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. 1992 Oct;12(10):4553–4561. doi: 10.1128/mcb.12.10.4553

Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.

D E Quelle 1, F W Quelle 1, D M Wojchowski 1
PMCID: PMC360382  PMID: 1406645

Abstract

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.

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Selected References

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  1. Bazan J. F. Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6934–6938. doi: 10.1073/pnas.87.18.6934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carroll M. P., Spivak J. L., McMahon M., Weich N., Rapp U. R., May W. S. Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation. J Biol Chem. 1991 Aug 15;266(23):14964–14969. [PubMed] [Google Scholar]
  3. Chen W. J., Goldstein J. L., Brown M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed] [Google Scholar]
  4. Cosman D., Lyman S. D., Idzerda R. L., Beckmann M. P., Park L. S., Goodwin R. G., March C. J. A new cytokine receptor superfamily. Trends Biochem Sci. 1990 Jul;15(7):265–270. doi: 10.1016/0968-0004(90)90051-c. [DOI] [PubMed] [Google Scholar]
  5. D'Andrea A. D., Fasman G. D., Lodish H. F. Erythropoietin receptor and interleukin-2 receptor beta chain: a new receptor family. Cell. 1989 Sep 22;58(6):1023–1024. doi: 10.1016/0092-8674(89)90499-6. [DOI] [PubMed] [Google Scholar]
  6. D'Andrea A. D., Lodish H. F., Wong G. G. Expression cloning of the murine erythropoietin receptor. Cell. 1989 Apr 21;57(2):277–285. doi: 10.1016/0092-8674(89)90965-3. [DOI] [PubMed] [Google Scholar]
  7. D'Andrea A. D., Yoshimura A., Youssoufian H., Zon L. I., Koo J. W., Lodish H. F. The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains. Mol Cell Biol. 1991 Apr;11(4):1980–1987. doi: 10.1128/mcb.11.4.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. D'Andrea A. D., Zon L. I. Erythropoietin receptor. Subunit structure and activation. J Clin Invest. 1990 Sep;86(3):681–687. doi: 10.1172/JCI114763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis S., Aldrich T. H., Valenzuela D. M., Wong V. V., Furth M. E., Squinto S. P., Yancopoulos G. D. The receptor for ciliary neurotrophic factor. Science. 1991 Jul 5;253(5015):59–63. doi: 10.1126/science.1648265. [DOI] [PubMed] [Google Scholar]
  10. Dexter T. M., Garland J., Scott D., Scolnick E., Metcalf D. Growth of factor-dependent hemopoietic precursor cell lines. J Exp Med. 1980 Oct 1;152(4):1036–1047. doi: 10.1084/jem.152.4.1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fukunaga R., Ishizaka-Ikeda E., Pan C. X., Seto Y., Nagata S. Functional domains of the granulocyte colony-stimulating factor receptor. EMBO J. 1991 Oct;10(10):2855–2865. doi: 10.1002/j.1460-2075.1991.tb07835.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gearing D. P., Comeau M. R., Friend D. J., Gimpel S. D., Thut C. J., McGourty J., Brasher K. K., King J. A., Gillis S., Mosley B. The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor. Science. 1992 Mar 13;255(5050):1434–1437. doi: 10.1126/science.1542794. [DOI] [PubMed] [Google Scholar]
  14. Gearing D. P., Thut C. J., VandeBos T., Gimpel S. D., Delaney P. B., King J., Price V., Cosman D., Beckmann M. P. Leukemia inhibitory factor receptor is structurally related to the IL-6 signal transducer, gp130. EMBO J. 1991 Oct;10(10):2839–2848. doi: 10.1002/j.1460-2075.1991.tb07833.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gironès N., Alverez E., Seth A., Lin I. M., Latour D. A., Davis R. J. Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis. J Biol Chem. 1991 Oct 5;266(28):19006–19012. [PubMed] [Google Scholar]
  16. Gliniak B. C., Rohrschneider L. R. Expression of the M-CSF receptor is controlled posttranscriptionally by the dominant actions of GM-CSF or multi-CSF. Cell. 1990 Nov 30;63(5):1073–1083. doi: 10.1016/0092-8674(90)90510-l. [DOI] [PubMed] [Google Scholar]
  17. Hatakeyama M., Mori H., Doi T., Taniguchi T. A restricted cytoplasmic region of IL-2 receptor beta chain is essential for growth signal transduction but not for ligand binding and internalization. Cell. 1989 Dec 1;59(5):837–845. doi: 10.1016/0092-8674(89)90607-7. [DOI] [PubMed] [Google Scholar]
  18. Hatakeyama M., Tsudo M., Minamoto S., Kono T., Doi T., Miyata T., Miyasaka M., Taniguchi T. Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNA's. Science. 1989 May 5;244(4904):551–556. doi: 10.1126/science.2785715. [DOI] [PubMed] [Google Scholar]
  19. Hayashida K., Kitamura T., Gorman D. M., Arai K., Yokota T., Miyajima A. Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9655–9659. doi: 10.1073/pnas.87.24.9655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hempstead B. L., Patil N., Thiel B., Chao M. V. Deletion of cytoplasmic sequences of the nerve growth factor receptor leads to loss of high affinity ligand binding. J Biol Chem. 1990 Jun 15;265(17):9595–9598. [PubMed] [Google Scholar]
  21. Jones S. S., D'Andrea A. D., Haines L. L., Wong G. G. Human erythropoietin receptor: cloning, expression, and biologic characterization. Blood. 1990 Jul 1;76(1):31–35. [PubMed] [Google Scholar]
  22. Kanakura Y., Druker B., Cannistra S. A., Furukawa Y., Torimoto Y., Griffin J. D. Signal transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins. Blood. 1990 Aug 15;76(4):706–715. [PubMed] [Google Scholar]
  23. Kitamura T., Hayashida K., Sakamaki K., Yokota T., Arai K., Miyajima A. Reconstitution of functional receptors for human granulocyte/macrophage colony-stimulating factor (GM-CSF): evidence that the protein encoded by the AIC2B cDNA is a subunit of the murine GM-CSF receptor. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5082–5086. doi: 10.1073/pnas.88.12.5082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klinken S. P., Nicola N. A., Johnson G. R. In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8506–8510. doi: 10.1073/pnas.85.22.8506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krantz S. B. Erythropoietin. Blood. 1991 Feb 1;77(3):419–434. [PubMed] [Google Scholar]
  26. 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]
  27. Li J. P., D'Andrea A. D., Lodish H. F., Baltimore D. Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor. Nature. 1990 Feb 22;343(6260):762–764. doi: 10.1038/343762a0. [DOI] [PubMed] [Google Scholar]
  28. Linnekin D., Farrar W. L. Signal transduction of human interleukin 3 and granulocyte-macrophage colony-stimulating factor through serine and tyrosine phosphorylation. Biochem J. 1990 Oct 15;271(2):317–324. doi: 10.1042/bj2710317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mayeux P., Casadevall N., Lacombe C., Muller O., Tambourin P. Solubilization and hydrodynamic characteristics of the erythropoietin receptor. Evidence for a multimeric complex. Eur J Biochem. 1990 Nov 26;194(1):271–278. doi: 10.1111/j.1432-1033.1990.tb19453.x. [DOI] [PubMed] [Google Scholar]
  30. Mayeux P., Lacombe C., Casadevall N., Chretien S., Dusanter I., Gisselbrecht S. Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins. J Biol Chem. 1991 Dec 5;266(34):23380–23385. [PubMed] [Google Scholar]
  31. Migliaccio A. R., Migliaccio G., D'Andrea A., Baiocchi M., Crotta S., Nicolis S., Ottolenghi S., Adamson J. W. Response to erythropoietin in erythroid subclones of the factor-dependent cell line 32D is determined by translocation of the erythropoietin receptor to the cell surface. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11086–11090. doi: 10.1073/pnas.88.24.11086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miura O., D'Andrea A., Kabat D., Ihle J. N. Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis. Mol Cell Biol. 1991 Oct;11(10):4895–4902. doi: 10.1128/mcb.11.10.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Miyazaki T., Maruyama M., Yamada G., Hatakeyama M., Taniguchi T. The integrity of the conserved 'WS motif' common to IL-2 and other cytokine receptors is essential for ligand binding and signal transduction. EMBO J. 1991 Nov;10(11):3191–3197. doi: 10.1002/j.1460-2075.1991.tb04881.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mogi T., Marti T., Khorana H. G. Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin. J Biol Chem. 1989 Aug 25;264(24):14197–14201. [PubMed] [Google Scholar]
  35. Murakami M., Narazaki M., Hibi M., Yawata H., Yasukawa K., Hamaguchi M., Taga T., Kishimoto T. Critical cytoplasmic region of the interleukin 6 signal transducer gp130 is conserved in the cytokine receptor family. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11349–11353. doi: 10.1073/pnas.88.24.11349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Patthy L. Homology of a domain of the growth hormone/prolactin receptor family with type III modules of fibronectin. Cell. 1990 Apr 6;61(1):13–14. doi: 10.1016/0092-8674(90)90208-v. [DOI] [PubMed] [Google Scholar]
  37. Prywes R., Livneh E., Ullrich A., Schlessinger J. Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. EMBO J. 1986 Sep;5(9):2179–2190. doi: 10.1002/j.1460-2075.1986.tb04482.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Quelle D. E., Wojchowski D. M. Localized cytosolic domains of the erythropoietin receptor regulate growth signaling and down-modulate responsiveness to granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4801–4805. doi: 10.1073/pnas.88.11.4801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Quelle F. W., Quelle D. E., Wojchowski D. M. Interleukin 3, granulocyte-macrophage colony-stimulating factor, and transfected erythropoietin receptors mediate tyrosine phosphorylation of a common cytosolic protein (pp100) in FDC-ER cells. J Biol Chem. 1992 Aug 25;267(24):17055–17060. [PubMed] [Google Scholar]
  40. Quelle F. W., Wojchowski D. M. Proliferative action of erythropoietin is associated with rapid protein tyrosine phosphorylation in responsive B6SUt.EP cells. J Biol Chem. 1991 Jan 5;266(1):609–614. [PubMed] [Google Scholar]
  41. Rozakis-Adcock M., Kelly P. A. Mutational analysis of the ligand-binding domain of the prolactin receptor. J Biol Chem. 1991 Sep 5;266(25):16472–16477. [PubMed] [Google Scholar]
  42. Sawyer S. T., Krantz S. B., Goldwasser E. Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells. J Biol Chem. 1987 Apr 25;262(12):5554–5562. [PubMed] [Google Scholar]
  43. Sawyer S. T. The two proteins of the erythropoietin receptor are structurally similar. J Biol Chem. 1989 Aug 5;264(22):13343–13347. [PubMed] [Google Scholar]
  44. Takaki S., Mita S., Kitamura T., Yonehara S., Yamaguchi N., Tominaga A., Miyajima A., Takatsu K. Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor. EMBO J. 1991 Oct;10(10):2833–2838. doi: 10.1002/j.1460-2075.1991.tb07832.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thies R. S., Webster N. J., McClain D. A. A domain of the insulin receptor required for endocytosis in rat fibroblasts. J Biol Chem. 1990 Jun 15;265(17):10132–10137. [PubMed] [Google Scholar]
  46. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Walker F., Nicola N. A., Metcalf D., Burgess A. W. Hierarchical down-modulation of hemopoietic growth factor receptors. Cell. 1985 Nov;43(1):269–276. doi: 10.1016/0092-8674(85)90032-7. [DOI] [PubMed] [Google Scholar]
  48. Watowich S. S., Yoshimura A., Longmore G. D., Hilton D. J., Yoshimura Y., Lodish H. F. Homodimerization and constitutive activation of the erythropoietin receptor. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2140–2144. doi: 10.1073/pnas.89.6.2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Winkelmann J. C., Penny L. A., Deaven L. L., Forget B. G., Jenkins R. B. The gene for the human erythropoietin receptor: analysis of the coding sequence and assignment to chromosome 19p. Blood. 1990 Jul 1;76(1):24–30. [PubMed] [Google Scholar]
  50. Yang Y. C., Ciarletta A. B., Temple P. A., Chung M. P., Kovacic S., Witek-Giannotti J. S., Leary A. C., Kriz R., Donahue R. E., Wong G. G. Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell. 1986 Oct 10;47(1):3–10. doi: 10.1016/0092-8674(86)90360-0. [DOI] [PubMed] [Google Scholar]
  51. Yoshimura A., D'Andrea A. D., Lodish H. F. Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4139–4143. doi: 10.1073/pnas.87.11.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yoshimura A., Lodish H. F. In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130. Mol Cell Biol. 1992 Feb;12(2):706–715. doi: 10.1128/mcb.12.2.706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoshimura A., Zimmers T., Neumann D., Longmore G., Yoshimura Y., Lodish H. F. Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor. J Biol Chem. 1992 Jun 5;267(16):11619–11625. [PubMed] [Google Scholar]
  54. Zon L. I., Moreau J. F., Koo J. W., Mathey-Prevot B., D'Andrea A. D. The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein. Mol Cell Biol. 1992 Jul;12(7):2949–2957. doi: 10.1128/mcb.12.7.2949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. de Vos A. M., Ultsch M., Kossiakoff A. A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992 Jan 17;255(5042):306–312. doi: 10.1126/science.1549776. [DOI] [PubMed] [Google Scholar]

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