Research Project
3083171
The binding of erythropoietin to its receptor results in the activation of one or more signal pathways resulting in erythroid cell proliferation and differentiation. The identification of those characteristics of the erythropoietin receptor necessary for signal initiation will increase our understanding of erythropoiesis. This proposal will employ site specific antipeptide antibodies to the cytosolic portion of the receptor and oligonucleotide directed mutagenesis to probe the receptor's structural and functional characteristics. Antipeptide antibodies to several hydrophilic domains of the murine erythropoietin receptor cytosolic region have been raised in rabbits. These affinity purified antibodies identify both the recombinant receptor protein transfected into COS-7 cells and the native erythropoietin receptor in transformed and normal murine erythroid cells. These CYTO-Abs will be used to probe the receptor and perturb its function in cell-free and intact cell receptor assays. The cell free assay is based on the demonstration that activation of the receptor in isolated erythroid membranes results in the rapid dephosphorylation of a 43kDa protein, pp43. Perturbation of receptor function in the absence or presence of erythropoietin with these CYTO-Abs will permit presumptive identification of receptor domains relevant to phosphorylation or dephosphorylation of pp43. Activation of the receptor on intact erythroid cells results in a cascade of phosphorylation and dephosphorylation of cytosolic protein substrates and in increases in steady state levels of c-myc MRMA and decreases in steady state levels of c-myb mRNA. Antibodies will be microinjected into erythroid cells and perturbation of receptor function will be monitored by observing the effect on selective protein phosphorylation/dephosphorylation and cMYC and cMYB expression. The resulting structural and functional map of the receptors cytosolic region will be delineated further by mutagenesis of selected amino acid residues. Mutated recombinant receptor will be transfected into functional erythroid cells and the effect of receptor mutation on both cell free and intact cell receptor signaling will be determined. The results of these studies will provide important new information on the signaling pathways activated by the erythropoietin receptor and may result ultimately in the design of new therapeutic tools for the treatment of disorders of erythropoiesis.
