G-PROTEIN-COUPLED RECEPTORS IN NORMAL HUMAN ERYTHROID PROGENITOR CELLS

Human erythroid progenitor cells were isolated from peripheral blood o f healthy donors and amplified in a suspension culture system using re combinant growth factors (stem cell factor, interleukin-3, granulocyte -macrophage colony-stimulating factor and erythropoietin) as well as c onditioned medium from a human bone marrow stroma cell line to support cell proliferation. After 6-8 days of culture, the cell population co nsisted mainly of erythroid colony-forming cells (burst-forming units, BFU-Es and colony-forming units, CFU-Es). In these cells, we studied ligand-induced changes in intracellular Ca2+ concentration ([Ca2+](i)) and cAMP formation as the primary effector systems of guanine nucleot ide-binding protein (G protein)-coupled receptors. The results confirm ed the functional expression of receptors for adenosine (type A(2B)), prostaglandin E(1) and isoprenaline (beta-adrenoceptor), all of which stimulated adenylyl cyclase, as well as for ADP (purinoceptor types P- 2T and P-2U), platelet-activating factor and thrombin all of which cau sed a transient increase in [Ca2+](i). The efficacy of adenosine and p rostaglandin E(1) in stimulating cAMP formation was more than 5 times higher than that of isoprenaline, suggesting a low beta-adrenoceptor d ensity. The response to adenosine and isoprenaline decreased by 80 and 55% respectively during maturation into the proerythroblast stage. Si milarly, thapsigargin-sensitive intracellular Ca2+ stores and ligand-i nduced Ca2+ release declined by about 60% during the CFU-E-to-erythrob last transition. The overall functional expression pattern of G protei n-coupled receptors differed from that in human erythroleukaemia cell lines or from that in platelets. Primary culture systems for nontransf ormed cells, such as the one presented here, thus will be indispensabl e for the study of the functional role of G protein-dependent signalli ng during haematopoiesis.