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-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.

Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain

“…The human EpoR is not activated by gp55‐P (Showers et al ., 1993; Hoatlin et al ., 1995); the murine and human EpoRs differ only in three positions in the transmembrane domain and are 82% identical overall (Jones et al ., 1990). Strikingly, mutation of Leu238 to Ser in the transmembrane domain rendered the human EpoR sensitive to activation by gp55‐P.…”

“…The human EpoR is not activated by gp55-P (Showers et al, 1993;; the murine and human EpoRs differ only in three positions in the transmembrane domain and are 82% identical overall (Jones et al, 1990). Strikingly, mutation of Leu238 to Ser in the transmembrane domain rendered the human EpoR sensitive to activation by gp55-P. Conversely, mutation of Ser238 of the murine EpoR to Leu abolished activation by gp55-P. Mutation of Ser238 to Ala did not abolish activation, nor did mutation of other hydroxyl-containing amino acid residues to Ala or Thr (Figure 3A and B).…”

“…Only gp55‐P and not gp55‐A can activate the murine EpoR to support proliferation of cell lines and to induce Epo‐independent red cell formation from early erythroid progenitors. The human EpoR binds to but is not activated by gp55‐P and cannot support Epo‐independent proliferation of cell lines (Showers et al ., 1993; Hoatlin and Kabat, 1995).…”