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.