Korzeniowski Marek K, Baird Barbara, Holowka David
Department of Chemistry and Chemical Biology Cornell University, Ithaca, NY 14853.
AIMS Biophys. 2016;3(1):99-118. doi: 10.3934/biophy.2016.1.99. Epub 2016 Feb 28.
Oligomerization of the Ca sensor, STIM1, in the endoplasmic reticulum (ER) membrane, caused by depletion of ER Ca stores, results in STIM1 coupling to the plasma membrane Ca channel protein, Orai1, to activate Ca influx in a process known as store-operated Ca 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 Ca 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 Ca store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.
内质网（ER）钙库耗竭导致内质网膜中钙传感器STIM1寡聚化，使得STIM1与质膜钙通道蛋白Orai1偶联，从而在一个称为钙库操纵性钙内流的过程中激活钙内流。我们使用基于荧光测定法的荧光共振能量转移（FRET）来监测在转染了含有选定截断、缺失和点突变的STIM1构建体的COS7细胞中STIM1寡聚化的变化，这些构建体在腔面（N端）或胞质（C端）末端用供体和受体荧光蛋白进行标记。我们从C端对STIM1进行连续截断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的一个促进寡聚化的片段，并且结果表明在CAD/SOAR（1 - 448）之后立即截断会导致基础胞质钙显著升高以及STIM1自发聚集。我们发现CAD/SOAR（449 - 462）C端的一个14个氨基酸的序列可防止COS7细胞中STIM1的自发聚集和激活。响应钙库耗竭时，不含CAD/SOAR的C端标记的STIM1与含CAD/SOAR的STIM1构建体聚集在一起。然而，这些供体 - 受体对的FRET并没有受到刺激而增加，反而表现出FRET降低，这与全长STIM1中受刺激的构象延伸一致。我们发现这个14个氨基酸的序列在此过程中起调节作用。总体而言，我们的FRET结果为活细胞中钙库耗竭刺激STIM1胞质段在寡聚化过程中发生构象延伸提供了证据。
