Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells

Abstract

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 erythropoietin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein α subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G(αi2), α(i3), α(s), and α(q/11), but not α(i1) and α(o). Terminal differentiation was associated with a selective loss (≃ 80%) of G(αi3) and an increase in a truncated cytosolic form of G(αi2), while the membrane levels of α(i2), α(q/11), and α(s) did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for α(s), G3 cells contained significantly higher levels of the different G protein α subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G(αi3) may affect cellular responses that are mediated via P(2T) purine or thrombin receptors.