title: Agonist-induced desensitization of the mu opioid receptor is determined by threonine 394 preceded by acidic amino acids in the COOH-terminal tail.
abstract: To identify the structural determinants necessary for mu opioid receptor desensitization, we serially ablated potential phosphorylation sites in the carboxyl tail of the receptor and examined their effects on [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO)-induced desensitization. First, we replaced Thr394 with alanine (T394A) and stably expressed this mutant receptor in Chinese hamster ovary cells. The T394A receptor did not desensitize after 1 h of treatment with DAMGO, indicating that Thr394 is required for agonist-induced early desensitization. To test whether Thr394 was the only residue necessary, we investigated the importance of 7 potential phosphorylation sites between residues 363 and 383, which were all replaced by alanines with the Thr394 maintained. This mutant (AT) showed partial loss of desensitization (30%), which was attributable to the Ala mutation at Thr383, since complete desensitization was achieved by restoring Thr383 (ATT). These results suggest that Thr394 is the primary recognition site for G protein-coupled receptor kinases, but Thr383 is also required for complete agonist-induced desensitization. The specificity of Thr394 as the primary initiation site appears to be dependent on the preceding acidic amino acid stretch, because in a mutant in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines (EQ), agonist-induced desensitization was completely abolished, identical to the T394A mutant.
authors.affiliation: Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Y
authors.initials: Y
authors.lastname: Pak
authors.forename: B F
authors.initials: BF
authors.lastname: O'Dowd
authors.forename: S R
authors.initials: SR
authors.lastname: George
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009270:Naloxone / Q000378:metabolism; D017354:Point Mutation; D051381:Rats; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013912:Threonine; D014162:Transfection
chemical_list: D004745:Enkephalins; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D013912:Threonine; D009270:Naloxone; D012694:Serine; D000409:Alanine