Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB2 receptor
Paul Gouldson, Bernard Calandra, Pascale Legoux, Aimée Kernéis, Murielle Rinaldi-Carmona, Francis Barth, Gérard Le Fur, Pascual Ferrara, David Shire
We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)-pyrazole-3-carboxamide) on the human cannabinoid CB2 receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser161 and Ser165, were singly mutated to the cognate cannabinoid CB1 receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB2 receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB2 receptor-specific residue, Cys175 resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.