Expression of the chicken hepatic glycoprotein receptor in Xenopus oocytes: conservation of ligand and receptor targeting signals.
We have obtained expression of the beta-N-acetylglucosamine-binding receptor from chicken hepatocytes in Xenopus oocytes by injecting mRNA synthesized in vitro from a full length cDNA cloned into an expression vector (Mellow et al: J. Biol Chem 263: 5468-5473, 1988). Immunoprecipitation of the receptor after labeling of oocytes with [35S]-methionine for times ranging from 6 to 72 h revealed 4-5 closely spaced bands of 25-30 kDa after SDS-PAGE. Although these bands were largely resistant to endoglycosidase H cleavage, endoglycosidase F reduced the size of all bands to a single species at 23-24 kDa, indicating that they resulted from heterogeneity in glycosylation of a single polypeptide. Incubation of oocytes expressing this receptor with [125I]-GlcNAc-BSA resulted in 1.8 to 10 x higher levels of cell-associated ligand in mRNA-injected vs. water-injected control oocytes, 2-35% of cell-associated counts was removed by EGTA rinse at 20 degrees C, suggesting that most ligand was inaccessible (presumably intracellular). Immunoprecipitation of sucrose gradient fractions detected receptor molecules predominantly in a light organelle at 1.09-1.12 g/cc (the density of early endosomes and plasma membrane vesicles), with no evidence of the receptor in much heavier yolk platelet fractions even in the presence of ligand. In contrast, internalized [125I]-GlcNAc-BSA was found either at the top of the gradients or in organelles at 1.09-1.17 g/cc and in yolk platelets. TCA precipitation indicated that much intracellular ligand was degraded to acid-soluble fragments. Addition of vitellogenin (the yolk protein precursor) to the medium together with the [125I]-GlcNAc-BSA shifted much of the ligand into yolk platelets. These data indicate that the chicken glycoprotein receptor expressed in oocytes mediates binding and internalization of this ligand into an organelle in which ligand-receptor dissociation occurs, allowing for separation of these two molecules into different compartments. The behavior of ligand in Xenopus oocytes expressing the chicken receptor closely resembles its behavior in hepatocytes.
Wall
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DA
Department of Drug Delivery, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.
Holguin
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D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D003001:Cloning, Molecular; D004530:Egg Yolk / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D006031:Glycosylation; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D009865:Oocytes; D011971:Receptors, Immunologic / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014415:Tunicamycin / Q000494:pharmacology; D014819:Vitellogenins / Q000494:pharmacology; D014981:Xenopus
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D008024:Ligands; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D014819:Vitellogenins; D014415:Tunicamycin