Mutagenesis of a single amino acid in the rat μ-opioid receptor discriminates ligand binding

To investigate the role of Asp(114) in the cloned rat mu-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant mu receptor D114N. The wild-type mu receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The mu-selective agonists [D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin and morphine and the endogenous peptides Met-enkephalin and beta-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type mu receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant mu receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N mu receptors to inhibit adenylyl cyclase. Although the Asp(114) to Asn(114) mutation reduced full-agonist binding, mutation of His(297) to Asn(297) in the mu receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the mu receptor than do the prototypical full agonists.