
change this residue to Arg, Gln, and Glu and still detect WIN 55,212-2 binding activity to CB1 (Chin et al., 1998). Consequently, many
investigators have emphasized the involvement of amino acid residues in
other TMs for the interaction with AAIs (Abood et al., 1998; Song and
Reggio, 1998). For example, molecular modeling studies have pointed to the potential role of residues such as Phe and Tyr in TM5 of CB2 that
could stabilize an interaction with WIN 55,212-2 through aromatic
stacking. However, the data presented here have shown that the residues
of TM3 also contribute substantially to the binding of AAIs, and
residues other than Lys192 in this TM appear critical.
Although our conceptual strategy was to switch the entire TM3 domain from CB1 to CB2, in effect, this amounts to only six amino acid changes. However, modeling indicates that these changes are largely clustered in one area of TM3, as seen in the helical net representation (Fig. 1B). This cluster is positioned proximal to and on the same helical face as Lys192, which has been implicated as a key residue for the binding of other classes of cannabinoid ligands. This intriguing observation therefore suggests that this cluster is very likely to constitute a part of the ligand-binding pocket for the cannabinoid receptor. To assess the contribution of the individual residue differences in this region in TM3, two single-site mutations were made in which a residue in CB1 was exchanged for the corresponding residue from CB2: G195S and A198M. The A198M receptor was indistinguishable from wild-type CB1 in WIN 55,212-2 binding. In contrast, the G195S receptor exhibited enhanced WIN 55,212-2 binding, suggesting that this residue plays a critical role in the CB2-like binding characteristics of the CB1/2(TM3) chimeric receptor with AAIs.
The change from Gly to Ser could alter the interaction with AAIs by
introducing a new hydrogen-bonding group. Previous SAR studies
demonstrated that the removal of the carbonyl oxygen of AAIs did not
affect their binding to CB1 (Kumar et al., 1995; Reggio et al., 1998),
suggesting that this moiety is not required for the binding of AAIs to
CB1. However, these AAI analogs lacking the carbonyl oxygen displayed a
7- to 10-fold loss of binding affinity for CB2 compared with the
binding of WIN 55,212-2 and, thus, loss of selectivity for CB2 (Reggio
et al., 1998). This suggested the possibility that hydrogen bonding of
WIN 55,212-2 occurs with the amino acid residues of CB2, which may be
absent in CB1. Indeed, this may explain the higher binding affinity to CB2, and the change from Gly to Ser in our receptor may provide an
additional contact point in the receptor for such interactions.
The parallelism of the results from the cAMP assays and the data from
the binding assays suggest that the effects caused by the TM3 switch
are mainly on the ligand-binding sites. For CP 55,940, the
EC50 value for inhibition of cAMP accumulation is similar for the chimera and wild-type receptors, and an enhancement with the chimera is observed only with the AAIs. Unlike the K192E mutant of CB1 in which the mutational effect propagates from the TM
region to the G protein-coupling region (Chin et al., 1998), the G
protein-coupling efficiency does not appear to be affected by the
mutations in TM3 generated here.
Coupled with previous studies identifying the involvement of Lys192 in
binding other cannabinoid agonists (Song and Bonner, 1996; Chin et al.,
1998), we demonstrate that this region of TM3 of the cannabinoid
receptor is also critical for binding AAIs. This indicates that
different classes of ligands for the cannabinoid receptor have at least
partially overlapping binding pockets, although the functional groups
within these domains that are involved in making key contacts may be
ligand specific. This information is important for the development of
future generations of ligands with enhanced binding affinity and
receptor subtype selectivity.
| |
Acknowledgments |
|---|
We thank Pfizer, Inc., for providing us with CP 55,940.
| |
Footnotes |
|---|
Accepted for publication July 8, 1999.
Received for publication March 15, 1999.
1 This work was supported in part by the Critical Technologies Program of Connecticut Innovations, Inc., and grants from the National Institute on Drug Abuse (DA09158) and General Medical Science (GM37639) (all to D.A.K.) and by a grant from the National Institute on Drug Abuse (DA03590) (to J.W.H.).
2 Present address: Department of Biochemistry, Stockholm University, 106 91 Stockholm, Sweden.
Send reprint requests to: Dr. Debra A. Kendall, Department of Molecular and Cell Biology, Box U-44, 75 North Eagleville Rd., University of Connecticut, Storrs, CT 06269-3044. E-mail: kendall{at}uconnvm.uconn.edu
| |
Abbreviations |
|---|
THC, 9-tetrahydrocannabinol;
TM, transmembrane region;
AAI, aminoalkyindole;
CB1, central cannabinoid
receptor;
CB2, peripheral cannabinoid receptor;
CP 55,940, ()-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol;
WIN 55,212-2, (R)-(+)-[2.3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthalenyl)methanone
monomethanesulfonate;
SR 141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
hydrochloride;
JWH 015, 1-propyl-2-methyl-3-(1-naphthoyl)indole;
JWH 018, 1-pentyl-3-(1-naphthoyl)indole;
(R)-methanandamide, (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide;
MEM, minimal essential medium;
CHO, Chinese hamster ovary.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
)-9-Tetrahydrocannabinol antagonizes the peripheral cannabinoid receptor-mediated inhibition of adenylyl cyclase.
J Biol Chem
271:
9902-9905This article has been cited by other articles:
|
|
 |
|
  I. A. Khasabova, S. G. Khasabov, C. Harding-Rose, L. G. Coicou, B. A. Seybold, A. E. Lindberg, C. D. Steevens, D. A. Simone, and V. S. Seybold A Decrease in Anandamide Signaling Contributes to the Maintenance of Cutaneous Mechanical Hyperalgesia in a Model of Bone Cancer Pain J. Neurosci., October 29, 2008; 28(44): 11141 - 11152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Jeske, A. M. Patwardhan, N. Gamper, T. J. Price, A. N. Akopian, and K. M. Hargreaves Cannabinoid WIN 55,212-2 Regulates TRPV1 Phosphorylation in Sensory Neurons J. Biol. Chem., October 27, 2006; 281(43): 32879 - 32890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Andersson, A. M. D'Antona, D. A. Kendall, G. Von Heijne, and C.-N. Chin Membrane Assembly of the Cannabinoid Receptor 1: Impact of a Long N-Terminal Tail Mol. Pharmacol., September 1, 2003; 64(3): 570 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Sim-Selley and B. R. Martin Effect of Chronic Administration of R-(+)-[2,3-Dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone Mesylate (WIN55,212-2) or Delta 9-Tetrahydrocannabinol on Cannabinoid Receptor Adaptation in Mice J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 36 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Howlett, F. Barth, T. I. Bonner, G. Cabral, P. Casellas, W. A. Devane, C. C. Felder, M. Herkenham, K. Mackie, B. R. Martin, et al. International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors Pharmacol. Rev., June 1, 2002; 54(2): 161 - 202. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
),
the CB2 receptor (
), and the CB1/2(TM3) receptor (
). Binding
assays were performed over a concentration range of 0.2 to 50 nM
[3H]WIN 55,212-2. Specific binding is the difference
between the binding in the absence and presence of 2 µM unlabeled WIN
55,212-2. Saturation binding isotherms were analyzed with nonlinear
regression analysis as described in Materials and
Methods. Data presented are the mean ± S.E. of three
independent experiments, with each performed in duplicate.
