SIGNAL TRANSDUCTION MECHANISMS OF ERYTHROPOIETIN

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The long term goal of studies proposed here is to determine the signal transduction mechanisms through which growth factors stimulate hematopoietic proliferation and differentiation. This knowledge is essential to understanding disorders of hematopoietic regulation including aplastic anemia and leukemia. The major goal of this project is to understand the mechanisms through which erythropoietin (Epo) regulates ion channels during erythroid differentiation. This system is a model to delineate the immediate signalling events which follow interaction of erythropoietin with its receptor on normal cells. The mechanisms of regulation of the calcium channel will be examined at the single cell level on normal human BFU-E derived erythroblasts at different stages of differentiation with plasma membrane patch-clamp methodology, microinjection, and quantitative fluorescence microscopy coupled digital video imaging. The following specific aims will be addressed: Specific Aim 1: Electrophysiologic characterization of the Epo- regulatable calcium channel. We have previously shown that Epo induces an increase in cytosolic [Cai] in day 10 erythroblasts which results from Ca++ influx. Calcium current will be isolated on day 10 BFU-E derived erythroblasts with the nystatin perforated membrane patch, and the voltage dependence of these channels and influence of Epo on membrane hyperpolarization determined. Cell-free, inside-out patches, cell- attached patches, and the nystatin perforated vesicle will be employed to further characterize single Ca++ channels and measure Ca++ channel density. Specific Aim 2: Determination of the signalling mechanisms through which erythropoietin regulates calcium channels. A. To determine whether GTP- binding proteins modulate the calcium channel GTPgammaS, GDPbetaS or antibodies to Gialpha1,2,3, Goalpha, or p21 ras will be microinjected into day 10 cells to influence the Epo-stimulated [Cai] rise. Appropriate alpha subunits or p21 ras will be microinjected to reconstitute the Epo response. B. To explore the role of EPo-modulated protein phosphorylation, erythroblasts will be treated with specific inhibitors for serine/threonine or tyrosine kinases or phosphatases and [Cai] measured. C. To determine whether inositol phosphate hydrolysis is involved, IP3 and IP4 will be microinjected and [Cai] measured. Specific Aim 3: Exploration of differences in the signalling mechanism of erythropoietin at different stages of erythroid maturation. Since day 7 BFU-E derived cells do not respond to Epo with an increase in [Cai], Gialpha1,2,3 ad Goalpha will be measured with immunoblot on day 7 and 10 cells. Ca++ channel density on day 7 cells will be measured with the cell-attached or nystatin perforated vesicle configuration. Differences in channel/receptor coupling will be explored with microinjection of activated alpha subunits or p21 ras.