Am J Physiol Cell Physiol. 2004 Dec;287(6):C1667-78. Epub 2004 Aug 25.

Erythropoietin-modulated calcium influx through TRPC2 is mediated by phospholipase Cgamma and IP3R.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Tong, Q., Chu, X., Cheung, J. Y., Conrad, K., Stahl, R., Barber, D. L., Mignery, G., Miller, B. A.,
["Department of Pediatrics, Milton S. Hershey Medical Center, PO Box 850, Hershey, PA 17033, USA."]
In the present study, we examined the mechanisms through which erythropoietin (Epo) activates the calcium-permeable transient receptor potential protein channel (TRPC)2. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in intracellular calcium concentration ([Ca(2+)](i)). This increase in [Ca(2+)](i) was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U-73122 but not by the inactive analog U-73343, demonstrating the requirement for PLC activity in Epo-modulated Ca(2+) influx in primary erythroid cells. To determine whether PLC is involved in the activation of TRPC2 by Epo, cell models were used to examine this interaction. Single CHO-S cells that expressed transfected Epo receptor (Epo-R) and TRPC2 were identified, and [Ca(2+)](i) was quantitated. Epo-induced Ca(2+) influx through TRPC2 was inhibited by pretreatment with U-73122 or by downregulation of PLCgamma1 by RNA interference. PLC activation results in the production of inositol 1,4,5-trisphosphate (IP(3)), and TRPC2 has IP(3) receptor (IP(3)R) binding sites. To determine whether IP(3)R is involved in Epo-R signaling, TRPC2 mutants were prepared with partial or complete deletions of the COOH-terminal IP(3)R binding domains. In cells expressing TRPC2 IP(3)R binding mutants and Epo-R, no significant increase in [Ca(2+)](i) was observed after Epo stimulation. TRPC2 coassociated with Epo-R, PLCgamma, and IP(3)R, and the association between TRPC2 and IP(3)R was disrupted in these mutants. Our data demonstrate that Epo-R modulates TRPC2 activation through PLCgamma; that interaction of IP(3)R with TRPC2 is required; and that Epo-R, TRPC2, PLCgamma, and IP(3)R interact to form a signaling complex.
PMID: 15329338External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Epo-R Inference Prediction 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 IP3R2 Inference Prediction 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PLCƣ1 Inference Prediction 15329338
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  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
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Epo-R Co-immunoprecipitation CHO-S Mouse Full-length Mouse Full-length 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Epo-R Co-immunoprecipitation Mouse erythroblasts 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 IP3R2 Co-immunofluorescence staining Mouse splenic erythroblast 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 IP3R2 Co-immunoprecipitation CHO-S Mouse Full-length Rat Full-length 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PLCƣ1 Co-immunoprecipitation CHO-S Mouse Full-length Rat Full-length 15329338
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PLCƣ1 Co-immunoprecipitation Mouse erythroblasts 15329338
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPC2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 IP3R2 Co-immunoprecipitation Mouse 1001-1036 Rat Not determined 15329338
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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