STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain.
STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain.
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DOI：
10.3934/biophy.2016.1.99
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发表时间：
2016
期刊：
影响因子：
1.5
通讯作者：
Holowka D
中科院分区：
文献类型：
--
作者：
Korzeniowski MK;Baird B;Holowka D
内质网（ER）膜中的Ca 2+传感器STIM 1的寡聚化由ER Ca 2+储存的耗尽引起，导致STIM 1与质膜Ca 2+通道蛋白Orai 1偶联，以在称为储存操作的Ca 2+进入的过程中激活Ca 2+内流。我们使用基于荧光的荧光共振能量转移（FRET）来监测STIM 1寡聚化的COS 7细胞中的STIM 1寡聚化的变化，该COS 7细胞用含有选定的截短、缺失和点突变的STIM 1构建体转染，并在管腔（N-末端）或细胞质（C-末端）末端用供体和受体荧光蛋白标记。我们从C末端连续截短STIM 1的结果支持先前的证据，即CRAC激活结构域（CAD/SOAR，人类序列342-448）是STIM 1的寡聚体促进片段，并且它们表明，在CAD/SOAR（1-448）之后的截短导致基底细胞质Ca 2+显著升高和自发的STIM 1聚集。我们发现CAD/SOAR（449-462）C末端的14个氨基酸序列阻止了COS 7细胞中STIM 1的自发聚集和激活。响应于存储耗尽，C-末端标记的STIM 1没有CAD/SOAR集群与CAD/SOAR含有STIM 1结构。然而，这些供体-受体对不经历FRET的刺激增加，而是表现出FRET的减少，这与全长STIM 1中的刺激构象延伸一致。我们发现14个氨基酸序列在这个过程中起着调节作用。总的来说，我们的FRET结果提供的证据表明，在活细胞中，Ca 2+存储耗尽刺激构象延伸的细胞质段的STIM 1，伴随其寡聚化。
Oligomerization of the Ca2+ sensor, STIM1, in the endoplasmic reticulum (ER) membrane, caused by depletion of ER Ca2+ stores, results in STIM1 coupling to the plasma membrane Ca2+ channel protein, Orai1, to activate Ca2+ influx in a process known as store-operated Ca2+ entry. We use fluorimetry-based fluorescence resonance energy transfer (FRET) to monitor changes in STIM1 oligomerization in COS7 cells transfected with STIM1 constructs containing selected truncations, deletions, and point mutations, and labeled with donor and acceptor fluorescent proteins at either the luminal (N-terminal) or the cytoplasmic (C-terminal) ends. Our results with sequential truncations of STIM1 from the C-terminus support previous evidence that the CRAC activation domain (CAD/SOAR, human sequence 342–448) is an oligomer-promoting segment of STIM1, and they show that truncation just after CAD/SOAR (1–448) causes significantly elevated basal cytoplasmic Ca2+ and spontaneous STIM1 clustering. We find that a 14 amino acid sequence just C-terminal of CAD/SOAR (449–462) prevents spontaneous clustering and activation of STIM1 in COS7 cells. In response to store depletion, C-terminally labeled STIM1 without CAD/SOAR clusters together with CAD/SOAR-containing STIM1 constructs. However, these donor-acceptor pairs do not undergo a stimulated increase in FRET, exhibiting instead a decrease in FRET consistent with a stimulated conformational extension in full length STIM1. We find that the 14 amino acid sequence plays a regulatory role in this process. Overall, our FRET results provide evidence in live cells that Ca2+ store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.