Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Nov 1;327(Pt 3):859–865. doi: 10.1042/bj3270859

Superagonistic behaviour of epidermal growth factor/transforming growth factor-alpha chimaeras: correlation with receptor routing after ligand-induced internalization.

A E Lenferink 1, R H Kramer 1, M J van Vugt 1, M Königswieser 1, P P Di Fiore 1, E J van Zoelen 1, M L van de Poll 1
PMCID: PMC1218868  PMID: 9581567

Abstract

Human epidermal growth factor (EGF) and human transforming growth factor alpha (TGF-alpha) are structurally related polypeptide growth factors that exert their mitogenic activity through interaction with a common cell-surface receptor, the epidermal growth factor receptor (EGFR). The biological effect induced by these two ligands is quantitatively similar in most cases; in some test systems, however, TGF-alpha functions as a more potent form of EGF. In this study, we have compared EGF, TGF-alpha and ten previously described chimaeras of these two ligands in terms of their ability to generate a mitogenic response in cells carrying the human EGFR, and observed that three of the mutant growth factors (E3T, E4T and T3E4T) are mitogenic at concentrations 10-fold lower than that of either wild-type EGF or TGF-alpha. No difference in tyrosine kinase activity of the receptor towards an external substrate was observed after binding of the various mutants. It has been established before [Ebner and Derynck (1991) Cell Regulation 2, 599-612] that EGF and TGF-alpha differ in the processing of the receptor-ligand complex after internalization, as a result of their different pH sensitivities of receptor binding. Similar measurements on our chimaeric mutants revealed that the above superagonists show an enhanced pH dependence of binding in comparison with EGF. Furthermore, induction of receptor recycling by these superagonists is largely comparable with that induced by TGF-alpha. No superagonistic behaviour was observed on a cell-line containing an EGFR/erbB-2 chimaera which does not show ligand-induced internalization. These data show that EGF/TGFalpha chimaeras can be more active than the naturally occurring ligands, and that receptor recycling after ligand-induced internalization seems to be a prerequisite for this phenomenon.

Full Text

The Full Text of this article is available as a PDF (791.2 KB).

Selected References

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

  1. Baass P. C., Di Guglielmo G. M., Authier F., Posner B. I., Bergeron J. J. Compartmentalized signal transduction by receptor tyrosine kinases. Trends Cell Biol. 1995 Dec;5(12):465–470. doi: 10.1016/s0962-8924(00)89116-3. [DOI] [PubMed] [Google Scholar]
  2. Barrandon Y., Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor. Cell. 1987 Sep 25;50(7):1131–1137. doi: 10.1016/0092-8674(87)90179-6. [DOI] [PubMed] [Google Scholar]
  3. Basu S. K., Goldstein J. L., Anderson R. G., Brown M. S. Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell. 1981 May;24(2):493–502. doi: 10.1016/0092-8674(81)90340-8. [DOI] [PubMed] [Google Scholar]
  4. Boukamp P., Petrussevska R. T., Breitkreutz D., Hornung J., Markham A., Fusenig N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988 Mar;106(3):761–771. doi: 10.1083/jcb.106.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Campion S. R., Niyogi S. K. Interaction of epidermal growth factor with its receptor. Prog Nucleic Acid Res Mol Biol. 1994;49:353–383. doi: 10.1016/s0079-6603(08)60055-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Di Guglielmo G. M., Baass P. C., Ou W. J., Posner B. I., Bergeron J. J. Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. EMBO J. 1994 Sep 15;13(18):4269–4277. doi: 10.1002/j.1460-2075.1994.tb06747.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ebner R., Derynck R. Epidermal growth factor and transforming growth factor-alpha: differential intracellular routing and processing of ligand-receptor complexes. Cell Regul. 1991 Aug;2(8):599–612. doi: 10.1091/mbc.2.8.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Felder S., Miller K., Moehren G., Ullrich A., Schlessinger J., Hopkins C. R. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 1990 May 18;61(4):623–634. doi: 10.1016/0092-8674(90)90474-s. [DOI] [PubMed] [Google Scholar]
  10. French A. R., Tadaki D. K., Niyogi S. K., Lauffenburger D. A. Intracellular trafficking of epidermal growth factor family ligands is directly influenced by the pH sensitivity of the receptor/ligand interaction. J Biol Chem. 1995 Mar 3;270(9):4334–4340. doi: 10.1074/jbc.270.9.4334. [DOI] [PubMed] [Google Scholar]
  11. Gladhaug I. P., Christoffersen T. Rapid constitutive internalization and externalization of epidermal growth factor receptors in isolated rat hepatocytes. Monensin inhibits receptor externalization and reduces the capacity for continued endocytosis of epidermal growth factor. J Biol Chem. 1988 Sep 5;263(25):12199–12203. [PubMed] [Google Scholar]
  12. Groenen L. C., Nice E. C., Burgess A. W. Structure-function relationships for the EGF/TGF-alpha family of mitogens. Growth Factors. 1994;11(4):235–257. doi: 10.3109/08977199409010997. [DOI] [PubMed] [Google Scholar]
  13. Honegger A., Dull T. J., Bellot F., Van Obberghen E., Szapary D., Schmidt A., Ullrich A., Schlessinger J. Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites. EMBO J. 1988 Oct;7(10):3045–3052. doi: 10.1002/j.1460-2075.1988.tb03169.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hynes N. E., Stern D. F. The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta. 1994 Dec 30;1198(2-3):165–184. doi: 10.1016/0304-419x(94)90012-4. [DOI] [PubMed] [Google Scholar]
  15. Karunagaran D., Tzahar E., Beerli R. R., Chen X., Graus-Porta D., Ratzkin B. J., Seger R., Hynes N. E., Yarden Y. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J. 1996 Jan 15;15(2):254–264. [PMC free article] [PubMed] [Google Scholar]
  16. Kienhuis C. B., Heuvel J. J., Ross H. A., Swinkels L. M., Foekens J. A., Benraad T. J. Six methods for direct radioiodination of mouse epidermal growth factor compared: effect of nonequivalence in binding behavior between labeled and unlabeled ligand. Clin Chem. 1991 Oct;37(10 Pt 1):1749–1755. [PubMed] [Google Scholar]
  17. Kramer R. H., Lenferink A. E., van Bueren-Koornneef I. L., van der Meer A., van de Poll M. L., van Zoelen E. J. Identification of the high affinity binding site of transforming growth factor-alpha (TGF-alpha) for the chicken epidermal growth factor (EGF) receptor using EGF/TGF-alpha chimeras. J Biol Chem. 1994 Mar 25;269(12):8708–8711. [PubMed] [Google Scholar]
  18. Lax I., Bellot F., Howk R., Ullrich A., Givol D., Schlessinger J. Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules. EMBO J. 1989 Feb;8(2):421–427. doi: 10.1002/j.1460-2075.1989.tb03393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lax I., Johnson A., Howk R., Sap J., Bellot F., Winkler M., Ullrich A., Vennstrom B., Schlessinger J., Givol D. Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha. Mol Cell Biol. 1988 May;8(5):1970–1978. doi: 10.1128/mcb.8.5.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee J., Dull T. J., Lax I., Schlessinger J., Ullrich A. HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor. EMBO J. 1989 Jan;8(1):167–173. doi: 10.1002/j.1460-2075.1989.tb03361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Massagué J. Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors in human placenta membranes and A431 cells. J Biol Chem. 1983 Nov 25;258(22):13614–13620. [PubMed] [Google Scholar]
  22. Nesterov A., Lysan S., Vdovina I., Nikolsky N., Fujita D. J. Phosphorylation of the epidermal growth factor receptor during internalization in A-431 cells. Arch Biochem Biophys. 1994 Sep;313(2):351–359. doi: 10.1006/abbi.1994.1398. [DOI] [PubMed] [Google Scholar]
  23. Nilsson B., Abrahmsén L. Fusions to staphylococcal protein A. Methods Enzymol. 1990;185:144–161. doi: 10.1016/0076-6879(90)85015-g. [DOI] [PubMed] [Google Scholar]
  24. Pinkas-Kramarski R., Soussan L., Waterman H., Levkowitz G., Alroy I., Klapper L., Lavi S., Seger R., Ratzkin B. J., Sela M. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J. 1996 May 15;15(10):2452–2467. [PMC free article] [PubMed] [Google Scholar]
  25. Puddicombe S. M., Wood L., Chamberlin S. G., Davies D. E. The interaction of an epidermal growth factor/transforming growth factor alpha tail chimera with the human epidermal growth factor receptor reveals unexpected complexities. J Biol Chem. 1996 Nov 29;271(48):30392–30397. doi: 10.1074/jbc.271.48.30392. [DOI] [PubMed] [Google Scholar]
  26. Reddy C. C., Niyogi S. K., Wells A., Wiley H. S., Lauffenburger D. A. Engineering epidermal growth factor for enhanced mitogenic potency. Nat Biotechnol. 1996 Dec;14(13):1696–1699. doi: 10.1038/nbt1296-1696. [DOI] [PubMed] [Google Scholar]
  27. Schreiber A. B., Winkler M. E., Derynck R. Transforming growth factor-alpha: a more potent angiogenic mediator than epidermal growth factor. Science. 1986 Jun 6;232(4755):1250–1253. doi: 10.1126/science.2422759. [DOI] [PubMed] [Google Scholar]
  28. Sorkin A., Di Fiore P. P., Carpenter G. The carboxyl terminus of epidermal growth factor receptor/erbB-2 chimerae is internalization impaired. Oncogene. 1993 Nov;8(11):3021–3028. [PubMed] [Google Scholar]
  29. Sorkin A., Waters C. M. Endocytosis of growth factor receptors. Bioessays. 1993 Jun;15(6):375–382. doi: 10.1002/bies.950150603. [DOI] [PubMed] [Google Scholar]
  30. Strauch K. L., Johnson K., Beckwith J. Characterization of degP, a gene required for proteolysis in the cell envelope and essential for growth of Escherichia coli at high temperature. J Bacteriol. 1989 May;171(5):2689–2696. doi: 10.1128/jb.171.5.2689-2696.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walker F., Nice E., Fabri L., Moy F. J., Liu J. F., Wu R., Scheraga H. A., Burgess A. W. Resistance to receptor-mediated degradation of a murine epidermal growth factor analogue (EGF-Val-47) potentiates its mitogenic activity. Biochemistry. 1990 Nov 27;29(47):10635–10640. doi: 10.1021/bi00499a009. [DOI] [PubMed] [Google Scholar]
  33. Wiley H. S., Herbst J. J., Walsh B. J., Lauffenburger D. A., Rosenfeld M. G., Gill G. N. The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor. J Biol Chem. 1991 Jun 15;266(17):11083–11094. [PubMed] [Google Scholar]
  34. Winkler M. E., Bringman T., Marks B. J. The purification of fully active recombinant transforming growth factor alpha produced in Escherichia coli. J Biol Chem. 1986 Oct 15;261(29):13838–13843. [PubMed] [Google Scholar]
  35. Zapf A., Hsu D., Olefsky J. M. Comparison of the intracellular itineraries of insulin-like growth factor-I and insulin and their receptors in Rat-1 fibroblasts. Endocrinology. 1994 Jun;134(6):2445–2452. doi: 10.1210/endo.134.6.8194471. [DOI] [PubMed] [Google Scholar]
  36. van Zoelen E. J., Kramer R. H., van Reen M. M., Veerkamp J. H., Ross H. A. An exact general analysis of ligand binding displacement and saturation curves. Biochemistry. 1993 Jun 22;32(24):6275–6280. doi: 10.1021/bi00075a022. [DOI] [PubMed] [Google Scholar]
  37. van Zoelen E. J., van Oostwaard T. M., de Laat S. W. Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor. J Biol Chem. 1986 Apr 15;261(11):5003–5009. [PubMed] [Google Scholar]
  38. van de Poll M. L., Lenferink A. E., van Vugt M. J., Jacobs J. J., Janssen J. W., Joldersma M., van Zoelen E. J. A single amino acid exchange, Arg-45 to Ala, generates an epidermal growth factor (EGF) mutant with high affinity for the chicken EGF receptor. J Biol Chem. 1995 Sep 22;270(38):22337–22343. doi: 10.1074/jbc.270.38.22337. [DOI] [PubMed] [Google Scholar]
  39. van de Poll M. L., van Vugt M. J., Lenferink A. E., van Zoelen E. J. Insertion of Argos sequences into the B-loop of epidermal growth factor results in a low-affinity ligand with strong agonistic activity. Biochemistry. 1997 Jun 17;36(24):7425–7431. doi: 10.1021/bi970227f. [DOI] [PubMed] [Google Scholar]

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

RESOURCES