Abstract
In Chinese hamster ovary (CHO) fibroblast cells the protein kinase C activating phorbol ester, phorbol myristate acetate (PMA), stimulates an increase in cell surface transferrin receptor (TR) expression by increasing the exocytic rate of the recycling pathway. The human TR expressed in CHO cells is similarly affected by PMA treatment. A mutant human TR in which the major protein kinase C phosphorylation site, serine 24, has been replaced with the non-phosphorylatable amino acid glycine has been constructed to investigate the role of receptor phosphorylation in the PMA induced up-regulation. The Gly-24- substituted receptor binds, internalizes, and recycles Tf. Furthermore, the altered receptor mediates cellular Fe accumulation from diferric- Tf, thereby fulfilling the receptor's major biological role. The Gly-24 TR behaves identically to the wild-type TR when cells are treated with PMA. Therefore, Ser-24 phosphorylation is not required for the PMA- induced redistribution of the human TR expressed in CHO cells. The increased TR expression on the cell surface after PMA treatment results from an increase in the rate of exocytosis of the recycling receptors. No change in the endocytic rate or the size of the recycling receptor pool was observed. These results indicate that the PMA effect on the TR surface expression may result from a more general perturbation of membrane trafficking rather than a specific modulation of the TR.
Full Text
The Full Text of this article is available as a PDF (819.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bridges K. R., Cudkowicz A. Effect of iron chelators on the transferrin receptor in K562 cells. J Biol Chem. 1984 Nov 10;259(21):12970–12977. [PubMed] [Google Scholar]
- Buys S. S., Keogh E. A., Kaplan J. Fusion of intracellular membrane pools with cell surfaces of macrophages stimulated by phorbol esters and calcium ionophores. Cell. 1984 Sep;38(2):569–576. doi: 10.1016/0092-8674(84)90511-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Davis R. J., Johnson G. L., Kelleher D. J., Anderson J. K., Mole J. E., Czech M. P. Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C. J Biol Chem. 1986 Jul 5;261(19):9034–9041. [PubMed] [Google Scholar]
- 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]
- Fallon R. J., Schwartz A. L. Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line. J Biol Chem. 1986 Nov 15;261(32):15081–15089. [PubMed] [Google Scholar]
- Huebers H. A., Finch C. A. The physiology of transferrin and transferrin receptors. Physiol Rev. 1987 Apr;67(2):520–582. doi: 10.1152/physrev.1987.67.2.520. [DOI] [PubMed] [Google Scholar]
- Klausner R. D., Harford J., van Renswoude J. Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester. Proc Natl Acad Sci U S A. 1984 May;81(10):3005–3009. doi: 10.1073/pnas.81.10.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- May W. S., Jacobs S., Cuatrecasas P. Association of phorbol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2016–2020. doi: 10.1073/pnas.81.7.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- May W. S., Sahyoun N., Jacobs S., Wolf M., Cuatrecasas P. Mechanism of phorbol diester-induced regulation of surface transferrin receptor involves the action of activated protein kinase C and an intact cytoskeleton. J Biol Chem. 1985 Aug 5;260(16):9419–9426. [PubMed] [Google Scholar]
- 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]
- 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]
- Rao K. K., Shapiro D., Mattia E., Bridges K., Klausner R. Effects of alterations in cellular iron on biosynthesis of the transferrin receptor in K562 cells. Mol Cell Biol. 1985 Apr;5(4):595–600. doi: 10.1128/mcb.5.4.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao K., Harford J. B., Rouault T., McClelland A., Ruddle F. H., Klausner R. D. Transcriptional regulation by iron of the gene for the transferrin receptor. Mol Cell Biol. 1986 Jan;6(1):236–240. doi: 10.1128/mcb.6.1.236. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Tanner L. I., Lienhard G. E. Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization. J Biol Chem. 1987 Jul 5;262(19):8975–8980. [PubMed] [Google Scholar]
- Thompson L. H., Baker R. M. Isolation of mutants of cultured mammalian cells. Methods Cell Biol. 1973;6:209–281. doi: 10.1016/s0091-679x(08)60052-7. [DOI] [PubMed] [Google Scholar]
- Trowbridge I. S., Newman R. A., Domingo D. L., Sauvage C. Transferrin receptors: structure and function. Biochem Pharmacol. 1984 Mar 15;33(6):925–932. doi: 10.1016/0006-2952(84)90447-7. [DOI] [PubMed] [Google Scholar]
- Ward J. H., Jordan I., Kushner J. P., Kaplan J. Heme regulation of HeLa cell transferrin receptor number. J Biol Chem. 1984 Nov 10;259(21):13235–13240. [PubMed] [Google Scholar]
- 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]
- Wiley H. S., Kaplan J. Epidermal growth factor rapidly induces a redistribution of transferrin receptor pools in human fibroblasts. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7456–7460. doi: 10.1073/pnas.81.23.7456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamashiro D. J., Tycko B., Fluss S. R., Maxfield F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 1984 Jul;37(3):789–800. doi: 10.1016/0092-8674(84)90414-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]