Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts of erythropoietin

Barbara Miller, K. Foster, J. D. Robishaw, C. F. Whitfield, L. Bell, J. Y. Cheung

Research output: Contribution to journalArticle

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Abstract

Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Ca(c)]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Ca(c)], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Ca(c)] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Ca(c)] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of G(iα) (1,2, and 3) and G(oα). G(iα)1 or G(iα)3 and G(iα)2 were identified but no G(oα) was found. To examine the influence of Epo on adenylate cyclase activity,day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP)production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Ca(c)] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutryl cAMP failed to increase [Ca(c)]. In summary, pertussis toxin blocks the increase in [Ca(c)] in erythroblasts after Epo stimulation, suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were G(iα) 1, 2, or 3, but not G(oα).

Original languageEnglish (US)
Pages (from-to)486-492
Number of pages7
JournalBlood
Volume77
Issue number3
StatePublished - Jan 1 1991

Fingerprint

Erythroblasts
Pertussis Toxin
Erythropoietin
Guanosine Triphosphate
Carrier Proteins
Calcium
Adenylyl Cyclases
Erythroid Precursor Cells
Membranes
Cyclic AMP
Fluorescence microscopy
Protein Subunits
Theophylline
Fluorescence Microscopy
GTP-Binding Proteins
Hot Temperature
Imaging techniques
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Cite this

Miller, B., Foster, K., Robishaw, J. D., Whitfield, C. F., Bell, L., & Cheung, J. Y. (1991). Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts of erythropoietin. Blood, 77(3), 486-492.
Miller, Barbara ; Foster, K. ; Robishaw, J. D. ; Whitfield, C. F. ; Bell, L. ; Cheung, J. Y. / Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts of erythropoietin. In: Blood. 1991 ; Vol. 77, No. 3. pp. 486-492.
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abstract = "Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Ca(c)]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Ca(c)], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Ca(c)] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Ca(c)] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of G(iα) (1,2, and 3) and G(oα). G(iα)1 or G(iα)3 and G(iα)2 were identified but no G(oα) was found. To examine the influence of Epo on adenylate cyclase activity,day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP)production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Ca(c)] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutryl cAMP failed to increase [Ca(c)]. In summary, pertussis toxin blocks the increase in [Ca(c)] in erythroblasts after Epo stimulation, suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were G(iα) 1, 2, or 3, but not G(oα).",
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Miller, B, Foster, K, Robishaw, JD, Whitfield, CF, Bell, L & Cheung, JY 1991, 'Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts of erythropoietin', Blood, vol. 77, no. 3, pp. 486-492.

Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts of erythropoietin. / Miller, Barbara; Foster, K.; Robishaw, J. D.; Whitfield, C. F.; Bell, L.; Cheung, J. Y.

In: Blood, Vol. 77, No. 3, 01.01.1991, p. 486-492.

Research output: Contribution to journalArticle

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T1 - Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts of erythropoietin

AU - Miller, Barbara

AU - Foster, K.

AU - Robishaw, J. D.

AU - Whitfield, C. F.

AU - Bell, L.

AU - Cheung, J. Y.

PY - 1991/1/1

Y1 - 1991/1/1

N2 - Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Ca(c)]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Ca(c)], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Ca(c)] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Ca(c)] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of G(iα) (1,2, and 3) and G(oα). G(iα)1 or G(iα)3 and G(iα)2 were identified but no G(oα) was found. To examine the influence of Epo on adenylate cyclase activity,day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP)production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Ca(c)] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutryl cAMP failed to increase [Ca(c)]. In summary, pertussis toxin blocks the increase in [Ca(c)] in erythroblasts after Epo stimulation, suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were G(iα) 1, 2, or 3, but not G(oα).

AB - Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Ca(c)]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Ca(c)], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Ca(c)] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Ca(c)] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat-inactivated PT. These observations provided strong evidence that a PT-sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of G(iα) (1,2, and 3) and G(oα). G(iα)1 or G(iα)3 and G(iα)2 were identified but no G(oα) was found. To examine the influence of Epo on adenylate cyclase activity,day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP)production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Ca(c)] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutryl cAMP failed to increase [Ca(c)]. In summary, pertussis toxin blocks the increase in [Ca(c)] in erythroblasts after Epo stimulation, suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were G(iα) 1, 2, or 3, but not G(oα).

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