Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Apr 15;25(8):1585–1590. doi: 10.1093/nar/25.8.1585

A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage.

C Souriau 1, P Fort 1, P Roux 1, O Hartley 1, M P Lefranc 1, M Weill 1
PMCID: PMC146613  PMID: 9092666

Abstract

Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

Full Text

The Full Text of this article is available as a PDF (129.4 KB).

Selected References

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

  1. Boonstra J., Rijken P., Humbel B., Cremers F., Verkleij A., van Bergen en Henegouwen P. The epidermal growth factor. Cell Biol Int. 1995 May;19(5):413–430. doi: 10.1006/cbir.1995.1086. [DOI] [PubMed] [Google Scholar]
  2. Campion S. R., Biamonti C., Montelione G. T., Niyogi S. K. The role of asparagine-32 in forming the receptor-binding epitope of human epidermal growth factor. Protein Eng. 1993 Aug;6(6):651–659. doi: 10.1093/protein/6.6.651. [DOI] [PubMed] [Google Scholar]
  3. Campion S. R., Tadaki D. K., Niyogi S. K. Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification. J Cell Biochem. 1992 Sep;50(1):35–42. doi: 10.1002/jcb.240500108. [DOI] [PubMed] [Google Scholar]
  4. Clackson T., Wells J. A. In vitro selection from protein and peptide libraries. Trends Biotechnol. 1994 May;12(5):173–184. doi: 10.1016/0167-7799(94)90079-5. [DOI] [PubMed] [Google Scholar]
  5. Di Fiore P. P., Pierce J. H., Fleming T. P., Hazan R., Ullrich A., King C. R., Schlessinger J., Aaronson S. A. Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell. 1987 Dec 24;51(6):1063–1070. doi: 10.1016/0092-8674(87)90592-7. [DOI] [PubMed] [Google Scholar]
  6. Dudgeon T. J., Cooke R. M., Baron M., Campbell I. D., Edwards R. M., Fallon A. Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance. FEBS Lett. 1990 Feb 26;261(2):392–396. doi: 10.1016/0014-5793(90)80600-n. [DOI] [PubMed] [Google Scholar]
  7. Engler D. A., Campion S. R., Hauser M. R., Cook J. S., Niyogi S. K. Critical functional requirement for the guanidinium group of the arginine 41 side chain of human epidermal growth factor as revealed by mutagenic inactivation and chemical reactivation. J Biol Chem. 1992 Feb 5;267(4):2274–2281. [PubMed] [Google Scholar]
  8. Fan Z., Baselga J., Masui H., Mendelsohn J. Antitumor effect of anti-epidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res. 1993 Oct 1;53(19):4637–4642. [PubMed] [Google Scholar]
  9. Fan Z., Masui H., Altas I., Mendelsohn J. Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 1993 Sep 15;53(18):4322–4328. [PubMed] [Google Scholar]
  10. Fu X. Y., Zhang J. J. Transcription factor p91 interacts with the epidermal growth factor receptor and mediates activation of the c-fos gene promoter. Cell. 1993 Sep 24;74(6):1135–1145. doi: 10.1016/0092-8674(93)90734-8. [DOI] [PubMed] [Google Scholar]
  11. Gill G. N., Kawamoto T., Cochet C., Le A., Sato J. D., Masui H., McLeod C., Mendelsohn J. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Jun 25;259(12):7755–7760. [PubMed] [Google Scholar]
  12. Griffiths A. D., Williams S. C., Hartley O., Tomlinson I. M., Waterhouse P., Crosby W. L., Kontermann R. E., Jones P. T., Low N. M., Allison T. J. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 1994 Jul 15;13(14):3245–3260. doi: 10.1002/j.1460-2075.1994.tb06626.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gritz L., Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983 Nov;25(2-3):179–188. doi: 10.1016/0378-1119(83)90223-8. [DOI] [PubMed] [Google Scholar]
  14. Heldin C. H. Dimerization of cell surface receptors in signal transduction. Cell. 1995 Jan 27;80(2):213–223. doi: 10.1016/0092-8674(95)90404-2. [DOI] [PubMed] [Google Scholar]
  15. Herbst R., Lammers R., Schlessinger J., Ullrich A. Substrate phosphorylation specificity of the human c-kit receptor tyrosine kinase. J Biol Chem. 1991 Oct 25;266(30):19908–19916. [PubMed] [Google Scholar]
  16. Hommel U., Harvey T. S., Driscoll P. C., Campbell I. D. Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha. J Mol Biol. 1992 Sep 5;227(1):271–282. doi: 10.1016/0022-2836(92)90697-i. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Matsunami R. K., Yette M. L., Stevens A., Niyogi S. K. Mutational analysis of leucine 47 in human epidermal growth factor. J Cell Biochem. 1991 Jul;46(3):242–249. doi: 10.1002/jcb.240460307. [DOI] [PubMed] [Google Scholar]
  20. Maxwell I. H., Harrison G. S., Wood W. M., Maxwell F. A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Biotechniques. 1989 Mar;7(3):276–280. [PubMed] [Google Scholar]
  21. Mendelsohn J. Epidermal growth factor receptor as a target for therapy with antireceptor monoclonal antibodies. J Natl Cancer Inst Monogr. 1992;(13):125–131. [PubMed] [Google Scholar]
  22. Oka T., Sakamoto S., Miyoshi K., Fuwa T., Yoda K., Yamasaki M., Tamura G., Miyake T. Synthesis and secretion of human epidermal growth factor by Escherichia coli. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7212–7216. doi: 10.1073/pnas.82.21.7212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Riechmann L., Weill M. Phage display and selection of a site-directed randomized single-chain antibody Fv fragment for its affinity improvement. Biochemistry. 1993 Aug 31;32(34):8848–8855. doi: 10.1021/bi00085a016. [DOI] [PubMed] [Google Scholar]
  24. Riedel H., Massoglia S., Schlessinger J., Ullrich A. Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1477–1481. doi: 10.1073/pnas.85.5.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Scott J. K., Smith G. P. Searching for peptide ligands with an epitope library. Science. 1990 Jul 27;249(4967):386–390. doi: 10.1126/science.1696028. [DOI] [PubMed] [Google Scholar]
  26. Smith G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985 Jun 14;228(4705):1315–1317. doi: 10.1126/science.4001944. [DOI] [PubMed] [Google Scholar]
  27. Smith G. P., Scott J. K. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 1993;217:228–257. doi: 10.1016/0076-6879(93)17065-d. [DOI] [PubMed] [Google Scholar]
  28. Tadaki D. K., Niyogi S. K. The functional importance of hydrophobicity of the tyrosine at position 13 of human epidermal growth factor in receptor binding. J Biol Chem. 1993 May 15;268(14):10114–10119. [PubMed] [Google Scholar]
  29. Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986 Aug 15;46(4):567–574. doi: 10.1016/0092-8674(86)90882-2. [DOI] [PubMed] [Google Scholar]
  30. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  31. Velu T. J., Beguinot L., Vass W. C., Willingham M. C., Merlino G. T., Pastan I., Lowy D. R. Epidermal-growth-factor-dependent transformation by a human EGF receptor proto-oncogene. Science. 1987 Dec 4;238(4832):1408–1410. doi: 10.1126/science.3500513. [DOI] [PubMed] [Google Scholar]
  32. Wagner B. J., Hayes T. E., Hoban C. J., Cochran B. H. The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO J. 1990 Dec;9(13):4477–4484. doi: 10.1002/j.1460-2075.1990.tb07898.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yarden Y., Harari I., Schlessinger J. Purification of an active EGF receptor kinase with monoclonal antireceptor antibodies. J Biol Chem. 1985 Jan 10;260(1):315–319. [PubMed] [Google Scholar]
  34. Yarden Y. Receptor-like oncogenes: functional analysis through novel experimental approaches. Mol Immunol. 1990 Dec;27(12):1319–1324. doi: 10.1016/0161-5890(90)90037-z. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES