Abstract
The mu opiate receptor is a principal brain site for activities of morphine, other opiate drugs, and opioid peptides in modulating pain and altering mood. Recent cloning of cDNAs encoding rat and human mu receptors 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-terminal 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 deletion of 33 C-terminal amino acids yielded a receptor at which morphine, but not the substituted enkephalin DAMGO ([D-Ala2,MePhe4,Glyol5]enkephalin), inhibited adenylate cyclase. Alanine substitution for each charged TM residue in the N-terminally deleted receptor reduced affinities for morphine, DAMGO, and the opiate antagonist naloxone. Replacement of TM II Asp114 with asparagine or glutamic acid increased mu receptor affinity for naloxone. TM II and TM III glutamic acid substitutions for Asp114 and Asp147 reduced agonist binding affinities but allowed full inhibition of adenylate cyclase at high agonist concentrations. TM VI histidine substitution with alanine yielded a receptor that produced almost twice the cyclase inhibition displayed by the wild type receptor in parallel transient expression assays. These findings underscore the importance of charged residues in TM II, III, and VI for different receptor functions and the modest involvement of extensive portions of N- and C-terminal receptor domains in these processes.
Highlights
From the Molecular Neurobiology Branch and Office of the Director, Intramural Research Program, National Institute on Drug Abuse and the Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224
~l~lenkephalini)n, hibited adenylate cyclase.Alanine tors, reveals that each is a member of the seven-transmemsubstitution for each charged TM residue in the N-ter- brane domain G protein-linked receptor family[1,16,17,18,19,20,21]
We report the resultof initial char- against a glutaraldehydeconjugate of the C-terminal 18 amino acids of acterization of the structurelfunction relationships of the p opi- the rat pORrleceptor and keyhole limpet hemocyanin recognized p opiate receptor (pOR)
Summary
Limpet hemocyanin using glutaraldehyde as described previously [33]. Three rabbits were immunized with subdermal and intramuscular in-. Control ing each mutation were isolated by agarose gel electrophoresis and experiments included staining of paraformaldehyde-fixed brain secsubcloned into AN64 previously digested with the appropriate restric- tions in the samefashion, use of preimmune sera, omission of secondary tion enzymes, and thesubcloned fragments were resequenced to verify antibodies, preincubation in which the C-terminal peptide or an irrelthe absence of inadvertant mutations. Binding densities were normalized to total protein concentration, determined by the Bradford Both the wild type p opiate receptor, pOR1, and the N-terminal deletion mutant AN64 (termed WT,; see below)expressed robust binding ofDAMGO and naloxone as well as method (Bio-Rad). COS cells were combined with various labeled and morphine- and DAMGO-mediated inhibition of forskolin-
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