The central and peripheral cannabinoid receptors (CB1 and CB2): Structural characterization and active site elucidation using covalent probes

Date of Completion

January 2007


Chemistry, Pharmaceutical




Activation of different classes of plasma membrane receptors regulates the activity of practically every cell of the body. The vast majority of these receptors belong to the super family of G protein coupled receptors (GPCRs). Human CB1 and CB2, like other GPCRs, have a similar integral membrane topology. The transmembrane domains (TMs) are predicted to be those regions that are involved in ligand-receptor interaction. Although the two subtypes share affinity for a few cannabinergic ligands and areas that are important for ligand binding and receptor subtype specificity have been identified, the actual ligand-binding sites remain unknown and we cannot assume ligands bind in the same manner to both receptor subtypes because of the non-conserved amino acid sequence and their contrasting physiological roles. Hence, determination of the ligand receptor-binding motifs will facilitate the design of receptor-selective, bioactive, and safe synthetic cannabinoids. ^ In order to do this we need proof that CB1 and CB2 bind the same ligands differently. This can be achieved by using high affinity covalent ligands that have similar affinity towards both the CB1 and CB2 receptors and identifying key amino acid residues in the two receptors that interact with the covalent moieties of these ligands. This approach has been termed as Ligand Based Structural Biology by our lab. ^ With the peripheral Cannabinoid Receptor CB2, biochemical techniques were used to identify the ligand binding motif. This involved a number of technically-demanding experimental steps like functional over-expression of CB2 cannabinoid receptors in insect cells using the baculovirus system, interaction of covalent cannabinergic ligands with CB2 receptor preparations, purification of the ligand-receptor covalent complex using affinity chromatography and identification of the amino acid residue(s) through mass spectroscopic analysis of the digested fragments. Using this method, the binding motifs of AM1336 (CB2 antagonist) and AM841 (CB2 agonist) have been characterized. ^ The central cannabinoid receptor (CB1), is more difficult to purify, so site-directed mutagenesis and subsequent biochemical testing were used to identify the ligand binding motifs. In order to do this a global set of cysteine substitutions (cysteine to serine) in the full length and truncated human CB1 receptors was generated and saturation binding assays were performed to assess the expression levels. CB1 selective covalent probes were used to label the receptor and binding assays with [ 3H] CP55940 were undertaken to assess the extent to which, if any, the mutations would alter the covalent binding ability of the cannabinergic ligands. Using this methodology, the cysteine in the transmembrane helix 6 (C6.47(355)) has been identified as the site of covalent attachment of AM3677, an anandamide analog and the cysteine in transmembrane helix 7 (C7.42(386)) as the site of covalent attachment of AM4073, a classical cannabinoid. ^ In conclusion, the work presented here demonstrates for the first time a complete peptide mapping of a GPCR beyond rhodopsin using nanoLC coupled to the 4000 Q-Trap MS and the high accuracy mass measurement LTQ-FT MS and a MALDI-TOF. Also, the key binding motifs for different classes of cannabinergic ligands with respect to both the central and peripheral cannabinoid receptors have been identified and unequivocally proved. And this work has laid the foundation for future experiments to characterize the binding site and ultimately produce sufficient amounts of purified protein for crystallization and NMR studies. ^