BETA-[H-3]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-[H-3] funaltre xamine (beta-[H-3]FNA) covalently labeled mu-opioid receptors with hig h specificity in bovine striatal membranes. Beta-[H-3]FNA-labeled mu-o pioid receptors migrated as a broad band with a molecular mass range o f 68-97 kDa. It is controversial whether beta-FNA binds irreversibly t o mu-opioid receptors in other species. In this study, we demonstrated that beta-[H-3]FNA also labeled mu-opioid receptors with high specifi city in brain membranes of the guinea pig, rat, and mouse. Sodium dode cyl sulfate-polyacrylamide gel electrophoresis and fluorography reveal ed that in each species beta-[H-3]FNA specifically bound to a protein in which labeling was greatly reduced by naloxone. These labeled recep tors had broad molecular mass ranges, and the molecular masses were di fferent 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-Acetylglucosam ine eluted a peak of radioactivity. Sodium dodecyl sulfate-polyacrylam ide gel electrophoresis and fluorography showed that in all four speci es the mu receptor was the only protein labeled with beta-[H-3]FNA in the WGL eluate. The molecular masses of labeled mu-opioid receptors we re 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 n ature of the carbohydrate moieties linked to the receptor protein and whether the species variation in the molecular mass was due to variabl e degrees of glycosylation. The bovine WGL eluate was treated with var ious glycosidases. Neuraminidase treatment decreased the receptor mole cular mass by 6-7 kDa, whereas alpha-mannosidase had no effect. Remova l of N-linked carbohydrates at asparagine residues by peptide-N4-N-ace tyl-beta-glucosaminyl]asparagine amidase (N-Glycanase) resulted in a m uch sharper specifically labeled protein band of 43 kDa. These results indicate that mu-opioid receptors are heavily glycosylated and the ma jor carbohydrate moieties are of the complex type, N-linked to asparag ine. After the WGL eluates for the four species were treated with N-Gl ycanase, the labeled receptors became much sharper bands with very sim ilar molecular masses, i.e., 43 kDa for the cow and guinea pig, 39 kDa for the rat, and and 40 kDa for the mouse. These results indicate tha t the variation among species in the molecular mass of beta-[H-3] FNA- labeled mu-opioid receptors is mainly the result of variable degrees o f glycosylation.