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Indu S Ambudkar - One of the best experts on this subject based on the ideXlab platform.
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functional communication between ip3r and STIM2 at sub threshold stimuli is a critical checkpoint for initiation of soce
bioRxiv, 2021Co-Authors: Moaz Ahmad, Hwei Ling Ong, Gayeon Son, Hassan Saadi, Mohamed Trebak, Zahra Shokatian, Lara E Terry, David I Yule, Indu S AmbudkarAbstract:Abstract Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters which co- ordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2-N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry. Significance STIM proteins sense decreases in [Ca2+]ER and cluster in endoplasmic reticulum (ER)-plasma membrane (PM) junctions where they recruit and activate Orai1. While STIM1 clustering requires substantial [Ca2+]ER decrease, STIM2 displays pre-clustering under resting conditions and regulates basal Ca2+ entry. The mechanism(s) underlying constitutive clustering of STIM2 is not known. We show herein that endogenous STIM2 assembles as mobile and immobile clusters and that Orai1 is recruited to the latter. Anchoring of STIM2 clusters is triggered by decreases in local [Ca2+]ER that is mediated by ambient activity of IP3R and sensed by the STIM2 N-terminus. This functional link between IP3R and STIM2 governs constitutive STIM2 clustering and ensures coupling of [Ca2+]ER decrease at sub-threshold stimuli with activation of Ca2+ entry.
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STIM2 targets orai1 stim1 to the akap79 signaling complex and confers coupling of ca2 entry with nfat1 activation
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Gayeon Son, Stefan Feske, Rajesh Bhardwaj, Krishna P Subedi, Hwei Ling Ong, Lucile Noyer, Hassan Saadi, Changyu Zheng, Indu S AmbudkarAbstract:The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.
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cross talk between n terminal and c terminal domains in stromal interaction molecule 2 STIM2 determines enhanced STIM2 sensitivity
Journal of Biological Chemistry, 2019Co-Authors: Scott M Emrich, Yandong Zhou, Krishna P Subedi, Ryan E Yoast, Ping Xin, Xuexin Zhang, Trayambak Pathak, Robert M Nwokonko, Maxime Gueguinou, Indu S AmbudkarAbstract:Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1−/−, STIM2−/−, and STIM1/2−/− knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB–activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.
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STIM2 induces activated conformation of stim1 to control orai1 function in er pm junctions
Cell Reports, 2018Co-Authors: Krishna P Subedi, Hwei Ling Ong, Gayeon Son, Xibao Liu, Indu S AmbudkarAbstract:Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells.
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assembly of er pm junctions a critical determinant in the regulation of soce and trpc1
Advances in Experimental Medicine and Biology, 2017Co-Authors: Krishna P Subedi, Hwei Ling Ong, Indu S AmbudkarAbstract:Store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, is activated when ER-[Ca2+] is decreased. SOCE is mediated via the primary channel, Orai1, as well as others such as TRPC1. STIM1 and STIM2 are ER-Ca2+ sensor proteins that regulate Orai1 and TRPC1. SOCE requires assembly of STIM proteins with the plasma membrane channels which occurs within distinct regions in the cell that have been termed as endoplasmic reticulum (ER)-plasma membrane (PM) junctions. The PM and ER are in close proximity to each other within this region, which allows STIM1 in the ER to interact with and activate either Orai1 or TRPC1 in the plasma membrane. Activation and regulation of SOCE involves dynamic assembly of various components that are involved in mediating Ca2+ entry as well as those that determine the formation and stabilization of the junctions. These components include proteins in the cytosol, ER and PM, as well as lipids in the PM. Recent studies have also suggested that SOCE and its components are compartmentalized within ER-PM junctions and that this process might require remodeling of the plasma membrane lipids and reorganization of structural and scaffolding proteins. Such compartmentalization leads to the generation of spatially- and temporally-controlled Ca2+signals that are critical for regulating many downstream cellular functions.
Donald L. Gill - One of the best experts on this subject based on the ideXlab platform.
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Orai channel C-terminal peptides are key modulators of STIM-Orai coupling and calcium signal generation
'Elsevier BV', 2021Co-Authors: James H. Baraniak, Yandong Zhou, Youjun Wang, Mohamed Trebak, Robert M Nwokonko, Michelle R. Jennette, Sarah A. Kazzaz, Jazmin M. Stenson, Abigale L. Whitsell, Donald L. GillAbstract:Summary: Junctional coupling between endoplasmic reticulum (ER) Ca2+-sensor STIM proteins and plasma membrane (PM) Orai channels mediates Ca2+ signals in most cells. We reveal that PM-tethered, fluorescently tagged C-terminal M4x (fourth transmembrane helix contains a cytoplasmic C-terminal extension) peptides from Orai channels undergo a Leu-specific signature of direct interaction with the STIM1 Orai-activating region (SOAR), exactly mimicking STIM1 binding to gate Orai channels. The 20-amino-acid Orai3-M4x peptide associates avidly with STIM1 within ER-PM junctions, functions to competitively block native Ca2+ signals, and mediates a key modification of STIM-Orai coupling induced by 2-aminoethoxydiphenyl borate. By blocking STIM-Orai coupling, the Orai3-M4x peptide reveals the critical role of Orai channels in driving Ca2+ oscillatory signals and transcriptional control through NFAT. The M4x peptides interact independently with SOAR dimers consistent with unimolecular coupling between Orai subunits and STIM1 dimers. We reveal the critical role of M4x helices in defining the coupling interface between STIM and Orai proteins to mediate store-operated Ca2+ signals
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distinct orai coupling domains in stim1 and STIM2 define the orai activating site
Nature Communications, 2014Co-Authors: Xizhuo Wang, Yandong Zhou, Youjun Wang, Jonathan Soboloff, Eunan Hendron, Salvatore Mancarella, Mark Andrake, Brad S Rothberg, Donald L. GillAbstract:STIM1 and STIM2 are widely expressed endoplasmic reticulum (ER) Ca(2+) sensor proteins able to translocate within the ER membrane to physically couple with and gate plasma membrane Orai Ca(2+) channels. Although they are structurally similar, we reveal critical differences in the function of the short STIM-Orai-activating regions (SOAR) of STIM1 and STIM2. We narrow these differences in Orai1 gating to a strategically exposed phenylalanine residue (Phe-394) in SOAR1, which in SOAR2 is substituted by a leucine residue. Remarkably, in full-length STIM1, replacement of Phe-394 with the dimensionally similar but polar histidine head group prevents both Orai1 binding and gating, creating an Orai1 non-agonist. Thus, this residue is critical in tuning the efficacy of Orai activation. While STIM1 is a full Orai1-agonist, leucine-replacement of this crucial residue in STIM2 endows it with partial agonist properties, which may be critical for limiting Orai1 activation stemming from its enhanced sensitivity to store-depletion.
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distinct roles of stim1 and STIM2 c terminal orai coupling domains
Biophysical Journal, 2013Co-Authors: Xizhuo Wang, Youjun Wang, Eunan Hendron, Donald L. GillAbstract:Store-operated Ca2+ entry (SOCe) is essential for Ca2+ homeostasis and signaling. SOCe is mediated by STIM proteins which function as ER Ca2+ store sensors, coupling with and activating PM Orai Ca2+ channels. While STIM1-Orai1 coupling is well characterized, the coupling between STIM2 and Orai1 shows some important functional differences from STIM1. The molecular basis of these differences remains uncharacterized. We examined the STIM2 C-terminal (S2-Ct) region that has considerable homology with the known functional coupling domains of S1-Ct. We examined the comparative functions of STIM1 and STIM2 fragments using a combination of Ca2+ imaging, patch-clamp current analysis, and analysis of the pharmacological modifier, 2-APB. Deletion of the “variable” C-terminal region (534-833) immediately downstream from the STIM-Orai activating region of STIM2 (SOAR2; 435-533) from either whole STIM2 or S2-Ct, had little effect on the activation of Orai1 channels. Similarly, deletion from S2-Ct of the N-terminal region (325-433) upstream from SOAR2, had little effect on Orai1-activation by S2-Ct. Thus the cytosolic regions outside SOAR2 seem to be less important for mediating STIM2 coupling to and activate Orai1. Interestingly, SOAR2 expression alone is sufficient to mimic some of the different coupling properties that distinguish full length STIM2 from STIM1, including the poor intrinsic coupling to activate Orai1 and the strong enhancement of Orai1 activation induced by 2-APB. To gain further insights on how the two SOAR domains couple and activate Orai1, we constructed a series of SOAR1 and SOAR2 chimeras. Using these chimeras, our results reveal that the Sα1-Sα3 helices in the SOAR molecules are important for defining the distinct Orai1 activating properties of STIM1 and STIM2.
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abstract 13362 stim1 and STIM2 are required in smooth muscle development and function
Circulation, 2011Co-Authors: Salvatore Mancarella, Hui Gao, Rosario Scalia, Steven R Houser, Donald L. GillAbstract:STIM1 and STIM2 control Ca2+ signaling in several cell types by activating the store operated Ca2+ entry (SOCE). Whereas their role and functions are well studied in immune cells, their role in vas...
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the calcium store sensor stim1 reciprocally controls orai and cav1 2 channels
Science, 2010Co-Authors: Youjun Wang, Jonathan Soboloff, Xiang D. Tang, Eunan Hendron, Salvatore Mancarella, Xiaoxiang Deng, Satoru Eguchi, Donald L. GillAbstract:Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (CaV1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with CaV1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both CaV1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed CaV1.2 and Orai channels has major implications for Ca2+ signal generation in excitable and nonexcitable cells.
Peter B. Stathopulos - One of the best experts on this subject based on the ideXlab platform.
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Synergistic stabilization by nitrosoglutathione-induced thiol modifications in the stromal interaction molecule-2 luminal domain suppresses basal and store operated calcium entry
Scientific Reports, 2020Co-Authors: Matthew J. Novello, Qingping Feng, Mengqi Zhang, Peter B. StathopulosAbstract:Stromal interaction molecule−1 and −2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca^2+) sensing proteins that, together with Orai1-composed Ca^2+ channels on the plasma membrane (PM), regulate intracellular Ca^2+ levels. Recent evidence suggests that S -nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca^2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca^2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca^2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S -Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca^2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca^2+ and SOCE regulation.
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the 2β splice variation alters the structure and function of the stromal interaction molecule coiled coil domains
International Journal of Molecular Sciences, 2018Co-Authors: Steve Chung, Mengqi Zhang, Peter B. StathopulosAbstract:Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.
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Structural elements of stromal interaction molecule function.
Cell Calcium, 2018Co-Authors: Matthew J. Novello, Qingping Feng, Mitsuhiko Ikura, Peter B. StathopulosAbstract:Abstract Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.
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auto inhibitory role of the ef sam domain of stim proteins in store operated calcium entry
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Peter B. Stathopulos, Rainer Schindl, Christoph Romanin, Guangyao Li, Mitsuhiko IkuraAbstract:Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca2+) sensors that differentially regulate plasma membrane Ca2+ release activated Ca2+ channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca2+-binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54° pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing “super-unstable” or “super-stable” STIM1/STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca2+- and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca2+ entry by STIM1 and STIM2.
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stromal interaction molecule stim 1 and STIM2 calcium sensing regions exhibit distinct unfolding and oligomerization
2009Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Stromal interactionmolecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2 ) entry as well as basal cytoplasmic Ca2 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 -motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2 levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2 -permeable channels on the plasma membrane. Here, we show that the kinetic half-timeof conversion fromamonomeric to a steadyoligomeric state is>70 shorter for STIM1EF-SAMthan STIM2 under similar conditions. Urea-induced rates of unfolding forSTIM1EF-SAMare>3 quickerwhencomparedwithSTIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specificN-terminal residues beyond EF-SAMcan influence the stability of this region.We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles inCa2 homeostasis and signaling.
Stefan Feske - One of the best experts on this subject based on the ideXlab platform.
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STIM2 targets orai1 stim1 to the akap79 signaling complex and confers coupling of ca2 entry with nfat1 activation
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Gayeon Son, Stefan Feske, Rajesh Bhardwaj, Krishna P Subedi, Hwei Ling Ong, Lucile Noyer, Hassan Saadi, Changyu Zheng, Indu S AmbudkarAbstract:The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.
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stim1 and STIM2 mediate cancer induced inflammation in t cell acute lymphoblastic leukemia
Cell Reports, 2018Co-Authors: Shella Saint Fleurlominy, Martin Vaeth, Mate Maus, Ingo Lange, Isabelle Zee, David Suh, Cynthia Liu, Anastasia N Tikhonova, Iannis Aifantis, Stefan FeskeAbstract:T cell acute lymphoblastic leukemia (T-ALL) is commonly associated with activating mutations in the NOTCH1 pathway. Recent reports have shown a link between NOTCH1 signaling and intracellular Ca2+ homeostasis in T-ALL. Here, we investigate the role of store-operated Ca2+ entry (SOCE) mediated by the Ca2+ channel ORAI1 and its activators STIM1 and STIM2 in T-ALL. Deletion of STIM1 and STIM2 in leukemic cells abolishes SOCE and significantly prolongs the survival of mice in a NOTCH1-dependent model of T-ALL. The survival advantage is unrelated to the leukemic cell burden but is associated with the SOCE-dependent ability of malignant T lymphoblasts to cause inflammation in leukemia-infiltrated organs. Mice with STIM1/STIM2-deficient T-ALL show a markedly reduced necroinflammatory response in leukemia-infiltrated organs and downregulation of signaling pathways previously linked to cancer-induced inflammation. Our study shows that leukemic T lymphoblasts cause inflammation of leukemia-infiltrated organs that is dependent on SOCE.
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STIM2 enhances receptor stimulated ca2 signaling by promoting recruitment of stim1 to the endoplasmic reticulum plasma membrane junctions
Science Signaling, 2015Co-Authors: Hwei Ling Ong, Stefan Feske, Kwong Tai Cheng, Xibao Liu, Changyu Zheng, Lorena Brito De Souza, Corinne M Goldsmith, Indu S AmbudkarAbstract:A central component of receptor-evoked Ca(2+) signaling is store-operated Ca(2+) entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca(2+). We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca(2+) signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with STIM1 mutant lacking the polybasic region STIM1ΔK resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER Ca(2+) stores are mildly depleted, thus increasing the sensitivity of Ca(2+) signaling to agonists.
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cd4 and cd8 t cell dependent antiviral immunity requires stim1 and STIM2
Journal of Clinical Investigation, 2014Co-Authors: Patrick J. Shaw, Martin Vaeth, Carl Weidinger, Kevin Luethy, Susan M Kaech, Stefan FeskeAbstract:Calcium signaling is critical for lymphocyte function, and intracellular Ca2+ concentrations are regulated by store-operated Ca2+ entry (SOCE) through Ca2+ release–activated Ca2+ (CRAC) channels. In patients, loss-of-function mutations in CRAC channel components ORAI1 and STIM1 abolish SOCE and are associated with recurrent and chronic viral infections. Here, using mice with conditional deletion of Stim1 and its homolog STIM2 in T cells, we determined that both components are required for the maintenance of virus-specific memory CD8+ T cells and recall responses following secondary infection. In the absence of STIM1 and STIM2, acute viral infections became chronic. Early during infection, STIM1 and STIM2 were required for the differentiation of naive CD8+ T cells into fully functional cytolytic effector cells and mediated the production of cytokines and prevented cellular exhaustion in viral-specific CD8+ effector T cells. Importantly, memory and recall responses by CD8+ T cells required expression of STIM1 and STIM2 in CD4+ T cells. CD4+ T cells lacking STIM1 and STIM2 were unable to provide “help” to CD8+ T cells due to aberrant regulation of CD40L expression. Together, our data indicate that STIM1, STIM2, and CRAC channel function play distinct but synergistic roles in CD4+ and CD8+ T cells during antiviral immunity.
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Molecular regulation of CRAC channels and their role in lymphocyte function
Cellular and Molecular Life Sciences, 2013Co-Authors: Patrick J. Shaw, Markus Hoth, Stefan FeskeAbstract:Calcium (Ca^2+) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca^2+ entry (SOCE) through Ca^2+ release-activated Ca^2+ (CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins that form the channel pore and are activated by stromal interaction molecules (STIM) 1 and 2. Located in the membrane of the endoplasmic reticulum, STIM1 and STIM2 have the dual function of sensing the intraluminal Ca^2+ concentration in the ER and to activate CRAC channels. A decrease in the ER’s Ca^2+ concentration induces STIM multimerization and translocation into puncta close to the plasma membrane where they bind to and activate ORAI channels. Since the identification of ORAI and STIM genes as the principal mediators of CRAC channel function, substantial advances have been achieved in understanding the molecular regulation and physiological role of CRAC channels in cells of the immune system and other organs. In this review, we discuss the mechanisms that regulate CRAC channel function and SOCE, the role of recently identified proteins and mechanisms that modulate the activation of ORAI/STIM proteins and the consequences of CRAC channel dysregulation for lymphocyte function and immunity.
Mitsuhiko Ikura - One of the best experts on this subject based on the ideXlab platform.
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Structural elements of stromal interaction molecule function.
Cell Calcium, 2018Co-Authors: Matthew J. Novello, Qingping Feng, Mitsuhiko Ikura, Peter B. StathopulosAbstract:Abstract Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.
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auto inhibitory role of the ef sam domain of stim proteins in store operated calcium entry
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Peter B. Stathopulos, Rainer Schindl, Christoph Romanin, Guangyao Li, Mitsuhiko IkuraAbstract:Stromal interaction molecules (STIM)s function as endoplasmic reticulum calcium (Ca2+) sensors that differentially regulate plasma membrane Ca2+ release activated Ca2+ channels in various cells. To probe the structural basis for the functional differences between STIM1 and STIM2 we engineered a series of EF-hand and sterile α motif (SAM) domain (EF-SAM) chimeras, demonstrating that the STIM1 Ca2+-binding EF-hand and the STIM2 SAM domain are major contributors to the autoinhibition of oligomerization in each respective isoform. Our nuclear magnetic resonance (NMR) derived STIM2 EF-SAM structure provides a rationale for an augmented stability, which involves a 54° pivot in the EF-hand:SAM domain orientation permissible by an expanded nonpolar cleft, ionic interactions, and an enhanced hydrophobic SAM core, unique to STIM2. Live cells expressing “super-unstable” or “super-stable” STIM1/STIM2 EF-SAM chimeras in the full-length context show a remarkable correlation with the in vitro data. Together, our data suggest that divergent Ca2+- and SAM-dependent stabilization of the EF-SAM fold contributes to the disparate regulation of store-operated Ca2+ entry by STIM1 and STIM2.
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stromal interaction molecule stim 1 and STIM2 calcium sensing regions exhibit distinct unfolding and oligomerization
2009Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Stromal interactionmolecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2 ) entry as well as basal cytoplasmic Ca2 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 -motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2 levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2 -permeable channels on the plasma membrane. Here, we show that the kinetic half-timeof conversion fromamonomeric to a steadyoligomeric state is>70 shorter for STIM1EF-SAMthan STIM2 under similar conditions. Urea-induced rates of unfolding forSTIM1EF-SAMare>3 quickerwhencomparedwithSTIM2, coherent with partial unfolding-coupled aggregation. Additionally, we demonstrate that the isoform-specificN-terminal residues beyond EF-SAMcan influence the stability of this region.We postulate that distinct oligomerization dynamics of STIM isoforms have evolved to adapt to differential roles inCa2 homeostasis and signaling.
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Stromal interaction molecule (STIM) 1 and STIM2 calcium sensing regions exhibit distinct unfolding and oligomerization kinetics.
The Journal of biological chemistry, 2008Co-Authors: Peter B. Stathopulos, Le Zheng, Mitsuhiko IkuraAbstract:Abstract Stromal interaction molecules (STIM) 1 and STIM2 are regulators of store-operated calcium (Ca2+) entry as well as basal cytoplasmic Ca2+ 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 α-motif (SAM) domains (EF-SAM), which are responsible for sensing changes in Ca2+ levels and initiating oligomerization. STIM oligomerization is a key induction step in the activation of Ca2+-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 >70× shorter for STIM1 EF-SAM than STIM2 under similar conditions. Urea-induced rates of unfolding for STIM1 EF-SAM are >3× 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 Ca2+ homeostasis and signaling.
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biophysical characterization of the ef hand and sam domain containing ca2 sensory region of stim1 and STIM2
Biochemical and Biophysical Research Communications, 2008Co-Authors: Peter B. Stathopulos, Mitsuhiko IkuraAbstract:Abstract Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER)-membrane associated Ca2+ sensor which activates store-operated Ca2+ entry (SOCE). The homologue, STIM2 possesses a high sequence identity to STIM1 (∼61%), while its role in SOCE seems to be distinct from that of STIM1. In order to understand the underlying mechanism for the functional differences between STIM1 and STIM2, we investigated the biophysical properties of the luminal Ca2+-binding region which contains an EF-hand motif and a sterile α-motif (SAM) domain (hereafter called EF–SAM; residues 58–201 in STIM1 and 149–292 in STIM2). STIM2 EF–SAM has a low apparent Ca2+-binding affinity (Kd ∼0.5 mM), which is similar to that reported for STIM1 EF–SAM. In the presence of Ca2+, STIM2 EF–SAM is monomeric and well-folded, analogous to what was previously observed for STIM1 EF–SAM. In contrast to apo STIM1 EF–SAM, apo STIM2 EF–SAM is more structurally stable and does not readily aggregate. Our circular dichroism (CD) data demonstrate the existence of a long-lived, well-folded monomeric state for apo STIM2 EF–SAM, together with a less α-helical/partially unfolded aggregated state which is detectable only at higher protein concentrations and higher temperatures. Our biophysical studies reveal a structural stability difference in the EF–SAM region between STIM1 and STIM2, which may account for their different biological functions.