hu-243 and glyceryl-2-arachidonate

Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB(2) cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB(1) and CB(2) cannabinoid receptors but not in many other GPCRs) of the human CB(2) 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 mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB(2) receptor.

Topics: Amino Acid Substitution; Animals; Arachidonic Acids; Benzoxazines; Binding Sites; Binding, Competitive; Cannabinoids; COS Cells; Dronabinol; Endocannabinoids; Excitatory Amino Acid Antagonists; Glycerides; Humans; Morpholines; Mutagenesis, Site-Directed; Naphthalenes; Protein Structure, Tertiary; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction; Structure-Activity Relationship; Transfection; Tryptophan