A LYSINE RESIDUE OF THE CANNABINOID RECEPTOR IS CRITICAL FOR RECEPTORRECOGNITION BY SEVERAL AGONISTS BUT NOT WIN55212-2

Lys(192) in the third transmembrane domain of the human CB1 cannabinoi d receptor was converted to an alanine to study its role in receptor r ecognition and activation by agonists. HU-210, CP-55940, WIN55212-2, a nd anandamide, four cannabinoid agonists with distinct chemical struct ures, were used to characterize the wild-type and the mutant receptors . In human embryonal kidney 293 cells stably expressing the wild-type receptor, specific binding to [H-3]WIN55212-2 and inhibition of cAMP a ccumulation by cannabinoid agonists were demonstrated, with different ligands exhibiting the expected rank orders of potency and stereoselec tivity in competition binding and functional assays. In cells expressi ng the mutant receptor, the binding affinity of the receptor for [H-3] WIN55212-2 was only slightly affected (the K-d for the mutant receptor was twice that of the wild-type), and the ability of WIN55212-2 to in hibit cAMP accumulation was unchanged. However, HU-210, CP-55940, and anandamide were unable to compete for [H-3]WIN55212-2 binding to the m utant receptor. In addition, the potencies of HU-210, CP-55940, and an andamide in inhibiting cAMP accumulation were reduced by >100-fold. Th ese results demonstrate that Lys(192) is critical for receptor binding by HU-210, CP-55940, and anandamide. Because Lys(192) is not importan t for receptor binding and activation by WIN55212-2, WIN55212-2 must i nteract with the cannabinoid receptor through at least one point of in teraction that is distinct from those of the three other agonists.