Search tips
Search criteria 


Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. 1993 January 1; 120(1): 85–93.
PMCID: PMC2119487

Consumption of EGF by A431 cells: evidence for receptor recycling


We examined the extent of EGF consumption by EGFR in A431 cells. When 125I-EGF was added to A431 cell cultures at low or high density, at concentrations which corresponded to 10-fold excess of ligand over receptor on the cell surface, most of the 125I-EGF was consumed within 2 h. The amounts of 125I-EGF consumed were much greater than available EGFR on the A431 cells, by a factor of 6.5 in low-density cultures and 5.8 in high-density cultures. When the concentration of 125I-EGF was increased in low density cultures, further consumption of 125I-EGF by the A431 cells was greatly reduced, partially due to a rapid down regulation of EGFR. However, when higher concentrations of 125I-EGF were added to high density cultures, with reduced receptor down regulation, the cells continued to consume a large fraction of the EGF in the culture medium. The consumption of 125I-EGF by these cultures was in excellent agreement with the measured amount of ligand internalized into the cell. EGF consumption was far in excess of the number of EGFR down regulated or degraded. Only a minor portion of the EGFR could have been replaced during the assay period by synthesis of new EGFR or from the intracellular pool of EGFR, and the fluid-phase uptake of EGF is only temporarily increased by exposure to EGF. Our results demonstrate that EGFR in high density A431 cell cultures recycled many times. The apparent level of recycling was dependent upon the concentration of EGF and followed Michaelis-Menton kinetics for ligand concentrations as high as 215 nM. At this EGF concentration, high-density cultures consumed 45 EGF molecules per receptor over a period of 6 h.

Full Text

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

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Arsenis G, Hayes GR, Livingston JN. Insulin receptor cycling and insulin action in the rat adipocyte. J Biol Chem. 1985 Feb 25;260(4):2202–2207. [PubMed]
  • Barnes DW. Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture. J Cell Biol. 1982 Apr;93(1):1–4. [PMC free article] [PubMed]
  • Bjorge JD, Kudlow JE. Epidermal growth factor receptor synthesis is stimulated by phorbol ester and epidermal growth factor. Evidence for a common mechanism. J Biol Chem. 1987 May 15;262(14):6615–6622. [PubMed]
  • Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem. 1987;56:881–914. [PubMed]
  • Carpenter G, Cohen S. Epidermal growth factor. J Biol Chem. 1990 May 15;265(14):7709–7712. [PubMed]
  • Carpentier JL, Gorden P, Anderson RG, Goldstein JL, Brown MS, Cohen S, Orci L. Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts. J Cell Biol. 1982 Oct;95(1):73–77. [PMC free article] [PubMed]
  • Clark AJ, Ishii S, Richert N, Merlino GT, Pastan I. Epidermal growth factor regulates the expression of its own receptor. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8374–8378. [PubMed]
  • Cowley GP, Smith JA, Gusterson BA. Increased EGF receptors on human squamous carcinoma cell lines. Br J Cancer. 1986 Feb;53(2):223–229. [PMC free article] [PubMed]
  • Derynck R. Transforming growth factor-alpha: structure and biological activities. J Cell Biochem. 1986;32(4):293–304. [PubMed]
  • Derynck R, Goeddel DV, Ullrich A, Gutterman JU, Williams RD, Bringman TS, Berger WH. 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]
  • Dunn WA, Hubbard AL. Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics. J Cell Biol. 1984 Jun;98(6):2148–2159. [PMC free article] [PubMed]
  • Dunn WA, Connolly TP, Hubbard AL. Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol. 1986 Jan;102(1):24–36. [PMC free article] [PubMed]
  • Earp HS, Austin KS, Blaisdell J, Rubin RA, Nelson KG, Lee LW, Grisham JW. Epidermal growth factor (EGF) stimulates EGF receptor synthesis. J Biol Chem. 1986 Apr 15;261(11):4777–4780. [PubMed]
  • Fabricant RN, De Larco JE, Todaro GJ. Nerve growth factor receptors on human melanoma cells in culture. Proc Natl Acad Sci U S A. 1977 Feb;74(2):565–569. [PubMed]
  • Felder S, Miller K, Moehren G, Ullrich A, Schlessinger J, Hopkins CR. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 1990 May 18;61(4):623–634. [PubMed]
  • Gill GN, Lazar CS. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells. Nature. 1981 Sep 24;293(5830):305–307. [PubMed]
  • Gladhaug IP, Christoffersen T. Kinetics of epidermal growth factor binding and processing in isolated intact rat hepatocytes. Dynamic externalization of receptors during ligand internalization. Eur J Biochem. 1987 Apr 15;164(2):267–275. [PubMed]
  • Glenney JR, Jr, Chen WS, Lazar CS, Walton GM, Zokas LM, Rosenfeld MG, Gill GN. 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. [PubMed]
  • Haigler H, Ash JF, Singer SJ, 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. [PubMed]
  • Haigler HT, McKanna JA, 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. [PMC free article] [PubMed]
  • Haigler HT, McKanna JA, Cohen S. Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor. J Cell Biol. 1979 Oct;83(1):82–90. [PMC free article] [PubMed]
  • Haigler HT, Maxfield FR, Willingham MC, 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]
  • Hendler FJ, Ozanne BW. Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest. 1984 Aug;74(2):647–651. [PMC free article] [PubMed]
  • Honegger AM, Dull TJ, Felder S, Van Obberghen E, Bellot F, Szapary D, Schmidt A, Ullrich A, Schlessinger J. Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell. 1987 Oct 23;51(2):199–209. [PubMed]
  • Jäckle S, Runquist EA, Miranda-Brady S, Havel RJ. Trafficking of the epidermal growth factor receptor and transferrin in three hepatocytic endosomal fractions. J Biol Chem. 1991 Jan 25;266(3):1396–1402. [PubMed]
  • Kawamoto T, Sato JD, Le A, Polikoff J, Sato GH, Mendelsohn J. Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1337–1341. [PubMed]
  • Krupp MN, Connolly DT, Lane MD. Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts. J Biol Chem. 1982 Oct 10;257(19):11489–11496. [PubMed]
  • Kudlow JE, Cheung CY, Bjorge JD. Epidermal growth factor stimulates the synthesis of its own receptor in a human breast cancer cell line. J Biol Chem. 1986 Mar 25;261(9):4134–4138. [PubMed]
  • Lai WH, Cameron PH, Wada I, Doherty JJ, 2nd, Kay DG, Posner BI, Bergeron JJ. Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo. J Cell Biol. 1989 Dec;109(6 Pt 1):2741–2749. [PMC free article] [PubMed]
  • Lin CR, Chen WS, Kruiger W, Stolarsky LS, Weber W, Evans RM, Verma IM, Gill GN, Rosenfeld MG. Expression cloning of human EGF receptor complementary DNA: gene amplification and three related messenger RNA products in A431 cells. Science. 1984 May 25;224(4651):843–848. [PubMed]
  • Lund KA, Opresko LK, Starbuck C, Walsh BJ, Wiley HS. Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization. J Biol Chem. 1990 Sep 15;265(26):15713–15723. [PubMed]
  • Marshall S, Green A, Olefsky JM. Evidence for recycling of insulin receptors in isolated rat adipocytes. J Biol Chem. 1981 Nov 25;256(22):11464–11470. [PubMed]
  • Masui H, Kawamoto T, Sato JD, Wolf B, Sato G, Mendelsohn J. Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 1984 Mar;44(3):1002–1007. [PubMed]
  • McKanna JA, Haigler HT, 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. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Sato JD, Kawamoto T, Le AD, Mendelsohn J, Polikoff J, Sato GH. Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med. 1983 Dec;1(5):511–529. [PubMed]
  • Schlessinger J, Shechter Y, Willingham MC, 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. [PubMed]
  • Sorkin A, Krolenko S, Kudrjavtceva N, Lazebnik J, Teslenko L, Soderquist AM, Nikolsky N. Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways. J Cell Biol. 1991 Jan;112(1):55–63. [PMC free article] [PubMed]
  • Sorkin A, Waters C, Overholser KA, Carpenter G. Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis. J Biol Chem. 1991 May 5;266(13):8355–8362. [PubMed]
  • Stoscheck CM, Carpenter G. Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells. J Cell Physiol. 1984 Sep;120(3):296–302. [PubMed]
  • Stoscheck CM, Soderquist AM, Carpenter G. Biosynthesis of the epidermal growth factor receptor in cultured human cells. Endocrinology. 1985 Feb;116(2):528–535. [PubMed]
  • Sunada H, Magun BE, Mendelsohn J, MacLeod CL. Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3825–3829. [PubMed]
  • Sunada H, Peacock J, Mendelsohn J. Ligand induced internalization of epidermal growth factor receptors by A431 cells decreases at high cell densities in culture. Growth Factors. 1991;5(1):45–55. [PubMed]
  • Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. [PubMed]
  • Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature. 309(5967):418–425. [PubMed]
  • Van de Vijver MJ, Kumar R, Mendelsohn J. Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly. J Biol Chem. 1991 Apr 25;266(12):7503–7508. [PubMed]
  • Wiley HS. Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system. J Cell Biol. 1988 Aug;107(2):801–810. [PMC free article] [PubMed]
  • Xu YH, Ishii S, Clark AJ, Sullivan M, Wilson RK, Ma DP, Roe BA, Merlino GT, Pastan I. Human epidermal growth factor receptor cDNA is homologous to a variety of RNAs overproduced in A431 carcinoma cells. Nature. 309(5971):806–810. [PubMed]

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