Hirschler-Laszkiewicz Iwona, Tong Qin, Conrad Kathleen, Zhang Wenyi, Flint Wesley W, Barber Alistair J, Barber Dwayne L, Cheung Joseph Y, Miller Barbara A
Department of Pediatrics, the Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
J Biol Chem. 2009 Feb 13;284(7):4567-81. doi: 10.1074/jbc.M804734200. Epub 2008 Dec 13.
Regulation of intracellular calcium (Ca(2+)) 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+) 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+) 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+) 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+). 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+), 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.
促红细胞生成素（Epo）对细胞内钙（Ca(2+)）的调节是控制红系祖细胞增殖和分化信号通路的重要组成部分，但调节机制尚不清楚。瞬时受体电位通道（TRPC）超家族的两个成员TRPC3和同源的TRPC6在正常人类红系前体细胞中表达。在此我们发现，在红系分化过程中TRPC3表达增加而TRPC6表达减少。这与Epo刺激后Ca(2+)显著更大幅度的增加相关，表明TRPC3/TRPC6的比例具有重要生理意义。在异源表达TRPC和促红细胞生成素受体（Epo-R）的HEK 293T细胞中，Epo通过TRPC3而非TRPC6刺激Ca(2+)增加。用TRPC6的C末端替换TRPC3的C末端（TRPC3-C6C）导致Epo无法激活。相反，用TRPC3的C末端替换TRPC6的C末端（TRPC6-C3C）导致Epo刺激后Ca(2+)增加。替换N末端没有影响。TRPC3 C末端671至746位氨基酸之间的结构域对Epo反应至关重要。Epo-R和磷脂酶Cγ与TRPC3相关联，而这些相互作用在TRPC6和TRPC3-C6C嵌合体中显著减少。TRPC3和TRPC6形成异源四聚体。TRPC6或C3/C6嵌合体与TRPC3和Epo-R共表达可抑制Epo刺激的Ca(2+)增加。在异源表达系统中，Epo刺激可增加TRPC3的细胞表面表达，而这被TRPC6抑制。然而，在原代成红细胞中，在Epo刺激Ca(2+)增加的成红细胞中未观察到TRPC3细胞表面表达增加，表明TRPC3增加的膜插入并非必需。这些数据表明TRPC6调节Epo对TRPC3的激活。内源性地，TRPC6对TRPC3的调节可能主要通过调节信号机制，包括减少TRPC6与磷脂酶Cγ和Epo-R的相互作用。
