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. 1989 Aug;8(8):2231–2240. doi: 10.1002/j.1460-2075.1989.tb08347.x

Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor.

E Alvarez 1, N Gironès 1, R J Davis 1
PMCID: PMC401153  PMID: 2507316

Abstract

The human transferrin receptor is expressed as a disulfide-linked dimer at the cell surface. The sites of intermolecular disulfide bonds are Cys-89 and Cys-98. We have examined the functional significance of the covalent dimeric structure of the transferrin receptor by substitution of Cys-89 and Cys-98 with serine residues. Wild-type and mutated transferrin receptors were expressed in Chinese hamster ovary cells (clone TF-) that lack detectable endogenous transferrin receptors. The rates of receptor endocytosis and recycling were measured and the accumulation of iron by cells incubated with [59Fe]diferric transferrin was investigated. No significant differences between these rates were observed when cells expressing wild-type and mutated receptors were compared. The structure of the mutant receptor lacking intermolecular disulfide bonds was investigated. The presence of a population of mutant receptors with a non-covalent dimeric structure was indicated by cross-linking studies using diferric [125I]transferrin and the bifunctional reagent disuccinimidyl suberimidate. However, sucrose density gradient sedimentation analysis of Triton X-100 solubilized transferrin receptors demonstrated that the mutant receptor existed as a monomer in the absence of diferric transferrin and as an apparent dimer in the presence of this receptor ligand. We conclude that the covalent dimeric structure of the transferrin receptor is not required for the expression of the dimeric state and functional activity of the receptor.

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Selected References

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

  1. 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]
  2. Ciechanover A., Schwartz A. L., Dautry-Varsat A., Lodish H. F. Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem. 1983 Aug 25;258(16):9681–9689. [PubMed] [Google Scholar]
  3. Davis R. J., Corvera S., Czech M. P. Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane. J Biol Chem. 1986 Jul 5;261(19):8708–8711. [PubMed] [Google Scholar]
  4. Davis R. J., Czech M. P. Amiloride directly inhibits growth factor receptor tyrosine kinase activity. J Biol Chem. 1985 Feb 25;260(4):2543–2551. [PubMed] [Google Scholar]
  5. Davis R. J., Czech M. P. Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor. EMBO J. 1986 Apr;5(4):653–658. doi: 10.1002/j.1460-2075.1986.tb04263.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis R. J., Faucher M., Racaniello L. K., Carruthers A., Czech M. P. Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms. J Biol Chem. 1987 Sep 25;262(27):13126–13134. [PubMed] [Google Scholar]
  7. Davis R. J., Meisner H. Regulation of transferrin receptor cycling by protein kinase C is independent of receptor phosphorylation at serine 24 in Swiss 3T3 fibroblasts. J Biol Chem. 1987 Nov 25;262(33):16041–16047. [PubMed] [Google Scholar]
  8. 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]
  9. Jing S. Q., Trowbridge I. S. Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J. 1987 Feb;6(2):327–331. doi: 10.1002/j.1460-2075.1987.tb04758.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Klausner R. D., van Renswoude J., Kempf C., Rao K., Bateman J. L., Robbins A. R. Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis. J Cell Biol. 1984 Mar;98(3):1098–1101. doi: 10.1083/jcb.98.3.1098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kühn L. C., McClelland A., Ruddle F. H. Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell. 1984 May;37(1):95–103. doi: 10.1016/0092-8674(84)90304-0. [DOI] [PubMed] [Google Scholar]
  12. Lamb J. E., Ray F., Ward J. H., Kushner J. P., Kaplan J. Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells. J Biol Chem. 1983 Jul 25;258(14):8751–8758. [PubMed] [Google Scholar]
  13. Lesley J. F., Schulte R. J. Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies. Mol Cell Biol. 1985 Aug;5(8):1814–1821. doi: 10.1128/mcb.5.8.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lesley J. F., Schulte R. J. Selection of cell lines resistant to anti-transferrin receptor antibody: evidence for a mutation in transferrin receptor. Mol Cell Biol. 1984 Sep;4(9):1675–1681. doi: 10.1128/mcb.4.9.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. May W. S., Jr, Cuatrecasas P. Transferrin receptor: its biological significance. J Membr Biol. 1985;88(3):205–215. doi: 10.1007/BF01871086. [DOI] [PubMed] [Google Scholar]
  16. McClelland A., Kühn L. C., Ruddle F. H. The human transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell. 1984 Dec;39(2 Pt 1):267–274. doi: 10.1016/0092-8674(84)90004-7. [DOI] [PubMed] [Google Scholar]
  17. McGraw T. E., Dunn K. W., Maxfield F. R. Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J Cell Biol. 1988 Apr;106(4):1061–1066. doi: 10.1083/jcb.106.4.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McGraw T. E., Greenfield L., Maxfield F. R. Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor. J Cell Biol. 1987 Jul;105(1):207–214. doi: 10.1083/jcb.105.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Newman R., Domingo D., Trotter J., Trowbridge I. Selection and properties of a mouse L-cell transformant expressing human transferrin receptor. Nature. 1983 Aug 18;304(5927):643–645. doi: 10.1038/304643a0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Raso V., Basala M. A highly cytotoxic human transferrin-ricin A chain conjugate used to select receptor-modified cells. J Biol Chem. 1984 Jan 25;259(2):1143–1149. [PubMed] [Google Scholar]
  23. Rothenberger S., Iacopetta B. J., Kühn L. C. Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site. Cell. 1987 May 8;49(3):423–431. doi: 10.1016/0092-8674(87)90295-9. [DOI] [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schneider C., Sutherland R., Newman R., Greaves M. Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9. J Biol Chem. 1982 Jul 25;257(14):8516–8522. [PubMed] [Google Scholar]
  26. Sutherland R., Delia D., Schneider C., Newman R., Kemshead J., Greaves M. Ubiquitous cell-surface glycoprotein on tumor cells is proliferation-associated receptor for transferrin. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4515–4519. doi: 10.1073/pnas.78.7.4515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trowbridge I. S., Lesley J., Schulte R. Murine cell surface transferrin receptor: studies with an anti-receptor monoclonal antibody. J Cell Physiol. 1982 Sep;112(3):403–410. doi: 10.1002/jcp.1041120314. [DOI] [PubMed] [Google Scholar]
  28. Trowbridge I. S., Lopez F. Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits human tumor cell growth in vitro. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1175–1179. doi: 10.1073/pnas.79.4.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Turkewitz A. P., Amatruda J. F., Borhani D., Harrison S. C., Schwartz A. L. A high yield purification of the human transferrin receptor and properties of its major extracellular fragment. J Biol Chem. 1988 Jun 15;263(17):8318–8325. [PubMed] [Google Scholar]
  30. Turkewitz A. P., Schwartz A. L., Harrison S. C. A pH-dependent reversible conformational transition of the human transferrin receptor leads to self-association. J Biol Chem. 1988 Nov 5;263(31):16309–16315. [PubMed] [Google Scholar]
  31. 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]
  32. Yarden Y., Schlessinger J. Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry. 1987 Mar 10;26(5):1443–1451. doi: 10.1021/bi00379a035. [DOI] [PubMed] [Google Scholar]
  33. Zerial M., Suomalainen M., Zanetti-Schneider M., Schneider C., Garoff H. Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells. EMBO J. 1987 Sep;6(9):2661–2667. doi: 10.1002/j.1460-2075.1987.tb02557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]
  35. van Driel I. R., Davis C. G., Goldstein J. L., Brown M. S. Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain. J Biol Chem. 1987 Nov 25;262(33):16127–16134. [PubMed] [Google Scholar]

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