Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- 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 erythropoetin(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 alpha 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 alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha 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 alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.
Kesselring
F
F
Department of Pharmacology, Universität Bern, Switzerland.
Spicher
K
K
Porzig
H
H
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D000242:Cyclic AMP / Q000096:biosynthesis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004357:Drug Synergism; D015672:Erythroid Precursor Cells / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000145:classification / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000473:pathology*; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis*; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D037342:Pertussis Toxin; D011943:Receptors, Adrenergic, beta / Q000032:analysis / Q000187:drug effects; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D009363:Neoplasm Proteins; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D004921:Erythropoietin; D000246:Adenosine Diphosphate Ribose; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D037342:Pertussis Toxin; D013917:Thrombin; D019204:GTP-Binding Proteins; D007545:Isoproterenol; D004121:Dimethyl Sulfoxide