STRUCTURE AND FUNCTION OF THE ERYTHROPOIETIN RECEPTOR

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We propose to continue our studies of the physical interaction between the erythropoietin receptor (EPO-R) and the Friend Spleen Focus-Forming Virus envelope protein, gp55 (D'Andrea, 1989a; Li, 1990). The interaction of these two polypeptides results in the constitutive activation of the EPO-R and represents a novel mechanism of retroviral leukemogenesis. Our work is health related since other retroviruses may induce hematopoietic malignancies by analogous mechanisms. Also, the activation of the EPO-R by EPO itself has become a standard treatment for various disease related or treatment induced anemias. During Project I of the proposed 5 year study period we will delineate the domains of the EPO-R which are important in signal transduction and in interaction with the gp55. Using a functional assay, the ability of the wild-type EPO-R cDNA to confer EPO dependence on an IL-3 dependent cell, we will screen mutants of the EPO-R for their ability to confer EPO dependence. Using a coimmunoprecipitation assay, we will delineate the EPO-R domain required for binding to the gp55. Chimeric receptors, containing regions of the EPO-R and regions of either the IL-2R beta chain or the IL-3R alpha chain, will further define these two functional domains. Also, using these chimeric receptor mutants we will study the homodimerization of the EPO-R and the amino acid residues which mediate this process. Project II will focus on the functional interaction of gp55 and EPO-R. We will study the normal and abnormal biosynthesis, carbohydrate processing, and ligand-induced phosphorylation of the EPO-R and the effects of gp55 binding. Other subunits of the EPO-R will be identified by crosslinking and coimmunoprecipitation, and various cloning strategies will be used to isolate the cDNAs encoding these other subunits.