Ligand Binding and Modulation of Cyclic AMP Levels Depend on the Chemical Nature of Residue 192 of the Human Cannabinoid Receptor 1

Abstract

The human cannabinoid receptor associated with the CNS (CB1) binds Δ9‐tetrahydrocannabinol, the psychoactive component of marijuana, and other cannabimimetic compounds. This receptor is a member of the seven transmembrane domain G protein‐coupled receptor family and mediates its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor activation requires identification of the active site residues and their role. Lys192 of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, we generated mutant receptors in which this amino acid was changed to either Arg (K192R), Gln (K192Q), or Glu (K192E). Wild‐type and mutant receptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP‐55,940 and the aminoalkylindole WIN 55,212‐2. We found that only the most conservative change of Lys to Arg allowed retention of binding affinity to CP‐55,940, whereas WIN 55,212‐2 bound to all of the mutant receptors in the same range as it bound the wild type. Analysis of the ligand‐induced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212‐2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this study indicate that binding of CP‐55,940 is highly sensitive to the chemical nature of residue 192. In contrast, although this residue is not critical for WIN 55,212‐2 binding, the data suggest a role for Lys192 in WIN 55,212‐2‐induced receptor activation.

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

“…6B) These results suggest that K3.28(192) is important to the binding of SR141716A at WT CB1, but this residue is not important to the binding of VCHSR at WT CB1. Residue K3.28(192) has previously been shown to be a very important residue for agonist binding at CB1 (Song and Bonner, 1996;Chin et al, 1998). The results reported herein are the first demonstration that this residue is also important for inverse agonist binding at CB1.…”

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

“…Lys3.28(192) was used as the primary interaction site in CB 1 docking studies of HU210 and CP55,940 reported here based upon the K3.28(192)A mutation results of Song and Bonner (1996) and supported by subsequent studies (Chin et al, 1998). These studies showed a complete loss of HU210 and CP55,940 (but not WIN 55,212-2) binding and a Ͼ100-fold increase in EC 50 value for receptor activation upon this mutation.…”

“…Computational and mutational studies have identified several key amino acid residues in discrete noncontiguous regions of the TMHs that contribute in forming ligand binding sites (Abood, 2005;Reggio, 2005). These include but are not limited to the Lys3.28 (192), involved in the binding of anandamide, CP55,940, HU210, and SR141716A but not WIN55,212-2 (Song and Bonner, 1996;Chin et al, 1998;Hurst et al, 2002) via a hydrogen bonding interaction with the ligand. An aromatic microdomain contributed by Phe3.36,Trp4.64,Tyr5.39,Trp5.43,and Trp6.48 is involved in the binding of WIN55,212-2 and SR141716A (McAllister et al, 2002(McAllister et al, , 2003(McAllister et al, , 2004.…”

Mutations of CB1 T210 Produce Active and Inactive Receptor Forms: Correlations with Ligand Affinity, Receptor Stability, and Cellular Localization

“…Two endocannabinoid agonist analogues, arachidonyl-2-chloroethylamine (ACEA) and (R)-(+)-methanandamide, which are believed to share some of the same receptor contact points as CP55940 and HU-210 (22), were also tested; the T210I receptor was found to have a 3-4-fold enhancement in affinity for these ligands relative to that of wild-type CB 1 ( Table 1). The aminoalkylindole, (R)-(+)-WIN55212-2, is thought to have contact points with the receptor different from those of the compounds mentioned above, as shown by both modeling studies (35) and mutagenesis studies (26). Regardless, the T210I receptor displayed a similar change in its affinity for (R)-(+)-WIN55212-2 ( Table 1).…”