Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Feb;13(2):739–748. doi: 10.1128/mcb.13.2.739

Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line.

M O Showers 1, J C DeMartino 1, Y Saito 1, A D D'Andrea 1
PMCID: PMC358956  PMID: 8423798

Abstract

The Friend spleen focus-forming virus (SFFV) gp55 glycoprotein binds to the erythropoietin receptor (EPO-R), causing constitutive receptor signaling and the first stage of Friend erythroleukemia. We have used three independent strategies to further define this transforming molecular interaction. First, using a retroviral selection strategy, we have isolated the cDNAs encoding three fusion polypeptides containing regions of both EPO-R and gp55. These fusion proteins, like full-length gp55, transformed the Ba/F3 factor-dependent hematopoietic cell line and localized the transforming activity of gp55 to its transmembrane domain. Second, we have isolated a mutant of gp55 (F-gp55-M1) which binds, but fails to activate, EPO-R. We have compared the transforming activity of this gp55 mutant with the EPO-R-gp55 fusion proteins and with other variants of gp55, including wild-type polycythemia Friend gp55 and Rauscher gp55. All of the fusion polypeptides and mutant gp55 polypeptides were expressed at comparable levels, and all coimmunoprecipitated with wild-type EPO-R, but only the Friend gp55 and the EPO-R-gp55 fusion proteins constitutively activated wild-type EPO-R. Third, we have examined the specificity of the EPO-R-gp55 interaction by comparing the differential activation of murine and human EPO-R by gp55. Wild-type gp55 had a highly specific interaction with murine EPO-R; gp55 bound, but did not activate, human EPO-R.

Full text

PDF
748

Images in this article

741Image
on p.741
742Image
on p.742
744Image
on p.744
745Image
on p.745

Selected References

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

  1. Aizawa S., Suda Y., Furuta Y., Yagi T., Takeda N., Watanabe N., Nagayoshi M., Ikawa Y. Env-derived gp55 gene of Friend spleen focus-forming virus specifically induces neoplastic proliferation of erythroid progenitor cells. EMBO J. 1990 Jul;9(7):2107–2116. doi: 10.1002/j.1460-2075.1990.tb07379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amanuma H., Katori A., Obata M., Sagata N., Ikawa Y. Complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of Friend spleen focus-forming virus. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3913–3917. doi: 10.1073/pnas.80.13.3913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amanuma H., Watanabe N., Nishi M., Ikawa Y. Requirement of the single base insertion at the 3' end of the env-related gene of Friend spleen focus-forming virus for pathogenic activity and its effect on localization of the glycoprotein product (gp55). J Virol. 1989 Nov;63(11):4824–4833. doi: 10.1128/jvi.63.11.4824-4833.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ben-David Y., Bernstein A. Friend virus-induced erythroleukemia and the multistage nature of cancer. Cell. 1991 Sep 6;66(5):831–834. doi: 10.1016/0092-8674(91)90428-2. [DOI] [PubMed] [Google Scholar]
  5. Bestwick R. K., Boswell B. A., Kabat D. Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat. J Virol. 1984 Sep;51(3):695–705. doi: 10.1128/jvi.51.3.695-705.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bestwick R. K., Kozak S. L., Kabat D. Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5404–5408. doi: 10.1073/pnas.85.15.5404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Casadevall N., Lacombe C., Muller O., Gisselbrecht S., Mayeux P. Multimeric structure of the membrane erythropoietin receptor of murine erythroleukemia cells (Friend cells). Cross-linking of erythropoietin with the spleen focus-forming virus envelope protein. J Biol Chem. 1991 Aug 25;266(24):16015–16020. [PubMed] [Google Scholar]
  8. Chung S. W., Wolff L., Ruscetti S. K. Transmembrane domain of the envelope gene of a polycythemia-inducing retrovirus determines erythropoietin-independent growth. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7957–7960. doi: 10.1073/pnas.86.20.7957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark S. P., Mak T. W. Complete nucleotide sequence of an infectious clone of Friend spleen focus-forming provirus: gp55 is an envelope fusion glycoprotein. Proc Natl Acad Sci U S A. 1983 Aug;80(16):5037–5041. doi: 10.1073/pnas.80.16.5037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Daley G. Q., Van Etten R. A., Jackson P. K., Bernards A., Baltimore D. Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line. Mol Cell Biol. 1992 Apr;12(4):1864–1871. doi: 10.1128/mcb.12.4.1864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gliniak B. C., Kabat D. Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor. J Virol. 1989 Sep;63(9):3561–3568. doi: 10.1128/jvi.63.9.3561-3568.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoatlin M. E., Kozak S. L., Lilly F., Chakraborti A., Kozak C. A., Kabat D. Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9985–9989. doi: 10.1073/pnas.87.24.9985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Kilpatrick D. R., Srinivas R. V., Compans R. W. The spleen focus-forming virus envelope glycoprotein is defective in oligomerization. J Biol Chem. 1989 Jun 25;264(18):10732–10737. [PubMed] [Google Scholar]
  20. Li J. P., Baltimore D. Mechanism of leukemogenesis induced by mink cell focus-forming murine leukemia viruses. J Virol. 1991 May;65(5):2408–2414. doi: 10.1128/jvi.65.5.2408-2414.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li J. P., Bestwick R. K., Spiro C., Kabat D. The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor. J Virol. 1987 Sep;61(9):2782–2792. doi: 10.1128/jvi.61.9.2782-2792.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Longmore G. D., Lodish H. F. An activating mutation in the murine erythropoietin receptor induces erythroleukemia in mice: a cytokine receptor superfamily oncogene. Cell. 1991 Dec 20;67(6):1089–1102. doi: 10.1016/0092-8674(91)90286-8. [DOI] [PubMed] [Google Scholar]
  24. Machida C. A., Bestwick R. K., Kabat D. A weakly pathogenic Rauscher spleen focus-forming virus mutant that lacks the carboxyl-terminal membrane anchor of its envelope glycoprotein. J Virol. 1985 Mar;53(3):990–993. doi: 10.1128/jvi.53.3.990-993.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Majumdar M. K., Cho C. L., Fox M. T., Eckner K. L., Kozak S., Kabat D., Geib R. W. Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia. J Virol. 1992 Jun;66(6):3652–3660. doi: 10.1128/jvi.66.6.3652-3660.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Marchuk D., Drumm M., Saulino A., Collins F. S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991 Mar 11;19(5):1154–1154. doi: 10.1093/nar/19.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  29. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruscetti S. K., Janesch N. J., Chakraborti A., Sawyer S. T., Hankins W. D. Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line. J Virol. 1990 Mar;64(3):1057–1062. doi: 10.1128/jvi.64.3.1057-1062.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ruscetti S., Wolff L. Spleen focus-forming virus: relationship of an altered envelope gene to the development of a rapid erythroleukemia. Curr Top Microbiol Immunol. 1984;112:21–44. doi: 10.1007/978-3-642-69677-0_2. [DOI] [PubMed] [Google Scholar]
  32. Sitbon M., d'Auriol L., Ellerbrok H., André C., Nishio J., Perryman S., Pozo F., Hayes S. F., Wehrly K., Tambourin P. Substitution of leucine for isoleucine in a sequence highly conserved among retroviral envelope surface glycoproteins attenuates the lytic effect of the Friend murine leukemia virus. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5932–5936. doi: 10.1073/pnas.88.13.5932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Srinivas R. V., Compans R. W. Membrane association and defective transport of spleen focus-forming virus glycoproteins. J Biol Chem. 1983 Dec 10;258(23):14718–14724. [PubMed] [Google Scholar]
  34. Srinivas R. V., Kilpatrick D. R., Tucker S., Rui Z., Compans R. W. The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus. J Virol. 1991 Oct;65(10):5272–5280. doi: 10.1128/jvi.65.10.5272-5280.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Watanabe N., Nishi M., Ikawa Y., Amanuma H. A deletion in the Friend spleen focus-forming virus env gene is necessary for its product (gp55) to be leukemogenic. J Virol. 1990 Jun;64(6):2678–2686. doi: 10.1128/jvi.64.6.2678-2686.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Watanabe N., Nishi M., Ikawa Y., Amanuma H. Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3' half of the env gene. J Virol. 1991 Jan;65(1):132–137. doi: 10.1128/jvi.65.1.132-137.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Wolff L., Scolnick E., Ruscetti S. Envelope gene of the Friend spleen focus-forming virus: deletion and insertions in 3' gp70/p15E-encoding region have resulted in unique features in the primary structure of its protein product. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4718–4722. doi: 10.1073/pnas.80.15.4718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Yoshimura A., Longmore G., Lodish H. F. Point mutation in the exoplasmic domain of the erythropoietin receptor resulting in hormone-independent activation and tumorigenicity. Nature. 1990 Dec 13;348(6302):647–649. doi: 10.1038/348647a0. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES