**3. STIM1-ORAI1-mediated Ca2+ influx**

The mechanism of activation has been well documented for the complex STIM1-ORAI1. At resting state, STIM1 is distributed on the surface of the ER, where its cytosolic domain is folded in a tight state due to an intramolecular clamp between domains CC1a1 and CC3 [13]. The inactive and resting STIM1 is a dimer [14], and the activation of Ca2+-release from intracellular stores due to the activation of the phosphoinositide pathway, leads to the transient depletion of Ca2+ levels within the ER. The consequent Ca2+ dissociation from the intraluminal domain of STIM1 triggers the activation of the protein in a more extended state that lets STIM1 to form oligomers [15]. In contrast to what it has been observed for inactive STIM1, which shows a high mobility on the ER surface while it is bound to EB1 and microtubules, STIM1 oligomers are quite immobile when they reach ER-PM junctions. This oligomerization has been extensively documented when STIM1 is targeted with fluorescent tags (**Figure 1**).

[27], supporting a model in which Ca2+/CaM binds to the N-terminus of ORAI1 to trigger

**Figure 1.** HEK293 cells stably expressing STIM1-GFP were incubated in Hank's balanced salt solution (HBSS) (left) or in Ca2+-free HBSS with 1 μM thapsigargin (right) to trigger Ca2+ store depletion. After 10-min incubation cells were fixed and visualized under wide-field fluorescence microscopy. In control cells (left panel), STIM1-GFP showed a localization that matched with endoplasmic reticulum. Thapsigargin induced aggregation of STIM1-GFP revealed by the clustering of GFP fluorescence (right panel), a result that demonstrated that the recombinant protein STIM1-GFP was sensitive to

Regulation of Calcium Signaling by STIM1 and ORAI1 http://dx.doi.org/10.5772/intechopen.78587 7

The large Rab GTPase CRACR2A mediates another mechanism that controls and prevents excessive Ca2+ entry. At low intracellular Ca2+ levels, CRACR2A enhances the binding of STIM1 to ORAI1, but at higher [Ca2+]i, that is, after ORAI1 activation, CRACR2A dissociates from ORAI1, inhibiting SOCE [28]. ORAI1 residues involved in the binding to CRACR2A are the same as those that bind Ca2+/CaM, so Ca2+/CaM and Ca2+-free CRACR2A are competitors

STIMATE, a protein encoded by TMEM110 gene, is an ER-resident protein and a modulator for the activity of the STIM1-ORAI1 complex [29]. When Ca2+ dissociates from STIM1, the conformational change to a more extended structure of STIM1 facilitates the binding between STIMATE and STIM1-CC1 domain, avoiding the inhibition of CAD domain. This is the reason why STIMATE promotes the full extended conformation and the formation of STIM1 clustering at ER-PM junctions. In addition, the protein SARAF has been described as a negative regulator of SOCE [30]. As an ER membrane-resident protein, SARAF associates with STIM1 to promote Ca2+-dependent inactivation of SOCE. In this regard, a conserved STIM1 (448–530) C-terminal inhibitory domain (CTID) has been reported to regulate Ca2+-dependent inhibition [31]. CTID shows the capability to promote access of SARAF to the STIM1-ORAI1 activation

Additional regulators of the complex STIM1-ORAI1 have been reported, including septins [32]

in ER-PM junctions before store-depletion and facilitate STIM1 targeting to these junctions, where STIM1 recruits ORAI1. On the other hand, RASSF4 (RAS association domain family 4) also regulates SOCE by affecting the translocation of STIM1 to ER-PM junctions. Finally, a recent report has shown that ORAI1, as well as STIM1 phosphorylated at ERK1/2-target sites, are recruited at the leading edge of migrating cells, where ORAI1 binds cortactin, a regulator

) polarize

and RASSF4 [33]. Septin filaments and phosphatidylinositol-4,5-bisphosphate (PIP<sup>2</sup>

region (SOAR or CAD), thus promoting inactivation of SOCE.

channel inactivation.

store depletion (reprinted from reference [25]).

for ORAI1.

The binding of STIM1 to the microtubule plus-end tracking protein EB1, ensures the targeting of STIM1 to ER-PM junctions [16]. However, this binding to EB1 is not required for the activation of ORAI1. STIM1 dissociates from EB1 by a mechanism regulated by the phosphorylation of a set of serine residues (Ser575, Ser608, and Ser621) adjacent to the EB1-binding site [17]. This STIM1 phosphorylation is mediated by the kinases ERK1/2, which become activated in the absence of Ca2+-influx by the activation of tyrosine kinase receptors at the plasma membrane [18–20]. Thus, STIM1 bound to EB1 travels to ER-PM junctions [6], but it dissociates from EB1 to bind to ORAI1 [17]. The physical interaction between STIM1 and ORAI1 is fully required for gating the Ca2+ channel. This interaction is mediated by the aforementioned CRAC activation domain (CAD) of STIM1 and both the cytosolic C-terminus and the N-terminus of ORAI1 [21], although the binding to the N-terminus is slightly weaker [22]. The stoichiometry of the complex STIM1-ORAI1 is also in the center of debate, but the current accepted proposal supports a 1:1 to 2:1 ratio in order to activate the channel [23, 24].

The high selectivity of ORAI1 for Ca2+ is due to the pore design, with a selectivity filter mediated by the acidic glutamate residue E106 at the first transmembrane domain [26]. Once Ca2+ influx is activated, a negative feedback controls the excessive Ca2+ entry, and Ca2+/calmodulin mediates this mechanism of inactivation. A short domain (residues 470–491) with seven acidic amino acids, close to the CAD binding domain, is directly involved in the Ca2+-dependent inactivation [27]. Also in this report, Mullins et al. identified a membrane-proximal N-terminal domain of ORAI1 (residues 68–91) that binds calmodulin (CaM) in a Ca2+-dependent manner

(~500 μM) is twofold higher than that of STIM1 [8], suggesting that STIM2 becomes activated with smaller changes in intraluminal Ca2+ levels, whereas STIM1 activates Ca2+ entry upon

ORAI1 is a plasma membrane protein with four transmembrane domains with the N- and C-termini oriented to the cytosol. The Ca2+ channel is formed by a hexamer of ORAI1 monomers, with the Ca2+ pore in the center of the hexamer [10, 11]. Both the N- and C-terminal domains are involved in the binding to STIM1 [12]. The paralogues ORAI2 and ORAI3 share 63% and 58% sequence identity with ORAI1, being the extracellular loop 3 that connects TM

The mechanism of activation has been well documented for the complex STIM1-ORAI1. At resting state, STIM1 is distributed on the surface of the ER, where its cytosolic domain is folded in a tight state due to an intramolecular clamp between domains CC1a1 and CC3 [13]. The inactive and resting STIM1 is a dimer [14], and the activation of Ca2+-release from intracellular stores due to the activation of the phosphoinositide pathway, leads to the transient depletion of Ca2+ levels within the ER. The consequent Ca2+ dissociation from the intraluminal domain of STIM1 triggers the activation of the protein in a more extended state that lets STIM1 to form oligomers [15]. In contrast to what it has been observed for inactive STIM1, which shows a high mobility on the ER surface while it is bound to EB1 and microtubules, STIM1 oligomers are quite immobile when they reach ER-PM junctions. This oligomerization has been extensively documented when STIM1 is targeted with fluorescent tags (**Figure 1**).

The binding of STIM1 to the microtubule plus-end tracking protein EB1, ensures the targeting of STIM1 to ER-PM junctions [16]. However, this binding to EB1 is not required for the activation of ORAI1. STIM1 dissociates from EB1 by a mechanism regulated by the phosphorylation of a set of serine residues (Ser575, Ser608, and Ser621) adjacent to the EB1-binding site [17]. This STIM1 phosphorylation is mediated by the kinases ERK1/2, which become activated in the absence of Ca2+-influx by the activation of tyrosine kinase receptors at the plasma membrane [18–20]. Thus, STIM1 bound to EB1 travels to ER-PM junctions [6], but it dissociates from EB1 to bind to ORAI1 [17]. The physical interaction between STIM1 and ORAI1 is fully required for gating the Ca2+ channel. This interaction is mediated by the aforementioned CRAC activation domain (CAD) of STIM1 and both the cytosolic C-terminus and the N-terminus of ORAI1 [21], although the binding to the N-terminus is slightly weaker [22]. The stoichiometry of the complex STIM1-ORAI1 is also in the center of debate, but the current accepted proposal sup-

The high selectivity of ORAI1 for Ca2+ is due to the pore design, with a selectivity filter mediated by the acidic glutamate residue E106 at the first transmembrane domain [26]. Once Ca2+ influx is activated, a negative feedback controls the excessive Ca2+ entry, and Ca2+/calmodulin mediates this mechanism of inactivation. A short domain (residues 470–491) with seven acidic amino acids, close to the CAD binding domain, is directly involved in the Ca2+-dependent inactivation [27]. Also in this report, Mullins et al. identified a membrane-proximal N-terminal domain of ORAI1 (residues 68–91) that binds calmodulin (CaM) in a Ca2+-dependent manner

more severe conditions [9].

6 Calcium and Signal Transduction

domain 3 and 4, significantly larger in ORAI3.

**3. STIM1-ORAI1-mediated Ca2+ influx**

ports a 1:1 to 2:1 ratio in order to activate the channel [23, 24].

**Figure 1.** HEK293 cells stably expressing STIM1-GFP were incubated in Hank's balanced salt solution (HBSS) (left) or in Ca2+-free HBSS with 1 μM thapsigargin (right) to trigger Ca2+ store depletion. After 10-min incubation cells were fixed and visualized under wide-field fluorescence microscopy. In control cells (left panel), STIM1-GFP showed a localization that matched with endoplasmic reticulum. Thapsigargin induced aggregation of STIM1-GFP revealed by the clustering of GFP fluorescence (right panel), a result that demonstrated that the recombinant protein STIM1-GFP was sensitive to store depletion (reprinted from reference [25]).

[27], supporting a model in which Ca2+/CaM binds to the N-terminus of ORAI1 to trigger channel inactivation.

The large Rab GTPase CRACR2A mediates another mechanism that controls and prevents excessive Ca2+ entry. At low intracellular Ca2+ levels, CRACR2A enhances the binding of STIM1 to ORAI1, but at higher [Ca2+]i, that is, after ORAI1 activation, CRACR2A dissociates from ORAI1, inhibiting SOCE [28]. ORAI1 residues involved in the binding to CRACR2A are the same as those that bind Ca2+/CaM, so Ca2+/CaM and Ca2+-free CRACR2A are competitors for ORAI1.

STIMATE, a protein encoded by TMEM110 gene, is an ER-resident protein and a modulator for the activity of the STIM1-ORAI1 complex [29]. When Ca2+ dissociates from STIM1, the conformational change to a more extended structure of STIM1 facilitates the binding between STIMATE and STIM1-CC1 domain, avoiding the inhibition of CAD domain. This is the reason why STIMATE promotes the full extended conformation and the formation of STIM1 clustering at ER-PM junctions. In addition, the protein SARAF has been described as a negative regulator of SOCE [30]. As an ER membrane-resident protein, SARAF associates with STIM1 to promote Ca2+-dependent inactivation of SOCE. In this regard, a conserved STIM1 (448–530) C-terminal inhibitory domain (CTID) has been reported to regulate Ca2+-dependent inhibition [31]. CTID shows the capability to promote access of SARAF to the STIM1-ORAI1 activation region (SOAR or CAD), thus promoting inactivation of SOCE.

Additional regulators of the complex STIM1-ORAI1 have been reported, including septins [32] and RASSF4 [33]. Septin filaments and phosphatidylinositol-4,5-bisphosphate (PIP<sup>2</sup> ) polarize in ER-PM junctions before store-depletion and facilitate STIM1 targeting to these junctions, where STIM1 recruits ORAI1. On the other hand, RASSF4 (RAS association domain family 4) also regulates SOCE by affecting the translocation of STIM1 to ER-PM junctions. Finally, a recent report has shown that ORAI1, as well as STIM1 phosphorylated at ERK1/2-target sites, are recruited at the leading edge of migrating cells, where ORAI1 binds cortactin, a regulator of plasma membrane ruffling [34]. This membrane ruffling is the reorganization of the cortical cytoskeleton required for the formation of filopodia and lamellipodia, and STIM1-KO (knockout) and ORAI1-KO cells, engineered by CRISPR/Cas9 genome editing, showed defective membrane ruffling and largely diminished cell migration [34], demonstrating that Ca2+ influx through STIM1-activated ORAI1 is essential for these events.

kinases [34], where phospho-STIM1 acts in cooperation with ORAI1 to regulate the Ca2+ influx that rules cell migration. Consequently, phospho-STIM1 is an effector of ERK1/2 and an essential mediator for the activation of Ca2+ influx upon stimulation of cells with IGF-1 [20],

Regulation of Calcium Signaling by STIM1 and ORAI1 http://dx.doi.org/10.5772/intechopen.78587 9

Other MAPKs, such as p38 MAPK, have been shown to regulate SOCE, although by an indirect mechanism. Transforming growth factor beta (TGFβ) regulates megakaryocyte maturation and platelet formation by upregulating the expression of the serum-glucocorticoid inducible kinase SGK1 [44, 45], which is p38 MAPK-dependent [46]. SGK1 stimulates nuclear translocation of transcription factor NF-κB, which upregulates ORAI1 expression, increasing SOCE. This increase was demonstrated to be sensitive to p38 MAPK inhibition, SGK1 inhibition, and NF-κB inhibition, demonstrating the role of p38 MAPK in the upregulation of SOCE [47]. Another proposal was reported by Sundivakkam et al. [48], who reported that p38 MAPK directly phosphorylates STIM1. In this report, it was shown that pharmacological inhibition of p38 MAPK increased SOCE and that p38β knockdown prevented STIM1 phosphorylation and potentiated SOCE. However, this report did not identify the phosphorylated Ser/Thr residue(s), since the findings were based on the use of a phospho-Ser antibody [48],

The crosstalk between SOCE and cAMP-activated pathways has been investigated thoroughly since the molecular description of STIM1 and ORAI1. For instance, the Ca2+/CaM-stimulated adenylyl cyclase 8 (AC8) was found to be activated by SOCE and co-localized with STIM1 and ORAI1 in lipid rafts [49]. Interestingly, other authors found that lowering the concentration of free Ca2+ within the ER led to recruitment of adenylyl cyclases, enhancing the production of cAMP with the subsequent PKA activation, being this action independent of the [Ca2+]i [50]. Because activation of STIM1 and translocation to ER-PM junctions were required for coupling ER-Ca2+ depletion and adenylyl cyclase activity, without altering [Ca2+]i, those authors proposed the occurrence of a pathway termed store-operated cAMP signaling (SOcAMPS), a pathway that was later confirmed for other AC isoforms, such as AC3 [51]. More recently, it was confirmed that other Ca2+ channels, including TRPC1, were also involved in the activation of ACs [52]. It has been reported that STIM1 interacts with the plasma membrane adenylyl cyclase 6 to regulate melanogenesis [53], and this interaction is mediated by the Ser/Pro-rich C-terminal region of STIM1. These reports, together with the finding that ORAI1- and SOCEdeficient fibroblasts showed impaired cAMP production and cAMP-dependent signaling [54], strongly support the direct relationship between Ca2+ depletion at ER stores, STIM1 acti-

As for other pathways, the regulation between STIM1 and PKA seems to be reciprocal. In addition to the CRAC channel ORAI1, the plasma membrane-resident STIM1 activates storeindependent arachidonic acid regulated Ca2+ (ARC) channels, and this activation depends on the phosphorylation of STIM1 at Thr389 by PKA, which requires the scaffold protein AKAP79 [55]. This phosphorylation triggers a structural change in the SOAR region of STIM1 (also

known as CAD) being essential for the selective activation of ARC channels [56].

or EGF [18, 19, 34].

**4.2. cAMP and PKA**

but not a site-specific phospho-specific antibody.

vation, and enhanced production of cAMP.
