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. 1989 Nov 1;109(5):2495–2507. doi: 10.1083/jcb.109.5.2495

Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors

PMCID: PMC2115872  PMID: 2553748

Abstract

Many cell types display two classes of epidermal growth factor receptor (EGFR) as judged from EGF binding studies; i.e., a major class of low affinity EGFR and a minor class of high affinity EGFR. We have studied their respective contribution to the cascade of events elicited by EGF in human A431 carcinoma cells, using anti-EGFR mAb 2E9. This antibody specifically blocks EGF binding to low affinity EGFR, without activating receptors in intact cells, and thus enables us to study the effects of exclusive EGF binding to high affinity EGFR. We show that blocking of low affinity EGFR by mAb 2E9 has almost no effect on the activation of the receptor protein-tyrosine kinase by EGF, suggesting that EGFR kinase activation occurs exclusively through the subclass of high affinity EGFR (5-10%). In addition, we provide evidence that high affinity EGFR exists both in monomeric and dimeric forms, and that cross-phosphorylation of low affinity EGFR by high affinity EGFR may take place in dimers of both receptor types. We demonstrate that the following early cellular response to EGF are also unimpaired in the presence of mAb 2E9: (a) inositol phosphate production, (b) release of Ca2+ from intracellular stores, (c) rise in intracellular pH, (d) phosphorylation of EGF on threonine residue 654, (e) induction of c-fos gene expression, and (f) alteration in cell morphology. As possible nonspecific side effects, we observed that the EGF induced Ca2+ influx and fluid-phase pinocytosis were inhibited in A431 cells in the presence of mAb 2E9. We conclude, therefore, that the activation of the EGFR signal transduction cascade can occur completely through exclusive binding of EGF to the subclass of high affinity EGFR.

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Selected References

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  1. 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]
  2. Beemon K., Hunter T. Characterization of Rous sarcoma virus src gene products synthesized in vitro. J Virol. 1978 Nov;28(2):551–566. doi: 10.1128/jvi.28.2.551-566.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Behzadian M. A., Shimizu N. Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor. Cell Struct Funct. 1985 Sep;10(3):219–232. doi: 10.1247/csf.10.219. [DOI] [PubMed] [Google Scholar]
  4. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  5. Boonstra J., Mummery C. L., Feyen A., de Hoog W. J., van der Saag P. T., de Laat S. W. Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP. J Cell Physiol. 1987 Jun;131(3):409–417. doi: 10.1002/jcp.1041310313. [DOI] [PubMed] [Google Scholar]
  6. Boonstra J., Mummery C. L., van der Saag P. T., de Laat S. W. Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters. J Cell Physiol. 1985 Jun;123(3):347–352. doi: 10.1002/jcp.1041230309. [DOI] [PubMed] [Google Scholar]
  7. Boonstra J., van Belzen N., van Maurik P., Hage W. J., Blok F. J., Wiegant F. A., Verkleij A. J. Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy, surface replication, freeze-etching and label fracture. J Microsc. 1985 Oct;140(Pt 1):119–129. doi: 10.1111/j.1365-2818.1985.tb02667.x. [DOI] [PubMed] [Google Scholar]
  8. Bravo R., Burckhardt J., Curran T., Müller R. Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. EMBO J. 1985 May;4(5):1193–1197. doi: 10.1002/j.1460-2075.1985.tb03759.x. [DOI] [PMC free article] [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., Rees A. R., Gregoriou M., Kris R., Schlessinger J., Orci L. Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells. Exp Cell Res. 1986 Oct;166(2):312–326. doi: 10.1016/0014-4827(86)90479-9. [DOI] [PubMed] [Google Scholar]
  11. Chen W. S., Lazar C. S., Poenie M., Tsien R. Y., Gill G. N., Rosenfeld M. G. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. 1987 Aug 27-Sep 2Nature. 328(6133):820–823. doi: 10.1038/328820a0. [DOI] [PubMed] [Google Scholar]
  12. Chinkers M., McKanna J. A., Cohen S. Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth factors. J Cell Biol. 1979 Oct;83(1):260–265. doi: 10.1083/jcb.83.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chinkers M., McKanna J. A., Cohen S. Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor. J Cell Biol. 1981 Feb;88(2):422–429. doi: 10.1083/jcb.88.2.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  15. Cochet C., Gill G. N., Meisenhelder J., Cooper J. A., Hunter T. C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Feb 25;259(4):2553–2558. [PubMed] [Google Scholar]
  16. Cochet C., Kashles O., Chambaz E. M., Borrello I., King C. R., Schlessinger J. Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol Chem. 1988 Mar 5;263(7):3290–3295. [PubMed] [Google Scholar]
  17. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  18. Coussens L., Yang-Feng T. L., Liao Y. C., Chen E., Gray A., McGrath J., Seeburg P. H., Libermann T. A., Schlessinger J., Francke U. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science. 1985 Dec 6;230(4730):1132–1139. doi: 10.1126/science.2999974. [DOI] [PubMed] [Google Scholar]
  19. Defize L. H., Arndt-Jovin D. J., Jovin T. M., Boonstra J., Meisenhelder J., Hunter T., de Hey H. T., de Laat S. W. A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover. J Cell Biol. 1988 Sep;107(3):939–949. doi: 10.1083/jcb.107.3.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Defize L. H., Moolenaar W. H., van der Saag P. T., de Laat S. W. Dissociation of cellular responses to epidermal growth factor using anti-receptor monoclonal antibodies. EMBO J. 1986 Jun;5(6):1187–1192. doi: 10.1002/j.1460-2075.1986.tb04345.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Defize L. H., Mummery C. L., Moolenaar W. H., de Laat S. W. Antireceptor antibodies in the study of EGF-receptor interaction. Cell Differ. 1987 Mar;20(2-3):87–102. doi: 10.1016/0045-6039(87)90423-4. [DOI] [PubMed] [Google Scholar]
  22. Downward J., Parker P., Waterfield M. D. Autophosphorylation sites on the epidermal growth factor receptor. Nature. 1984 Oct 4;311(5985):483–485. doi: 10.1038/311483a0. [DOI] [PubMed] [Google Scholar]
  23. Fabricant R. N., De Larco J. E., Todaro G. J. Nerve growth factor receptors on human melanoma cells in culture. Proc Natl Acad Sci U S A. 1977 Feb;74(2):565–569. doi: 10.1073/pnas.74.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fanger B. O., Austin K. S., Earp H. S., Cidlowski J. A. Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors. Biochemistry. 1986 Oct 21;25(21):6414–6420. doi: 10.1021/bi00369a011. [DOI] [PubMed] [Google Scholar]
  25. Fanger B. O., Viceps-Madore D., Cidlowski J. A. Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids. Arch Biochem Biophys. 1984 Nov 15;235(1):141–149. doi: 10.1016/0003-9861(84)90262-5. [DOI] [PubMed] [Google Scholar]
  26. Friedman B., Frackelton A. R., Jr, Ross A. H., Connors J. M., Fujiki H., Sugimura T., Rosner M. R. Tumor promoters block tyrosine-specific phosphorylation of the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1984 May;81(10):3034–3038. doi: 10.1073/pnas.81.10.3034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gill G. N., Santon J. B., Bertics P. J. Regulatory features of the epidermal growth factor receptor. J Cell Physiol Suppl. 1987;Suppl 5:35–41. doi: 10.1002/jcp.1041330408. [DOI] [PubMed] [Google Scholar]
  28. Gooi H. C., Hounsell E. F., Lax I., Kris R. M., Libermann T. A., Schlessinger J., Sato J. D., Kawamoto T., Mendelsohn J., Feizi T. The carbohydrate specificities of the monoclonal antibodies 29.1, 455 and 3C1B12 to the epidermal growth factor receptor of A431 cells. Biosci Rep. 1985 Jan;5(1):83–94. doi: 10.1007/BF01117444. [DOI] [PubMed] [Google Scholar]
  29. Gregorou M., Rees A. R. Properties of a monoclonal antibody to epidermal growth factor receptor with implications for the mechanism of action of EGF. EMBO J. 1984 May;3(5):929–937. doi: 10.1002/j.1460-2075.1984.tb01910.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Hepler J. R., Nakahata N., Lovenberg T. W., DiGuiseppi J., Herman B., Earp H. S., Harden T. K. Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells. J Biol Chem. 1987 Mar 5;262(7):2951–2956. [PubMed] [Google Scholar]
  32. Honegger A. M., Dull T. J., Felder S., Van Obberghen E., Bellot F., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. doi: 10.1016/0092-8674(87)90147-4. [DOI] [PubMed] [Google Scholar]
  33. Honegger A. M., Kris R. M., Ullrich A., Schlessinger J. Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation. Proc Natl Acad Sci U S A. 1989 Feb;86(3):925–929. doi: 10.1073/pnas.86.3.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Honegger A. M., Szapary D., Schmidt A., Lyall R., Van Obberghen E., Dull T. J., Ullrich A., Schlessinger J. A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis. Mol Cell Biol. 1987 Dec;7(12):4568–4571. doi: 10.1128/mcb.7.12.4568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Hunter T., Cooper J. A. Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell. 1981 Jun;24(3):741–752. doi: 10.1016/0092-8674(81)90100-8. [DOI] [PubMed] [Google Scholar]
  36. Hunter T., Ling N., Cooper J. A. Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature. 1984 Oct 4;311(5985):480–483. doi: 10.1038/311480a0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. King A. C., Cuatrecasas P. Resolution of high and low affinity epidermal growth factor receptors. Inhibition of high affinity component by low temperature, cycloheximide, and phorbol esters. J Biol Chem. 1982 Mar 25;257(6):3053–3060. [PubMed] [Google Scholar]
  39. King C. R., Borrello I., Bellot F., Comoglio P., Schlessinger J. Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3. EMBO J. 1988 Jun;7(6):1647–1651. doi: 10.1002/j.1460-2075.1988.tb02991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. King C. S., Cooper J. A. Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation. J Biol Chem. 1986 Aug 5;261(22):10073–10078. [PubMed] [Google Scholar]
  41. Kris R. M., Lax I., Gullick W., Waterfield M. D., Ullrich A., Fridkin M., Schlessinger J. Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein. Cell. 1985 Mar;40(3):619–625. doi: 10.1016/0092-8674(85)90210-7. [DOI] [PubMed] [Google Scholar]
  42. Livneh E., Prywes R., Kashles O., Reiss N., Sasson I., Mory Y., Ullrich A., Schlessinger J. Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. J Biol Chem. 1986 Sep 25;261(27):12490–12497. [PubMed] [Google Scholar]
  43. Moolenaar W. H., Aerts R. J., Tertoolen L. G., de Laat S. W. The epidermal growth factor-induced calcium signal in A431 cells. J Biol Chem. 1986 Jan 5;261(1):279–284. [PubMed] [Google Scholar]
  44. Moolenaar W. H., Bierman A. J., Tilly B. C., Verlaan I., Defize L. H., Honegger A. M., Ullrich A., Schlessinger J. A point mutation at the ATP-binding site of the EGF-receptor abolishes signal transduction. EMBO J. 1988 Mar;7(3):707–710. doi: 10.1002/j.1460-2075.1988.tb02866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Moolenaar W. H. Effects of growth factors on intracellular pH regulation. Annu Rev Physiol. 1986;48:363–376. doi: 10.1146/annurev.ph.48.030186.002051. [DOI] [PubMed] [Google Scholar]
  46. Prywes R., Livneh E., Ullrich A., Schlessinger J. Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. EMBO J. 1986 Sep;5(9):2179–2190. doi: 10.1002/j.1460-2075.1986.tb04482.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Sato J. D., Kawamoto T., Le A. D., Mendelsohn J., Polikoff J., Sato G. H. Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med. 1983 Dec;1(5):511–529. [PubMed] [Google Scholar]
  49. Schreiber A. B., Lax I., Yarden Y., Eshhar Z., Schlessinger J. Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7535–7539. doi: 10.1073/pnas.78.12.7535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shechter Y., Hernaez L., Cuatrecasas P. Epidermal growth factor: biological activity requires persistent occupation of high-affinity cell surface receptors. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5788–5791. doi: 10.1073/pnas.75.12.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shoyab M., De Larco J. E., Todaro G. J. Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature. 1979 May 31;279(5712):387–391. doi: 10.1038/279387a0. [DOI] [PubMed] [Google Scholar]
  52. Soderquist A. M., Carpenter G. Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function. J Biol Chem. 1984 Oct 25;259(20):12586–12594. [PubMed] [Google Scholar]
  53. Stern D. F., Kamps M. P. EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions. EMBO J. 1988 Apr;7(4):995–1001. doi: 10.1002/j.1460-2075.1988.tb02906.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sturani E., Zippel R., Toschi L., Morello L., Comoglio P. M., Alberghina L. Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells. Mol Cell Biol. 1988 Mar;8(3):1345–1351. doi: 10.1128/mcb.8.3.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tilly B. C., van Paridon P. A., Verlaan I., de Laat S. W., Moolenaar W. H. Epidermal-growth-factor-induced formation of inositol phosphates in human A431 cells. Differences from the effect of bradykinin. Biochem J. 1988 Jun 15;252(3):857–863. doi: 10.1042/bj2520857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wheeler L. A., Sachs G., De Vries G., Goodrum D., Woldemussie E., Muallem S. Manoalide, a natural sesterterpenoid that inhibits calcium channels. J Biol Chem. 1987 May 15;262(14):6531–6538. [PubMed] [Google Scholar]
  57. Whiteley B., Glaser L. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor. J Cell Biol. 1986 Oct;103(4):1355–1362. doi: 10.1083/jcb.103.4.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wiegant F. A., Blok F. J., Defize L. H., Linnemans W. A., Verkley A. J., Boonstra J. Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells. J Cell Biol. 1986 Jul;103(1):87–94. doi: 10.1083/jcb.103.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]

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