CHANGES IN G-PROTEIN PATTERN AND IN G-PROTEIN-DEPENDENT SIGNALING DURING ERYTHROPOIETIN-INDUCED AND DIMETHYLSULFOXIDE-INDUCED DIFFERENTIATION OF MURINE ERYTHROLEUKEMIA-CELLS

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an eryth ropoetin(Epo)-sensitive, murine erythroleukemia cell line, Clone 6C8 s howed terminal erythroid differentiation in response to a combined tre atment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation, We measur ed G protein alpha subunit levels by toxin-catalyzed adenosine diphosp hate (ADP)-ribosylation with [P-32]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera, N ative RED-1 cells expressed G(alpha 12), alpha(13), alpha(s), and alph a(q/11), but not alpha(11) and alpha(o). Terminal differentiation was associated with a selective loss (approximate to 80%) of G(alpha i3) a nd an increase in a truncated cytosolic form of G(alpha 12), while the membrane levels of alpha(12), alpha(q/11), and alpha(s) did not chang e significantly. Treatment of G3 cells with the inducers was without e ffect on G protein abundance. However, except for alpha(s), G3 cells c ontained significantly higher levels of the different G protein alpha subunits tested, Stimulation of G protein-coupled receptors by thrombi n and ADP caused a pertussis toxin (PTX)-inhibitable transient increas e in intracellular Ca2+ that was markedly reduced in differentiated ce lls, In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sen sitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine mo nophosphate (cAMP) formation. Our results show that specific alteratio ns in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal rel ationship, The loss of G(alpha 13) may affect cellular responses that are mediated via P-2T purine or thrombin receptors. (C) 1994 by The Am erican Society of Hematology.