G-protein alpha subunit Gi alpha 2 mediates erythropoietin signal transduction in human erythroid precursors

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, GTP gamma S or GDP beta S, which maintain the a subunit in an activated or inactivated state, respectively. [Ca-i] increased significantly in a dose-dependent manner after microinjection of GTP gamma S. However, injection of GDP beta S 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, alpha or beta gamma transducin subunits were purified and microinjected as a sink for beta gamma or alpha subunits in the erythroblast, respectively. Transducin beta gamma, but not alpha, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the alpha subunit. Microinjected antibodies to Gi alpha 2, but not Gi alpha 1 or Gi alpha 3, blocked the erythropoietin-stimulated [Ca-i] rise, identifying Gi alpha 2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Gi alpha 2, but not Gi alpha 1 or Gi alpha 3 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 Gi alpha 2 is required in erythropoietin modulation of [Ca-i] via influx through calcium channels.