The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3
The regulatory domain of the inositol 1,4,5-trisphosphate receptor is necessary to keep the channel domain closed: possible physiological significance of specific cleavage by caspase 3
复制标题
DOI：
10.1042/bj20030599
复制
发表时间：
2004-01-15
影响因子：
4.1
通讯作者：
Mikoshiba, K
中科院分区：
文献类型：
--
作者：
Nakayama, T;Hattori, M;Mikoshiba, K
1，4，5-三磷酸莫西醇受体(IP(3)R1)是一种细胞内钙通道蛋白，在产生复杂的钙信号模式中起着关键作用。IP(3)R1由三个结构域组成：配体结合域、调节域和通道域。为了研究这些结构域在其门控机制中的功能，以及在细胞凋亡过程中观察到的caspase 3特异性切割的生理意义，我们利用了不同的绿色荧光蛋白(GFP)标记的IP(3)R1结构。GFP标记的全长IP(3)R1或IP(3)R1在HeLa中缺失配体结合区的表达而COS-7细胞对thapsigargin诱导的IP3生成激动剂ATP和Ca~(2+)泄漏的反应几乎没有影响。另一方面，在表达caspase-3裂解形式(GFP-IP(3)R1-Casp)或单独表达通道结构域(GFP-IP(3)R1-ES)的细胞中，ATP和thapsigargin都不能诱导细胞内钙离子浓度的增加。有趣的是，在这些细胞中，通常可以观察到商店操作的(类)钙离子进入，而与thapsigargin预处理无关。这些发现表明，在静息状态下，表达GFP-IP(3)R1-Casp或GFP-IP(3)R1-ES的细胞的钙储备库几乎是空的，并且这些蛋白不断地释放钙。因此，我们认为IP(3)R1的通道结构域倾向于保持开放，并且IP(3)R1的大调节域对于保持通道结构域的关闭是必要的。因此，半胱氨酸天冬氨酸蛋白酶3对IP(3)R1的切割可能是导致细胞内钙离子浓度升高的原因之一。最后，GFP-IP(3)R1-CASP或GFP-IP(3)R1-ES可以作为一种新的耗竭细胞内钙储存的工具。
The type 1 mositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca2+ channel protein that plays crucial roles in generating complex Ca2+ signalling patterns. Ip(3)R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa. and COS-7 cells had little effect on cells' responsiveness to an IP3-generating agonist ATP and Ca2+ leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca2+ concentration. Interestingly, store-operated (-like) Ca2+ entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca2+ stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca2+. We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca2+ concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca2+ stores.