Abstract
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.
Highlights
Regulation of intracellular calcium by erythropoietin is a signaling mechanism controlling the proliferation and differentiation of erythroid progenitors and precursors (1– 4)
TRPC3 is the first human transient receptor potential (TRP) channel shown to be regulated by a hematopoietic growth factor, erythropoietin (26), the homologous TRPC6 does not respond to Epo stimulation (3)
Our first finding is that TRPC3 expression increases and TRPC6 expression decreases during erythroid differentiation as cells mature from CD34ϩ cells to erythrocytes, resulting in a significant increase in the TRPC3/TRPC6 ratio (Fig. 1). [Ca2ϩ]i increased in response to Epo stimulation as the TRPC3/TRPC6 ratio increased, until the late stages of erythroid differentiation when erythropoietin receptor (Epo-R) expression was reduced
Summary
Erythropoietin; Epo-R, erythropoietin receptor; PLC␥, phospholipase C␥; [Ca2ϩ]i, intracellular calcium; TRP, transient receptor potential; TRPC, transient receptor potential channel; BFU-E, burst-forming unit-erythroid; HRP, horseradish peroxidase; IP3R, inositol 1,4,5-trisphosphate receptor; FRAP, fluorescence recovery after photobleaching; PBS, phosphate-buffered saline; h, human; CMV, cytomegalovirus; GFP, green fluorescent protein; ECL, enhanced chemiluminescence; BFP, blue fluorescent protein. The transient receptor potential (TRP) channel TRPC3 was recently shown to be expressed on primary human erythroid cells and is an important calcium channel regulated by Epo (26). We recently demonstrated that the Epo-stimulated increase in [Ca2ϩ]i through TRPC3 originated primarily from extracellular calcium influx, is mediated through PLC␥, and required interaction of PLC␥ and IP3R with TRPC3 (26). These studies revealed that domains in the TRPC6 C terminus are responsible for the lack of response to Epo and that the TRPC6 C terminus has much weaker interactions with PLC␥ and Epo-R than does TRPC3 These data demonstrate that TRPC6 has a role in regulating TRPC3 function in response to Epo. Epo stimulation enhanced heterologous TRPC3 insertion into the plasma membrane, and this was inhibited by TRPC6, a significant increase in Epo-stimulated cell surface expression of endogenous TRPC3 was not required for channel activation by Epo
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
