Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Feb 1;102(2):500–509. doi: 10.1083/jcb.102.2.500

Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells

PMCID: PMC2114073  PMID: 2868013

Abstract

We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti- receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF- dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beardmore J., Hopkins C. R. Uptake and intracellular processing of epidermal-growth-factor-receptor complexes. Biochem Soc Trans. 1984 Apr;12(2):165–168. doi: 10.1042/bst0120165. [DOI] [PubMed] [Google Scholar]
  2. Beguinot L., Lyall R. M., Willingham M. C., Pastan I. Down-regulation of the epidermal growth factor receptor in KB cells is due to receptor internalization and subsequent degradation in lysosomes. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2384–2388. doi: 10.1073/pnas.81.8.2384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boonstra J., van Maurik P., Defize L. H., de Laat S. W., Leunissen J. L., Verkley A. J. Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling. Eur J Cell Biol. 1985 Mar;36(2):209–216. [PubMed] [Google Scholar]
  4. 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]
  5. Carpenter G., Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979;48:193–216. doi: 10.1146/annurev.bi.48.070179.001205. [DOI] [PubMed] [Google Scholar]
  6. Carpenter G. Properties of the receptor for epidermal growth factor. Cell. 1984 Jun;37(2):357–358. doi: 10.1016/0092-8674(84)90365-9. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Faulk W. P., Taylor G. M. An immunocolloid method for the electron microscope. Immunochemistry. 1971 Nov;8(11):1081–1083. doi: 10.1016/0019-2791(71)90496-4. [DOI] [PubMed] [Google Scholar]
  9. Fredman P., Richert N. D., Magnani J. L., Willingham M. C., Pastan I., Ginsburg V. A monoclonal antibody that precipitates the glycoprotein receptor for epidermal growth factor is directed against the human blood group H type 1 antigen. J Biol Chem. 1983 Sep 25;258(18):11206–11210. [PubMed] [Google Scholar]
  10. Geoghegan W. D., Ackerman G. A. Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J Histochem Cytochem. 1977 Nov;25(11):1187–1200. doi: 10.1177/25.11.21217. [DOI] [PubMed] [Google Scholar]
  11. Gill G. N., Kawamoto T., Cochet C., Le A., Sato J. D., Masui H., McLeod C., Mendelsohn J. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Jun 25;259(12):7755–7760. [PubMed] [Google Scholar]
  12. Gooi H. C., Schlessinger J., Lax I., Yarden Y., Libermann T. A., Feizi T. Monoclonal antibody reactive with the human epidermal-growth-factor receptor recognizes the blood-group-A antigen. Biosci Rep. 1983 Nov;3(11):1045–1052. doi: 10.1007/BF01121031. [DOI] [PubMed] [Google Scholar]
  13. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  14. Haigler H. T., McKanna J. A., Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol. 1979 May;81(2):382–395. doi: 10.1083/jcb.81.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haigler H. T., McKanna J. A., Cohen S. Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor. J Cell Biol. 1979 Oct;83(1):82–90. doi: 10.1083/jcb.83.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harding C., Heuser J., Stahl P. Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding. Eur J Cell Biol. 1984 Nov;35(2):256–263. [PubMed] [Google Scholar]
  18. Harding C., Heuser J., Stahl P. Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. J Cell Biol. 1983 Aug;97(2):329–339. doi: 10.1083/jcb.97.2.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herman B., Albertini D. F. A time-lapse video image intensification analysis of cytoplasmic organelle movements during endosome translocation. J Cell Biol. 1984 Feb;98(2):565–576. doi: 10.1083/jcb.98.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herman B., Albertini D. F. The intracellular movement of endocytic vesicles in cultured granulosa cells. Cell Motil. 1982;2(6):583–597. doi: 10.1002/cm.970020607. [DOI] [PubMed] [Google Scholar]
  21. Hopkins C. R. Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells. Cell. 1983 Nov;35(1):321–330. doi: 10.1016/0092-8674(83)90235-0. [DOI] [PubMed] [Google Scholar]
  22. Hopkins C. R., Trowbridge I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. doi: 10.1083/jcb.97.2.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Horisberger M., Rosset J. Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem. 1977 Apr;25(4):295–305. doi: 10.1177/25.4.323352. [DOI] [PubMed] [Google Scholar]
  24. Hunter T. The epidermal growth factor receptor gene and its product. Nature. 1984 Oct 4;311(5985):414–416. doi: 10.1038/311414a0. [DOI] [PubMed] [Google Scholar]
  25. Kawamoto T., Mendelsohn J., Le A., Sato G. H., Lazar C. S., Gill G. N. Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells. J Biol Chem. 1984 Jun 25;259(12):7761–7766. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Oliver C., Hand A. R. Enzyme modulation of the Golgi apparatus and GERL: a cytochemical study of parotid acinar cells. J Histochem Cytochem. 1983 Aug;31(8):1041–1048. doi: 10.1177/31.8.6134769. [DOI] [PubMed] [Google Scholar]
  29. Robinson J. M., Karnovsky M. J. Ultrastructural localization of several phosphatases with cerium. J Histochem Cytochem. 1983 Oct;31(10):1197–1208. doi: 10.1177/31.10.6309949. [DOI] [PubMed] [Google Scholar]
  30. Schlessinger J., Shechter Y., Willingham M. C., Pastan I. Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2659–2663. doi: 10.1073/pnas.75.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schreiber A. B., Libermann T. A., Lax I., Yarden Y., Schlessinger J. Biological role of epidermal growth factor-receptor clustering. Investigation with monoclonal anti-receptor antibodies. J Biol Chem. 1983 Jan 25;258(2):846–853. [PubMed] [Google Scholar]
  32. 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]
  33. Tokuyasu K. T. A study of positive staining of ultrathin frozen sections. J Ultrastruct Res. 1978 Jun;63(3):287–307. doi: 10.1016/s0022-5320(78)80053-7. [DOI] [PubMed] [Google Scholar]
  34. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  35. Waterfield M. D., Mayes E. L., Stroobant P., Bennet P. L., Young S., Goodfellow P. N., Banting G. S., Ozanne B. A monoclonal antibody to the human epidermal growth factor receptor. J Cell Biochem. 1982;20(2):149–161. doi: 10.1002/jcb.240200207. [DOI] [PubMed] [Google Scholar]
  36. Willingham M. C., Haigler H. T., Fitzgerald D. J., Gallo M. G., Rutherford A. V., Pastan I. H. The morphologic pathway of binding and internalization of epidermal growth factor in cultured cells. Studies on A431, KB, and 3T3 cells, using multiple methods of labelling. Exp Cell Res. 1983 Jun;146(1):163–175. doi: 10.1016/0014-4827(83)90334-8. [DOI] [PubMed] [Google Scholar]
  37. Willingham M. C., Pastan I. H. Transit of epidermal growth factor through coated pits of the Golgi system. J Cell Biol. 1982 Jul;94(1):207–212. doi: 10.1083/jcb.94.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES