Erythropoietin-modulated calcium influx through TRPC2 is mediated by phospholipase Cγ and IP 3 R

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 PLCγ1 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, PLCγ, 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 PLCγ; that interaction of IP 3 R with TRPC2 is required; and that Epo-R, TRPC2, PLCγ, and IP 3 R interact to form a signaling complex.