Erythropoietin-modulated calcium influx through TRPC2 is mediated by phospholipase Cγ and IP3R

Qin Tong, Xin Chu, Joseph Y. Cheung, Kathleen Conrad, Richard Stahl, Dwayne L. Barber, Gregory Mignery, Barbara A. Miller

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


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 [Ca2+]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 Ca2+ 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 Ca2+ influx through TRPC2 was inhibited by pretreatment with U-73122 or by downregulation of PLCγ1 by RNA interference. PLC activation results in the production of inositol 1,4,5-trisphosphate (IP3), and TRPC2 has IP3 receptor (IP3R) binding sites. To determine whether IP3R is involved in Epo-R signaling, TRPC2 mutants were prepared with partial or complete deletions of the COOH-terminal IP3R binding domains. In cells expressing TRPC2 IP3R binding mutants and Epo-R, no significant increase in [Ca2+]i was observed after Epo stimulation. TRPC2 coassociated with Epo-R, PLCγ, and IP3R, and the association between TRPC2 and IP3R was disrupted in these mutants. Our data demonstrate that Epo-R modulates TRPC2 activation through PLCγ; that interaction of IP3R with TRPC2 is required; and that Epo-R, TRPC2, PLOγ, and IP3R interact to form a signaling complex.

Original languageEnglish (US)
Pages (from-to)C1667-C1678
JournalAmerican Journal of Physiology - Cell Physiology
Issue number6 56-6
StatePublished - Dec 2004

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology


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