STIM1的激活受CRAC激活域附近的14个氨基酸序列的调控。

IF 1.1 Q4 BIOPHYSICS AIMS Biophysics Pub Date : 2016-01-01 Epub Date: 2016-02-28 DOI:10.3934/biophy.2016.1.99
Marek K Korzeniowski, Barbara Baird, David Holowka
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引用次数: 8

摘要

内质网(ER)膜中Ca2+传感器STIM1的寡聚化,是由ER Ca2+存储的消耗引起的,导致STIM1偶联到质膜Ca2+通道蛋白Orai1,激活Ca2+内流,这一过程被称为存储操作的Ca2+进入。我们使用基于荧光学的荧光共振能量转移(FRET)来监测COS7细胞中STIM1寡聚化的变化,这些细胞转染了含有选定截断、缺失和点突变的STIM1构建体,并在管腔(n端)或细胞质(c端)端标记了供体和受体荧光蛋白。我们从c端连续截断STIM1的结果支持了先前的证据,即CRAC激活域(CAD/SOAR,人类序列342-448)是STIM1的寡聚促进片段,并且他们表明,在CAD/SOAR(1-448)之后的截断导致基底细胞质Ca2+显著升高和自发的STIM1聚集。我们发现CAD/SOAR(449-462)的c端有一个14个氨基酸的序列,可以阻止COS7细胞中STIM1的自发聚集和激活。为了应对存储枯竭,c末端标记了不含CAD/SOAR簇的STIM1和含有CAD/SOAR的STIM1构建体。然而,这些供体-受体对并没有经历FRET的刺激增加,而是表现出FRET的减少,这与全长STIM1中受刺激的构象延伸一致。我们发现14个氨基酸序列在这一过程中起调控作用。总的来说,我们的FRET结果在活细胞中提供了Ca2+存储耗尽刺激STIM1细胞质段的构象延伸并伴随其寡聚化的证据。
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STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain.

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.

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来源期刊
AIMS Biophysics
AIMS Biophysics BIOPHYSICS-
CiteScore
2.40
自引率
20.00%
发文量
16
审稿时长
8 weeks
期刊介绍: AIMS Biophysics is an international Open Access journal devoted to publishing peer-reviewed, high quality, original papers in the field of biophysics. We publish the following article types: original research articles, reviews, editorials, letters, and conference reports. AIMS Biophysics welcomes, but not limited to, the papers from the following topics: · Structural biology · Biophysical technology · Bioenergetics · Membrane biophysics · Cellular Biophysics · Electrophysiology · Neuro-Biophysics · Biomechanics · Systems biology
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