G-protein α subunit Giα2 mediates erythropoietin signal transduction in human erythroid precursors

Barbara Miller, Laurie Bell, Carl A. Hansen, Janet D. Robishaw, Maurine E. Linder, Joseph Y. Cheung

Research output: Contribution to journalArticle

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Abstract

Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPγS or GDPβS, which maintain the α subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPγS. However, injection of GDPβS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, α or βγ transducin subunits were purified and microinjected as a sink for βγ or α subunits in the erythroblast, respectively. Transducin βγ, but not α, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the α subunit. Microinjected antibodies to Giα2, but not Giα1 or Giα3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Giα2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Giα2, but not Giα1 or Giα3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G- proteins in hematopoietic cells and show that Giα2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.

Original languageEnglish (US)
Pages (from-to)1728-1736
Number of pages9
JournalJournal of Clinical Investigation
Volume98
Issue number8
DOIs
StatePublished - Oct 15 1996

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Protein Subunits
Erythropoietin
GTP-Binding Proteins
Signal Transduction
Carrier Proteins
Microinjections
Erythroblasts
Transducin
Calcium
Pertussis Toxin
Erythroid Precursor Cells
Fura-2
Calcium Channels
Guanosine Triphosphate
Fluorescence
Injections
Antibodies

All Science Journal Classification (ASJC) codes

  • Medicine(all)

Cite this

Miller, Barbara ; Bell, Laurie ; Hansen, Carl A. ; Robishaw, Janet D. ; Linder, Maurine E. ; Cheung, Joseph Y. / G-protein α subunit Giα2 mediates erythropoietin signal transduction in human erythroid precursors. In: Journal of Clinical Investigation. 1996 ; Vol. 98, No. 8. pp. 1728-1736.
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abstract = "Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPγS or GDPβS, which maintain the α subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPγS. However, injection of GDPβS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, α or βγ transducin subunits were purified and microinjected as a sink for βγ or α subunits in the erythroblast, respectively. Transducin βγ, but not α, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the α subunit. Microinjected antibodies to Giα2, but not Giα1 or Giα3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Giα2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Giα2, but not Giα1 or Giα3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G- proteins in hematopoietic cells and show that Giα2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.",
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G-protein α subunit Giα2 mediates erythropoietin signal transduction in human erythroid precursors. / Miller, Barbara; Bell, Laurie; Hansen, Carl A.; Robishaw, Janet D.; Linder, Maurine E.; Cheung, Joseph Y.

In: Journal of Clinical Investigation, Vol. 98, No. 8, 15.10.1996, p. 1728-1736.

Research output: Contribution to journalArticle

TY - JOUR

T1 - G-protein α subunit Giα2 mediates erythropoietin signal transduction in human erythroid precursors

AU - Miller, Barbara

AU - Bell, Laurie

AU - Hansen, Carl A.

AU - Robishaw, Janet D.

AU - Linder, Maurine E.

AU - Cheung, Joseph Y.

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N2 - Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPγS or GDPβS, which maintain the α subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPγS. However, injection of GDPβS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, α or βγ transducin subunits were purified and microinjected as a sink for βγ or α subunits in the erythroblast, respectively. Transducin βγ, but not α, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the α subunit. Microinjected antibodies to Giα2, but not Giα1 or Giα3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Giα2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Giα2, but not Giα1 or Giα3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G- proteins in hematopoietic cells and show that Giα2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.

AB - Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPγS or GDPβS, which maintain the α subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPγS. However, injection of GDPβS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, α or βγ transducin subunits were purified and microinjected as a sink for βγ or α subunits in the erythroblast, respectively. Transducin βγ, but not α, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the α subunit. Microinjected antibodies to Giα2, but not Giα1 or Giα3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Giα2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Giα2, but not Giα1 or Giα3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G- proteins in hematopoietic cells and show that Giα2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.

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