Thr353, located within the COOH-terminal tail of the delta opiate receptor, is involved in receptor down-regulation.
Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down-regulation is not known. In order to address this, we generated a number of mutants of the delta opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down-regulation. The receptor with a mutation of Thr353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr353 is critical for the agonist-mediated down-regulation of the delta opiate receptor.
Cvejic
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S
Department of Pharmacology, New York University Medical Center, New York, New York 10016, USA.
Trapaidze
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Cyr
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Devi
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D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002999:Clone Cells; D006224:Cricetinae; D004174:Diprenorphine / Q000378:metabolism; D015536:Down-Regulation; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017465:Receptors, Opioid, delta / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016324:Sequence Tagged Sites; D013912:Threonine; D014162:Transfection
D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D004174:Diprenorphine; D013912:Threonine; D016308:Enkephalin, Leucine-2-Alanine; D000409:Alanine