The Leu/Val(6.51) Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ(9)-Tetrahydrocannabinol

[Image: see text] (−)-Δ(9)-trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, mediates its action by binding to two members of the G-protein-coupled receptor (GPCR) family: the cannabinoid CB(1) (CB(1)R) and CB(2) (CB(2)R) receptors. Molecular dynamics simulations showed that the pentyl chain of THC could adopts an I-shape conformation, filling an intracellular cavity between Phe(3.36) and Trp(6.48) for initial agonist-induced receptor activation, in CB(1)R but not in CB(2)R. This cavity opens to the five-carbon chain of THC by the conformational change of the γ-branched, flexible, Leu(6.51) side chain of CB(1)R, which is not feasible by the β-branched, mode rigid, Val(6.51) side chain of CB(2)R. In agreement with our computational results, THC could not decrease the forskolin-induced cAMP levels in cells expressing mutant CB(1)R(L6.51V) receptor but could activate the mutant CB(2)R(V6.51L) receptor as efficiently as wild-type CB(1)R. Additionally, JWH-133, a full CB(2)R agonist, contains a branched dimethyl moiety in the ligand chain that bridges Phe(3.36) and Val(6.51) for receptor activation. In this case, the substitution of Val(6.51) to Leu in CB(2)R makes JWH-133 unable to activate CB(2)R(V6.51L). In conclusion, our combined computational and experimental results have shown that the amino acid at position 6.51 is a key additional player in the initial mechanism of activation of GPCRs that recognize signaling molecules derived from lipid species.