LIGAND-BINDING AND MODULATION OF CYCLIC-AMP LEVELS DEPEND ON THE CHEMICAL NATURE OF RESIDUE-192 OF THE HUMAN CANNABINOID RECEPTOR-1

The human cannabinoid receptor associated with the CNS (CB1) binds Del ta(9)-tetrahydrocannabinol, the psychoactive component of marijuana, a nd other cannabimimetic compounds. This receptor is a member of the se ven transmembrane domain G protein-coupled receptor family and mediate s its effects through inhibition of adenylyl cyclase. An understanding of the molecular mechanisms involved in ligand binding and receptor a ctivation requires identification of the active site residues and thei r role. Lys(192) of the third transmembrane domain of the receptor is noteworthy because it is the only nonconserved, charged residue in the transmembrane region. To investigate the properties of this residue, which are important for both ligand binding and receptor activation, w e generated mutant receptors in which this amino acid was changed to e ither Arg (K192R), Gln (K192Q), or Glu (K192E). Wild-type and mutant-r eceptors were stably expressed in Chinese hamster ovary cells and were evaluated in binding assays with the bicyclic cannabinoid CP-55,940 a nd the aminoalkylindole WIN 55,212-2. We found that only the most cons ervative change of Lys to Arg allowed retention of binding affinity to CP-55,940, whereas WIN 55,212-2 bound to all of the mutant receptors in,the same range as it bound the wild type. Analysis of the ligand-in duced inhibition of cyclic AMP production in cells expressing each of the receptors gave an EC50 value for each agonist that was comparable to its binding affinity, with one exception. Although the mutant K192E receptor displayed similar binding affinity as the wild type with WIN 55,212-2, an order of magnitude difference was observed for the EC50 for cyclic AMP inhibition with this compound. The results of this stud y indicate that binding of CP-55,940 is highly sensitive to the chemic al nature of residue 192. In contrast, although this residue is not cr itical for WIN 55,212-2 binding, the data suggest a role for Lys(192) in WIN 55,212-2-induced receptor activation.