Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1983 Aug 1;97(2):508–521. doi: 10.1083/jcb.97.2.508

Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells

PMCID: PMC2112524  PMID: 6309862

Abstract

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half- life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR- gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome- like elements. Extracting ATR-gold complexes with Triton X after a 30- min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

Full Text

The Full Text of this article is available as a PDF (5.8 MB).

Selected References

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

  1. AWAI M., BROWN E. B. Studies of the metabolism of I-131-labeled human transferrin. J Lab Clin Med. 1963 Mar;61:363–396. [PubMed] [Google Scholar]
  2. Abrahamson D. R., Rodewald R. Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine. J Cell Biol. 1981 Oct;91(1):270–280. doi: 10.1083/jcb.91.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ackerman G. A., Wolken K. W. Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells. J Histochem Cytochem. 1981 Oct;29(10):1137–1149. doi: 10.1177/29.10.6271866. [DOI] [PubMed] [Google Scholar]
  4. Anderson R. G., Brown M. S., Beisiegel U., Goldstein J. L. Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J Cell Biol. 1982 Jun;93(3):523–531. doi: 10.1083/jcb.93.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Anderson R. G., Brown M. S., Goldstein J. L. Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells. J Cell Biol. 1981 Feb;88(2):441–452. doi: 10.1083/jcb.88.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Anderson R. G., Brown M. S., Goldstein J. L. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. doi: 10.1016/0092-8674(77)90022-8. [DOI] [PubMed] [Google Scholar]
  7. Basu S. K., Goldstein J. L., Anderson R. G., Brown M. S. Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell. 1981 May;24(2):493–502. doi: 10.1016/0092-8674(81)90340-8. [DOI] [PubMed] [Google Scholar]
  8. Beisiegel U., Schneider W. J., Goldstein J. L., Anderson R. G., Brown M. S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia. J Biol Chem. 1981 Nov 25;256(22):11923–11931. [PubMed] [Google Scholar]
  9. Bleil J. D., Bretscher M. S. Transferrin receptor and its recycling in HeLa cells. EMBO J. 1982;1(3):351–355. doi: 10.1002/j.1460-2075.1982.tb01173.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown M. S., Goldstein J. L. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3330–3337. doi: 10.1073/pnas.76.7.3330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Debanne M. T., Evans W. H., Flint N., Regoeczi E. Receptor-rich intracellular membrane vesicles transporting asialotransferrin and insulin in liver. Nature. 1982 Jul 22;298(5872):398–400. doi: 10.1038/298398a0. [DOI] [PubMed] [Google Scholar]
  12. Doyle D., Hou E., Warren R. Transfer of the hepatocyte receptor for serum asialo-glycoproteins to the plasma membrane of a fibroblast. Acquisition of the hepatocyte receptor functions by mouse L-cells. J Biol Chem. 1979 Aug 10;254(15):6853–6856. [PubMed] [Google Scholar]
  13. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  14. Enns C. A., Sussman H. H. Physical characterization of the transferrin receptor in human placentae. J Biol Chem. 1981 Oct 10;256(19):9820–9823. [PubMed] [Google Scholar]
  15. Faulk W. P., Taylor G. M. An immunocolloid method for the electron microscope. Immunochemistry. 1971 Nov;8(11):1081–1083. doi: 10.1016/0019-2791(71)90496-4. [DOI] [PubMed] [Google Scholar]
  16. Fehlmann M., Carpentier J. L., Van Obberghen E., Freychet P., Thamm P., Saunders D., Brandenburg D., Orci L. Internalized insulin receptors are recycled to the cell surface in rat hepatocytes. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5921–5925. doi: 10.1073/pnas.79.19.5921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fernandez-Pol J. A., Klos D. J. Isolation and characterization of normal rat kidney cell membrane proteins with affinity for transferrin. Biochemistry. 1980 Aug 19;19(17):3904–3912. doi: 10.1021/bi00558a003. [DOI] [PubMed] [Google Scholar]
  18. Geisow M. J., D'Arcy Hart P., Young M. R. Temporal changes of lysosome and phagosome pH during phagolysosome formation in macrophages: studies by fluorescence spectroscopy. J Cell Biol. 1981 Jun;89(3):645–652. doi: 10.1083/jcb.89.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Geoghegan W. D., Ackerman G. A. Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J Histochem Cytochem. 1977 Nov;25(11):1187–1200. doi: 10.1177/25.11.21217. [DOI] [PubMed] [Google Scholar]
  20. Geuze H. J., Slot J. W., Strous G. J., Lodish H. F., Schwartz A. L. Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis. Cell. 1983 Jan;32(1):277–287. doi: 10.1016/0092-8674(83)90518-4. [DOI] [PubMed] [Google Scholar]
  21. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  22. Goldstein J. L., Anderson R. G., Brown M. S. Receptor-mediated endocytosis and the cellular uptake of low density lipoprotein. Ciba Found Symp. 1982;(92):77–95. doi: 10.1002/9780470720745.ch5. [DOI] [PubMed] [Google Scholar]
  23. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Haigler H. T., McKanna J. A., 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. doi: 10.1083/jcb.83.1.82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Hamilton T. A., Wada H. G., Sussman H. H. Identification of transferrin receptors on the surface of human cultured cells. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6406–6410. doi: 10.1073/pnas.76.12.6406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Helenius A., Marsh M. Endocytosis of enveloped animal viruses. Ciba Found Symp. 1982;(92):59–76. doi: 10.1002/9780470720745.ch4. [DOI] [PubMed] [Google Scholar]
  30. Hopkins C. R., Boothroyd B., Gregory H. Early events following the binding of epidermal growth factor to surface receptors on ovarian granulosa cells. Eur J Cell Biol. 1981 Jun;24(2):259–265. [PubMed] [Google Scholar]
  31. Huet C., Ash J. F., Singer S. J. The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts. Cell. 1980 Sep;21(2):429–438. doi: 10.1016/0092-8674(80)90479-1. [DOI] [PubMed] [Google Scholar]
  32. Karin M., Mintz B. Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem cells. J Biol Chem. 1981 Apr 10;256(7):3245–3252. [PubMed] [Google Scholar]
  33. Khan M. N., Posner B. I., Verma A. K., Khan R. J., Bergeron J. J. Intracellular hormone receptors: evidence for insulin and lactogen receptors in a unique vesicle sedimenting in lysosome fractions of rat liver. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4980–4984. doi: 10.1073/pnas.78.8.4980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Maxfield F. R. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. doi: 10.1083/jcb.95.2.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Nunez M. T., Glass J., Cole E. S. the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes. Biochim Biophys Acta. 1981 Feb 18;673(1):137–146. [PubMed] [Google Scholar]
  38. Octave J. N., Schneider Y. J., Crichton R. R., Trouet A. Transferrin uptake by cultured rat embryo fibroblasts. The influence of temperature and incubation time, subcellular distribution and short-term kinetic studies. Eur J Biochem. 1981 Apr;115(3):611–618. [PubMed] [Google Scholar]
  39. Omary M. B., Trowbridge I. S. Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem. 1981 Dec 25;256(24):12888–12892. [PubMed] [Google Scholar]
  40. Omary M. B., Trowbridge I. S. Covalent binding of fatty acid to the transferrin receptor in cultured human cells. J Biol Chem. 1981 May 25;256(10):4715–4718. [PubMed] [Google Scholar]
  41. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Seligman P. A., Schleicher R. B., Allen R. H. Isolation and characterization of the transferrin receptor from human placenta. J Biol Chem. 1979 Oct 25;254(20):9943–9946. [PubMed] [Google Scholar]
  43. Stahl P., Schlesinger P. H., Sigardson E., Rodman J. S., Lee Y. C. Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: characterization and evidence for receptor recycling. Cell. 1980 Jan;19(1):207–215. doi: 10.1016/0092-8674(80)90402-x. [DOI] [PubMed] [Google Scholar]
  44. Tolson N. D., Boothroyd B., Hopkins C. R. Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A-coated gold in conjunction with biotin and fc-bearing ligands. J Microsc. 1981 Aug;123(Pt 2):215–226. doi: 10.1111/j.1365-2818.1981.tb01296.x. [DOI] [PubMed] [Google Scholar]
  45. Trowbridge I. S., Omary M. B. Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin. Proc Natl Acad Sci U S A. 1981 May;78(5):3039–3043. doi: 10.1073/pnas.78.5.3039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tycko B., Maxfield F. R. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 1982 Mar;28(3):643–651. doi: 10.1016/0092-8674(82)90219-7. [DOI] [PubMed] [Google Scholar]
  47. Wada H. G., Hass P. E., Sussman H. H. Transferrin receptor in human placental brush border membranes. Studies on the binding of transferrin to placental membrane vesicles and the identification of a placental brush border glycoprotein with high affinity for transferrin. J Biol Chem. 1979 Dec 25;254(24):12629–12635. [PubMed] [Google Scholar]
  48. Wall D. A., Wilson G., Hubbard A. L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. doi: 10.1016/0092-8674(80)90116-6. [DOI] [PubMed] [Google Scholar]
  49. Willingham M. C., Pastan I. H. Transit of epidermal growth factor through coated pits of the Golgi system. J Cell Biol. 1982 Jul;94(1):207–212. doi: 10.1083/jcb.94.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Willingham M. C., Pastan I. The receptosome: an intermediate organelle of receptor mediated endocytosis in cultured fibroblasts. Cell. 1980 Aug;21(1):67–77. doi: 10.1016/0092-8674(80)90115-4. [DOI] [PubMed] [Google Scholar]
  51. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES