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. 1981 Jan;78(1):445–449. doi: 10.1073/pnas.78.1.445

Genetics of cell surface receptors for bioactive polypeptides: variants of Swiss/3T3 fibroblasts resistant to a cytotoxic chimeric insulin.

W K Miskimins, N Shimizu
PMCID: PMC319070  PMID: 6264443

Abstract

Insulin crosslinked to the catalytic fragment A of diphtheria toxin has previously been shown to be cytotoxic to insulin-responsive mouse BALBc/3T3 cells but noncytotoxic to insulin-nonresponsive variant IN-2 cells [Miskimins, W. K. & Shimizu, N. (1979) Biochem. Biophys. Res. Commun. 91, 143-151]. We have now used this cytotoxic chimeric insulin to select resistant variants of Swiss/3T3 fibroblasts. Of eight resistant colonies isolated, two proved to be deficient in insulin receptor and exhibited altered morphologies and growth rates. These variants, however, retained the ability to bind and respond to epidermal growth factor. Thus, this selection technique is effective for the isolation of variants specifically related to cellular binding and processing of insulin. Such variants will be advantageous for the genetic and biochemical characterization of the mechanism of insulin's action.

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Selected References

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

  1. Catt K. J., Harwood J. P., Aguilera G., Dufau M. L. Hormonal regulation of peptide receptors and target cell responses. Nature. 1979 Jul 12;280(5718):109–116. doi: 10.1038/280109a0. [DOI] [PubMed] [Google Scholar]
  2. Dorland R. B., Middlebrook J. L., Leppla S. H. Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells. J Biol Chem. 1979 Nov 25;254(22):11337–11342. [PubMed] [Google Scholar]
  3. Fox C. F., Das M. Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis. J Supramol Struct. 1979;10(2):199–214. doi: 10.1002/jss.400100210. [DOI] [PubMed] [Google Scholar]
  4. Gorden P., Carpentier J. L., Freychet P., LeCam A., Orci L. Intracellular translocation of iodine-125-labeled insulin: direct demonstration in isolated hepatocytes. Science. 1978 May 19;200(4343):782–785. doi: 10.1126/science.644321. [DOI] [PubMed] [Google Scholar]
  5. Kahn C. R., Baird K. The fate of insulin bound to adipocytes. Evidence for compartmentalization and processing. J Biol Chem. 1978 Jul 25;253(14):4900–4906. [PubMed] [Google Scholar]
  6. Kahn C. R. Membrane receptors for hormones and neurotransmitters. J Cell Biol. 1976 Aug;70(2 Pt 1):261–286. doi: 10.1083/jcb.70.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Leppla S., Dorland R. B., Middlebrook J. L. Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J Biol Chem. 1980 Mar 25;255(6):2247–2250. [PubMed] [Google Scholar]
  8. Miskimins W. K., Shimizu N. Synthesis of a cytotoxic insulin cross-linked to diphtheria toxin fragment A capable of recognizing insulin receptors. Biochem Biophys Res Commun. 1979 Nov 14;91(1):143–151. doi: 10.1016/0006-291x(79)90595-3. [DOI] [PubMed] [Google Scholar]
  9. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  10. Pruss R. M., Herschman H. R. Variants of 3T3 cells lacking mitogenic response to epidermal growth factor. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3918–3921. doi: 10.1073/pnas.74.9.3918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sandvig K., Olsnes S., Pihl A. Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells. J Biol Chem. 1976 Jul 10;251(13):3977–3984. [PubMed] [Google Scholar]
  12. Savion N., Vlodavsky I., Gospodarowicz D. Role of the degradation process in the mitogenic effect of epidermal growth factor. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1466–1470. doi: 10.1073/pnas.77.3.1466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schlessinger J., Shechter Y., Willingham M. C., Pastan I. Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2659–2663. doi: 10.1073/pnas.75.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shimizu N., Behzadian M. A., Shimizu Y. Genetics of cell surface receptors for bioactive polypeptides: binding of epidermal growth factor is associated with the presence of human chromosome 7 in human-mouse cell hybrids. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3600–3604. doi: 10.1073/pnas.77.6.3600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shimizu N., Miskimins W. K., Shimizu Y. A cytotoxic epidermal growth factor cross-linked to diphtheria toxin A-fragment. FEBS Lett. 1980 Sep 8;118(2):274–278. doi: 10.1016/0014-5793(80)80237-7. [DOI] [PubMed] [Google Scholar]
  16. Shimizu Y., Shimizu N. Genetics of cell-surface receptors for bioactive polypeptides: a variant of mouse BALBc/3T3 fibroblasts possessing altered insulin-binding ability. Somatic Cell Genet. 1980 Sep;6(5):583–601. doi: 10.1007/BF01538639. [DOI] [PubMed] [Google Scholar]
  17. Siminovitch L. On the nature of hereditable variation in cultured somatic cells. Cell. 1976 Jan;7(1):1–11. doi: 10.1016/0092-8674(76)90249-x. [DOI] [PubMed] [Google Scholar]

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