The binding of iodinated erythropoietin (EP) to receptors in EP- responsive and nonresponsive mouse erythroid cells and membranes from mouse and human placentas will be characterized. A system of isolating immature erythroblasts which respond to EP by late erythroid maturation in vitro has been developed in this laboratory. These cells have receptors for EP with higher and lower affinity for the hormone. Structure of the EP receptor in these EP-responsive cells as identified by cross-linking to 125I-EP will be compared to the structure of EP receptors found in human erythroid cells and mouse erythroid cells which are not responsive to EP as well as to the EP- receptors found in mouse and human placentas. Possible differences in the structure of the receptors which differ in affinities for EP will be examined. The subunit structure of the receptor for EP will be examined by peptide mapping to investigate the relatedness of the 100 kDa and 85 kDa receptor proteins. Solubilization of the EP receptor will be studied. Reconstitution of solubilized receptors into artificial membranes will be carried out to assay for the presence of soluble receptors for EP and determine the effects of solubilization on the subunit structure of the receptor. Purification of the receptor will be attempted by conventional means and affinity chromatography. Purification of cross-linked receptors will also be carried out in the event that a function receptor for EP cannot be purified. Monoclonal and polyvalent antibodies will be raised against the receptor proteins. The metabolism of the EP receptor in cells will be examined. Autoradiography at the level of the electron microscope of the intracellular transit of 125I-EP will be examined. The cell will be perturbed with inhibitors of protein synthesis, glycosylation, and cell surface digestion by proteinases to test indirectly if the receptors for EP are recycled within the cell. Receptors cross-linked by photoreactive conjugates of EP and antibodies raised against the receptor will be used to probe if the receptor is recycled after binding EP. The research described here will contribute to the understanding of the mechanism by which EP controls the later stages of maturation of erythroid progenitor cells and may help understand the molecular basis of diseases such as erythroleukemia and polycythemia vera as well as anemias due to chronic disease or aplasia. Understanding the mechanism of action of EP is particularly important with the ongoing clinical trials of recombinant EP in patients with anemia.
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