-MU OPIATE RECEPTOR - CHARGED TRANSMEMBRANE DOMAIN AMINO-ACIDS ARE CRITICAL FOR AGONIST RECOGNITION AND INTRINSIC ACTIVITY

The mu opiate receptor is a principal brain site for activities of mor phine, other opiate drugs, and opioid peptides in modulating pain and altering mood. Recent cloning of cDNAs encoding rat and human mu recep tors reveals charged amino acid residues within putative transmembrane domains (TMs) II, III, and VI, a substantial N-terminal extracellular domain, and a C-terminal intracellular domain. Deletion of 64 N-termi nal amino acids produced little effect on receptor function (Wang, J. B., Imai, Y., Eppler, C. M., Gregor, P., Spivak, C. E., and Uhl, G. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10230-10234). Further dele tion of 33 C-terminal amino acids yielded a receptor at which morphine , but not the substituted enkephalin DAMGO ([D-Ala(2),MePhe(4),Gly-ol( 5)]enkephalin), inhibited adenylate cyclase. Alanine substitution for each charged TM residue in the N-terminally deleted receptor reduced a ffinities for morphine, DAMGO, and the opiate antagonist naloxone. Rep lacement of TM II Asp(114) with asparagine or glutamic acid increased p receptor affinity for naloxone. TM II and TM III glutamic acid subst itutions for Asp(114) and Asp(147) reduced agonist binding affinities but allowed full inhibition of adenylate cyclase at high agonist conce ntrations. TM VI histidine substitution with alanine yielded a recepto r that produced almost twice the cyclase inhibition displayed by the w ild type receptor in parallel transient expression assays. These findi ngs underscore the importance of charged residues in TM II, III, and V I for different receptor functions and the modest involvement of exten sive portions of N and C-terminaI receptor domains in these processes.