Ligands of structurally diverse natures are able to bind at the CB 1 cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB 1 agonists. To test the importance of these residues for receptor recognition, recombinant human CB 1 receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5Ј-3-O-(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of(1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (Ϫ)-11␤-hydroxy-3-(1Ј,1Ј-dimethylheptyl)hexahydrocannabinol (AM4056) and (Ϫ)-11-hydroxydimethylheptyl-⌬ 8 -tetrahydrocannabinol (HU210) were drastically reduced (50-to 100-fold) at the S7.39A mutant. Likewise, the EC 50 for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by Ͼ200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB 1 receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB 1 receptor. Our modeling studies predict that Ser7.39 in a gϪ1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding.The CB 1 cannabinoid receptor is a member of the G-protein coupled receptor (GPCR) family 1A, which includes the CB 2 receptor and the prototype rhodopsin (Howlett et al., 2002;Reggio, 2005). The human CB 1 and CB 2 receptors share only 44% amino acid overall homology, with a higher homology (68%) within the seven transmembrane domains (Munro et al., 1993). Both the CB 1 and CB 2 receptors share common signal transduction pathways, such as negative modulation of adenylyl cyclase activity (Felder et al., 1995) and also share certain common structural features with rhodopsin, including an extracellularly oriented N terminus, an intracellular carboxyl terminus, and hydrophobic transmembrane helices (TMHs).Although neither CB 1 nor CB 2 proteins have been crystallized, the crystal structure of rhodopsin (Palczewski et al., 2000) serves as a valuable template to model the putative CB 1 ligand binding domains. Ligands of structural diverse This study was supported by National Institutes of Health grants DA09978 and DA05274 (to M.E.A.), DA00489 and DA039434 (to P.H.R.), and DA09158 (to A.M. and M.E.A.).Article, publication date, and citation information can be found at

The biarylpyrazole, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 1) has been shown to act as an inverse agonist/antagonist at the cannabinoid CB1 receptor. Our previous mutant cycle study suggested that K3.28(192) is involved in a direct interaction with the C-3 substituent of 1 in wild-type (WT) CB1.(1) However, these results did not establish what part of the C-3 substituent of 1 is involved in the K3.28(192) hydrogen bond, the carboxamide oxygen or the piperidine nitrogen. Furthermore, our previous calcium channel assay results for 5-(4- chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole (VCHSR; 2) (an analogue of 1 that lacks hydrogen-bonding capability in its C-3 substituent) showed that this compound acts as a neutral antagonist, a result that is in contrast to 1, which acts as an inverse agonist in this same assay.(1) These results suggested a relationship between biarylpyrazole interaction with K3.28(192) at CB1 and inverse agonism, but these results were for a single pair of compounds (1 and 2). The work presented here was designed to test two hypotheses derived from our modeling and mutant cycle results. The hypotheses are as follows: (1) it is the carboxamide oxygen of the C-3 substituent of 1 that interacts directly with K3.28(192) and (2) the interaction with K3.28(192) is crucial for the production of inverse agonism for biarylpyrazoles such as 1. To determine whether the carboxamide oxygen or the piperidine nitrogen of the C-3 substituent may be the interaction site for K3.28(192), we designed, synthesized, and evaluated a new set of analogues of 1 (3-6, Chart 1) in which modifications only to the C-3 substituent of 1 have been made. In each case, the modifications that were made preserved the geometry of this substituent in 1. The absence of the piperidine nitrogen was not found to affect affinity, whereas the absence of the carboxamide oxygen resulted in a reduction in affinity. CB1 docking studies in an inactive state model of CB1 resulted in the trend, 3,1<5,4<2<6 for ligand/CB1 interaction energies. This trend was consistent with the trend in WT CB1 Ki values versus [3H]CP55,940 reported here. In calcium channel assays, all analogues with carboxamide oxygens (1, 3, and 4) were found to be inverse agonists, whereas those that lacked this group (2, 5, and 6) were found to be neutral antagonists. Taken together, these results support the hypothesis that it is the carboxamide oxygen of the C-3 substituent of 1 that engages in a hydrogen bond with K3.28(192) in WT CB1. Furthermore, functional results for 1-6 support the hypothesis that the interaction of 1 with K3.28(192) may be key to its inverse agonism.

The intracellular C-terminal helix 8 (H8) of the CB 1 cannabinoid receptor deviates from the highly conserved NPXXY(X) 5,6 F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB 1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB 2 sequence. The two mutant receptors differed from wild type ( 2؉ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to G␣ i3 but not G␣ 0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by G␣ i3 . Furthermore, G␣ i3 but not G␣ 0A enhanced basal facilitation ratio, suggesting that G␣ i3 is responsible for CB 1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with G␣ i1 or G␣ i2 but not with G␣ i3 . Molecular dynamics simulations of WT CB 1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X) 5,6 L contributes to maximal activity of the CB 1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.

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