Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPγS or GDPβS, which maintain the α subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPγS. However, injection of GDPβS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, α or βγ transducin subunits were purified and microinjected as a sink for βγ or α subunits in the erythroblast, respectively. Transducin βγ, but not α, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the α subunit. Microinjected antibodies to Giα2, but not Giα1 or Giα3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Giα2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Giα2, but not Giα1 or Giα3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G- proteins in hematopoietic cells and show that Giα2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.
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