Abstract
Ca2+ release-activated Ca2+ (CRAC) channels elevate cytoplasmic Ca2+ concentration, which is essential for T cell activation, differentiation and effector functions. T cell receptor stimulation induces depletion of the endoplasmic reticulum (ER) Ca2+ stores, which is sensed by stromal interaction molecule 1 (STIM1). STIM1 translocates to the ER-plasma membrane (PM) junctions to interact with ORAI1, the pore subunit of the CRAC channels. Here, we show that two members of the extended synaptotagmin (E-Syt) family, E-Syt1, and the short isoform of E-Syt2 (E-Syt2S), contribute to activation of CRAC channels in T cells. Knockdown or deletion of both ESYT1 and ESYT2 reduced store-operated Ca2+ entry (SOCE) and ORAI1-STIM1 clustering in Jurkat T cells. Further, depletion of E-Syts in primary T cells decreased Ca2+ entry and cytokine production. While the ER-PM junctions were reduced in both HeLa and Jurkat T cells deleted for ESYT1 and ESYT2, SOCE was impaired only in Jurkat T cells, suggesting that the membrane-tethering function of E-Syts is distinct from their role in SOCE. Mechanistically, E-Syt2S, the predominant isoform of E-Syt2 in T cells, recruited STIM1 to the junctions via a direct interaction. This study demonstrates a membrane-tethering-independent role of E-Syts in activation of CRAC channels in T cells.
Highlights
Ca2+ release-activated Ca2+ (CRAC) channels mediate a sustained increase in cytoplasmic Ca2+ concentration that is essential for T cell activation
To identify components of the endoplasmic reticulum (ER)-plasma membrane (PM) junctions where ORAI1 and stromal interaction molecule 1 (STIM1) interact, we carried out large-scale protein affinity purification using cells stably expressing low levels of FLAG-tagged STIM1 (Fig. 1A)
We found that extended synaptotagmin (E-Syt), especially E-Syt2S, the short isoform of E-Syt[2], play a direct role in the recruitment of STIM1 to the junctions via protein interaction, and regulate store-operated Ca2+ entry (SOCE) in T cells
Summary
Ca2+ release-activated Ca2+ (CRAC) channels mediate a sustained increase in cytoplasmic Ca2+ concentration that is essential for T cell activation. Junctate-junctophilin complex is localized at the ER-PM junctions, and after sensing ER C a2+ depletion via its ER-luminal C a2+-binding motif, junctate recruits STIM1 into the junctions Other molecules such as TMEM110 (alternatively, STIM-activating enhancer) and SARAF (SOCE-associated regulatory factor) have been identified as components of the ER-PM junctions in T c ells[8,9,10]. We show that E-Syt[1] and a short isoform of E-Syt[2], E-Syt2S, have a compensatory role in CRAC channel activation in T cells In addition to their conserved membrane-tethering function, E-Syt[1] and E-Syt2S affect Ca2+ signaling in T cells by regulating the recruitment of STIM1 to the ER-PM junctions. Our results show that the ER-PM junctions in T cells consist of unique members (i.e., E-Syt2S) that play specialized roles in regulation of Ca2+ signaling
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