The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin

Iwona Hirschler-Laszkiewicz, Qin Tong, Kathleen Waybill, Kathleen Conrad, Kerry Keefer, Wenyi Zhang, Shu-jen Chen, Joseph Y. Cheung, Barbara Miller

Research output: Contribution to journalArticle

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Abstract

Modulation of intracellular calcium ([Ca 2+] i) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normalhumanerythroid precursors, but Epo stimulates an increase in [Ca 2+] i through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca 2+] i. Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.

Original languageEnglish (US)
Pages (from-to)30636-30646
Number of pages11
JournalJournal of Biological Chemistry
Volume286
Issue number35
DOIs
StatePublished - Sep 2 2011

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Transient Receptor Potential Channels
AMP-Activated Protein Kinases
Erythropoietin
Protein Binding
Chemical activation
Binding Sites
Substitution reactions
Association reactions
Amino Acids
Leucine Zippers
Phosphorylation
Amino Acid Substitution
Cell membranes
Cytoskeleton
Cell Membrane
Modulation
Calcium

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{2f0320c7551643c687167fa9f3c419ed,
title = "The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin",
abstract = "Modulation of intracellular calcium ([Ca 2+] i) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normalhumanerythroid precursors, but Epo stimulates an increase in [Ca 2+] i through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca 2+] i. Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.",
author = "Iwona Hirschler-Laszkiewicz and Qin Tong and Kathleen Waybill and Kathleen Conrad and Kerry Keefer and Wenyi Zhang and Shu-jen Chen and Cheung, {Joseph Y.} and Barbara Miller",
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language = "English (US)",
volume = "286",
pages = "30636--30646",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
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The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin. / Hirschler-Laszkiewicz, Iwona; Tong, Qin; Waybill, Kathleen; Conrad, Kathleen; Keefer, Kerry; Zhang, Wenyi; Chen, Shu-jen; Cheung, Joseph Y.; Miller, Barbara.

In: Journal of Biological Chemistry, Vol. 286, No. 35, 02.09.2011, p. 30636-30646.

Research output: Contribution to journalArticle

TY - JOUR

T1 - The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin

AU - Hirschler-Laszkiewicz, Iwona

AU - Tong, Qin

AU - Waybill, Kathleen

AU - Conrad, Kathleen

AU - Keefer, Kerry

AU - Zhang, Wenyi

AU - Chen, Shu-jen

AU - Cheung, Joseph Y.

AU - Miller, Barbara

PY - 2011/9/2

Y1 - 2011/9/2

N2 - Modulation of intracellular calcium ([Ca 2+] i) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normalhumanerythroid precursors, but Epo stimulates an increase in [Ca 2+] i through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca 2+] i. Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.

AB - Modulation of intracellular calcium ([Ca 2+] i) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normalhumanerythroid precursors, but Epo stimulates an increase in [Ca 2+] i through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca 2+] i. Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.

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U2 - 10.1074/jbc.M111.238360

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JO - Journal of Biological Chemistry

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