In Friend erythroleukemia, the membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus (SFFV) binds as an agonist to murine erythropoietin receptors (EpoR) to stimulate erythroblastosis. We recently cloned the human Epo gene and produced helper-free retrovirions that encode Epo, murine EpoR or gp55. Coinfection of murine IL-3 dependent cells with the EpoR virus plus either of the other viruses was able to convert the cells to factor-independent proliferation. Interestingly, erythroblasts of mice homozygous for Fv-2r have severely reduced mitogenic response to Epo; and these mice are therefore resistant to both Friend and to Epo virus- induced erythroleukemias. Mapping indicated that EpoR and Fv-2 genes are unlinked. We proposed: (1) Use genetic and biochemical methods to analyze Fv-2 role in EpoR signal transduction. Does Fv-2 encode a second EpoR subunit or a transducin? Molecularly clone Fv-2. (2) Friend erythroleukemia is considered restricted to mice. Why? Cannot gp55 bind to nonmurine EpoR? Or, do other species have an Fv-2rr-like phenotype? Clone human EpoR. Analyze mitogenic interactions of gp55 and Epo with human and murine EpoR in factor-dependent cells from humans or mice. (3) Use methods including mutagenesis to identify active sites for binding and mitogenesis on Epo and EpoR. (4) Produce a soluble derivative of EpoR in substantial amounts. Study Epo-sEpoR interaction by physical methods including cocrystallization and X-ray diffraction. Use sEpoR for a sensitive Epo assay. Does sEpoR cure Friend erythroleukemia? (5) Analyze EpoR processing and mitogenic signalling. This project is important for understanding hematopoietic growth factor receptors in leukemias and it provides a unique model for learning at the molecular level how host genetic variation (e.g., Fv-2) controls susceptibility to an oncogenic protein.
