Beta-[3H]funaltrexamine-labeled mu-opioid receptors: species variations in molecular mass and glycosylation by complex-type, N-linked oligosaccharides.
We previously showed that under defined conditions beta-[3H]funaltrexamine (beta-[3H]FNA) covalently labeled mu-opioid receptors with high specificity in bovine striatal membranes. beta-[3H]FNA-labeled mu-opioid receptors migrated as a broad band with a molecular mass range of 68-97 kDa. It is controversial whether beta-FNA binds irreversibly to mu-opioid receptors in other species. In this study, we demonstrated that beta-[3H]FNA also labeled mu-opioid receptors with high specificity in brain membranes of the guinea pig, rat, and mouse. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed that in each species beta-[3H]FNA specifically bound to a protein in which labeling was greatly reduced by naloxone. These labeled receptors had broad molecular mass ranges, and the molecular masses were different among these species, in the order of cow > guinea pig > rat > mouse. Membranes were subjected to solubilization with 2% Triton X-100 and wheat germ lectin (WGL) affinity chromatography. N-Acetylglucosamine eluted a peak of radioactivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that in all four species the mu receptor was the only protein labeled with beta-[3H]FNA in the WGL eluate. The molecular masses of labeled mu-opioid receptors were 70-88 kDa (median, 77 kDa) for the cow, 66-80 kDa (median, 72 kDa) for the guinea pig, 60-75 kDa (median, 67 kDa) for the rat, and 60-72 kDa (median, 66 kDa) for the mouse. In addition, we investigated the nature of the carbohydrate moieties linked to the receptor protein and whether the species variation in the molecular mass was due to variable degrees of glycosylation. The bovine WGL eluate was treated with various glycosidases. Neuraminidase treatment decreased the receptor molecular mass by 6-7 kDa, whereas alpha-mannosidase had no effect. Removal of N-linked carbohydrates at asparagine residues by peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase (N-Glycanase) resulted in a much sharper specifically labelled protein band of 43 kDa. These results indicate that mu-opioid receptors are heavily glycosylated and the major carbohydrate moieties are of the complex type, N-linked to asparagine. After the WGL eluates for the four species were treated with N-Glycanase, the labeled receptors became much sharper bands with very similar molecular masses, i.e., 43 kDa for the cow and guinea pig, 39 kDa for the rat, and and 40 kDa for the mouse.(ABSTRACT TRUNCATED AT 400 WORDS)
Liu-Chen
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Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Chen
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Phillips
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D000581:Amidohydrolases / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002417:Cattle; D002846:Chromatography, Affinity; D003342:Corpus Striatum / Q000378:metabolism / Q000648:ultrastructure; D005470:Fluorometry; D006031:Glycosylation; D006168:Guinea Pigs; D008361:Mannosidases / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008970:Molecular Weight; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000378:metabolism; D009439:Neuraminidase / Q000494:pharmacology; D009844:Oligosaccharides / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D017207:Rats, Sprague-Dawley; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility; D013045:Species Specificity; D014316:Tritium; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D017450:Receptors, Opioid, mu; D014316:Tritium; D009271:Naltrexone; C024524:beta-funaltrexamine; D008361:Mannosidases; D009439:Neuraminidase; D043323:alpha-Mannosidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase