Location and function of STIM1 in the activation of Ca2+ entry signals
Location and function of STIM1 in the activation of Ca2+ entry signals
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DOI：
10.1074/jbc.m802239200
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发表时间：
2008-09-19
影响因子：
4.8
通讯作者：
Gill, Donald L.
中科院分区：
文献类型：
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作者：
Hewavitharana, Thamara;Deng, Xiaoxiang;Gill, Donald L.
大多数细胞中，存储操纵通道 (SOC) 会介导 Ca2+ 进入信号，以响应内质网 (ER) Ca2+ 耗尽。 STIM1 通过其 EF 手 Ca2+ 结合基序感知内质网腔内 Ca2+ 的减少，并在近质膜 (PM) 内质网连接处聚集，从而激活功能性 SOC PM Orai1。 STIM1 也存在于 PM 中，但其作用尚不清楚。使用表达缺乏 Ca2+ 结合的 STIM1-D76A/E87A EF-hand 突变体 (STIM1(EF)) 的稳定 EF20 HEK293 细胞系检查 STIM1 介导的偶联。尽管有 Ca2+ 的组成性进入，EF20 细胞仍能存活，因此可以在不消耗 ER Ca2+ 的情况下研究 SOC 激活。 STIM1(EF)仅存在于稳定的近PM连接中，比STIM1(WT)形成的连接大3.5倍。表达 STIMEF 的细胞具有正常的 ER Ca2+ 水平，但 ER Ca2+ 泄漏显着减少。 EF20 细胞中抗凋亡 Bcl-2 蛋白（BCl-2、MCL-1、BCL-XL）的表达增加了 2 倍，可能反映了 EF20 细胞的存活，但不能解释 ER Ca2+ 渗漏减少。表达 STIM1(WT) 或 STIM1(EF) 的细胞的表面生物素化和链霉亲和素下拉揭示了两种蛋白质的强烈 PM 相互作用。虽然可以清楚地检测到 STIM1(WT) 的表面表达，但在细胞表面检测不到 STIM1(EF)。因此，Ca2+结合缺陷型STIM1(EF)突变体仅存在于近PM连接内的聚集体中，但与STIM1WT不同，它不会被运输到PM。尽管未插入 PM，但外部应用单克隆抗 N 端 STIM1 抗体可阻断 STIMEF 介导的 Ca2+ 组成型进入，但仅限于表达内源性 STIM1(WT) 的细胞，而不是缺乏 STIMWT 的 DT40 STIM1 敲除细胞。这表明 PM-STIM1 可能在 SOC 激活中发挥调节作用。
Store-operated channels (SOCs) mediate Ca2+ entry signals in response to endoplasmic reticulum (ER) Ca2+ depletion in most cells. STIM1 senses decreased ER luminal Ca2+ through its EF-hand Ca2+-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca2+ binding. EF20 cells were viable despite constitutive Ca2+ entry, allowing study of SOC activation without depleting ER Ca2+. STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIMEF-expressing cells had normal ER Ca2+ levels but substantially reduced ER Ca2+ leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca2+ leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca2+ binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIMEF-mediated Ca2+ entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIMWT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.