Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1988 May;62(5):1634–1639. doi: 10.1128/jvi.62.5.1634-1639.1988

Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.

L Ellis 1, A Levitan 1, M H Cobb 1, P Ramos 1
PMCID: PMC253191  PMID: 2833613

Abstract

The human insulin receptor (IR) is a transmembrane glycoprotein, whose cytoplasmic domain contains an insulin-activated protein-tyrosine kinase (EC 2.7.1.112). By the use of an appropriately engineered baculovirus expression vector, a soluble cytoplasmic derivative of this domain was expressed in the insect cell line Spodoptera frugiperda (Sf9). At 24 to 48 h after Sf9 cells were infected with recombinant virus, a protein of the size expected for this domain (approximately 48 kilodaltons) constituted a major band when total cell lysates of metabolically labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. This protein (designated AchIRPTK) was immunoprecipitated by three monoclonal antibodies, each of which recognizes a distinct antigenic site of the IR cytoplasmic domain and requires the native structure of the protein for recognition and one of which binds at or near the physiologically relevant site(s) of IR autophosphorylation. In vivo, AchIRPTK was phosphorylated on both tyrosine and serine residues. When affinity purified, the kinase was active in vitro; it autophosphorylated exclusively on tyrosine residues, and phosphorylated the exogenous substrates histone H2b and poly(Glu-Tyr). The expression of an active IR protein-tyrosine kinase molecule in this heterologous cell system provides an efficient experimental method for producing this domain in quantity for enzymatic and structural studies.

Full text

PDF
1634

Images in this article

1636Image
on p.1636
1636Image
on p.1636
1637Image
on p.1637
1637Image
on p.1637
1638Image
on p.1638

Selected References

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

  1. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  2. Ebina Y., Ellis L., Jarnagin K., Edery M., Graf L., Clauser E., Ou J. H., Masiarz F., Kan Y. W., Goldfine I. D. The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell. 1985 Apr;40(4):747–758. doi: 10.1016/0092-8674(85)90334-4. [DOI] [PubMed] [Google Scholar]
  3. Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
  4. Ellis L., Morgan D. O., Clauser E., Edery M., Jong S. M., Wang L. H., Roth R. A., Rutter W. J. Mechanisms of receptor-mediated transmembrane communication. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 2):773–784. doi: 10.1101/sqb.1986.051.01.090. [DOI] [PubMed] [Google Scholar]
  5. Ellis L., Morgan D. O., Clauser E., Roth R. A., Rutter W. J. A membrane-anchored cytoplasmic domain of the human insulin receptor mediates a constitutively elevated insulin-independent uptake of 2-deoxyglucose. Mol Endocrinol. 1987 Jan;1(1):15–24. doi: 10.1210/mend-1-1-15. [DOI] [PubMed] [Google Scholar]
  6. Ellis L., Morgan D. O., Jong S. M., Wang L. H., Roth R. A., Rutter W. J. Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5101–5105. doi: 10.1073/pnas.84.15.5101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis L., Morgan D. O., Koshland D. E., Jr, Clauser E., Moe G. R., Bollag G., Roth R. A., Rutter W. J. Linking functional domains of the human insulin receptor with the bacterial aspartate receptor. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8137–8141. doi: 10.1073/pnas.83.21.8137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  9. Kahn C. R. The molecular mechanism of insulin action. Annu Rev Med. 1985;36:429–451. doi: 10.1146/annurev.me.36.020185.002241. [DOI] [PubMed] [Google Scholar]
  10. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miyamoto C., Smith G. E., Farrell-Towt J., Chizzonite R., Summers M. D., Ju G. Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1985 Oct;5(10):2860–2865. doi: 10.1128/mcb.5.10.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Morgan D. O., Ho L., Korn L. J., Roth R. A. Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase. Proc Natl Acad Sci U S A. 1986 Jan;83(2):328–332. doi: 10.1073/pnas.83.2.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Morgan D. O., Roth R. A. Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain. Biochemistry. 1986 Mar 25;25(6):1364–1371. doi: 10.1021/bi00354a026. [DOI] [PubMed] [Google Scholar]
  14. Rosen O. M. After insulin binds. Science. 1987 Sep 18;237(4821):1452–1458. doi: 10.1126/science.2442814. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Smith G. E., Ju G., Ericson B. L., Moschera J., Lahm H. W., Chizzonite R., Summers M. D. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8404–8408. doi: 10.1073/pnas.82.24.8404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Smith G. E., Summers M. D., Fraser M. J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983 Dec;3(12):2156–2165. doi: 10.1128/mcb.3.12.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  19. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES