Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin

Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Ca_c]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Ca_c], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Ca_c] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Ca_c] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunobloted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of G_iα (1,2, and 3) and G_oα. G_iα1 or G_iα3 and G_iα2 were identified but no G_oα was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Ca_c] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutryl cAMP failed to increase [Ca_c]. In summary, pertussis toxin blocks the increase in [Ca_c] in erythroblasts after Epo stimulation, suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were G_iα 1, 2, or 3, but not G_oα.