Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1985 Jan 1;225(1):85–94. doi: 10.1042/bj2250085

Functional receptors for epidermal growth factor in an epithelial-cell line derived from the rat small intestine.

J Blay, K D Brown
PMCID: PMC1144556  PMID: 2983665

Abstract

Epidermal growth factor (EGF) regulates the proliferation of cells of a rat intestinal epithelial-cell line (RIE-1) in culture. Confluent RIE-1 cells were stimulated to proliferate by EGF with a half-maximal effect at 1-2 ng/ml. In contrast, the growth of sparse RIE-1 cells was inhibited by the growth factor. Binding studies at 4 degrees C with 125I-EGF identified two classes of binding sites for EGF on RIE-1 cells, one of high affinity (KD = 1.8 X 10(-10)M; 1.8 X 10(4) receptors/cell) and one of lower affinity (KD = 5.2 X 10(-9)M; 6.3 X 10(4) receptors/cell). After binding to the cells at 37 degrees C, 125I-EGF was rapidly internalized and subsequently degraded. Degradation products were released into the medium after a lag of 15-30 min. The degradation of 125I-EGF did not occur at 4 degrees C and was inhibited at 37 degrees C by chloroquine, methylamine or NH4Cl, but not by colchicine. Exposure of RIE-1 cells to EGF caused a time- and dose-dependent loss of EGF receptors from the cell surface. The recovery of receptors after the removal of EGF was retarded in the absence of serum and prevented by the presence of cycloheximide or actinomycin D. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis separation of the 125I-EGF-receptor complex from RIE-1 cells after covalently cross-linking with disuccinimidyl suberate indicated a receptor of Mr congruent to 160 000. The demonstration of functional EGF receptors in this cell line provides further evidence that EGF may regulate intestinal-epithelial-cell physiology.

Full text

PDF
85

Images in this article

92Fig. 8.
on p.92

Selected References

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

  1. Aharonov A., Pruss R. M., Herschman H. R. Epidermal growth factor. Relationship between receptor regulation and mitogenesis in 3T3 cells. J Biol Chem. 1978 Jun 10;253(11):3970–3977. [PubMed] [Google Scholar]
  2. Al-Nafussi A. I., Wright N. A. The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents. Virchows Arch B Cell Pathol Incl Mol Pathol. 1982;40(1):63–69. doi: 10.1007/BF02932851. [DOI] [PubMed] [Google Scholar]
  3. Baker J. B., Simmer R. L., Glenn K. C., Cunningham D. D. Thrombin and epidermal growth factor become linked to cell surface receptors during mitogenic stimulation. Nature. 1979 Apr 19;278(5706):743–745. doi: 10.1038/278743a0. [DOI] [PubMed] [Google Scholar]
  4. Beaulieu J. F., Calvert R. The effect of epidermal growth factor (EGF) on the differentiation of the rough endoplasmic reticulum in fetal mouse small intestine in organ culture. J Histochem Cytochem. 1981 Jun;29(6):765–770. doi: 10.1177/29.6.6265548. [DOI] [PubMed] [Google Scholar]
  5. Blay J., Brown K. D. Characterization of an epithelioid cell line derived from rat small intestine: demonstration of cytokeratin filaments. Cell Biol Int Rep. 1984 Jul;8(7):551–560. doi: 10.1016/0309-1651(84)90054-7. [DOI] [PubMed] [Google Scholar]
  6. Bower J. M., Camble R., Gregory H., Gerring E. L., Willshire I. R. The inhibition of gastric acid secretion by epidermal growth factor. Experientia. 1975 Jul 15;31(7):825–826. doi: 10.1007/BF01938488. [DOI] [PubMed] [Google Scholar]
  7. Brown K. D., Dicker P., Rozengurt E. Inhibition of epidermal growth factor binding to surface receptors by tumor promotors. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1037–1043. doi: 10.1016/0006-291x(79)90221-3. [DOI] [PubMed] [Google Scholar]
  8. Brown K. D., Friedkin M., Rozengurt E. Colchicine inhibits epidermal growth factor degradation in 3T3 cells. Proc Natl Acad Sci U S A. 1980 Jan;77(1):480–484. doi: 10.1073/pnas.77.1.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brown K. D., Holley R. W. Epidermal growth factor and the control of proliferation of Balb 3T3 and benzo[a]pyrene-transformed Balb 3T3 cells. J Cell Physiol. 1979 Jul;100(1):139–146. doi: 10.1002/jcp.1041000114. [DOI] [PubMed] [Google Scholar]
  10. Brown K. D., Yeh Y. C., Holley R. W. Binding, internalization, and degradation of epidermal growth factor by balb 3T3 and BP3T3 cells: relationship to cell density and the stimulation of cell proliferation. J Cell Physiol. 1979 Aug;100(2):227–238. doi: 10.1002/jcp.1041000204. [DOI] [PubMed] [Google Scholar]
  11. Byyny R. L., Orth D. N., Cohen S., Doyne E. S. Epidermal growth factor: effects of androgens and adrenergic agents. Endocrinology. 1974 Sep;95(3):776–782. doi: 10.1210/endo-95-3-776. [DOI] [PubMed] [Google Scholar]
  12. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Collins M. K., Sinnett-Smith J. W., Rozengurt E. Platelet-derived growth factor treatment decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells. J Biol Chem. 1983 Oct 10;258(19):11689–11693. [PubMed] [Google Scholar]
  15. Comens P. G., Simmer R. L., Baker J. B. Direct linkage of 125I-EGF to cell surface receptors. A useful artifact of chloramine-T treatment. J Biol Chem. 1982 Jan 10;257(1):42–45. [PubMed] [Google Scholar]
  16. Dembiński A., Gregory H., Konturek S. J., Polański M. Trophic action of epidermal growth factor on the pancreas and gastroduodenal mucosa in rats. J Physiol. 1982 Apr;325:35–42. doi: 10.1113/jphysiol.1982.sp014133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Elder J. B., Ganguli P. C., Gillespie I. E., Gerring E. L., Gregory H. Effect of urogastrone on gastric secretion and plasma gastrin levels in normal subjects. Gut. 1975 Nov;16(11):887–893. doi: 10.1136/gut.16.11.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Elder J. B., Williams G., Lacey E., Gregory H. Cellular localisation of human urogastrone/epidermal growth factor. Nature. 1978 Feb 2;271(5644):466–467. doi: 10.1038/271466a0. [DOI] [PubMed] [Google Scholar]
  19. Feldman E. J., Aures D., Grossman M. I. Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of mouse. Proc Soc Exp Biol Med. 1978 Dec;159(3):400–402. doi: 10.3181/00379727-159-40357. [DOI] [PubMed] [Google Scholar]
  20. Forgue-Lafitte M. E., Laburthe M., Chamblier M. C., Moody A. J., Rosselin G. Demonstration of specific receptors for EGF--urogastrone in isolated rat intestinal epithelial cells. FEBS Lett. 1980 Jun 2;114(2):243–246. doi: 10.1016/0014-5793(80)81125-2. [DOI] [PubMed] [Google Scholar]
  21. Gresik E. W., van der Noen H., Barka T. Epidermal growth factor-like material in rat submandibular gland. Am J Anat. 1979 Sep;156(1):83–89. doi: 10.1002/aja.1001560108. [DOI] [PubMed] [Google Scholar]
  22. Grotendorst G. R. Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation, and tumor promoters. Cell. 1984 Feb;36(2):279–285. doi: 10.1016/0092-8674(84)90221-6. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Heitz P. U., Kasper M., van Noorden S., Polak J. M., Gregory H., Pearse A. G. Immunohistochemical localisation of urogastrone to human duodenal and submandibular glands. Gut. 1978 May;19(5):408–413. doi: 10.1136/gut.19.5.408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hirata Y., Orth D. N. Concentrations of epidermal growth factor, nerve growth factor, and submandibular gland renin in male and female mouse tissue and fluids. Endocrinology. 1979 Dec;105(6):1382–1387. doi: 10.1210/endo-105-6-1382. [DOI] [PubMed] [Google Scholar]
  26. Johnson L. R., Guthrie P. D. Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor. Am J Physiol. 1980 Jan;238(1):G45–G49. doi: 10.1152/ajpgi.1980.238.1.G45. [DOI] [PubMed] [Google Scholar]
  27. Kirkland W. L., Yang N. S., Jorgensen T., Longley C., Furmanski P. Growth of normal and malignant human mammary epithelial cells in culture. J Natl Cancer Inst. 1979 Jul;63(1):29–41. [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Linsley P. S., Blifeld C., Wrann M., Fox C. F. Direct linkage of epidermal growth factor to its receptor. Nature. 1979 Apr 19;278(5706):745–748. doi: 10.1038/278745a0. [DOI] [PubMed] [Google Scholar]
  30. Malo C., Ménard D. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology. 1982 Jul;83(1 Pt 1):28–35. [PubMed] [Google Scholar]
  31. Moore J. B., Jr Purification and partial characterization of epidermal growth factor isolated from the male rat submaxillary gland. Arch Biochem Biophys. 1978 Jul;189(1):1–7. doi: 10.1016/0003-9861(78)90106-6. [DOI] [PubMed] [Google Scholar]
  32. Oka Y., Ghishan F. K., Greene H. L., Orth D. N. Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine. Endocrinology. 1983 Mar;112(3):940–944. doi: 10.1210/endo-112-3-940. [DOI] [PubMed] [Google Scholar]
  33. Olsen P. S., Nexø E. Quantitation of epidermal growth factor in the rat. Identification and partial characterization of duodenal EGF. Scand J Gastroenterol. 1983 Sep;18(6):771–776. doi: 10.3109/00365528309182093. [DOI] [PubMed] [Google Scholar]
  34. Osborene C. K., Hamilton B., Nover M. Receptor binding and processing of epidermal growth factor by human breast cancer cells. J Clin Endocrinol Metab. 1982 Jul;55(1):86–93. doi: 10.1210/jcem-55-1-86. [DOI] [PubMed] [Google Scholar]
  35. Phillips P. D., Kuhnle E., Cristofalo V. J. [125I]EGF binding ability is stable throughout the replicative life-span of WI-38 cells. J Cell Physiol. 1983 Mar;114(3):311–316. doi: 10.1002/jcp.1041140309. [DOI] [PubMed] [Google Scholar]
  36. Quaroni A., May R. J. Establishment and characterizaton of intestinal epithelial cell cultures. Methods Cell Biol. 1980;21B:403–427. [PubMed] [Google Scholar]
  37. Richards R. C., Beardmore J. M., Brown P. J., Molloy C. M., Johnson P. M. Epidermal growth factor receptors on isolated human placental syncytiotrophoblast plasma membrane. Placenta. 1983 Apr-Jun;4(2):133–138. doi: 10.1016/s0143-4004(83)80026-5. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Savage C. R., Jr, Inagami T., Cohen S. The primary structure of epidermal growth factor. J Biol Chem. 1972 Dec 10;247(23):7612–7621. [PubMed] [Google Scholar]
  40. Scheving L. A., Yeh Y. C., Tsai T. H., Scheving L. E. Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the duodenum, jejunum, ileum, caecum, colon, and rectum of the adult male mouse. Endocrinology. 1980 May;106(5):1498–1503. doi: 10.1210/endo-106-5-1498. [DOI] [PubMed] [Google Scholar]
  41. Scheving L. A., Yeh Y. C., Tsai T. H., Scheving L. E. Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the tongue, esophagus, and stomach of the adult male mouse. Endocrinology. 1979 Dec;105(6):1475–1480. doi: 10.1210/endo-105-6-1475. [DOI] [PubMed] [Google Scholar]
  42. Thorell J. I., Johansson B. G. Enzymatic iodination of polypeptides with 125I to high specific activity. Biochim Biophys Acta. 1971 Dec 28;251(3):363–369. doi: 10.1016/0005-2795(71)90123-1. [DOI] [PubMed] [Google Scholar]
  43. Thornburg W., Matrisian L., Magun B., Koldovský O. Gastrointestinal absorption of epidermal growth factor in suckling rats. Am J Physiol. 1984 Jan;246(1 Pt 1):G80–G85. doi: 10.1152/ajpgi.1984.246.1.G80. [DOI] [PubMed] [Google Scholar]
  44. Valkirs G. E., Feher G. Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides. J Cell Biol. 1982 Oct;95(1):179–188. doi: 10.1083/jcb.95.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vlodavsky I., Brown K. D., Gospodarowicz D. A comparison of the binding of epidermal growth factor to cultured granulosa and luteal cells. J Biol Chem. 1978 May 25;253(10):3744–3750. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES