Project: Research project

Project Details


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

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

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.
Effective start/end date3/1/942/28/95



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