Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Mar;8(3):1247–1252. doi: 10.1128/mcb.8.3.1247

Transforming growth factor alpha: mutation of aspartic acid 47 and leucine 48 results in different biological activities.

E Lazar 1, S Watanabe 1, S Dalton 1, M B Sporn 1
PMCID: PMC363269  PMID: 3285178

Abstract

To study the relationship between the primary structure of transforming growth factor alpha (TGF-alpha) and some of its functional properties (competition with epidermal growth factor (EGF) for binding to the EGF receptor and induction of anchorage-independent growth), we introduced single amino acid mutations into the sequence for the fully processed, 50-amino-acid human TGF-alpha. The wild-type and mutant proteins were expressed in a vector by using a yeast alpha mating pheromone promoter. Mutations of two amino acids that are conserved in the family of the EGF-like peptides and are located in the carboxy-terminal part of TGF-alpha resulted in different biological effects. When aspartic acid 47 was mutated to alanine or asparagine, biological activity was retained; in contrast, substitutions of this residue with serine or glutamic acid generated mutants with reduced binding and colony-forming capacities. When leucine 48 was mutated to alanine, a complete loss of binding and colony-forming abilities resulted; mutation of leucine 48 to isoleucine or methionine resulted in very low activities. Our data suggest that these two adjacent conserved amino acids in positions 47 and 48 play different roles in defining the structure and/or biological activity of TGF-alpha and that the carboxy terminus of TGF-alpha is involved in interactions with cellular TGF-alpha receptors. The side chain of leucine 48 appears to be crucial either indirectly in determining the biologically active conformation of TGF-alpha or directly in the molecular recognition of TGF-alpha by its receptor.

Full text

PDF
1249

Selected References

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

  1. Anzano M. A., Roberts A. B., Smith J. M., Sporn M. B., De Larco J. E. Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type alpha and type beta transforming growth factors. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6264–6268. doi: 10.1073/pnas.80.20.6264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blomquist M. C., Hunt L. T., Barker W. C. Vaccinia virus 19-kilodalton protein: relationship to several mammalian proteins, including two growth factors. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7363–7367. doi: 10.1073/pnas.81.23.7363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Bringman T. S., Lindquist P. B., Derynck R. Different transforming growth factor-alpha species are derived from a glycosylated and palmitoylated transmembrane precursor. Cell. 1987 Feb 13;48(3):429–440. doi: 10.1016/0092-8674(87)90194-2. [DOI] [PubMed] [Google Scholar]
  5. COHEN S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem. 1962 May;237:1555–1562. [PubMed] [Google Scholar]
  6. Carpenter G., Cohen S. Epidermal growth factor. Annu Rev Biochem. 1979;48:193–216. doi: 10.1146/annurev.bi.48.070179.001205. [DOI] [PubMed] [Google Scholar]
  7. Chang W., Upton C., Hu S. L., Purchio A. F., McFadden G. The genome of Shope fibroma virus, a tumorigenic poxvirus, contains a growth factor gene with sequence similarity to those encoding epidermal growth factor and transforming growth factor alpha. Mol Cell Biol. 1987 Jan;7(1):535–540. doi: 10.1128/mcb.7.1.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooke R. M., Wilkinson A. J., Baron M., Pastore A., Tappin M. J., Campbell I. D., Gregory H., Sheard B. The solution structure of human epidermal growth factor. 1987 May 28-Jun 3Nature. 327(6120):339–341. doi: 10.1038/327339a0. [DOI] [PubMed] [Google Scholar]
  9. Derynck R., Goeddel D. V., Ullrich A., Gutterman J. U., Williams R. D., Bringman T. S., Berger W. H. Synthesis of messenger RNAs for transforming growth factors alpha and beta and the epidermal growth factor receptor by human tumors. Cancer Res. 1987 Feb 1;47(3):707–712. [PubMed] [Google Scholar]
  10. Derynck R., Roberts A. B., Winkler M. E., Chen E. Y., Goeddel D. V. Human transforming growth factor-alpha: precursor structure and expression in E. coli. Cell. 1984 Aug;38(1):287–297. doi: 10.1016/0092-8674(84)90550-6. [DOI] [PubMed] [Google Scholar]
  11. Gregory H. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature. 1975 Sep 25;257(5524):325–327. doi: 10.1038/257325a0. [DOI] [PubMed] [Google Scholar]
  12. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hitzeman R. A., Chang C. N., Matteucci M., Perry L. J., Kohr W. J., Wulf J. J., Swartz J. R., Chen C. Y., Singh A. Construction of expression vectors for secretion of human interferons by yeast. Methods Enzymol. 1986;119:424–433. doi: 10.1016/0076-6879(86)19062-8. [DOI] [PubMed] [Google Scholar]
  14. Hollenberg M. D., Gregory H. Epidermal growth factor-urogastrone: biological activity and receptor binding of derivatives. Mol Pharmacol. 1980 May;17(3):314–320. [PubMed] [Google Scholar]
  15. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee D. C., Rochford R., Todaro G. J., Villarreal L. P. Developmental expression of rat transforming growth factor-alpha mRNA. Mol Cell Biol. 1985 Dec;5(12):3644–3646. doi: 10.1128/mcb.5.12.3644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marquardt H., Hunkapiller M. W., Hood L. E., Todaro G. J. Rat transforming growth factor type 1: structure and relation to epidermal growth factor. Science. 1984 Mar 9;223(4640):1079–1082. doi: 10.1126/science.6320373. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  20. Montelione G. T., Wüthrich K., Nice E. C., Burgess A. W., Scheraga H. A. Identification of two anti-parallel beta-sheet conformations in the solution structure of murine epidermal growth factor by proton magnetic resonance. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8594–8598. doi: 10.1073/pnas.83.22.8594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Montelione G. T., Wüthrich K., Nice E. C., Burgess A. W., Scheraga H. A. Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5226–5230. doi: 10.1073/pnas.84.15.5226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Samsoondar J., Kobrin M. S., Kudlow J. E. Alpha-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. I. Purification from conditioned medium. J Biol Chem. 1986 Nov 5;261(31):14408–14413. [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Savage C. R., Jr, Hash J. H., Cohen S. Epidermal growth factor. Location of disulfide bonds. J Biol Chem. 1973 Nov 25;248(22):7669–7672. [PubMed] [Google Scholar]
  25. Shechter Y., Schlessinger J., Jacobs S., Chang K. J., Cuatrecasas P. Fluorescent labeling of hormone receptors in viable cells: preparation and properties of highly fluorescent derivatives of epidermal growth factor and insulin. Proc Natl Acad Sci U S A. 1978 May;75(5):2135–2139. doi: 10.1073/pnas.75.5.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Simpson R. J., Smith J. A., Moritz R. L., O'Hare M. J., Rudland P. S., Morrison J. R., Lloyd C. J., Grego B., Burgess A. W., Nice E. C. Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity. Eur J Biochem. 1985 Dec 16;153(3):629–637. doi: 10.1111/j.1432-1033.1985.tb09346.x. [DOI] [PubMed] [Google Scholar]
  27. Singh A., Lugovoy J. M., Kohr W. J., Perry L. J. Synthesis, secretion and processing of alpha-factor-interferon fusion proteins in yeast. Nucleic Acids Res. 1984 Dec 11;12(23):8927–8938. doi: 10.1093/nar/12.23.8927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Todaro G. J., Fryling C., De Larco J. E. Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5258–5262. doi: 10.1073/pnas.77.9.5258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Upton C., Macen J. L., McFadden G. Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha. J Virol. 1987 Apr;61(4):1271–1275. doi: 10.1128/jvi.61.4.1271-1275.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Watanabe S., Lazar E., Sporn M. B. Transformation of normal rat kidney (NRK) cells by an infectious retrovirus carrying a synthetic rat type alpha transforming growth factor gene. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1258–1262. doi: 10.1073/pnas.84.5.1258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. de Larco J. E., Todaro G. J. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):4001–4005. doi: 10.1073/pnas.75.8.4001. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES