Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Nov 15;352(Pt 1):99–108.

Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity.

M R Holbrook 1, L L Slakey 1, D J Gross 1
PMCID: PMC1221436  PMID: 11062062

Abstract

The epidermal growth factor receptor (EGFr), when expressed on the cell surface, has long been known to display two distinct affinities for epidermal growth factor (EGF) binding. In addition, the treatment of cells expressing the EGFr with phorbol esters has been shown to cause a loss of the high-affinity binding capacity of the receptor. In the present study, point mutations that alter acidic or phosphorylation sites have been made in an intracellular domain near Tyr-992 (residues 988-992) of the EGFr. Equilibrium (125)I-EGF binding studies demonstrate that the conversion of Tyr-992 into glutamate induces a 4-fold decrease in the EGFr apparent low-affinity dissociation constant, whereas the mutation of two acidic residues, Asp-988 and Glu-991, or the conversion of Tyr-992 into phenylalanine does not alter EGFr affinity. Phorbol ester treatment of EGFr-expressing Chinese hamster ovary cells results in a loss of high-affinity binding and an increase in the apparent low-affinity dissociation constant of the receptor, similar to the effect of a truncation mutant in which the C-terminal 190 residues are deleted. These results are examined in the context of a new model for regulation of the affinity of the EGFr for EGF in which a cytosolic particle stabilizes the high-affinity conformation of the EGFr and a rapid equilibrium exists between EGFr high-affinity and low-affinity conformations. This model demonstrates that the macroscopic affinities of the EGFr can differ from the affinities of individual EGFr molecules and provides a theoretical framework whereby the measured affinities of the EGFr are modulated by intracellular interactions.

Full Text

The Full Text of this article is available as a PDF (154.4 KB).

Selected References

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

  1. Bellot F., Moolenaar W., Kris R., Mirakhur B., Verlaan I., Ullrich A., Schlessinger J., Felder S. High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses. J Cell Biol. 1990 Feb;110(2):491–502. doi: 10.1083/jcb.110.2.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berkers J. A., van Bergen en Henegouwen P. M., Boonstra J. Three classes of epidermal growth factor receptors on HeLa cells. J Biol Chem. 1991 Jan 15;266(2):922–927. [PubMed] [Google Scholar]
  3. Bowen S., Stanley K., Selva E., Davis R. J. Constitutive phosphorylation of the epidermal growth factor receptor blocks mitogenic signal transduction. J Biol Chem. 1991 Jan 15;266(2):1162–1169. [PubMed] [Google Scholar]
  4. Chamberlin S. G., Davies D. E. A unified model of c-erbB receptor homo- and heterodimerisation. Biochim Biophys Acta. 1998 May 19;1384(2):223–232. doi: 10.1016/s0167-4838(97)00203-3. [DOI] [PubMed] [Google Scholar]
  5. Chang C. P., Lazar C. S., Walsh B. J., Komuro M., Collawn J. F., Kuhn L. A., Tainer J. A., Trowbridge I. S., Farquhar M. G., Rosenfeld M. G. Ligand-induced internalization of the epidermal growth factor receptor is mediated by multiple endocytic codes analogous to the tyrosine motif found in constitutively internalized receptors. J Biol Chem. 1993 Sep 15;268(26):19312–19320. [PubMed] [Google Scholar]
  6. Chen W. S., Lazar C. S., Lund K. A., Welsh J. B., Chang C. P., Walton G. M., Der C. J., Wiley H. S., Gill G. N., Rosenfeld M. G. Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell. 1989 Oct 6;59(1):33–43. doi: 10.1016/0092-8674(89)90867-2. [DOI] [PubMed] [Google Scholar]
  7. Chung J. C., Sciaky N., Gross D. J. Heterogeneity of epidermal growth factor binding kinetics on individual cells. Biophys J. 1997 Aug;73(2):1089–1102. doi: 10.1016/S0006-3495(97)78141-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Countaway J. L., Gironès N., Davis R. J. Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells. J Biol Chem. 1989 Aug 15;264(23):13642–13647. [PubMed] [Google Scholar]
  9. Davis R. J. Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine-protein kinase activity. J Biol Chem. 1988 Jul 5;263(19):9462–9469. [PubMed] [Google Scholar]
  10. Defize L. H., Boonstra J., Meisenhelder J., Kruijer W., Tertoolen L. G., Tilly B. C., Hunter T., van Bergen en Henegouwen P. M., Moolenaar W. H., de Laat S. W. Signal transduction by epidermal growth factor occurs through the subclass of high affinity receptors. J Cell Biol. 1989 Nov;109(5):2495–2507. doi: 10.1083/jcb.109.5.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Holbrook M. R., O'Donnell J. B., Jr, Slakey L. L., Gross D. J. Epidermal growth factor receptor internalization rate is regulated by negative charges near the SH2 binding site Tyr992. Biochemistry. 1999 Jul 20;38(29):9348–9356. doi: 10.1021/bi990195r. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Lemmon M. A., Bu Z., Ladbury J. E., Zhou M., Pinchasi D., Lax I., Engelman D. M., Schlessinger J. Two EGF molecules contribute additively to stabilization of the EGFR dimer. EMBO J. 1997 Jan 15;16(2):281–294. doi: 10.1093/emboj/16.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Mayo K. H., Nunez M., Burke C., Starbuck C., Lauffenburger D., Savage C. R., Jr Epidermal growth factor receptor binding is not a simple one-step process. J Biol Chem. 1989 Oct 25;264(30):17838–17844. [PubMed] [Google Scholar]
  17. McClure S. J., Robinson P. J. Dynamin, endocytosis and intracellular signalling (review). Mol Membr Biol. 1996 Oct-Dec;13(4):189–215. doi: 10.3109/09687689609160598. [DOI] [PubMed] [Google Scholar]
  18. Ringerike T., Stang E., Johannessen L. E., Sandnes D., Levy F. O., Madshus I. H. High-affinity binding of epidermal growth factor (EGF) to EGF receptor is disrupted by overexpression of mutant dynamin (K44A). J Biol Chem. 1998 Jul 3;273(27):16639–16642. doi: 10.1074/jbc.273.27.16639. [DOI] [PubMed] [Google Scholar]
  19. Roy L. M., Gittinger C. K., Landreth G. E. Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells. J Cell Physiol. 1989 Aug;140(2):295–304. doi: 10.1002/jcp.1041400215. [DOI] [PubMed] [Google Scholar]
  20. Sorkin A., Mazzotti M., Sorkina T., Scotto L., Beguinot L. Epidermal growth factor receptor interaction with clathrin adaptors is mediated by the Tyr974-containing internalization motif. J Biol Chem. 1996 Jun 7;271(23):13377–13384. doi: 10.1074/jbc.271.23.13377. [DOI] [PubMed] [Google Scholar]
  21. Van der Heyden M. A., Nievers M., Verkleij A. J., Boonstra J., Van Bergen en Henegouwen P. M. Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF. FEBS Lett. 1997 Jun 30;410(2-3):265–268. doi: 10.1016/s0014-5793(97)00599-1. [DOI] [PubMed] [Google Scholar]
  22. Walker F., Burgess A. W. Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor. J Biol Chem. 1991 Feb 15;266(5):2746–2752. [PubMed] [Google Scholar]
  23. Walton G. M., Chen W. S., Rosenfeld M. G., Gill G. N. Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates. J Biol Chem. 1990 Jan 25;265(3):1750–1754. [PubMed] [Google Scholar]
  24. 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]
  25. Wiley H. S. Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system. J Cell Biol. 1988 Aug;107(2):801–810. doi: 10.1083/jcb.107.2.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wofsy C., Goldstein B., Lund K., Wiley H. S. Implications of epidermal growth factor (EGF) induced egf receptor aggregation. Biophys J. 1992 Jul;63(1):98–110. doi: 10.1016/S0006-3495(92)81572-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yarden Y., Ullrich A. Molecular analysis of signal transduction by growth factors. Biochemistry. 1988 May 3;27(9):3113–3119. doi: 10.1021/bi00409a001. [DOI] [PubMed] [Google Scholar]
  28. den Hartigh J. C., van Bergen en Henegouwen P. M., Verkleij A. J., Boonstra J. The EGF receptor is an actin-binding protein. J Cell Biol. 1992 Oct;119(2):349–355. doi: 10.1083/jcb.119.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van Belzen N., Spaargaren M., Verkleij A. J., Boonstra J. Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering. J Cell Physiol. 1990 Nov;145(2):365–375. doi: 10.1002/jcp.1041450223. [DOI] [PubMed] [Google Scholar]
  30. van Bergen en Henegouwen P. M., Defize L. H., de Kroon J., van Damme H., Verkleij A. J., Boonstra J. Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells. J Cell Biochem. 1989 Apr;39(4):455–465. doi: 10.1002/jcb.240390411. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES