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

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-Ml) 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.The Friend and Rauscher murine leukemia virus complexes induce a rapid and fatal erythroleukemia in adult mice (4,30). It is the replication-defective spleen focus-forming virus (SFFV) contained in these complexes that initiates the disease. Both Friend and Rauscher SFFV encode a 55-kDa glycoprotein, gp55, which is necessary and sufficient for the initiation of leukemia (1). Friend SFFV gp55 (F-gp55), encoded by the polycythemia-inducing strain of SFFV, has recently been shown to bind to the normal erythropoietin receptor (EPO-R) expressed in infected erythroblasts, resulting in constitutive activation of EPO-R, EPO-independent growth of the infected cells, and the first stage of Friend erythroleukemia (4,17,22).Several features of the F-gp55 polypeptide affect its leukemogenic activity. F-gp55 is transport defective, with the majority of its expression remaining intracellular (16,19,35,40). Its cell surface form appears to be a prerequisite for its leukemogenicity (21). Also, it is the transmembrane region of F-gp55 that is required for its ability to generate EPOindependent erythroblasts in vivo (8,37,38).The precise molecular interaction between F-gp55 and EPO-R is poorly understood. Most of the EPO-R-gp55 complex is formed in the endoplasmic reticulum of infected cells (41), although cross-linked complexes of EPO-R and F-gp55 have also been detected at the cell surface (7). In addition, the transmembrane region of EPO-R is critical for its interaction with gp55, and at least one binding site for gp55 is present in the extracytoplasmic region of EPO-R (44). Furthermore, the molecular interactions bet...

A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.

“…We initially synthesized cDNA encoding a truncated form of murine EPO-R [EPO-R(T)], which lacks the critical signaltransducing domain previously described (13,17,30) was transfected into the ecotropic packaging cell line, E86, and a helper-free stock of PLSXN-EPO-R(T) was generated (38). The expression of full-length EPO-R and EPO-R(T) in infected Ba/F3 cells was confirmed by FACScan analysis (Fig.…”

“…While the bulk of the interaction between F-gp55 and EPO-R occurs in an intracellular compartment (44), the complex of F-gp55 and EPO-R has also been detected at the cell surface (5,16). We next tested EPO-R(T) for its ability to block F-gp55-mediated activation of wild-type EPO-R. Parental Ba/F3 cells, Ba/F3-EPO-R cells, or Ba/F3-EPO-R-EPO-R(T) cells were infected with a retrovirus encoding F-gp55 (38). Infected subclones were isolated by limiting dilution and analyzed for IL-3-dependent or EPO-dependent growth ( 3).…”

“…Selection with G418 (1.0 mg/ml) in 10% WEHI-3B-conditioned medium was initiated 48 h after electroporation. Selected cells were subcloned by limiting dilution, and individual subclones were expanded in G418-containing IL-3 medium and analyzed for the presence of the wild-type EPO-R or EPO-R(T) polypeptide by metabolic labeling and immunoprecipitation as previously described (38). Alternatively, Ba/F3 cells growing in IL-3 medium were infected with retroviral construct PLXSN-EPO-R(T) (13).…”

Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia

“…The murine EPOR, but not the human EPOR, is able to support gp55 P -mediated Ba/F3-cell proliferation. 31 Indeed, the structural basis for this difference has been localized to a single amino acid in the transmembrane domain of the EPOR. 36 We found that mice expressing the human EPOR instead of the murine EPOR were fully susceptible to Friend virus-induced erythroblastosis.…”

“…The human EPOR is ineffective in this context. 31 We studied mice with a targeted mutation of the murine Epor that were transgenic for the human EPOR (Epor Ϫ/Ϫ ;Tg(EPOR)). 21 …”