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. 1984 Jun 1;98(6):2148–2159. doi: 10.1083/jcb.98.6.2148

Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics

PMCID: PMC2113050  PMID: 6327725

Abstract

We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high- affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.

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Selected References

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  1. Aronson N. N., Jr, Dennis P. A., Dunn W. A. Metabolism of leupeptin and its effect on autophagy in the perfused rat liver. Acta Biol Med Ger. 1981;40(10-11):1531–1538. [PubMed] [Google Scholar]
  2. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  3. Beaufay H., Amar-Costesec A., Feytmans E., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J Cell Biol. 1974 Apr;61(1):188–200. doi: 10.1083/jcb.61.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burger R. L., Schneider R. J., Mehlman C. S., Allen R. H. Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12. J Biol Chem. 1975 Oct 10;250(19):7707–7713. [PubMed] [Google Scholar]
  6. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carpenter G. Solubilization of membrane receptor for epidermal growth factor. Life Sci. 1979 Apr 30;24(18):1691–1697. doi: 10.1016/0024-3205(79)90254-6. [DOI] [PubMed] [Google Scholar]
  8. Carpentier J. L., Gorden P., Barazzone P., Freychet P., Le Cam A., Orci L. Intracellular localization of 125I-labeled insulin in hepatocytes from intact rat liver. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2803–2807. doi: 10.1073/pnas.76.6.2803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Das M., Fox C. F. Molecular mechanism of mitogen action: processing of receptor induced by epidermal growth factor. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2644–2648. doi: 10.1073/pnas.75.6.2644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Desbuquois B., Lopez S., Burlet H. Ligand-induced translocation of insulin receptors in intact rat liver. J Biol Chem. 1982 Sep 25;257(18):10852–10860. [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. Fehlmann M., Carpentier J. L., Van Obberghen E., Freychet P., Thamm P., Saunders D., Brandenburg D., Orci L. Internalized insulin receptors are recycled to the cell surface in rat hepatocytes. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5921–5925. doi: 10.1073/pnas.79.19.5921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. FitzGerald D. J., Padmanabhan R., Pastan I., Willingham M. C. Adenovirus-induced release of epidermal growth factor and pseudomonas toxin into the cytosol of KB cells during receptor-mediated endocytosis. Cell. 1983 Feb;32(2):607–617. doi: 10.1016/0092-8674(83)90480-4. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Galloway C. J., Dean G. E., Marsh M., Rudnick G., Mellman I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334–3338. doi: 10.1073/pnas.80.11.3334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  19. Goldstein J. L., Basu S. K., Brunschede G. Y., Brown M. S. Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans. Cell. 1976 Jan;7(1):85–95. doi: 10.1016/0092-8674(76)90258-0. [DOI] [PubMed] [Google Scholar]
  20. Higa Y., Oshiro S., Kino K., Tsunoo H., Nakajima H. Catabolism of globin-haptoglobin in liver cells after intravenous administration of hemoglobin-haptoglobin to rats. J Biol Chem. 1981 Dec 10;256(23):12322–12328. [PubMed] [Google Scholar]
  21. Howell K. E., Ito A., Palade G. E. Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean? J Cell Biol. 1978 Nov;79(2 Pt 1):581–589. doi: 10.1083/jcb.79.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hubbard A. L., Cohn Z. A. The enzymatic iodination of the red cell membrane. J Cell Biol. 1972 Nov;55(2):390–405. doi: 10.1083/jcb.55.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Josefsberg Z., Posner B. I., Patel B., Bergeron J. J. The uptake of prolactin into female rat liver. Concentration of intact hormone in the Golgi apparatus. J Biol Chem. 1979 Jan 10;254(1):209–214. [PubMed] [Google Scholar]
  26. Kaplan J. Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages. Cell. 1980 Jan;19(1):197–205. doi: 10.1016/0092-8674(80)90401-8. [DOI] [PubMed] [Google Scholar]
  27. Khairallah E. A., Mortimore G. E. Assessment of protein turnover in perfused rat liver. Evidence for amino acid compartmentation from differential labeling of free and tRNA-gound valine. J Biol Chem. 1976 Mar 10;251(5):1375–1384. [PubMed] [Google Scholar]
  28. Krupp M. N., Connolly D. T., Lane M. D. Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts. J Biol Chem. 1982 Oct 10;257(19):11489–11496. [PubMed] [Google Scholar]
  29. Krupp M., Lane M. D. On the mechanism of ligand-induced down-regulation of insulin receptor level in the liver cell. J Biol Chem. 1981 Feb 25;256(4):1689–1694. [PubMed] [Google Scholar]
  30. LaBadie J. H., Chapman K. P., Aronson N. N., Jr Glycoprotein catabolism in rat liver: Lysosomal digestion of iodinated asialo-fetuin. Biochem J. 1975 Nov;152(2):271–279. doi: 10.1042/bj1520271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McKanna J. A., Haigler H. T., Cohen S. Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5689–5693. doi: 10.1073/pnas.76.11.5689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moriarity D. M., Savage C. R., Jr Interaction of epidermal growth factor with adult rat liver parenchymal cells in primary culture. Arch Biochem Biophys. 1980 Sep;203(2):506–518. doi: 10.1016/0003-9861(80)90208-8. [DOI] [PubMed] [Google Scholar]
  33. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  34. Ohkuma S., Moriyama Y., Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982 May;79(9):2758–2762. doi: 10.1073/pnas.79.9.2758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prieels J. P., Pizzo S. V., Glasgow L. R., Paulson J. C., Hill R. L. Hepatic receptor that specifically binds oligosaccharides containing fucosyl alpha1 leads to 3 N-acetylglucosamine linkages. Proc Natl Acad Sci U S A. 1978 May;75(5):2215–2219. doi: 10.1073/pnas.75.5.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roth T. F., Cutting J. A., Atlas S. B. Protein transport: a selective membrane mechanism. J Supramol Struct. 1976;4(4):527–548. doi: 10.1002/jss.400040413. [DOI] [PubMed] [Google Scholar]
  37. Savage C. R., Jr, Cohen S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J Biol Chem. 1972 Dec 10;247(23):7609–7611. [PubMed] [Google Scholar]
  38. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  39. Smith A., Morgan W. T. Hemopexin-mediated transport of heme into isolated rat hepatocytes. J Biol Chem. 1981 Nov 10;256(21):10902–10909. [PubMed] [Google Scholar]
  40. St Hilaire R. J., Jones A. L. Epidermal growth factor: its biologic and metabolic effects with emphasis on the hepatocyte. Hepatology. 1982 Sep-Oct;2(5):601–613. doi: 10.1002/hep.1840020515. [DOI] [PubMed] [Google Scholar]
  41. Stahl P., Schlesinger P. H., Sigardson E., Rodman J. S., Lee Y. C. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell. 1980 Jan;19(1):207–215. doi: 10.1016/0092-8674(80)90402-x. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Taylor J. M., Mitchell W. M., Cohen S. Epidermal growth factor. Physical and chemical properties. J Biol Chem. 1972 Sep 25;247(18):5928–5934. [PubMed] [Google Scholar]
  44. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Wiley H. S., Cunningham D. D. A steady state model for analyzing the cellular binding, internalization and degradation of polypeptide ligands. Cell. 1981 Aug;25(2):433–440. doi: 10.1016/0092-8674(81)90061-1. [DOI] [PubMed] [Google Scholar]

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