Residues Accessible in the Binding-Site Crevice of Transmembrane Helix 6 of the CB2 Cannabinoid Receptor

Abstract

We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [3H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59 and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a α-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59 and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors.

Recombinant Cannabinoid Type 2 Receptor in Liposome Model Activates G Protein in Response to Anionic Lipid Constituents

“…The ␣-helical domains of the receptor are shown as predicted by the CB 2 receptor homology model of Xie et al (45) derived from the crystal structure of bovine rhodopsin. The end of helix VI on the extracellular side is shown as predicted by Nebane et al (77). The snake plot is in reasonable agreement with the reported structure of the CB 2 receptor in a bilayer obtained in a molecular simulation (67).…”

Examining the critical roles of human CB2 receptor residues Valine 3.32 (113) and Leucine 5.41 (192) in ligand recognition and downstream signaling activities

“…Specifically, five hydrophobic residues were found to be in close contact with all three ligands during docking simulations: V113, L192, L201, V261 and W258. Indeed, L201, V261 and W258 have previously been implicated in the formation of the ligand binding pocket [23, 39]. A prior report affirms that residue 3.32 is occupied by a negatively charged amino acid in biogenic amine receptors (D2 Dopamine, β2 Adrenergic and m3 Muscaranic) and that it is essential for recognition and binding of endogenous amines [40].…”

Chromenopyrazole, a Versatile Cannabinoid Scaffold with in Vivo Activity in a Model of Multiple Sclerosis

“…Binding site anchoring interactions within the receptor for each ligand were based on earlier published docking studies for HU210 43 and for AM-841. 44,45 …”