Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Aug 1;107(2):801–810. doi: 10.1083/jcb.107.2.801

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

PMCID: PMC2115211  PMID: 3262110

Abstract

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple- affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Aharonov A., Pruss R. M., Herschman H. R. Epidermal growth factor. Relationship between receptor regulation and mitogenesis in 3T3 cells. J Biol Chem. 1978 Jun 10;253(11):3970–3977. [PubMed] [Google Scholar]
  2. Anderson R. G., Brown M. S., Goldstein J. L. Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells. J Cell Biol. 1981 Feb;88(2):441–452. doi: 10.1083/jcb.88.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnes D. W. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture. J Cell Biol. 1982 Apr;93(1):1–4. doi: 10.1083/jcb.93.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berg H. C., Purcell E. M. Physics of chemoreception. Biophys J. 1977 Nov;20(2):193–219. doi: 10.1016/S0006-3495(77)85544-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buss J. E., Chouvet C., Gill G. N. Comparison of protein phosphorylations in variant A431 cells with different growth responses to epidermal growth factor. J Cell Physiol. 1984 Jun;119(3):296–306. doi: 10.1002/jcp.1041190307. [DOI] [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., Cohen S. Human epidermal growth factor and the proliferation of human fibroblasts. J Cell Physiol. 1976 Jun;88(2):227–237. doi: 10.1002/jcp.1040880212. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Chatelier R. C., Ashcroft R. G., Lloyd C. J., Nice E. C., Whitehead R. H., Sawyer W. H., Burgess A. W. Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data. EMBO J. 1986 Jun;5(6):1181–1186. doi: 10.1002/j.1460-2075.1986.tb04344.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen S., Carpenter G., King L., Jr Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem. 1980 May 25;255(10):4834–4842. [PubMed] [Google Scholar]
  11. Cohen S. Purification of the receptor for epidermal growth factor from A-431 cells: its function as a tyrosyl kinase. Methods Enzymol. 1983;99:379–387. doi: 10.1016/0076-6879(83)99074-2. [DOI] [PubMed] [Google Scholar]
  12. Cohen S., Ushiro H., Stoscheck C., Chinkers M. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem. 1982 Feb 10;257(3):1523–1531. [PubMed] [Google Scholar]
  13. DeLisi C., Metzger H. Some physical chemical aspects of receptor-ligand interactions. Immunol Commun. 1976;5(5):417–436. doi: 10.3109/08820137609033858. [DOI] [PubMed] [Google Scholar]
  14. DeLisi C., Wiegel F. W. Effect of nonspecific forces and finite receptor number on rate constants of ligand--cell bound-receptor interactions. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5569–5572. doi: 10.1073/pnas.78.9.5569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Erickson J., Goldstein B., Holowka D., Baird B. The effect of receptor density on the forward rate constant for binding of ligands to cell surface receptors. Biophys J. 1987 Oct;52(4):657–662. doi: 10.1016/S0006-3495(87)83258-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gamou S., Kim Y. S., Shimizu N. Different responses to EGF in two human carcinoma cell lines, A431 and UCVA-1, possessing high numbers of EGF receptors. Mol Cell Endocrinol. 1984 Sep;37(2):205–213. doi: 10.1016/0303-7207(84)90053-4. [DOI] [PubMed] [Google Scholar]
  17. Gex-Fabry M., DeLisi C. Receptor-mediated endocytosis: a model and its implications for experimental analysis. Am J Physiol. 1984 Nov;247(5 Pt 2):R768–R779. doi: 10.1152/ajpregu.1984.247.5.R768. [DOI] [PubMed] [Google Scholar]
  18. Gill G. N., Lazar C. S. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature. 1981 Sep 24;293(5830):305–307. doi: 10.1038/293305a0. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Haigler H. T., Schlaepfer D. D., Burgess W. H. Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors. J Biol Chem. 1987 May 15;262(14):6921–6930. [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Hillman G. M., Schlessinger J. Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis. Biochemistry. 1982 Mar 30;21(7):1667–1672. doi: 10.1021/bi00536a030. [DOI] [PubMed] [Google Scholar]
  25. Hock R. A., Hollenberg M. D. Characterization of the receptor for epidermal growth factor-urogastrone in human placenta membranes. J Biol Chem. 1980 Nov 25;255(22):10731–10736. [PubMed] [Google Scholar]
  26. Hopkins C. R., Miller K., Beardmore J. M. Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake. J Cell Sci Suppl. 1985;3:173–186. doi: 10.1242/jcs.1985.supplement_3.17. [DOI] [PubMed] [Google Scholar]
  27. Kawamoto T., Sato J. D., Le A., Polikoff J., Sato G. H., Mendelsohn J. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1337–1341. doi: 10.1073/pnas.80.5.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Knauer D. J., Wiley H. S., Cunningham D. D. Relationship between epidermal growth factor receptor occupancy and mitogenic response. Quantitative analysis using a steady state model system. J Biol Chem. 1984 May 10;259(9):5623–5631. [PubMed] [Google Scholar]
  29. 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]
  30. Lin C. R., Chen W. S., Kruiger W., Stolarsky L. S., Weber W., Evans R. M., Verma I. M., Gill G. N., Rosenfeld M. G. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science. 1984 May 25;224(4651):843–848. doi: 10.1126/science.6326261. [DOI] [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. Moses A. C., Nissley S. P., Short P. A., Rechler M. M. Immunological cross-reactivity of multiplication-stimulating activity polypeptides. Eur J Biochem. 1980 Jan;103(2):401–408. doi: 10.1111/j.1432-1033.1980.tb04326.x. [DOI] [PubMed] [Google Scholar]
  33. Opresko L. K., Wiley H. S. Receptor-mediated endocytosis in Xenopus oocytes. I. Characterization of the vitellogenin receptor system. J Biol Chem. 1987 Mar 25;262(9):4109–4115. [PubMed] [Google Scholar]
  34. Opresko L. K., Wiley H. S. Receptor-mediated endocytosis in Xenopus oocytes. II. Evidence for two novel mechanisms of hormonal regulation. J Biol Chem. 1987 Mar 25;262(9):4116–4123. [PubMed] [Google Scholar]
  35. Pastan I. H., Willingham M. C. Receptor-mediated endocytosis of hormones in cultured cells. Annu Rev Physiol. 1981;43:239–250. doi: 10.1146/annurev.ph.43.030181.001323. [DOI] [PubMed] [Google Scholar]
  36. Rees A. R., Gregoriou M., Johnson P., Garland P. B. High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion. EMBO J. 1984 Aug;3(8):1843–1847. doi: 10.1002/j.1460-2075.1984.tb02057.x. [DOI] [PMC free article] [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. Sawyer S. T., Cohen S. Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells. Biochemistry. 1981 Oct 13;20(21):6280–6286. doi: 10.1021/bi00524a057. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Wank S. A., DeLisi C., Metzger H. Analysis of the rate-limiting step in a ligand-cell receptor interaction: the immunoglobulin E system. Biochemistry. 1983 Feb 15;22(4):954–959. doi: 10.1021/bi00273a038. [DOI] [PubMed] [Google Scholar]
  41. Ward J. H., Kushner J. P., Kaplan J. Regulation of HeLa cell transferrin receptors. J Biol Chem. 1982 Sep 10;257(17):10317–10323. [PubMed] [Google Scholar]
  42. Weber W., Bertics P. J., Gill G. N. Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation. J Biol Chem. 1984 Dec 10;259(23):14631–14636. [PubMed] [Google Scholar]
  43. 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]
  44. Wiley H. S., Cunningham D. D. Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface. J Cell Biochem. 1982;19(4):383–394. doi: 10.1002/jcb.240190407. [DOI] [PubMed] [Google Scholar]
  45. Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
  46. Wiley H. S., Kaplan J. Epidermal growth factor rapidly induces a redistribution of transferrin receptor pools in human fibroblasts. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7456–7460. doi: 10.1073/pnas.81.23.7456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wiley H. S., McKinley D. N. Assay of growth factor stimulation of fluid-phase endocytosis. Methods Enzymol. 1987;146:402–417. doi: 10.1016/s0076-6879(87)46042-4. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Zidovetzki R., Yarden Y., Schlessinger J., Jovin T. M. Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6981–6985. doi: 10.1073/pnas.78.11.6981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. van Opheusden J. H., Wiegel F. W., Goldstein B. Forward rate constants for receptor clusters. Variational methods for upper and lower bounds. Biophys Chem. 1984 Oct;20(3):237–248. doi: 10.1016/0301-4622(84)87028-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES