Erythropoietin is a prime regulator of red cell development. Substantial progress has been made in defining its structure and its physiological role in promoting the proliferation of committed erythroid progenitors and their terminal differentiation. However, very little is known about the cell surface receptor for this factor or its mechanism of action.
The aim of the proposed research is to characterize this receptor physicochemically and structurally, ultimately through gene cloning and analysis. To date, attempts to study the erythropoietin receptor have been confounded by the circumstance that erythropoietin is inactivated when labeled (radio) chemically at tyrosinyl or lysinyl residues. I now have identified carboxyl groups as a useful site for chemical labeling and have developed a fully bioactive biotin-(COOH)- erythropoietin for use as receptor ligand. Using this ligand (or erythropoietin directly radioiodinated at carboxyl groups), the kinetic and equilibrium binding properties of the receptor of murine CFU-e will be characterized. Through affinity crosslinking studies, the minimal molecular weight and subunit constitution of this receptor will be defined. Erythroleukemia cells also will be screened in order to identify a continuous cell line expressing the erythropoietin receptor at densities sufficiently high to provide for its purification. Purification of the erythropoietin receptor will proceed through its solubilization and affinity chromatography on immobilized hormone. Partial amino acid sequencing of receptor will provide for the preparation of oligonucleotide probes for use in gene and cDNA cloning. Alternative approaches to receptor gene cloning also will be pursued including total cellular DNA-mediated gene transfer, expression, and retrieval, and cloning by expression in E. coli of a receptor cDNA encoding a functional ligand binding domain. Knowledge of the structure of the erythropoietin receptor will provide valuable insight regarding its function as a mediator of erythriod cell growth and differentiation, its relatedness to other growth factor receptors, and its potential role as a proto- oncogenic product.
