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

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.

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

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.