Signal Transduction Mechanisms of Erythropoietin

Project: Research project

Project Details


The erythropoietin (Epo) receptor is a model for understanding signal transduction mechanisms of the
cytokine receptor superfamily. Calcium signaling plays an important role in cell proliferation and apoptosis,
yet little is known about regulation of ion channels by hematopoietic growth factors. Using quantitative
fluorescence microscopy and electrophysiology, our laboratory was the first to demonstrate that Epo
modulates the intracellular calcium concentration ([Ca2+] 0 through activation of members of the TRPC ion
channel family. The transient receptor potential (TRP) superfamily is a diverse group of voltage-independent
calcium permeable channels which are involved in sustained calcium entry in nonexcitable cells. TRPC3
and TRPC6 are members of the TRPC subfamily which are expressed in human erythroid cells. TRPC3 is
activated by Epo, while TRPC6 inhibits TRPC3 activation. TRPC2 is also activated by Epo in murine
erythroid cells but is a pseudogene in humans. In this grant, Specific Aim 1 will study the mechanisms by
which Epo regulates [Ca2*1i through activation of TRPC3. We will examine (1) mechanisms of TRPC3
tyrosine phosphorylation after Epo stimulation by identifying the involved kinase and key tyrosine residues on
TRPC3 which are phosphorylated and required for channel activation; (2) the role of phospholipase C and
IP3R in TRPC3 activation by Epo; and (3) the mechanisms through which Epo regulates TRPC3 cell surface
localization. Specific Aim 2 will examine the mechanisms through which TRPC6 inhibits TRPC3 activation by
Epo. TRPC3 and TRPC6 expression are regulated during normal erythroid differentiation. We will determine
whether TRPC6 inhibits TRPC3 membrane localization or tyrosine phosphorylation and identify channel
domains which are responsible for the different responses to Epo. Specific Aim 3 will examine the functional
importance of TRPC3 in ervthropoiesis using TRPC3 whole animal and tissue specific knockout mice and
TRPC2-/-TRPC3-/- double knockouts. Preliminary results show that TRPC2 knockout animals are protected
from hemolysis in response to oxidative stress. Understanding regulation of TRP channels by hematopoietic
growth factors including erythropoietin is of fundamental biological importance, and the knowledge gained
from these studies will be widely applicable to regulation of hematopoiesis by other members of the cytokine
receptor superfamily.
Effective start/end date7/20/076/30/08




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