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. 1986 Mar 1;102(3):932–942. doi: 10.1083/jcb.102.3.932

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes

PMCID: PMC2114122  PMID: 3512582

Abstract

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R- vesicles. After 2.5 min at 37 degrees C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37 degrees C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R- vesicles. A maximum of 30% of the ligand could be found in R- vesicles (14-44 min). When livers were maintained at 16 degrees C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R- vesicles at 37 degrees C remained there when livers were cooled to 16 degrees C. R- endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASOR-horseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R- structures were of lower density (1.09 g/cc), large (400- nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R- vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R- and represent the final prelysosomal compartment.

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Selected References

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  1. Bergeron J. J., Cruz J., Khan M. N., Posner B. I. Uptake of insulin and other ligands into receptor-rich endocytic components of target cells: the endosomal apparatus. Annu Rev Physiol. 1985;47:383–403. doi: 10.1146/annurev.ph.47.030185.002123. [DOI] [PubMed] [Google Scholar]
  2. Bridges K., Harford J., Ashwell G., Klausner R. D. Fate of receptor and ligand during endocytosis of asialoglycoproteins by isolated hepatocytes. Proc Natl Acad Sci U S A. 1982 Jan;79(2):350–354. doi: 10.1073/pnas.79.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carpentier J. L., Gorden P., Anderson R. G., Goldstein J. L., Brown M. S., Cohen S., Orci L. Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts. J Cell Biol. 1982 Oct;95(1):73–77. doi: 10.1083/jcb.95.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dickson R. B., Beguinot L., Hanover J. A., Richert N. D., Willingham M. C., Pastan I. Isolation and characterization of a highly enriched preparation of receptosomes (endosomes) from a human cell line. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5335–5339. doi: 10.1073/pnas.80.17.5335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drickamer K., Mamon J. F., Binns G., Leung J. O. Primary structure of the rat liver asialoglycoprotein receptor. Structural evidence for multiple polypeptide species. J Biol Chem. 1984 Jan 25;259(2):770–778. [PubMed] [Google Scholar]
  6. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  7. Dunn W. A., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Cell Biol. 1984 Jun;98(6):2148–2159. doi: 10.1083/jcb.98.6.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dunn W. A., LaBadie J. H., Aronson N. N., Jr Inhibition of 125I-asialofetuin catabolism by leupeptin in the perfused rat liver and in vivo. J Biol Chem. 1979 May 25;254(10):4191–4196. [PubMed] [Google Scholar]
  9. Dunn W. A., Wall D. A., Hubbard A. L. Use of isolated, perfused liver in studies of receptor-mediated endocytosis. Methods Enzymol. 1983;98:225–241. doi: 10.1016/0076-6879(83)98151-x. [DOI] [PubMed] [Google Scholar]
  10. Ehrenreich J. H., Bergeron J. J., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol. 1973 Oct;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  13. Geuze H. J., Slot J. W., Strous G. J., Luzio J. P., Schwartz A. L. A cycloheximide-resistant pool of receptors for asialoglycoproteins and mannose 6-phosphate residues in the Golgi complex of hepatocytes. EMBO J. 1984 Nov;3(11):2677–2685. doi: 10.1002/j.1460-2075.1984.tb02193.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geuze H. J., Slot J. W., Strous G. J., Schwartz A. L. The pathway of the asialoglycoprotein-ligand during receptor-mediated endocytosis: a morphological study with colloidal gold/ligand in the human hepatoma cell line, Hep G2. Eur J Cell Biol. 1983 Nov;32(1):38–44. [PubMed] [Google Scholar]
  15. Hanover J. A., Willingham M. C., Pastan I. Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes. Cell. 1984 Dec;39(2 Pt 1):283–293. doi: 10.1016/0092-8674(84)90006-0. [DOI] [PubMed] [Google Scholar]
  16. Harford J., Bridges K., Ashwell G., Klausner R. D. Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A pH-mediated nonlysosomal event. J Biol Chem. 1983 Mar 10;258(5):3191–3197. [PubMed] [Google Scholar]
  17. Harford J., Wolkoff A. W., Ashwell G., Klausner R. D. Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor. J Cell Biol. 1983 Jun;96(6):1824–1828. doi: 10.1083/jcb.96.6.1824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hubbard A. L., Bartles J. R., Braiterman L. T. Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies. J Cell Biol. 1985 Apr;100(4):1115–1125. doi: 10.1083/jcb.100.4.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hubbard A. L., Wall D. A., Ma A. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J Cell Biol. 1983 Jan;96(1):217–229. doi: 10.1083/jcb.96.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hubbard A. L., Wilson G., Ashwell G., Stukenbrok H. An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types. J Cell Biol. 1979 Oct;83(1):47–64. doi: 10.1083/jcb.83.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hudgin R. L., Pricer W. E., Jr, Ashwell G., Stockert R. J., Morell A. G. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem. 1974 Sep 10;249(17):5536–5543. [PubMed] [Google Scholar]
  22. Ito A., Palade G. E. Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes. Evidence obtained by immunoadsorption method. J Cell Biol. 1978 Nov;79(2 Pt 1):590–597. doi: 10.1083/jcb.79.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kawajiri K., Ito A., Omura T. Subfractionation of rat liver microsomes by immunoprecipitation and immunoadsorption methods. J Biochem. 1977 Mar;81(3):779–789. doi: 10.1093/oxfordjournals.jbchem.a131516. [DOI] [PubMed] [Google Scholar]
  24. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  27. Marsh M., Bolzau E., Helenius A. Penetration of Semliki Forest virus from acidic prelysosomal vacuoles. Cell. 1983 Mar;32(3):931–940. doi: 10.1016/0092-8674(83)90078-8. [DOI] [PubMed] [Google Scholar]
  28. Merisko E. M., Farquhar M. G., Palade G. E. Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells. J Cell Biol. 1982 Mar;92(3):846–857. doi: 10.1083/jcb.92.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miljanich G. P., Brasier A. R., Kelly R. B. Partial purification of presynaptic plasma membrane by immunoadsorption. J Cell Biol. 1982 Jul;94(1):88–96. doi: 10.1083/jcb.94.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Oka J. A., Weigel P. H. Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments. J Biol Chem. 1983 Sep 10;258(17):10253–10262. [PubMed] [Google Scholar]
  31. Pricer W. E., Jr, Ashwell G. Subcellular distribution of a mammalian hepatic binding protein specific for asialoglycoproteins. J Biol Chem. 1976 Dec 10;251(23):7539–7544. [PubMed] [Google Scholar]
  32. Pricer W. E., Jr, Ashwell G. The binding of desialylated glycoproteins by plasma membranes of rat liver. J Biol Chem. 1971 Aug 10;246(15):4825–4833. [PubMed] [Google Scholar]
  33. Quintart J., Courtoy P. J., Baudhuin P. Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins. J Cell Biol. 1984 Mar;98(3):877–884. doi: 10.1083/jcb.98.3.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Regoeczi E., Debanne M. T., Hatton M. C., Koj A. Elimination of asialofetuin and asialoorosomucoid by the intact rat. Quantitative aspects of the hepatic clearance mechanism. Biochim Biophys Acta. 1978 Jul 3;541(3):372–384. doi: 10.1016/0304-4165(78)90196-4. [DOI] [PubMed] [Google Scholar]
  35. Rekvig O. P., Hannestad K. Acid elution of blood group antibodies from intact erythrocytes. Vox Sang. 1977;33(5):280–285. doi: 10.1111/j.1423-0410.1977.tb04476.x. [DOI] [PubMed] [Google Scholar]
  36. Rodewald R. Distribution of immunoglobulin G receptors in the small intestine of the young rat. J Cell Biol. 1980 Apr;85(1):18–32. doi: 10.1083/jcb.85.1.18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rogers M. J., Strittmatter P. Lipid-protein interactions in the reconstitution of the microsomal reduced nicotinamide adenine dinucleotide-cytochrome b 5 reductase system. J Biol Chem. 1973 Feb 10;248(3):800–806. [PubMed] [Google Scholar]
  38. Roman L. M., Hubbard A. L. A domain-specific marker for the hepatocyte plasma membrane. II. Ultrastructural localization of leucine aminopeptidase to the bile canalicular domain of isolated rat liver plasma membranes. J Cell Biol. 1984 Apr;98(4):1488–1496. doi: 10.1083/jcb.98.4.1488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Roman L. M., Hubbard A. L. A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption. J Cell Biol. 1984 Apr;98(4):1497–1504. doi: 10.1083/jcb.98.4.1497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roman L. M., Hubbard A. L. A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain. J Cell Biol. 1983 Jun;96(6):1548–1558. doi: 10.1083/jcb.96.6.1548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Steer C. J., Ashwell G. Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem. 1980 Apr 10;255(7):3008–3013. [PubMed] [Google Scholar]
  42. Tanabe T., Pricer W. E., Jr, Ashwell G. Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J Biol Chem. 1979 Feb 25;254(4):1038–1043. [PubMed] [Google Scholar]
  43. Tolleshaug H. Binding and internalization of asialo-glycoproteins by isolated rat hepatocytes. Int J Biochem. 1981;13(1):45–51. doi: 10.1016/0020-711x(81)90135-x. [DOI] [PubMed] [Google Scholar]
  44. Tycko B., Keith C. H., Maxfield F. R. Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line. J Cell Biol. 1983 Dec;97(6):1762–1776. doi: 10.1083/jcb.97.6.1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wall D. A., Hubbard A. L. Galactose-specific recognition system of mammalian liver: receptor distribution on the hepatocyte cell surface. J Cell Biol. 1981 Sep;90(3):687–696. doi: 10.1083/jcb.90.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wall D. A., Hubbard A. L. Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments. J Cell Biol. 1985 Dec;101(6):2104–2112. doi: 10.1083/jcb.101.6.2104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wall D. A., Wilson G., Hubbard A. L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. doi: 10.1016/0092-8674(80)90116-6. [DOI] [PubMed] [Google Scholar]
  48. Warren R., Doyle D. Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes. J Biol Chem. 1981 Feb 10;256(3):1346–1355. [PubMed] [Google Scholar]
  49. Weigel P. H. Evidence that the hepatic asialoglycoprotein receptor is internalized during endocytosis and that receptor recycling can be uncoupled from endocytosis at low temperature. Biochem Biophys Res Commun. 1981 Aug 31;101(4):1419–1425. doi: 10.1016/0006-291x(81)91605-3. [DOI] [PubMed] [Google Scholar]
  50. Weigel P. H., Ray D. A., Oka J. A. Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells. Anal Biochem. 1983 Sep;133(2):437–449. doi: 10.1016/0003-2697(83)90106-9. [DOI] [PubMed] [Google Scholar]
  51. Wolkoff A. W., Klausner R. D., Ashwell G., Harford J. Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex. J Cell Biol. 1984 Feb;98(2):375–381. doi: 10.1083/jcb.98.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]

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