Abstract
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.
Highlights
The molecular basis for store-operated Ca2+ entry (SOCE) has been extensively investigated since the discoveries of the endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule 1 (STIM1) and the Ca2+-selective channel proteins, Orai1–3, which are activated by association with oligomerized STIM1 [3,4,5]
Wt STIM1 is most commonly observed in sheets independently of Orai1, and store depletion increases the size of clusters, which remain localized (Figure 1B, top right panels)
Our study with live cells provides evidence for a conformational extension in the cytoplasmic segment of STIM1 that is stimulated by depletion of Ca2+ from ER stores
Summary
The molecular basis for store-operated Ca2+ entry (SOCE) has been extensively investigated since the discoveries of the ER Ca2+ sensor, STIM1 (and its homologue, STIM2; [1,2]) and the Ca2+-selective channel proteins, Orai, which are activated by association with oligomerized STIM1 [3,4,5]. Several groups found that expression of a portion of this cytoplasmic region as a soluble protein spontaneously activates SOCE [8,9,12,13,14] This minimal segment, commonly referred to as SOAR (human sequence 344–442) [13] or CAD (342–448; [14]) (Figure 1A), is not active when expressed as part of full length STIM1 in the absence of store depletion, suggesting that it is normally sequestered prior to STIM1 activation and oligomerization [12,14]. Human STIM1 mutation 4EA in the Cα3 helix of the CC1 domain was found to elicit STIM1 activation [8], and two subsequent fluorescence resonance energy transfer (FRET) studies on isolated cytoplasmic segments of STIM1 provided evidence that activating mutations in the CC1 domain of STIM1 (4EA and L251S) cause structural extensions, suggesting that these conformational changes are involved in this functional STIM1-Orai coupling [9,10]
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