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. 1992 Jul;63(1):98–110. doi: 10.1016/S0006-3495(92)81572-2

Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

C Wofsy 1, B Goldstein 1, K Lund 1, H S Wiley 1
PMCID: PMC1262128  PMID: 1420877

Abstract

To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are also important in shaping the binding curves. Because we have controlled experimentally for many sources of receptor heterogeneity, we have limited the potential explanations for residual Scatchard plot curvature.

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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. Bertics P. J., Gill G. N. Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor. J Biol Chem. 1985 Nov 25;260(27):14642–14647. [PubMed] [Google Scholar]
  3. Biswas R., Basu M., Sen-Majumdar A., Das M. Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization. Biochemistry. 1985 Jul 2;24(14):3795–3802. doi: 10.1021/bi00335a056. [DOI] [PubMed] [Google Scholar]
  4. Böni-Schnetzler M., Pilch P. F. Mechanism of epidermal growth factor receptor autophosphorylation and high-affinity binding. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7832–7836. doi: 10.1073/pnas.84.22.7832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Carraway K. L., 3rd, Cerione R. A. Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events. J Biol Chem. 1991 May 15;266(14):8899–8906. [PubMed] [Google Scholar]
  7. Carraway K. L., 3rd, Koland J. G., Cerione R. A. Visualization of epidermal growth factor (EGF) receptor aggregation in plasma membranes by fluorescence resonance energy transfer. Correlation of receptor activation with aggregation. J Biol Chem. 1989 May 25;264(15):8699–8707. [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Cunningham B. C., Mulkerrin M. G., Wells J. A. Dimerization of human growth hormone by zinc. Science. 1991 Aug 2;253(5019):545–548. doi: 10.1126/science.1907025. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Dembo M., Goldstein B. Theory of equilibrium binding of symmetric bivalent haptens to cell surface antibody: application to histamine release from basophils. J Immunol. 1978 Jul;121(1):345–353. [PubMed] [Google Scholar]
  15. 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]
  16. Downward J., Waterfield M. D., Parker P. J. Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. J Biol Chem. 1985 Nov 25;260(27):14538–14546. [PubMed] [Google Scholar]
  17. 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]
  18. Fanger B. O., Stephens J. E., Staros J. V. High-yield trapping of EGF-induced receptor dimers by chemical cross-linking. FASEB J. 1989 Jan;3(1):71–75. doi: 10.1096/fasebj.3.1.2783412. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Glenney J. R., Jr, Chen W. S., Lazar C. S., Walton G. M., Zokas L. M., Rosenfeld M. G., Gill G. N. Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell. 1988 Mar 11;52(5):675–684. doi: 10.1016/0092-8674(88)90405-9. [DOI] [PubMed] [Google Scholar]
  21. Gullick W. J., Downward J., Waterfield M. D. Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function. EMBO J. 1985 Nov;4(11):2869–2877. doi: 10.1002/j.1460-2075.1985.tb04016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hatakeyama M., Tsudo M., Minamoto S., Kono T., Doi T., Miyata T., Miyasaka M., Taniguchi T. Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's. Science. 1989 May 5;244(4904):551–556. doi: 10.1126/science.2785715. [DOI] [PubMed] [Google Scholar]
  23. Heisermann G. J., Wiley H. S., Walsh B. J., Ingraham H. A., Fiol C. J., Gill G. N. Mutational removal of the Thr669 and Ser671 phosphorylation sites alters substrate specificity and ligand-induced internalization of the epidermal growth factor receptor. J Biol Chem. 1990 Aug 5;265(22):12820–12827. [PubMed] [Google Scholar]
  24. Heldin C. H., Ernlund A., Rorsman C., Rönnstrand L. Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation. J Biol Chem. 1989 May 25;264(15):8905–8912. [PubMed] [Google Scholar]
  25. 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]
  26. Honegger A., Dull T. J., Szapary D., Komoriya A., Kris R., Ullrich A., Schlessinger J. Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 1988 Oct;7(10):3053–3060. doi: 10.1002/j.1460-2075.1988.tb03170.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Koland J. G., Cerione R. A. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism. J Biol Chem. 1988 Feb 15;263(5):2230–2237. [PubMed] [Google Scholar]
  28. Lax I., Mitra A. K., Ravera C., Hurwitz D. R., Rubinstein M., Ullrich A., Stroud R. M., Schlessinger J. Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain. J Biol Chem. 1991 Jul 25;266(21):13828–13833. [PubMed] [Google Scholar]
  29. Levitzki A., Schlessinger J. Cooperativity in associating proteins. Monomer-dimer equilibrium coupled to ligand binding. Biochemistry. 1974 Dec 3;13(25):5214–5219. doi: 10.1021/bi00722a026. [DOI] [PubMed] [Google Scholar]
  30. Lin C. R., Chen W. S., Lazar C. S., Carpenter C. D., Gill G. N., Evans R. M., Rosenfeld M. G. Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms. Cell. 1986 Mar 28;44(6):839–848. doi: 10.1016/0092-8674(86)90006-1. [DOI] [PubMed] [Google Scholar]
  31. Lund K. A., Lazar C. S., Chen W. S., Walsh B. J., Welsh J. B., Herbst J. J., Walton G. M., Rosenfeld M. G., Gill G. N., Wiley H. S. Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation. J Biol Chem. 1990 Nov 25;265(33):20517–20523. [PubMed] [Google Scholar]
  32. 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]
  33. McCaffrey P. G., Friedman B., Rosner M. R. Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1984 Oct 25;259(20):12502–12507. [PubMed] [Google Scholar]
  34. Metzger H., Alcaraz G., Hohman R., Kinet J. P., Pribluda V., Quarto R. The receptor with high affinity for immunoglobulin E. Annu Rev Immunol. 1986;4:419–470. doi: 10.1146/annurev.iy.04.040186.002223. [DOI] [PubMed] [Google Scholar]
  35. Northwood I. C., Davis R. J. Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization. J Biol Chem. 1988 Jun 5;263(16):7450–7453. [PubMed] [Google Scholar]
  36. Northwood I. C., Davis R. J. Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization. J Biol Chem. 1988 Jun 5;263(16):7450–7453. [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. Schlessinger J. Allosteric regulation of the epidermal growth factor receptor kinase. J Cell Biol. 1986 Dec;103(6 Pt 1):2067–2072. doi: 10.1083/jcb.103.6.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schlessinger J. Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci. 1988 Nov;13(11):443–447. doi: 10.1016/0968-0004(88)90219-8. [DOI] [PubMed] [Google Scholar]
  40. Schreiner G. F., Unanue E. R. Membrane and cytoplasmic changes in B lymphocytes induced by ligand-surface immunoglobulin interaction. Adv Immunol. 1976;24:37–165. doi: 10.1016/s0065-2776(08)60329-6. [DOI] [PubMed] [Google Scholar]
  41. Slieker L. J., Lane M. D. Post-translational processing of the epidermal growth factor receptor. Glycosylation-dependent acquisition of ligand-binding capacity. J Biol Chem. 1985 Jan 25;260(2):687–690. [PubMed] [Google Scholar]
  42. Smith K. A. The interleukin 2 receptor. Annu Rev Cell Biol. 1989;5:397–425. doi: 10.1146/annurev.cb.05.110189.002145. [DOI] [PubMed] [Google Scholar]
  43. Sorkin A., Carpenter G. Dimerization of internalized epidermal growth factor receptors. J Biol Chem. 1991 Dec 5;266(34):23453–23460. [PubMed] [Google Scholar]
  44. Spaargaren M., Defize L. H., Boonstra J., de Laat S. W. Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase. J Biol Chem. 1991 Jan 25;266(3):1733–1739. [PubMed] [Google Scholar]
  45. Unkeless J. C., Fleit H., Mellman I. S. Structural Aspects and Heterogeneity of Immunoglobulin Fc Receptors. Adv Immunol. 1981;31:247–270. doi: 10.1016/s0065-2776(08)60922-0. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Yarden Y., Schlessinger J. Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry. 1987 Mar 10;26(5):1443–1451. doi: 10.1021/bi00379a035. [DOI] [PubMed] [Google Scholar]
  49. Yarden Y., Schlessinger J. Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation. Biochemistry. 1987 Mar 10;26(5):1434–1442. doi: 10.1021/bi00379a034. [DOI] [PubMed] [Google Scholar]

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