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. 1989 Dec 1;109(6):2741–2749. doi: 10.1083/jcb.109.6.2741

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo [published erratum appears in J Cell Biol 1990 Jan;110(1):following 227]

PMCID: PMC2115960  PMID: 2592403

Abstract

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

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Selected References

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  1. Baudhuin P., Evrard P., Berthet J. Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles. J Cell Biol. 1967 Jan;32(1):181–191. doi: 10.1083/jcb.32.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benveniste R., Carson S. A. Binding characteristics of epidermal growth factor receptors in male and female rat liver cell membrane preparations. Mol Cell Endocrinol. 1985 Jul;41(2-3):147–151. doi: 10.1016/0303-7207(85)90017-6. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bergeron J. J., Paiement J., Khan M. N., Smith C. E. Terminal glycosylation in rat hepatic Golgi fractions: heterogeneous locations for sialic acid and galactose acceptors and their transferases. Biochim Biophys Acta. 1985 Dec 19;821(3):393–403. doi: 10.1016/0005-2736(85)90043-4. [DOI] [PubMed] [Google Scholar]
  5. Bergeron J. J., Rachubinski R. A., Sikstrom R. A., Posner B. I., Paiement J. Galactose transfer to endogenous acceptors within Golgi fractions of rat liver. J Cell Biol. 1982 Jan;92(1):139–146. doi: 10.1083/jcb.92.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Brodsky F. M. Living with clathrin: its role in intracellular membrane traffic. Science. 1988 Dec 9;242(4884):1396–1402. doi: 10.1126/science.2904698. [DOI] [PubMed] [Google Scholar]
  8. Brown M. S., Anderson R. G., Goldstein J. L. Recycling receptors: the round-trip itinerary of migrant membrane proteins. Cell. 1983 Mar;32(3):663–667. doi: 10.1016/0092-8674(83)90052-1. [DOI] [PubMed] [Google Scholar]
  9. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem. 1987;56:881–914. doi: 10.1146/annurev.bi.56.070187.004313. [DOI] [PubMed] [Google Scholar]
  10. Carpentier J. L., White M. F., Orci L., Kahn R. C. Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells. J Cell Biol. 1987 Dec;105(6 Pt 1):2751–2762. doi: 10.1083/jcb.105.6.2751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cohen S., Fava R. A. Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells. J Biol Chem. 1985 Oct 5;260(22):12351–12358. [PubMed] [Google Scholar]
  12. Cuatrecasas P. Isolation of the insulin receptor of liver and fat-cell membranes (detergent-solubilized-( 125 I)insulin-polyethylene glycol precipitation-sephadex). Proc Natl Acad Sci U S A. 1972 Feb;69(2):318–322. doi: 10.1073/pnas.69.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Dunn W. A., Connolly T. P., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol. 1986 Jan;102(1):24–36. doi: 10.1083/jcb.102.1.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Karnovsky M. J. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol. 1967 Oct;35(1):213–236. doi: 10.1083/jcb.35.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kay D. G., Lai W. H., Uchihashi M., Khan M. N., Posner B. I., Bergeron J. J. Epidermal growth factor receptor kinase translocation and activation in vivo. J Biol Chem. 1986 Jun 25;261(18):8473–8480. [PubMed] [Google Scholar]
  20. Khan M. N., Baquiran G., Brule C., Burgess J., Foster B., Bergeron J. J., Posner B. I. Internalization and activation of the rat liver insulin receptor kinase in vivo. J Biol Chem. 1989 Aug 5;264(22):12931–12940. [PubMed] [Google Scholar]
  21. Khan M. N., Savoie S., Bergeron J. J., Posner B. I. Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures. J Biol Chem. 1986 Jun 25;261(18):8462–8472. [PubMed] [Google Scholar]
  22. King A. C., Cuatrecasas P. Peptide hormone-induced receptor mobility, aggregation, and internalization. N Engl J Med. 1981 Jul 9;305(2):77–88. doi: 10.1056/NEJM198107093050206. [DOI] [PubMed] [Google Scholar]
  23. King A. C., Willis R. A., Cuatrecasas P. Accumulation of epidermal growth factor within cells does not depend on receptor recycling. Biochem Biophys Res Commun. 1980 Dec 16;97(3):840–845. doi: 10.1016/0006-291x(80)91453-9. [DOI] [PubMed] [Google Scholar]
  24. Korc M., Magun B. E. Recycling of epidermal growth factor in a human pancreatic carcinoma cell line. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6172–6175. doi: 10.1073/pnas.82.18.6172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lai W. H., Guyda H. J., Bergeron J. J. Binding and internalization of epidermal growth factor in human term placental cells in culture. Endocrinology. 1986 Jan;118(1):413–423. doi: 10.1210/endo-118-1-413. [DOI] [PubMed] [Google Scholar]
  26. Lai W. H., Guyda H. J. Characterization and regulation of epidermal growth factor receptors in human placental cell cultures. J Clin Endocrinol Metab. 1984 Feb;58(2):344–352. doi: 10.1210/jcem-58-2-344. [DOI] [PubMed] [Google Scholar]
  27. Lyall R. M., Pastan I., Willingham M. C. EGF induces receptor down-regulation with no receptor recycling in KB cells. J Cell Physiol. 1985 Jan;122(1):166–170. doi: 10.1002/jcp.1041220125. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Murthy U., Basu M., Sen-Majumdar A., Das M. Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains. J Cell Biol. 1986 Aug;103(2):333–342. doi: 10.1083/jcb.103.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pearse B. M. Receptors compete for adaptors found in plasma membrane coated pits. EMBO J. 1988 Nov;7(11):3331–3336. doi: 10.1002/j.1460-2075.1988.tb03204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schaudies R. P., Gorman R. M., Savage C. R., Jr, Poretz R. D. Proteolytic processing of epidermal growth factor within endosomes. Biochem Biophys Res Commun. 1987 Mar 13;143(2):710–715. doi: 10.1016/0006-291x(87)91412-4. [DOI] [PubMed] [Google Scholar]
  32. Simionescu N., Simionescu M. Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. J Cell Biol. 1976 Sep;70(3):608–621. doi: 10.1083/jcb.70.3.608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Smith C. E., Paiement J., Bergeron J. J. Subcellular distribution of acid NADPase activity within the parenchymal cells of rat liver. J Histochem Cytochem. 1986 May;34(5):649–658. doi: 10.1177/34.5.3009603. [DOI] [PubMed] [Google Scholar]
  34. Sorkin A. D., Teslenko L. V., Nikolsky N. N. The endocytosis of epidermal growth factor in A431 cells: a pH of microenvironment and the dynamics of receptor complex dissociation. Exp Cell Res. 1988 Mar;175(1):192–205. doi: 10.1016/0014-4827(88)90266-2. [DOI] [PubMed] [Google Scholar]
  35. Stoscheck C. M., Carpenter G. Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts. J Cell Biol. 1984 Mar;98(3):1048–1053. doi: 10.1083/jcb.98.3.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Teslenko L. V., Kornilova E. S., Sorkin A. D., Nikolsky N. N. Recycling of epidermal growth factor in A431 cells. FEBS Lett. 1987 Aug 31;221(1):105–109. doi: 10.1016/0014-5793(87)80361-7. [DOI] [PubMed] [Google Scholar]
  37. Wiley H. S., VanNostrand W., McKinley D. N., Cunningham D. D. Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions. J Biol Chem. 1985 May 10;260(9):5290–5295. [PubMed] [Google Scholar]

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