Palmitoylation of the rat mu opioid receptor.
We examined whether the mu opioid receptor was palmitoylated and attempted to determine sites of palmitoylation. Following metabolic labeling with [3H]palmitic acid and immunoaffinity purification of the mu opioid receptor, SDS-PAGE and fluorography revealed a broad labeled band with Mr of approximately 80 kDa in CHO cells stably expressing the rat mu receptor, but not in CHO cells transfected with the vector alone, indicating that the mu receptor is palmitoylated. Activation of the receptor with morphine did not affect the extent of palmitoylation. Hydroxylamine or dithiothreitol treatment removed most of the radioactivity, demonstrating that [3H]palmitic acid is incorporated into Cys residue(s) via thioester bond(s). Surprisingly, mutations of the only two Cys residues in the C-terminal domain did not reduce [3H]palmitic acid incorporation significantly. Thus, unlike many G-protein coupled receptors, the palmitoylation site(s) of the rat mu opioid receptor do(es) not reside in the C-terminal domain.
Chen
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Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Shahabi
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Xu
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Liu-Chen
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine; D004229:Dithiothreitol / Q000494:pharmacology; D019811:Hydroxylamine / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009020:Morphine / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D010446:Peptide Fragments; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D019811:Hydroxylamine; D019308:Palmitic Acid; D009020:Morphine; D003545:Cysteine; D004229:Dithiothreitol