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. 1985 Dec;76(6):2144–2150. doi: 10.1172/JCI112220

Biosynthesis of the transferrin receptor in rabbit reticulocytes.

T M Cox, M W O'Donnell, P Aisen, I M London
PMCID: PMC424330  PMID: 3001142

Abstract

These studies were performed to determine whether the reticulocyte can synthesize its own transferrin receptor and, if so, whether synthesis is subject to translational control by intracellular heme. Reticulocytosis (20-35%) was produced by bleeding rabbits and the washed cells were incubated for 1-4 h at 37 degrees C in buffered nutritional medium containing L-[35S]methionine. After washing and detergent lysis in the presence of protease inhibitors, supernatant reticulocyte extracts were analyzed for transferrin receptors by immunoprecipitation with specific ovine receptor antibody raised against denatured rabbit transferrin receptor. Immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. Antibody, but not preimmune sheep immunoglobin, consistently precipitated a 35S-labeled protein with an Mr of 90,000 (reduced), coincident with bona fide receptor subunits purified by ligand-affinity chromatography. Incorporation of radioactive methionine was exclusively associated with receptor in reticulocyte stroma, and nascent receptor was not detected on free polyribosomes. Incorporation of radioactivity in the receptor moiety accounted for 0.1-0.2% of total incorporation into TCA insoluble cell protein. Treatment of the cells with 40 micrograms/ml cycloheximide markedly inhibited amino acid incorporation into the receptor, thus indicating de novo synthesis of receptor protein. On treatment of reticulocytes with 4,6 dioxoheptanoate to induce heme deficiency by diminishing the formation of intracellular heme, synthesis of the receptor was inhibited by greater than 50%; synthesis was restored to control rates on addition of 50 microM exogenous hemin. These findings indicate that the reticulocyte retains receptor mRNA and that synthesis of the receptor in erythroid cells is subject to translational regulation by intracellular heme.

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Selected References

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

  1. BRUNS G. P., LONDON I. M. THE EFFECT OF HEMIN ON THE SYNTHESIS OF GLOBIN. Biochem Biophys Res Commun. 1965 Jan 18;18:236–242. doi: 10.1016/0006-291x(65)90746-1. [DOI] [PubMed] [Google Scholar]
  2. Baker E., Shaw D. C., Morgan E. H. Isolation and characterization of rabbit serum and milk transferrins. Evidence for difference in sialic acid content only. Biochemistry. 1968 Apr;7(4):1371–1378. doi: 10.1021/bi00844a019. [DOI] [PubMed] [Google Scholar]
  3. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  4. Beutler E., West C., Blume K. G. The removal of leukocytes and platelets from whole blood. J Lab Clin Med. 1976 Aug;88(2):328–333. [PubMed] [Google Scholar]
  5. Beuzard Y., London I. M. The effects of hemin and double-stranded RNA on alpha and beta globin synthesis in reticulocyte and Krebs II ascites cell-free systems and the relationship of these effects to an initiation factor preparation. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2863–2866. doi: 10.1073/pnas.71.7.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beuzard Y., Rodvien R., London I. M. Effect of hemin on the synthesis of hemoglobin and other proteins in mammalian cells. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1022–1026. doi: 10.1073/pnas.70.4.1022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boyer S. H., Smith K. D., Noyes A. N., Mullen M. A. Immunological characterization of rabbit hemoglobin alpha and beta chain-synthesizing polysomes. J Biol Chem. 1974 Nov 25;249(22):7210–7219. [PubMed] [Google Scholar]
  8. Bruck C., Portetelle D., Glineur C., Bollen A. One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-Gel blue chromatography. J Immunol Methods. 1982 Sep 30;53(3):313–319. doi: 10.1016/0022-1759(82)90178-8. [DOI] [PubMed] [Google Scholar]
  9. Bulova S. I., Burka E. R. Biosynthesis of nonglobin protein by membrane-bound ribosomes in reticulocytes. J Biol Chem. 1970 Oct 10;245(19):4907–4912. [PubMed] [Google Scholar]
  10. Enns C. A., Sussman H. H. Similarities between the transferrin receptor proteins on human reticulocytes and human placentae. J Biol Chem. 1981 Dec 25;256(24):12620–12623. [PubMed] [Google Scholar]
  11. Iacopetta B. J., Morgan E. H., Yeoh G. C. Transferrin receptors and iron uptake during erythroid cell development. Biochim Biophys Acta. 1982 May 7;687(2):204–210. doi: 10.1016/0005-2736(82)90547-8. [DOI] [PubMed] [Google Scholar]
  12. JANDL J. H., INMAN J. K., SIMMONS R. L., ALLEN D. W. Transfer of iron from serum iron-binding protein to human reticulocytes. J Clin Invest. 1959 Jan 1;38(1 Pt 1):161–185. doi: 10.1172/JCI103786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. JANDL J. H., KATZ J. H. The plasma-to-cell cycle of transferrin. J Clin Invest. 1963 Mar;42:314–326. doi: 10.1172/JCI104718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Leibman A., Aisen P. Transferrin receptor of the rabbit reticulocyte. Biochemistry. 1977 Apr 5;16(7):1268–1272. doi: 10.1021/bi00626a004. [DOI] [PubMed] [Google Scholar]
  16. Levin D., Ranu R. S., Ernst V., London I. M. Regulation of protein synthesis in reticulocyte lysates: phosphorylation of methionyl-tRNAf binding factor by protein kinase activity of translational inhibitor isolated from hemedeficient lysates. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3112–3116. doi: 10.1073/pnas.73.9.3112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lodish H. F., Small B., Chang H. Maturation of rabbit reticulocytes: degradation of specific reticulocyte proteins. Dev Biol. 1975 Nov;47(1):59–67. doi: 10.1016/0012-1606(75)90263-8. [DOI] [PubMed] [Google Scholar]
  18. Marchalonis J. J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem J. 1969 Jun;113(2):299–305. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mattia E., Rao K., Shapiro D. S., Sussman H. H., Klausner R. D. Biosynthetic regulation of the human transferrin receptor by desferrioxamine in K562 cells. J Biol Chem. 1984 Mar 10;259(5):2689–2692. [PubMed] [Google Scholar]
  20. Matts R. L., Levin D. H., London I. M. Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis. Proc Natl Acad Sci U S A. 1983 May;80(9):2559–2563. doi: 10.1073/pnas.80.9.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nunez M. T., Glass J., Fischer S., Lavidor L. M., Lenk E. M., Robinson S. H. Transferrin receptors in developing murine erythroid cells. Br J Haematol. 1977 Aug;36(4):519–526. doi: 10.1111/j.1365-2141.1977.tb00992.x. [DOI] [PubMed] [Google Scholar]
  22. OUCHTERLONY O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1–78. [PubMed] [Google Scholar]
  23. Petryshyn R., Rosa F., Fagard R., Levin D., London I. M. Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases. Biochem Biophys Res Commun. 1984 Mar 30;119(3):891–899. doi: 10.1016/0006-291x(84)90857-x. [DOI] [PubMed] [Google Scholar]
  24. Ponka P., Wilczynska A., Schulman H. M. Iron utilization in rabbit reticulocytes. A study using succinylacetone as an inhibitor or heme synthesis. Biochim Biophys Acta. 1982 Feb 10;720(1):96–105. doi: 10.1016/0167-4889(82)90043-x. [DOI] [PubMed] [Google Scholar]
  25. Ranu R. S., London I. M. Regulation of protein synthesis in rabbit reticulocyte lysates: preparation of efficient protein synthesis lysates and the purification and characterization of the heme-regulated translational inhibitory protein kinase. Methods Enzymol. 1979;60:459–484. doi: 10.1016/s0076-6879(79)60045-9. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Schreml W., Burka E. R. Properties of membrane-bound ribosomes in reticulocytes. J Biol Chem. 1968 Jul 10;243(13):3573–3580. [PubMed] [Google Scholar]
  28. 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]
  29. Shields D., Blobel G. Efficient cleavage and segregation of nascent presecretory proteins in a reticulocyte lysate supplemented with microsomal membranes. J Biol Chem. 1978 Jun 10;253(11):3753–3756. [PubMed] [Google Scholar]
  30. 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]
  31. Walsh R. J., Thomas E. D., Chow S. K., Fluharty R. G., Finch C. A. Iron Metabolism. Heme Synthesis in Vitro by Immature Erythrocytes. Science. 1949 Oct 14;110(2859):396–398. doi: 10.1126/science.110.2859.396. [DOI] [PubMed] [Google Scholar]
  32. Witt D. P., Woodworth R. C. Identification of the transferrin receptor of the rabbit reticulocyte. Biochemistry. 1978 Sep 19;17(19):3913–3917. doi: 10.1021/bi00612a004. [DOI] [PubMed] [Google Scholar]

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