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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK046778-12
Application #
6909756
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1994-03-01
Project End
2007-03-31
Budget Start
2004-07-01
Budget End
2005-03-31
Support Year
12
Fiscal Year
2004
Total Cost
$195,921
Indirect Cost
Name
Pennsylvania State University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Cheung, Joseph Y; Miller, Barbara A (2017) Transient Receptor Potential-Melastatin Channel Family Member 2: Friend or Foe. Trans Am Clin Climatol Assoc 128:308-329
Bao, Lei; Chen, Shu-Jen; Conrad, Kathleen et al. (2016) Depletion of the Human Ion Channel TRPM2 in Neuroblastoma Demonstrates Its Key Role in Cell Survival through Modulation of Mitochondrial Reactive Oxygen Species and Bioenergetics. J Biol Chem 291:24449-24464
Hoffman, Nicholas E; Miller, Barbara A; Wang, JuFang et al. (2015) Ca²? entry via Trpm2 is essential for cardiac myocyte bioenergetics maintenance. Am J Physiol Heart Circ Physiol 308:H637-50
Miller, Barbara A; Hoffman, Nicholas E; Merali, Salim et al. (2014) TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria. J Biol Chem 289:7615-29
Chen, Shu-jen; Hoffman, Nicholas E; Shanmughapriya, Santhanam et al. (2014) A splice variant of the human ion channel TRPM2 modulates neuroblastoma tumor growth through hypoxia-inducible factor (HIF)-1/2?. J Biol Chem 289:36284-302
Chen, Shu-jen; Zhang, Wenyi; Tong, Qin et al. (2013) Role of TRPM2 in cell proliferation and susceptibility to oxidative stress. Am J Physiol Cell Physiol 304:C548-60
Miller, Barbara A; Wang, JuFang; Hirschler-Laszkiewicz, Iwona et al. (2013) The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 304:H1010-22
Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Keefer, Kerry et al. (2012) Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis. Exp Hematol 40:71-83
Hirschler-Laszkiewicz, Iwona; Tong, Qin; Waybill, Kathleen et al. (2011) The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin. J Biol Chem 286:30636-46
Hirschler-Laszkiewicz, Iwona; Tong, Qin; Conrad, Kathleen et al. (2009) TRPC3 activation by erythropoietin is modulated by TRPC6. J Biol Chem 284:4567-81

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