Stromal Interaction Molecule Proteins Research Articles

A Conserved, Lipid‐Mediated Sorting Mechanism of Yeast Ist2 and Mammalian STIM Proteins to the Peripheral ER

Sorting of yeast Ist2 to the plasma membrane (PM) or the cortical endoplasmic reticulum (ER) requires a cortical sorting signal (CSS(Ist2)) that interacts with lipids including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) at the PM. Here, we show that the expression of Ist2 in mammalian cells resulted in a peripheral patch-like localization without any detection of Ist2 at the cell surface. Attached to C-termini of mammalian integral membrane proteins, the CSS(Ist2) targeted these proteins to PM-associated domains of the ER and abolished trafficking via the classical secretory pathway. The interaction of integral membrane proteins with PI(4,5)P(2) at the PM created ER-PM contacts. This process is similar to the regulated coupling of ER domains to the PM via stromal interaction molecule (STIM) proteins during store-operated Ca(2+) entry (SOCE). The CSS(Ist2) and the C-terminus of the ER-located Ca(2+) sensor STIM2 were sufficient to bind PI(4,5)P(2) and PI(3,4,5)P(3) at the PM, showing that an evolutionarily conserved mechanism is involved in the sorting of integral membrane proteins to PM-associated domains of the ER. Yeast Ist2 and STIM2 share a common basic and amphipathic signal at their extreme C-termini. STIM1 showed binding preference for liposomes containing PI(4,5)P(2), suggesting a specific contribution of lipids to the recruitment of ER domains to the PM during SOCE.

Properties of Orai1 mediated store‐operated current depend on the expression levels of STIM1 and Orai1 proteins

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (I(CRAC)) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I(CRAC). In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I(CRAC). The results show that cells expressing low Orai1 : STIM1 ratios produce I(CRAC) with strong fast Ca2+-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I(CRAC) with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

M1689 The Endocrine Disruptor Bisphenol a (BPA) Decreases Paracellular Permeability in the Female Rat Colon At Environmentally Relevant Doses and Increases Tight Junction Protein Expression in CACO-2 Cells

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Our previous studies have shown the canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and regulates early mucosal restitution. Increased TRPC1 channel activity increases store-operated Ca2+ entry (SOCE) and enhances IEC migration during restitution. However, the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Recently, stromal interaction molecule 1 (STIM1) has been identified as a store Ca2+ sensor and can rapidly translocate to the plasma membrane (PM) where it interacts with and modulates Ca2+ channels. We hypothesized that STIM1 is crucial for stimulation of IEC migration after wounding by activating TRPC1 channel activity.Methods: Studies were conducted in IEC-Cdx2L1 cells, which represent differentiated IECs. STIM1 subcellular distribution was analyzed by surface biotinylation assays and immunohistochemical staining, while its functions were investigated by specific siRNA (siSTIM1) and ectopic overexpression of constitutively active STIM1 EF-hand mutants. [Ca]cyt was measured by fluorescence digital imaging analysis. Cell migration was measured in a model that mimics cell division-independent epithelial restitution. Results: IECs highly expressed STIM1 in cultured cells and in the intestinal mucosa of the rat. STIM1 translocation to the PM increased significantly within 10 min after wounding, peaked between 30 to 60 min, and then gradually returned to normal. Maximum increases in levels of STIM1 at PM were ~six times the prewounding control, which was associated with a dramatic increase in SOCE. There were no changes in total STIM1 protein levels after wounding. STIM1 directly interacted with TRPC1 in the PM as measured by immunoprecipitation assays. Ectopic expression of constitutively active STIM1 EF-hand mutants increased SOCE (by ~60%) and also stimulated IECmigration (by ~35%) after wounding. In contrast, STIM1 silencing by siSTIM1 decreased SOCE, inhibited IEC migration, and delayed epithelial restitution in cells overexpressing TRPC1. Levels of Ca2+ influx due to SOCE were decreased by ~70% in STIM1-silenced populations, whereas cell migration was inhibited by ~45%. Conclusions: These findings indicate that 1) wounding increases STIM1 translocation to the PM in IECs and 2) STIM1 at the PM interacts with and activates TRPC1, thus stimulating epithelial restitution as a result of increase in SOCE.

Stromal Interaction Molecule (STIM) 1 and STIM2 Calcium Sensing Regions Exhibit Distinct Unfolding and Oligomerization Kinetics

Stromal interaction molecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca(2+)) entry as well as basal cytoplasmic Ca(2+) levels in human cells. Despite a high sequence similarity (>65%) and analogous sequence-based domain architectures, STIM1 and STIM2 differentially influence these phenomena. Among all eukaryotes, the endoplasmic reticulum luminal portion of STIM proteins minimally encode EF-hand and sterile alpha-motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca(2+) levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca(2+)-permeable channels on the plasma membrane. Here, we show that the kinetic half-time of conversion from a monomeric to a steady oligomeric state is >70x shorter for STIM1 EF-SAM than STIM2 under similar conditions. Urea-induced rates of unfolding for STIM1 EF-SAM are >3x quicker when compared with STIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specific N-terminal residues beyond EF-SAM can influence the stability of this region. We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles in Ca(2+) homeostasis and signaling.

Differential dependence of store‐operated and excitation‐coupled Ca2+ entry in skeletal muscle on STIM1 and Orai1

In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.

Interactions, Functions, and Independence of Plasma Membrane STIM1 and TRPC1 in Vascular Smooth Muscle Cells

Stromal interaction molecule 1 (STIM1) is a predicted single membrane-spanning protein involved in store-operated calcium entry and interacting with ion channels including TRPC1. Here, we focus on endogenous STIM1 of modulated vascular smooth muscle cells, which exhibited a nonselective cationic current in response to store depletion despite strong buffering of intracellular calcium at the physiological concentration. STIM1 mRNA and protein were detected and suppressed by specific short interfering RNA. Calcium entry evoked by store depletion was partially inhibited by STIM1 short interfering RNA, whereas calcium release was unaffected. STIM1 short interfering RNA suppressed cell migration but not proliferation. Antibody that specifically bound STIM1 revealed constitutive extracellular N terminus of STIM1 and extracellular application of the antibody caused fast inhibition of the current evoked by store depletion. The antibody also inhibited calcium entry and cell migration but not proliferation. STIM1 interacted with TRPC1, and TRPC1 contributed partially to calcium entry and cationic current. However, the underlying processes could not be explained only by a STIM1-TRPC1 partnership because extracellular TRPC1 antibody suppressed cationic current only in a fraction of cells, TRPC1-containing channels were important for cell proliferation as well as migration, and cell surface localization studies revealed TRPC1 alone, as well as with STIM1. The data suggest a complex situation in which there is not only plasma membrane-spanning STIM1 that is important for cell migration and TRPC1-independent store-operated cationic current but also TRPC1-STIM1 interaction, a TRPC1-dependent component of store-operated current, and STIM1-independent TRPC1 linked to cell proliferation.

Stopping STIM Stops Strokes

Proper responses of platelets in the blood to various physiological signals are essential: Unresponsiveness prevents clotting at sites of wounds, but overactivity can contribute to heart attacks or stroke. Platelets respond to a number of factors, but the various signals all feed into activation of phospholipase C and consequent release of calcium from intracellular stores. Depletion of calcium from the intracellular stores then leads to influx of extracellular calcium. The STIM1 protein (stromal interaction molecule 1) functions in detection of store emptying and activation of store-operated channels that allow calcium entry. Varga-Szabo et al . used knockout mice in which the Stim 1 gene was disrupted to study the role of STIM1 in arterial thrombosis and ischemic brain infarction. The Stim 1 –/– animals died a few hours after birth, but the authors were able to transplant bone marrow from the knockout animals to lethally irradiated host mice to study platelet function. In two in vivo models of arterial thrombosis induced by injury, formation of occlusive thrombi was inhibited in the Stim 1 –/– animals. Similarly, in a model of ischemic brain infarction induced by restriction of blood flow through the middle cerebral artery, infarct size was reduced in the Stim 1 –/– animals. Most animals with the Stim 1 –/– platelets stopped bleeding from a wound at the tail tip within about 10 minutes, as did wild-type mice. The response was more variable in the Stim 1 –/– animals, however, and about 35% of the animals showed longer bleeding. Because of this relatively mild effect on bleeding, the authors propose that inhibitors of Stim 1 might be attractive as antithrombolitics that could help treat stroke patients without the unacceptable side effect of increased intracerebral hemorrhage. As a caveat, the authors do note that results in the mouse models may not necessarily be directly comparable to the situation in humans. D. Varga-Szabo, A. Braun, C. Kleinschnitz, M. Bender, I. Pleines, M. Pham, T. Renné, G. Stoll, B. Nieswandt, The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction. J. Exp. Med . 205 , 1583-1591 (2008). [Abstract] [Full Text]

Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes†

The process of capacitative or store-operated Ca(2+) entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca(2+) stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca(2+) entry is readily demonstrated with protocols that provide extensive Ca(2+) store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca(2+) influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca(2+) oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca(2+) oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca(2+) entry but only partially reduced the frequency of Ca(2+) oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. Our findings establish that store-operated Ca(2+) entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent.

Dynamic simulation of the effect of calcium-release activated calcium channel on cytoplasmic Ca 2+ oscillation

A mathematical model is proposed to illustrate the activation of STIM1 (stromal interaction molecule 1) protein, the assembly and activation of calcium-release activated calcium (CRAC) channels in T cells. In combination with De Young–Keizer–Li–Rinzel model, we successfully reproduce a sustained Ca 2+ oscillation in cytoplasm. Our results reveal that Ca 2+ oscillation dynamics in cytoplasm can be significantly affected by the way how the Orai1 CRAC channel are assembled and activated. A low sustained Ca 2+ influx is observed through the CRAC channels across the plasma membrane. In particular, our model shows that a tetrameric channel complex can effectively regulate the total quantity of the channels and the ratio of the active channels to the total channels, and a period of Ca 2+ oscillation about 29 s is in agreement with published experimental data. The bifurcation analyses illustrate the different dynamic properties between our mixed Ca 2+ feedback model and the single positive or negative feedback models.

STIM1 Knockdown Reveals That Store-operated Ca2+ Channels Located Close to Sarco/Endoplasmic Ca2+ ATPases (SERCA) Pumps Silently Refill the Endoplasmic Reticulum

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol.

STIM1 and the noncapacitative ARC channels

Our understanding of the nature and regulation of receptor-activated Ca 2+ entry in nonexcitable cells has recently undergone a radical change that began with the identification of the stromal interacting molecule proteins (e.g., STIM1) as playing a critical role in the regulation of the capacitative, or store-operated, Ca 2+ entry. As such, current models emphasize the role of STIM1 located in the endoplasmic reticulum membrane, where it senses the status of the intracellular Ca 2+ stores via a luminal N-terminal Ca 2+-binding EF-hand domain. Dissociation of Ca 2+ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca 2+ channels (e.g., CRAC channels). Thus, the specific dependence on store-depletion, and the role of the Ca 2+-binding EF-hand domain in this process, are critical to all current models of the action of STIM1 on Ca 2+ entry. However, until recently, the effects of STIM1 on other modes of receptor-activated Ca 2+ entry have not been examined. Surprisingly, we found that STIM1 exerts similar, although not identical, actions on the arachidonic acid-regulated Ca 2+-selective (ARC) channels—a widely expressed mode of agonist-activated Ca 2+ entry whose activation is completely independent of Ca 2+ store depletion. Regulation of the ARC channels by STIM1 is not only independent of store depletion, but also of the Ca 2+-binding function of the EF-hand, and translocation of STIM1 to the plasma membrane. Instead, it is the pool of STIM1 that constitutively resides in the plasma membrane that is critical for the regulation of the ARC channels. Thus, ARC channel activity is selectively inhibited by exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1. Similarly, introducing mutations in STIM1 that prevent the N-linked glycosylation-dependent constitutive expression of the protein in the plasma membrane specifically inhibits the activity of the ARC channels without affecting the CRAC channels. These studies demonstrate that STIM1 is a far more universal regulator of Ca 2+ entry pathways than previously assumed, and has multiple, and entirely distinct, modes of action. Precisely how this same protein can act in such separate and specific ways on these different pathways of agonist-activated Ca 2+entry remains an intriguing, yet currently unresolved, question.

STIM1 regulates Ca2+ entry via arachidonate‐regulated Ca2+‐selective (ARC) channels without store depletion or translocation to the plasma membrane

Recent studies have indicated a critical role for STIM (stromal interacting molecule) proteins in the regulation of the store-operated mode of receptor-activated Ca2+ entry. Current models emphasize the role of STIM located in the endoplasmic reticulum membrane, where a Ca2+-binding EF-hand domain within the N-terminal of the protein lies within the lumen and is thought to represent the sensor for the depletion of intracellular Ca2+ stores. Dissociation of Ca2+ from this domain induces the aggregation of STIM to regions of the ER immediately adjacent to the plasma membrane where it acts to regulate the activity of store-operated Ca2+ channels. However, the possible effects of STIM on other modes of receptor-activated Ca2+ entry have not been examined. Here we show that STIM1 also regulates the arachidonic-acid-regulated Ca2+-selective (ARC) channels - receptor-activated Ca2+ entry channels whose activation is entirely independent of store depletion. Regulation of the ARC channels by STIM1 does not involve dissociation of Ca2+ from the EF-hand, or any translocation of STIM1. Instead, a critical role of STIM1 resident in the plasma membrane is indicated. Thus, exposure of intact cells to an antibody targeting the extracellular N-terminal domain of STIM1 inhibits ARC channel activity without significantly affecting the store-operated channels. A similar specific inhibition of the ARC channels is seen in cells expressing a STIM1 construct in which the N-linked glycosylation sites essential for the constitutive cell surface expression of STIM1, were mutated. We conclude that, in contrast to store-operated channels, regulation of ARC channels by STIM1 depends exclusively on the pool of STIM1 constitutively residing in the plasma membrane. These data demonstrate that STIM1 is a more universal regulator of Ca2+ entry pathways than previously thought, and appears to have multiple modes of action.

Regulation of STIM1, store-operated Ca2+influx, and nitric oxide generation by retinoic acid in rat mesangial cells

It has been shown that store-operated Ca(2+) influx (SOC) plays critical roles in the activation of endothelial nitric oxide (NO) synthase (eNOS) and generation of NO in endothelial cells. Recent studies indicate stromal interaction molecule 1 (STIM1) is the molecule responsible for SOC activation following Ca(2+) depletion in the ER. Retinoic acids (RA) have beneficial effects in the treatment of renal diseases. The mechanism of the RA action is still largely unknown. In the current study, we used primary cultured rat mesangial cells to examine the effect of RA on SOC and STIM1. In these cells, BK caused concentration-dependent [Ca(2+)](i) mobilization. Treatment of the cells with RA, while it had no effect on the initial peak, reduced the plateau phase of BK-mediated [Ca(2+)](i) response, indicating the inhibition of SOC by RA. The level of STIM1 protein but not mRNA in RA-treated cells was significantly reduced. RA treatment did not affect TGF-beta-mediated gradual Ca(2+) influx which occurred by superoxide anion-mediated mechanism, indicating RA treatment specifically inhibited SOC in mesangial cells. RT-PCR and Western blot analysis demonstrated that eNOS was expressed in rat mesangial cells grown in media containing 11 and 30 but not 5.5 mM glucose. Downregulation of STIM1 protein and BK-induced SOC by RA treatment or STIM1 dsRNA were associated with abolished NO production. The 26S proteasome inhibitor lactacystin blocked the RA-mediated downregulation of BK-induced SOC, suggesting that ubiquitin-proteasome pathway may be involved in RA-mediated STIM1 protein downregulation in rat mesangial cells. Our data suggest that glucose-induced eNOS expression and NO production in mesangial cells may contribute to hyperfiltration in diabetes and RA may exert beneficial effects by downregulation of STIM1 and SOC in mesangial cells.

Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile α-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.