TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells

Qin Tong, Iwona Hirschler-Laszkiewicz, Wenyi Zhang, Kathleen Conrad, David W. Neagley, Dwayne L. Barber, Joseph Y. Cheung, Barbara A. Miller

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca2+]i) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca 2+]i, which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCγ1 by RNA interference inhibited the Epo-stimulated increase in [Ca2+]i in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCγ, and substitution of predicted PLCγ Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCγ and inhibited the rise in [Ca 2+]i. Substitution of Tyr226 alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca 2+]i but not the association of PLCγ with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP 3). To determine whether IP3 is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP3 receptor (IP3R) binding domains. In cells expressing TRPC3 with mutant IP3R binding sites and Epo receptor, interaction of IP3R with TRPC3 was abolished, and Epo-modulated increase in [Ca2+]i was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCγ-mediated process that requires interaction of PLCγ and IP3R with TRPC3. They also show that TRPC3 Tyr226 is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.

Original languageEnglish (US)
Pages (from-to)10385-10395
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number16
DOIs
StatePublished - Apr 18 2008

Fingerprint

Erythroid Cells
Type C Phospholipases
Calcium Channels
Erythropoietin
Inositol 1,4,5-Trisphosphate Receptors
Chemical activation
Erythropoietin Receptors
Substitution reactions
HEK293 Cells
Calcium
Binding Sites
Transient Receptor Potential Channels
Pseudogenes
Inositol 1,4,5-Trisphosphate
RNA Interference
Phenylalanine
Redundancy
Down-Regulation
Cells
Association reactions

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Tong, Qin ; Hirschler-Laszkiewicz, Iwona ; Zhang, Wenyi ; Conrad, Kathleen ; Neagley, David W. ; Barber, Dwayne L. ; Cheung, Joseph Y. ; Miller, Barbara A. / TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 16. pp. 10385-10395.
@article{6c3ed7e90e034c8e97acbc1e78cb8068,
title = "TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells",
abstract = "Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca2+]i) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca 2+]i, which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCγ1 by RNA interference inhibited the Epo-stimulated increase in [Ca2+]i in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCγ, and substitution of predicted PLCγ Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCγ and inhibited the rise in [Ca 2+]i. Substitution of Tyr226 alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca 2+]i but not the association of PLCγ with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP 3). To determine whether IP3 is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP3 receptor (IP3R) binding domains. In cells expressing TRPC3 with mutant IP3R binding sites and Epo receptor, interaction of IP3R with TRPC3 was abolished, and Epo-modulated increase in [Ca2+]i was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCγ-mediated process that requires interaction of PLCγ and IP3R with TRPC3. They also show that TRPC3 Tyr226 is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.",
author = "Qin Tong and Iwona Hirschler-Laszkiewicz and Wenyi Zhang and Kathleen Conrad and Neagley, {David W.} and Barber, {Dwayne L.} and Cheung, {Joseph Y.} and Miller, {Barbara A.}",
year = "2008",
month = "4",
day = "18",
doi = "10.1074/jbc.M710231200",
language = "English (US)",
volume = "283",
pages = "10385--10395",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "16",

}

TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells. / Tong, Qin; Hirschler-Laszkiewicz, Iwona; Zhang, Wenyi; Conrad, Kathleen; Neagley, David W.; Barber, Dwayne L.; Cheung, Joseph Y.; Miller, Barbara A.

In: Journal of Biological Chemistry, Vol. 283, No. 16, 18.04.2008, p. 10385-10395.

Research output: Contribution to journalArticle

TY - JOUR

T1 - TRPC3 is the erythropoietin-regulated calcium channel in human erythroid cells

AU - Tong, Qin

AU - Hirschler-Laszkiewicz, Iwona

AU - Zhang, Wenyi

AU - Conrad, Kathleen

AU - Neagley, David W.

AU - Barber, Dwayne L.

AU - Cheung, Joseph Y.

AU - Miller, Barbara A.

PY - 2008/4/18

Y1 - 2008/4/18

N2 - Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca2+]i) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca 2+]i, which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCγ1 by RNA interference inhibited the Epo-stimulated increase in [Ca2+]i in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCγ, and substitution of predicted PLCγ Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCγ and inhibited the rise in [Ca 2+]i. Substitution of Tyr226 alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca 2+]i but not the association of PLCγ with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP 3). To determine whether IP3 is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP3 receptor (IP3R) binding domains. In cells expressing TRPC3 with mutant IP3R binding sites and Epo receptor, interaction of IP3R with TRPC3 was abolished, and Epo-modulated increase in [Ca2+]i was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCγ-mediated process that requires interaction of PLCγ and IP3R with TRPC3. They also show that TRPC3 Tyr226 is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.

AB - Erythropoietin (Epo) stimulates a significant increase in the intracellular calcium concentration ([Ca2+]i) through activation of the murine transient receptor potential channel TRPC2, but TRPC2 is a pseudogene in humans. TRPC3 expression increases on normal human erythroid progenitors during differentiation. Here, we determined that erythropoietin regulates calcium influx through TRPC3. Epo stimulation of HEK 293T cells transfected with Epo receptor and TRPC3 resulted in a dose-dependent increase in [Ca 2+]i, which required extracellular calcium influx. Treatment with the phospholipase C (PLC) inhibitor U-73122 or down-regulation of PLCγ1 by RNA interference inhibited the Epo-stimulated increase in [Ca2+]i in TRPC3-transfected HEK 293T cells and in primary human erythroid precursors, demonstrating a requirement for PLC. TRPC3 associated with PLCγ, and substitution of predicted PLCγ Src homology 2 binding sites (Y226F, Y555F, Y648F, and Y674F) on TRPC3 reduced the interaction of TRPC3 with PLCγ and inhibited the rise in [Ca 2+]i. Substitution of Tyr226 alone with phenylalanine significantly reduced the Epo-stimulated increase in [Ca 2+]i but not the association of PLCγ with TRPC3. PLC activation results in production of inositol 1,4,5-trisphosphate (IP 3). To determine whether IP3 is involved in Epo activation of TRPC3, TRPC3 mutants were prepared with substitution or deletion of COOH-terminal IP3 receptor (IP3R) binding domains. In cells expressing TRPC3 with mutant IP3R binding sites and Epo receptor, interaction of IP3R with TRPC3 was abolished, and Epo-modulated increase in [Ca2+]i was reduced. Our data demonstrate that Epo modulates TRPC3 activation through a PLCγ-mediated process that requires interaction of PLCγ and IP3R with TRPC3. They also show that TRPC3 Tyr226 is critical in Epo-dependent activation of TRPC3. These data demonstrate a redundancy of TRPC channel activation mechanisms by widely different agonists.

UR - http://www.scopus.com/inward/record.url?scp=44849083569&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=44849083569&partnerID=8YFLogxK

U2 - 10.1074/jbc.M710231200

DO - 10.1074/jbc.M710231200

M3 - Article

C2 - 18276585

AN - SCOPUS:44849083569

VL - 283

SP - 10385

EP - 10395

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 16

ER -