J Biol Chem. 2009 Feb 13;284(7):4567-81. Epub 2008 Dec 13.

TRPC3 activation by erythropoietin is modulated by TRPC6.External

Hirschler-Laszkiewicz, I., Tong, Q., Conrad, K., Zhang, W., Flint, W. W., Barber, A. J., Barber, D. L., Cheung, J. Y., Miller, B. A.,
["Department of Pediatrics, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA."]
Regulation of intracellular calcium ([Ca(2+)](i)) by erythropoietin (Epo) is an essential part of signaling pathways controlling proliferation and differentiation of erythroid progenitors, but regulatory mechanisms are largely unknown. TRPC3 and the homologous TRPC6 are two members of the transient receptor potential channel (TRPC) superfamily that are expressed on normal human erythroid precursors. Here we show that TRPC3 expression increases but TRPC6 decreases during erythroid differentiation. This is associated with a significantly greater increase in [Ca(2+)](i) in response to Epo stimulation, suggesting that the ratio of TRPC3/TRPC6 is physiologically important. In HEK 293T cells heterologously expressing TRPC and erythropoietin receptor (Epo-R), Epo stimulated an increase in [Ca(2+)](i) through TRPC3 but not TRPC6. Replacement of the C terminus of TRPC3 with the TRPC6 C terminus (TRPC3-C6C) resulted in loss of activation by Epo. In contrast, substitution of the C terminus of TRPC6 with that of TRPC3 (TRPC6-C3C) resulted in an increase in [Ca(2+)](i) in response to Epo. Substitution of the N termini had no effect. Domains in the TRPC3 C terminus between amino acids 671 and 746 are critical for the response to Epo. Epo-R and phospholipase Cgamma associated with TRPC3, and these interactions were significantly reduced with TRPC6 and TRPC3-C6C chimeras. TRPC3 and TRPC6 form heterotetramers. Coexpression of TRPC6 or C3/C6 chimeras with TRPC3 and Epo-R inhibited the Epo-stimulated increase in [Ca(2+)](i). In a heterologous expression system, Epo stimulation increased cell surface expression of TRPC3, which was inhibited by TRPC6. However, in primary erythroblasts, an increase in TRPC3 cell surface expression was not observed in erythroblasts in which Epo stimulated an increase in [Ca(2+)](i), demonstrating that increased membrane insertion of TRPC3 is not required. These data demonstrate that TRPC6 regulates TRPC3 activation by Epo. Endogenously, regulation of TRPC3 by TRPC6 may primarily be through modulation of signaling mechanisms, including reduced interaction of TRPC6 with phospholipase Cgamma and Epo-R.
PMID: 19074769External
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Screening Toggle
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPC6 Link Epo-R Inference Prediction 19074769
TRPC6 Link PLCγ1 Inference Prediction 19074769
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Validation: In vivo validation Toggle
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPC3 Link Epo-R Co-immunoprecipitation HEK293T Human Full-length Human Full-length 19074769
TRPC3 Link Epo-R Co-immunoprecipitation TF-1 19074769
TRPC3 Link Epo-R Co-immunofluorescence staining HEK293T Human Full-length Human Full-length 19074769
TRPC3 Link PLCγ1 Co-immunoprecipitation HEK293T Human Full-length Rat Full-length 19074769
TRPC3 Link TRPC6 Co-immunoprecipitation HEK293T Human Full-length Human Full-length 19074769
TRPC3 Link TRPC6 Co-immunoprecipitation TF-1 19074769
TRPC6 Link Epo-R Co-immunoprecipitation HEK293T Human Full-length Human Full-length 19074769
TRPC6 Link PLCγ1 Co-immunoprecipitation HEK293T Human Full-length Rat Full-length 19074769
TRPC6 Link TRPC3 Co-immunoprecipitation HEK293T Human Full-length Human Full-length 19074769
TRPC6 Link TRPC3 Co-immunoprecipitation TF-1 19074769
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Characterization Toggle
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPC3 Link Epo-R Co-immunoprecipitation Human 671-746 Human Not determined 19074769
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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