Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 May 1;104(5):1291–1297. doi: 10.1083/jcb.104.5.1291

Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells

PMCID: PMC2114476  PMID: 3494733

Abstract

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone- receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Aizenbud B. M., Gershon N. D. Diffusion of molecules on biological membranes of nonplanar form. A theoretical study. Biophys J. 1982 Jun;38(3):287–293. doi: 10.1016/S0006-3495(82)84560-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axelrod D., Wight A., Webb W., Horwitz A. Influence of membrane lipids on acetylcholine receptor and lipid probe diffusion in cultured myotube membrane. Biochemistry. 1978 Aug 22;17(17):3604–3609. doi: 10.1021/bi00610a029. [DOI] [PubMed] [Google Scholar]
  3. Barak L. S., Webb W. W. Diffusion of low density lipoprotein-receptor complex on human fibroblasts. J Cell Biol. 1982 Dec;95(3):846–852. doi: 10.1083/jcb.95.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carpenter G., Lembach K. J., Morrison M. M., Cohen S. Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts. J Biol Chem. 1975 Jun 10;250(11):4297–4304. [PubMed] [Google Scholar]
  5. Chinkers M., McKanna J. A., Cohen S. Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth factors. J Cell Biol. 1979 Oct;83(1):260–265. doi: 10.1083/jcb.83.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Criado M., Vaz W. L., Barrantes F. J., Jovin T. M. Translational diffusion of acetylcholine receptor (monomeric and dimeric forms) of Torpedo marmorata reconstituted into phospholipid bilayers studied by fluorescence recovery after photobleaching. Biochemistry. 1982 Nov 9;21(23):5750–5755. doi: 10.1021/bi00266a004. [DOI] [PubMed] [Google Scholar]
  7. Haigler H. T., Maxfield F. R., Willingham M. C., Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980 Feb 25;255(4):1239–1241. [PubMed] [Google Scholar]
  8. Haigler H. T., McKanna J. A., Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol. 1979 May;81(2):382–395. doi: 10.1083/jcb.81.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haigler H., Ash J. F., Singer S. J., Cohen S. Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3317–3321. doi: 10.1073/pnas.75.7.3317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hillman G. M., Schlessinger J. Lateral diffusion of epidermal growth factor complexed to its surface receptors does not account for the thermal sensitivity of patch formation and endocytosis. Biochemistry. 1982 Mar 30;21(7):1667–1672. doi: 10.1021/bi00536a030. [DOI] [PubMed] [Google Scholar]
  11. Hunter T. The epidermal growth factor receptor gene and its product. Nature. 1984 Oct 4;311(5985):414–416. doi: 10.1038/311414a0. [DOI] [PubMed] [Google Scholar]
  12. Koppel D. E., Sheetz M. P. Fluorescence photobleaching does not alter the lateral mobility of erythrocyte membrane glycoproteins. Nature. 1981 Sep 10;293(5828):159–161. doi: 10.1038/293159a0. [DOI] [PubMed] [Google Scholar]
  13. Livneh E., Benveniste M., Prywes R., Felder S., Kam Z., Schlessinger J. Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility. J Cell Biol. 1986 Aug;103(2):327–331. doi: 10.1083/jcb.103.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McCloskey M. A., Liu Z. Y., Poo M. M. Lateral electromigration and diffusion of Fc epsilon receptors on rat basophilic leukemia cells: effects of IgE binding. J Cell Biol. 1984 Sep;99(3):778–787. doi: 10.1083/jcb.99.3.778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McKanna J. A., Haigler H. T., Cohen S. Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5689–5693. doi: 10.1073/pnas.76.11.5689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McLaughlin S., Poo M. M. The role of electro-osmosis in the electric-field-induced movement of charged macromolecules on the surfaces of cells. Biophys J. 1981 Apr;34(1):85–93. doi: 10.1016/S0006-3495(81)84838-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Peters R. Translational diffusion in the plasma membrane of single cells as studied by fluorescence microphotolysis. Cell Biol Int Rep. 1981 Aug;5(8):733–760. doi: 10.1016/0309-1651(81)90231-9. [DOI] [PubMed] [Google Scholar]
  18. Poo M. M., Poo W. J., Lam J. W. Lateral electrophoresis and diffusion of Concanavalin A receptors in the membrane of embryonic muscle cell. J Cell Biol. 1978 Feb;76(2):483–501. doi: 10.1083/jcb.76.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Poo M. In situ electrophoresis of membrane components. Annu Rev Biophys Bioeng. 1981;10:245–276. doi: 10.1146/annurev.bb.10.060181.001333. [DOI] [PubMed] [Google Scholar]
  20. Prywes R., Livneh E., Ullrich A., Schlessinger J. Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. EMBO J. 1986 Sep;5(9):2179–2190. doi: 10.1002/j.1460-2075.1986.tb04482.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rees A. R., Gregoriou M., Johnson P., Garland P. B. High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion. EMBO J. 1984 Aug;3(8):1843–1847. doi: 10.1002/j.1460-2075.1984.tb02057.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Saffman P. G., Delbrück M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Savage C. R., Jr, Cohen S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J Biol Chem. 1972 Dec 10;247(23):7609–7611. [PubMed] [Google Scholar]
  24. Schlessinger J., Webb W. W., Elson E. L., Metzger H. Lateral motion and valence of Fc receptors on rat peritoneal mast cells. Nature. 1976 Dec 9;264(5586):550–552. doi: 10.1038/264550a0. [DOI] [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. Smith L. M., Parce J. W., Smith B. A., McConnell H. M. Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4177–4179. doi: 10.1073/pnas.76.9.4177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith L. M., Rubenstein J. L., Parce J. W., McConnell H. M. Lateral diffusion of M-13 coat protein in mixtures of phosphatidylcholine and cholesterol. Biochemistry. 1980 Dec 9;19(25):5907–5911. doi: 10.1021/bi00566a037. [DOI] [PubMed] [Google Scholar]
  28. Tank D. W., Fredericks W. J., Barak L. S., Webb W. W. Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts. J Cell Biol. 1985 Jul;101(1):148–157. doi: 10.1083/jcb.101.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tank D. W., Wu E. S., Meers P. R., Webb W. W. Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration. Biophys J. 1982 Nov;40(2):129–135. doi: 10.1016/S0006-3495(82)84467-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vaz W. L., Criado M., Madeira V. M., Schoellmann G., Jovin T. M. Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching. Biochemistry. 1982 Oct 26;21(22):5608–5612. doi: 10.1021/bi00265a034. [DOI] [PubMed] [Google Scholar]
  31. Wier M. L., Edidin M. Effects of cell density and extracellular matrix on the lateral diffusion of major histocompatibility antigens in cultured fibroblasts. J Cell Biol. 1986 Jul;103(1):215–222. doi: 10.1083/jcb.103.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
  33. Wolf D. E., Edidin M., Dragsten P. R. Effect of bleaching light on measurements of lateral diffusion in cell membranes by the fluorescence photobleaching recovery method. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2043–2045. doi: 10.1073/pnas.77.4.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolf D. E., Handyside A. H., Edidin M. Effect of microvilli on lateral diffusion measurements made by the fluorescence photobleaching recovery technique. Biophys J. 1982 Jun;38(3):295–297. doi: 10.1016/S0006-3495(82)84561-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zidovetzki R., Yarden Y., Schlessinger J., Jovin T. M. Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6981–6985. doi: 10.1073/pnas.78.11.6981. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES