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. 1990 Mar 1;110(3):559–568. doi: 10.1083/jcb.110.3.559

Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts

PMCID: PMC2116031  PMID: 2307699

Abstract

Measurements of nucleocytoplasmic transport of fluorescent-labeled macromolecules were performed in both an EGF-nonresponsive mutant fibroblast line (3T3-NR6) and in the same cell line reconstituted with active EGF receptors derived from rat hepatic membrane fraction. Immunolocalization studies of exogenously incorporated EGF receptors in reconstituted 3T3-NR6 fibroblasts demonstrated predominantly intracellular localization. The EGF receptor constructs also showed EGF- stimulated incorporation of [3H]thymidine, providing biochemical evidence for functional integration of the exogenously supplied EGF receptors into the reconstituted fibroblasts. Additional support for the functional incorporation of receptor may be inferred from the enhanced cellular accumulation of 125I-EGF in cells treated with chloroquine and leupeptin. 125I-EGF binding and transnuclear macromolecular transport measurements in mutant and reconstituted cells, in conjunction with such measurements on nuclei isolated from these cells, provide data consistent with a growth factor/nuclear signaling mechanism dependent on the nuclear acquisition of EGF binding activity from the plasma membrane.

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Selected References

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  1. Baglia F. A., Maul G. G. Nuclear ribonucleoprotein release and nucleoside triphosphatase activity are inhibited by antibodies directed against one nuclear matrix glycoprotein. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2285–2289. doi: 10.1073/pnas.80.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishayee S., Feinman J., Pittenger M., Michael H., Das M. Cell surface insertion of exogenous epidermal growth factor receptors into receptor- mutant cells: demonstration of insertion in the absence of added fusogenic agents. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1893–1897. doi: 10.1073/pnas.79.6.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carpentier J. L., White M. F., Orci L., Kahn R. C. Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells. J Cell Biol. 1987 Dec;105(6 Pt 1):2751–2762. doi: 10.1083/jcb.105.6.2751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen W. S., Lazar C. S., Poenie M., Tsien R. Y., Gill G. N., Rosenfeld M. G. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. 1987 Aug 27-Sep 2Nature. 328(6133):820–823. doi: 10.1038/328820a0. [DOI] [PubMed] [Google Scholar]
  5. Dean R. T., Jessup W., Roberts C. R. Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. Biochem J. 1984 Jan 1;217(1):27–40. doi: 10.1042/bj2170027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dunn W. A., Connolly T. P., Hubbard A. L. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol. 1986 Jan;102(1):24–36. doi: 10.1083/jcb.102.1.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Escobedo J. A., Williams L. T. A PDGF receptor domain essential for mitogenesis but not for many other responses to PDGF. Nature. 1988 Sep 1;335(6185):85–87. doi: 10.1038/335085a0. [DOI] [PubMed] [Google Scholar]
  8. Fields A. P., Pettit G. R., May W. S. Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C. J Biol Chem. 1988 Jun 15;263(17):8253–8260. [PubMed] [Google Scholar]
  9. Glenney J. R., Jr, Chen W. S., Lazar C. S., Walton G. M., Zokas L. M., Rosenfeld M. G., Gill G. N. Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies. Cell. 1988 Mar 11;52(5):675–684. doi: 10.1016/0092-8674(88)90405-9. [DOI] [PubMed] [Google Scholar]
  10. Goldfine I. D., Smith G. J. Binding of insulin to isolated nuclei. Proc Natl Acad Sci U S A. 1976 May;73(5):1427–1431. doi: 10.1073/pnas.73.5.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harford J., Bridges K., Ashwell G., Klausner R. D. Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A pH-mediated nonlysosomal event. J Biol Chem. 1983 Mar 10;258(5):3191–3197. [PubMed] [Google Scholar]
  12. Hokin L. E. Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem. 1985;54:205–235. doi: 10.1146/annurev.bi.54.070185.001225. [DOI] [PubMed] [Google Scholar]
  13. James R., Bradshaw R. A. Polypeptide growth factors. Annu Rev Biochem. 1984;53:259–292. doi: 10.1146/annurev.bi.53.070184.001355. [DOI] [PubMed] [Google Scholar]
  14. Jiang L. W., Schindler M. Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape. J Cell Biol. 1988 Jan;106(1):13–19. doi: 10.1083/jcb.106.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson L. K., Vlodavsky I., Baxter J. D., Gospodarowicz D. Nuclear accumulation of epidermal growth factor in cultured rat pituitary cells. Nature. 1980 Sep 25;287(5780):340–343. doi: 10.1038/287340a0. [DOI] [PubMed] [Google Scholar]
  16. Lin P. H., Selinfreund R., Wakshull E., Wharton W. Rapid and efficient purification of plasma membrane from cultured cells: characterization of epidermal growth factor binding. Biochemistry. 1987 Feb 10;26(3):731–736. doi: 10.1021/bi00377a012. [DOI] [PubMed] [Google Scholar]
  17. Livneh E., Prywes R., Kashles O., Reiss N., Sasson I., Mory Y., Ullrich A., Schlessinger J. Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. J Biol Chem. 1986 Sep 25;261(27):12490–12497. [PubMed] [Google Scholar]
  18. Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
  19. Maul G. G. The nuclear and the cytoplasmic pore complex: structure, dynamics, distribution, and evolution. Int Rev Cytol Suppl. 1977;(6):75–186. [PubMed] [Google Scholar]
  20. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  21. Merion M., Sly W. S. The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J Cell Biol. 1983 Mar;96(3):644–650. doi: 10.1083/jcb.96.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murthy U., Basu M., Sen-Majumdar A., Das M. Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains. J Cell Biol. 1986 Aug;103(2):333–342. doi: 10.1083/jcb.103.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Podlecki D. A., Smith R. M., Kao M., Tsai P., Huecksteadt T., Brandenburg D., Lasher R. S., Jarett L., Olefsky J. M. Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects. J Biol Chem. 1987 Mar 5;262(7):3362–3368. [PubMed] [Google Scholar]
  24. 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]
  25. Purrello F., Burnham D. B., Goldfine I. D. Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1189–1193. doi: 10.1073/pnas.80.5.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rakowicz-Szulczynska E. M., Rodeck U., Herlyn M., Koprowski H. Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3728–3732. doi: 10.1073/pnas.83.11.3728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Savion N., Vlodavsky I., Gospodarowicz D. Nuclear accumulation of epidermal growth factor in cultured bovine corneal endothelial and granulosa cells. J Biol Chem. 1981 Feb 10;256(3):1149–1154. [PubMed] [Google Scholar]
  28. Schindler M., Jiang L. W. Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei. J Cell Biol. 1987 Apr;104(4):849–853. doi: 10.1083/jcb.104.4.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schindler M., Jiang L. W. Fluorescence redistribution after photobleaching as a tool for dissecting the control elements of nucleocytoplasmic transport. Methods Enzymol. 1987;141:447–459. doi: 10.1016/0076-6879(87)41091-4. [DOI] [PubMed] [Google Scholar]
  30. Schneider C. A., Lim R. W., Terwilliger E., Herschman H. R. Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA. Proc Natl Acad Sci U S A. 1986 Jan;83(2):333–336. doi: 10.1073/pnas.83.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schumm D. E., Webb T. E. Insulin-modulated transport of RNA from isolated live nuclei. Arch Biochem Biophys. 1981 Aug;210(1):275–279. doi: 10.1016/0003-9861(81)90190-9. [DOI] [PubMed] [Google Scholar]
  32. Smith R. M., Jarett L. Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process. Proc Natl Acad Sci U S A. 1987 Jan;84(2):459–463. doi: 10.1073/pnas.84.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sorkin A. D., Teslenko L. V., Nikolsky N. N. The endocytosis of epidermal growth factor in A431 cells: a pH of microenvironment and the dynamics of receptor complex dissociation. Exp Cell Res. 1988 Mar;175(1):192–205. doi: 10.1016/0014-4827(88)90266-2. [DOI] [PubMed] [Google Scholar]
  34. Swanson J. A., McNeil P. L. Nuclear reassembly excludes large macromolecules. Science. 1987 Oct 23;238(4826):548–550. doi: 10.1126/science.2443981. [DOI] [PubMed] [Google Scholar]
  35. Thompson D. M., Cochet C., Chambaz E. M., Gill G. N. Separation and characterization of a phosphatidylinositol kinase activity that co-purifies with the epidermal growth factor receptor. J Biol Chem. 1985 Jul 25;260(15):8824–8830. [PubMed] [Google Scholar]
  36. 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]
  37. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  38. Wahl M., Carpenter G. Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C. J Biol Chem. 1988 Jun 5;263(16):7581–7590. [PubMed] [Google Scholar]
  39. Wiley H. S., VanNostrand W., McKinley D. N., Cunningham D. D. Intracellular processing of epidermal growth factor and its effect on ligand-receptor interactions. J Biol Chem. 1985 May 10;260(9):5290–5295. [PubMed] [Google Scholar]
  40. Wong K. Y., Hawley D., Vigneri R., Goldfine I. D. Comparison of solubilized and purified plasma membrane and nuclear insulin receptors. Biochemistry. 1988 Jan 12;27(1):375–379. doi: 10.1021/bi00401a056. [DOI] [PubMed] [Google Scholar]
  41. Yankner B. A., Shooter E. M. Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1269–1273. doi: 10.1073/pnas.76.3.1269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yarden Y., Escobedo J. A., Kuang W. J., Yang-Feng T. L., Daniel T. O., Tremble P. M., Chen E. Y., Ando M. E., Harkins R. N., Francke U. Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986 Sep 18;323(6085):226–232. doi: 10.1038/323226a0. [DOI] [PubMed] [Google Scholar]

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