Functional role of tryptophan residues in the fourth transmembrane domain of the CB2 cannabinoid receptor

Several tryptophan (Trp) residues are conserved in G protein-coupled recept ors (GPCRs). Relatively little is known about the contribution of these res idues and especially of those in the fourth transmembrane domain in the fun ction of the CB2 cannabinoid receptor. Replacing W158 (very highly conserve d in GPCRs) and W172 (conserved in CB1 and CB2 cannabinoid receptors but no t in many other GPCRs) of the human CB2 receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutati ng W172 to A or L eliminated agonist binding, W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wi ld-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at posit ions 158 and 172 seem to have a critical, but different, role in cannabinoi d binding to the human CB2 receptor.