PDF(Pubmed)
Abstract：
Recently, we demonstrated that agonist-stimulated Ca2+ signaling involving IP3 receptors modulates ER export rates through activation of the penta-EF Hand proteins apoptosis-linked gene-2 (ALG-2) and peflin. It is unknown, however, whether IP3Rs and penta-EF proteins regulate ER export rates at steady state. Here we tested this idea in normal rat kidney epithelial cells by manipulation of IP3R isoform expression. Under standard growth conditions, spontaneous cytosolic Ca2+ oscillations occurred simultaneously in successive groups of contiguous cells, generating intercellular Ca2+ waves that moved across the monolayer periodically. Depletion of IP3R-3, typically the least promiscuous IP3R isoform, caused increased cell participation in intercellular Ca2+ waves in unstimulated cells. The increased spontaneous signaling was sufficient to cause increased ALG-2 and COPII coat subunit Sec31A and decreased peflin localization at ER exit sites, resulting in increased ER-to-Golgi transport of the COPII client cargo VSV-G. The elevated ER-to-Golgi transport caused greater concentration of VSV-G at ER exit sites and had reciprocal effects on transport of VSV-G and a bulk-flow cargo, though both cargos equally required Sec31A. Inactivation of client cargo sorting using 4-phenylbutyrate had opposing reciprocal effects on client and bulk-flow cargo and neutralized any effect of ALG-2 activation on transport. This work extends our knowledge of ALG-2 mechanisms and indicates that in normal rat kidney cells, IP3R isoforms regulate homeostatic Ca2+ signaling that helps determine the basal secretion rate and stringency of COPII-dependent cargo sorting.
摘要：
最近,我们证明，涉及IP3受体的激动剂刺激的Ca2信号传导通过激活penta-EFHand（PEF）蛋白凋亡相关基因2（ALG-2）和peflin来调节ER输出率。它是未知的，然而,IP3Rs和PEF蛋白是否在稳定状态下调节内质网出口率。在这里，我们通过操纵IP3R同工型表达在正常大鼠肾脏（NRK）上皮细胞中测试了这一想法。在标准生长条件下，在连续的连续细胞组中同时发生自发的胞浆Ca2振荡，产生周期性跨单层移动的细胞间Ca2波（ICW）。IP3R-3的消耗，通常是最不混杂的IP3R同工型，导致未刺激细胞中ICW的细胞参与增加。增加的自发信号足以引起增加的ALG-2和COPII包被亚基Sec31A，并减少急诊室出口(ERES)的PEFLIN定位，导致COPII客户货物VSV-G的ER到高尔基运输增加。ER到高尔基的转运增加导致VSV-G在ERES的浓度增加，并对VSV-G和散装货物的运输产生相互影响，虽然两种货物同样需要Sec31A。使用4-苯基丁酸酯（4-PBA）灭活客户货物分拣对客户和散装货物具有相反的相互影响，并且中和了ALG-2激活对运输的任何影响。这项工作扩展了我们对ALG-2机制的认识，并表明在NRK细胞中，IP3R同工型调节稳态Ca2信号，有助于确定COPII依赖性货物分选的基础分泌率和严格性。
