Ligand-Binding Architecture of Human CB2 Cannabinoid Receptor: Evidence for Receptor Subtype-Specific Binding Motif and Modeling GPCR Activation

Abstract

SUMMARY The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, “ligand-assisted protein structure” (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.

A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor

“…These studies have shown that the C-3 side chain of classical cannabinoids is aligned parallel with the membrane acyl chains (60,62) and that the fatty acid chain of endogenous cannabinoids orients parallel to membrane acyl chains with terminal methyl near the center of the bilayer (61). Cannabinoid ligand entry at the TMH6/7 interface is supported by isothiocyanate labeling studies of CB2 using the classical cannabinoid, AM841 functionalized at the C-3 dimethylheptyl side chain terminal carbon (1). Despite the fact that C7.42(288) faces into the CB2 binding pocket and would be a likely covalent attachment site if the ligand entered the CB2 binding pocket in the traditional way (from extracellular), AM841 was found to selectively label only one Cys residue, C6.47(257) (1).…”

“…A discussion of these differences as well as model construction details are provided in our recent paper (33). The resulting model has been tested via mutation (34), substituted cysteine accessibility (33, 35), and isothiocyanate labeling studies (1).…”

“…Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, AM841, enters the CB2 receptor via the lipid bilayer (1). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated.…”

(4-(Bis(4-Fluorophenyl)Methyl)Piperazin-1-yl)(Cyclohexyl)Methanone Hydrochloride (LDK1229): A New Cannabinoid CB1Receptor Inverse Agonist from the Class of Benzhydryl Piperazine Analogs

“…Instead, rhodopsin/opsin have been reported to have lipid portals that are used for entry and exit via the lipid bilayer for 11-cis-retinal/trans-retinal as they are shuttled into and out of the receptor (Hildebrand et al, 2009). There is evidence from simulations (Hurst et al, 2010) and from experimental covalent labeling studies (Picone et al, 2005;Pei et al, 2008) that the cannabinoid receptors also possess a portal between transmembrane helix (TMH) 6 and TMH7 through which ligands enter. 3) In addition, the cannabinoid receptors and rhodopsin share an unusual GWNC amino acid sequence motif at the extracellular end of TMH4.…”

G-Protein Coupled Receptors: Experimental and Computational Approaches

“…The 3D structures of CB1 and CB2 have also been modeled using Rhodopsin as template. (Pei, Y., Mercier, R. W., Anday, J. K., Thakur, G. A., Zvonok, A. M., Hurst, D. et al 2008) Another GPCR which has been characterized by homology modeling techniques was melatonin receptor. This receptor is responsible for the effects of melatonin, a compound taking part in resynchronization of biological rhythms such as sleep.…”