A Cytosolic STIM2 Preprotein Created by Signal Peptide Inefficiency Activates ORAI1 in a Store-independent Manner

Abstract

Calcium (Ca 2؉ ) influx through the plasma membrane storeoperated Ca 2؉ channel ORAI1 is controlled by Ca 2؉ sensors of the stromal interaction molecule (STIM) family. STIM1 responds to endoplasmic reticulum (ER) Ca 2؉ store depletion by redistributing and activating ORAI1 from regions of the ER juxtaposed to the plasma membrane. Unlike STIM1, STIM2 can regulate ORAI1 in a store-dependent and store-independent manner, but the mechanism by which this is achieved is unknown. Here we find that STIM2 is translated from a highly conserved methionine residue and is directed to the ER by an incredibly long 101-amino acid signal peptide. We find that although the majority of the total STIM2 population resides on the ER membrane, a second population escapes ER targeting to accumulate as a full-length preprotein in the cytosol, signal peptide intact. Unlike STIM2, preSTIM2 localizes to the inner leaflet of the plasma membrane where it interacts with ORAI1 to regulate basal Ca 2؉ concentration and Ca 2؉ -dependent gene transcription in a store-independent manner. Furthermore, a third protein comprising a fragment of the STIM2 signal peptide is released from the ER membrane into the cytosol where it regulates gene transcription in a Ca 2؉ -independent manner. This study establishes a new model for STIM2-mediated regulation of ORAI1 in which two distinct proteins, STIM2 and preSTIM2, control store-dependent and store-independent modes of ORAI1 activation.Calcium (Ca 2ϩ ) entry through store-operated Ca 2ϩ channels (SOCs) 2 is essential for gene transcription-induced regulation of cell differentiation and function (1). Stromal interaction molecule 1 (STIM1) plays a critical role in activating the plasma membrane (PM)-localized SOCs of the ORAI family (2-4). Localized predominantly on the endoplasmic reticulum (ER), STIM1 spans the membrane with the N terminus confined to the ER lumen and the longer C terminus facing the cytosol (5). Control over STIM1 activation is mediated by ER Ca 2ϩ store depletion; dissociation of Ca 2ϩ from the N-terminal EF hand Ca 2ϩ binding domain induces oligomerization, which drives STIM1 to precise locations on the ER juxtaposed to the PM (6). From there the cytosolic C terminus recruits ORAI1 by uncovering an integral activation domain (CRAC activation domain (CAD), also known as SOAR or OASF), which is also essential for channel gating (7-10). Ca 2ϩ influx is terminated by store refilling, which returns STIM1 to its resting conformation (11). The essential involvement of STIM1 in regulating the activity of SOCs has been demonstrated by experimental manipulation of STIM1 levels; depletion of STIM1 abrogates store-operated Ca 2ϩ entry (SOCe), overexpression increases SOCe (1, 12), and co-overexpression of STIM1 and ORAI1 results in massive Ca 2ϩ influx that is almost entirely controlled by ER store depletion (4).In contrast to STIM1, the function of its closely related homolog, STIM2 (13), is enigmatic. Despite almost complete structural conservation between STIM homologs, STIM2 depletion has littl...

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“…Immunolocalization revealed that cytosolic pre-STIM2 localizes to the plasma membrane, where it interacts with ORAI1, increasing basal Ca 2ϩ concentration in a manner that is independent of ER Ca 2ϩ store depletion (30). The increase in basal Ca 2ϩ concentration by pre-STIM2 results in the induction of NFAT and nuclear factor-B (NF-B)-mediated gene transcription (30). Further to this, a third population, which is actually a fragment of the STIM2 signal peptide, upon its release from the ER membrane into the cytoplasm, becomes a regulator of gene transcription in a Ca 2ϩ independent fashion (30).…”

“…The complexity of ORAI channel regulation is further highlighted by the ability of STIM2 to activate ORAI1 in a store-independent manner (30). STIM2 has now been reported to exist in distinct functional forms: the widely investigated STIM2 and another population, referred to as pre-STIM2, that is able to escape ER targeting (30).…”

“…Further to this, a third population, which is actually a fragment of the STIM2 signal peptide, upon its release from the ER membrane into the cytoplasm, becomes a regulator of gene transcription in a Ca 2ϩ independent fashion (30). Together, these findings support the repositioning of paralogs in protein families into subcellular compartments as an emerging method for the functional diversification of replicated genes (30). Given the role of SPCA2 regulation of ORAI1 in breast cancer, assessment of pre-STIM2 in cancers, including those aside from the breast would now seem a priority.…”

“…STIM2 has now been reported to exist in distinct functional forms: the widely investigated STIM2 and another population, referred to as pre-STIM2, that is able to escape ER targeting (30). Immunolocalization revealed that cytosolic pre-STIM2 localizes to the plasma membrane, where it interacts with ORAI1, increasing basal Ca 2ϩ concentration in a manner that is independent of ER Ca 2ϩ store depletion (30). The increase in basal Ca 2ϩ concentration by pre-STIM2 results in the induction of NFAT and nuclear factor-B (NF-B)-mediated gene transcription (30).…”

“…The increase in basal Ca 2ϩ concentration by pre-STIM2 results in the induction of NFAT and nuclear factor-B (NF-B)-mediated gene transcription (30). Further to this, a third population, which is actually a fragment of the STIM2 signal peptide, upon its release from the ER membrane into the cytoplasm, becomes a regulator of gene transcription in a Ca 2ϩ independent fashion (30). Together, these findings support the repositioning of paralogs in protein families into subcellular compartments as an emerging method for the functional diversification of replicated genes (30).…”