Erythropoietin-modulated calcium influx through TRPC2 is mediated by phospholipase Cgamma and IP3R.
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.
Department of Pediatrics, Milton S. Hershey Medical Center, PO Box 850, Hershey, PA 17033, USA.
Qin
Q
Tong
Xin
X
Chu
Joseph Y
JY
Cheung
Kathleen
K
Conrad
Richard
R
Stahl
Dwayne L
DL
Barber
Gregory
G
Mignery
Barbara A
BA
Miller
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000378:metabolism*; D006224:Cricetinae; D004900:Erythroblasts / Q000166:cytology / Q000502:physiology; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D051966:Phospholipase C gamma; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D050052:TRPC Cation Channels; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D017467:Receptors, Erythropoietin; D050052:TRPC Cation Channels; C491858:Trpc2 protein, mouse; D004921:Erythropoietin; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium