Signal Transduction Mechanisms of Erythropoietin

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DESCRIPTION (provided by applicant): Our long-term goal is to understand the signal transduction mechanisms through which growth factors regulate hematopoietic proliferation and differentiation. Erythropoietin (Epo) stimulates an increase in intracellular calcium at specific stages of erythroid differentiation, and we have now demonstrated that Epo modulates calcium influx through TRPC2, a member of the family of TRP channels involved in sustained calcium entry in non-excitable cells. The major goal of this renewal is to understand the mechanisms through which Epo regulates calcium influx through TRPC2 and the functional importance of TRPC2 in erythroid proliferation and differentiation. Specific Aim 1: Identification of the Mechanisms Throuqh Which the Erythropoietin Receptor (Epo-R) Modulates TRPC2 Channel Activation. The role of four signal transducers in Epo-dependent regulation of TRPC2 will be determined in primary erythroblasts: the adaptor proteins Nck1/2 and Grb7, phospholipase C/inositol 1,4,5 trisphosphate, diacylglycerol, and the G-protein subunit Gi(2. Membrane localization is critical to the function of TRPC. We will examine TRPC2 subcellular localization, and colocalization with Epo-R and specific transducers, using confocal microscopy and immunoprecipitation. We will also determine if TRPC2 forms functional multimers with other TRP channels expressed on erythroid cells. Specific Aim 2: Identification of the TRPC2 Domains Which are Involved in Epo Modulation of Calcium Influx. We have developed a model system to study regulation of TRPC using CHO cells cotransfected with EPO-R in a GFP-expressing vector and TRPC linked to BFP. Intracellular free calcium is measured in the same single cells with Fura Red and digital video imaging. We will use this system to identify the role of four specific TRPC2 domains in calcium influx: the calcium pore, the C-terminal calmodulin and IP3R binding sites, the leucine zipper motif and the nuclear localization signal. We will also identify functional differences in TRPC2 N-terminal splice variants expressed in erythroid cells. Specific Aim 3: Determination of the Function of TRPC2 in Erythroid Proliferation/Differentiation. TRPC2 knockout mice will be used to examine the rote of TRPC2 in viability, proliferation, apoptosis induction and cell cycle progression of primary erythroid cells.