Mutation Studies of Ser7.39 and Ser2.60 in the Human CB₁Cannabinoid Receptor: Evidence for a Serine-Induced Bend in CB₁Transmembrane Helix 7

Abstract

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

Dual Role of the Second Extracellular Loop of the Cannabinoid Receptor 1: Ligand Binding and Receptor Localization

“…It is possible that a very small population of folded mutant receptor could respond at a higher concentration of CP55940. All six residues identified here are defined in other studies as part of EC2 (Kapur et al, 2007;Padgett et al, 2008), yet clustered at either the N terminus (Trp255 and Asn256) or C terminus (Phe268, Pro269, His270, and Ile271) of the loop, suggesting that they may play a role as two units, rather than influence ligand binding through an interaction with a single amino acid functional group.…”

CB1 Receptor Allosteric Modulators Display Both Agonist and Signaling Pathway Specificity

“…Studies have highlighted differences in the ligand recognition site for WIN55212 (and other aminoalkylindoles) and that of structurally-distinct agonists, including CP55940 and anandamide (McAllister et al, 2003;Abood, 2005;Kapur et al, 2007). In particular, mutation of W5.43A results in a significant loss of affinity of WIN55212 and SR141716A, while the affinity of CP55940 was unaffected by this mutation (for review see Abood, 2005).…”

Mapping the Structural Requirements in the CB1Cannabinoid Receptor Transmembrane Helix II for Signal Transduction

“…Protein membrane preparations harvested from transfected HEK 293 cells were prepared and assayed as described previously (Kapur et al, 2007). In brief, binding assays (saturation and competition binding assays) were initiated by the addition of 50 g of membrane protein to siliconized glass tubes (to reduce nonspecific binding) containing [ 3 H]CP,55940 and an appropriate volume of binding buffer A (50 mM Tris base, 1 mM EDTA, 3 mM MgCl 2 , and 5 mg/ml BSA, pH 7.4) to bring the final volume to 500 l. Nonspecific binding was determined in the presence of excess (1 M), unlabeled CP,55940.…”