SERAF, a Novel Regulator of Store Operated Calcium Entry (original) (raw)
Related papers
SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling
Cell, 2012
Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca 2+ stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca 2+ release-activated Ca 2+ (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Using heterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca 2+ -dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca 2+ signals and determining the content of the major intracellular Ca 2+ stores, a role that is likely to be important in protecting cells from Ca 2+ overfilling.
Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry
Scientific reports, 2016
Ca(2+) influx by store-operated Ca(2+) channels is a major mechanism for intracellular Ca(2+) homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca(2+) store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 inter...
Bidirectional regulation of calcium release–activated calcium (CRAC) channel by SARAF
Journal of Cell Biology, 2021
Store-operated calcium entry (SOCE) through the Ca 2+ release-activated Ca 2+ (CRAC) channel is a central mechanism by which cells generate Ca 2+ signals and mediate Ca 2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca 2+ depletion, SARAF facilitates a conformational change in the ER Ca 2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475-483) and promotes initial activation of STIM1, its translocation to ER-plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca 2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca 2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca 2+ responses and downstream gene expression in cells.
STIM and Orai: the long-awaited constituents of store-operated calcium entry
Trends in Pharmacological Sciences, 2009
Rapid changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i are the most commonly used signals in biology to regulate a whole host of cellular functions including contraction, secretion and gene activation. A widely utilized form of Ca 2+ influx is termed store-operated Ca 2+ entry (SOCE) due to its control by the Ca 2+ content of the endoplasmic reticulum (ER). The underlying molecular mechanism of SOCE has eluded identification until recently when two groups of proteins, the ER Ca 2+ sensors, STIM1 and -2, and the plasma membrane channels, Orai1, -2 and -3 have been identified. These landmark discoveries have allowed impressive progress in clarifying how these proteins work in concert and what developmental and cellular processes require their participation most. As we begin to better understand the biology of the STIM and Orai proteins, the attention to the pharmacological tools to influence their functions quickly follow suit. This review will briefly summarize recent developments in this exciting area of Ca 2+ signaling.
Role of STIM and Orai proteins in the store-operated calcium signaling pathway
Cell Calcium, 2007
Ca 2+ signals are universal among cells in regulating a spectrum of cellular responses. Phospholipase C-coupled receptors activate two components of Ca 2+ signals-rapid Ca 2+ release from ER stores, followed by slower Ca 2+ entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca 2+ entry process remained until recently a molecular mystery. The recent discovery of the necessity for STIM1 and Orai proteins in this process has provided crucial information on the coupling mechanism between stores and PM Ca 2+ entry. STIM1 is a single spanning membrane protein with an unpaired Ca 2+ binding EF-hand and appears to function as the sensor of ER luminal Ca 2+ , and, through redistribution in the ER, transduces information directly to the PM. Orai1 is a tetra-spanning PM protein and functions as the highly Ca 2+ -selective channel in the PM that is gated through interactions with the store-activated ER Ca 2+ sensor. Recent evidence shows the two proteins together are necessary and sufficient for the function of store-operated Ca 2+ entry. However, many questions arise about how and where the interactions of the STIM1 and Orai1 proteins occur within cells. Here we discuss recent information and ideas about the coupling between these proteins that leads to store-operated channel activation.
Proceedings of The National Academy of Sciences, 2006
Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca 2؉ influx and Ca 2؉ release-activated Ca 2؉ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca 2؉ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca 2؉ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca 2؉ pump sarco-͞ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca 2؉ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.
STIM1, an essential and conserved component of store-operated Ca2+ channel function
The Journal of Cell Biology, 2005
Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conser...
Orai1 and STIM Reconstitute Store-operated Calcium Channel Function
Journal of Biological Chemistry, 2006
The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca 2؉ entry (SOCE) in human embryonic kidney 293 cells and the Ca 2؉ release-activated Ca 2؉ current (I CRAC ) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca 2؉ entry by up to 103-fold. This entry is entirely storedependent since the same coexpression causes no measurable store-independent Ca 2؉ entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (storeindependent) Ca 2؉ entry. STIM proteins are known to mediate Ca 2؉ store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca 2؉ entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.
STIM and Orai1 Variants in Store-Operated Calcium Entry
Frontiers in pharmacology, 2015
Store-operated Ca(2+) entry (SOCE) is an ubiquitous mechanism for Ca(2+) entry in eukaryotic cells. This route for Ca(2+) influx is regulated by the filling state of the intracellular Ca(2+) stores communicated to the plasma membrane channels by the proteins of the Stromal Interaction Molecule (STIM) family, STIM1, and STIM2. Store-dependent, STIM1-modulated, channels include the Ca(2+) release-activated Ca(2+) channels, comprised of subunits of Orai proteins, as well as the store-operated Ca(2+) (SOC) channels, involving Orai1, and members of the canonical transient receptor potential family of proteins. Recent studies have revealed the expression of splice variants of STIM1, STIM2, and Orai1 in different cell types. While certain variants are ubiquitously expressed, others, such as STIM1L, show a more restricted expression. The splice variants for STIM and Orai1 proteins exhibit significant functional differences and reveal that alternative splicing enhance the functional diversit...