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A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells.

Srikanth S, Jung HJ, Kim KD, Souda P, Whitelegge J, Gwack Y - Nat. Cell Biol. (2010)

Bottom Line: Studies using knockdown mediated by small interfering RNA (siRNA) and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion.Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca(2+) and induced cell death, suggesting its active interaction with CRAC channels.These observations implicate CRACR2A, a novel Ca(2+) binding protein that is highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, David Geffen School of Medicine at the University of California, Los Angeles, California 90095, USA.

ABSTRACT
Orai1 and STIM1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate store-operated Ca(2+) entry (SOCE) in immune cells. Although it is known that Orai1 and STIM1 co-cluster and physically interact to mediate SOCE, the cytoplasmic machinery modulating these functions remains poorly understood. We sought to find modulators of Orai1 and STIM1 using affinity protein purification and identified a novel EF-hand protein, CRACR2A (also called CRAC regulator 2A, EFCAB4B or FLJ33805). We show that CRACR2A interacts directly with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca(2+) concentrations. Studies using knockdown mediated by small interfering RNA (siRNA) and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion. Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca(2+) and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca(2+) binding protein that is highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.

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CRACR2A directly interacts with Orai and STIM1. (a) CRACR2A co-immunoprecipitates with Orai1 and STIM1. Left two panels: HeLa cells stably expressing either STIM1 (HeLa), or STIM1 and Orai1 (Orai1) were transfected with a plasmid encoding CRACR2A. After immunoprecipitation with anti-FLAG resin (Orai1) and elution with the FLAG peptide, eluted fractions were immunoblotted using anti-STIM1, anti-CRACR2A, and anti-FLAG (Orai1) antibodies. Right two panels: HeLa cells stably expressing STIM1 were transfected with plasmids encoding Myc-Orai1 alone (Vec) or together with FLAG-CRACR2A (CRACR2A). After immunoprecipitation with anti-FLAG resin (CRACR2A), precipitates were immunoblotted with anti-STIM1, anti-Myc (Orai1), and anti-FLAG (CRACR2A) antibodies. (b) CRACR2A directly binds to the N terminus of Orai1. Left panels: GST pulldown analysis was performed between full-length GST-Orai1 and 6× His-tagged CRACR2A or CRACR2AEF2MUT proteins in the presence or absence of 2 mM CaCl2 (left panels). Lower panel shows input levels of GST-Orai1. Right panels: pulldown analysis with GST-fused fragments of the N terminus (N-, amino acids 64–91), intracellular loop (intra, amino acids 137–173) and the C terminus (C-, amino acids 254–301) of Orai1 and full-length 6× His-tagged CRACR2A. Lower panel shows input levels of Orai1 fragments. All the recombinant proteins were purified from E. coli. (c) CRACR2A interacts with Orai2 and Orai3 proteins. HeLa cells stably expressing STIM1 were transfected with plasmids encoding FLAG-Orai2 (FO2, top) or FLAG-Orai3 (FO3, bottom) together with Myc-CRACR2A. Lysates were immunprecipitated with anti-FLAG resin and immunoblotted for detection of CRACR2A (anti-Myc, left, indicated with *) and Orai2 or Orai3 (anti-FLAG, right). NS, non-specific band. (d) Interaction between the cytoplasmic fragments of STIM1 and CRACR2A. A schematic of STIM1 with cytoplasmic domains of coiled-coil (CC, amino-acid residues 250–400), serine/threonine rich sequence (S/T, residues 400–600), and PEST sequence (residues 600–685) is shown. The CAD/SOAR fragment is indicated. Purified GST-fused STIM1 fragments were immunoblotted with anti-GST antibodies (left). Their interactions with purified full-length Orai1, CRACR2A, or STIM1 proteins were assessed by pulldown analysis in the absence (−Ca2+) or presence (+Ca2+) of 2 mM CaCl2. STIM1 fragments and CRACR2A were purified from E. coli, while full-length Orai1 and STIM1 proteins were purified from baculovirus infected insect cells. For full scans see Supplementary Information, Fig. S12.
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Figure 2: CRACR2A directly interacts with Orai and STIM1. (a) CRACR2A co-immunoprecipitates with Orai1 and STIM1. Left two panels: HeLa cells stably expressing either STIM1 (HeLa), or STIM1 and Orai1 (Orai1) were transfected with a plasmid encoding CRACR2A. After immunoprecipitation with anti-FLAG resin (Orai1) and elution with the FLAG peptide, eluted fractions were immunoblotted using anti-STIM1, anti-CRACR2A, and anti-FLAG (Orai1) antibodies. Right two panels: HeLa cells stably expressing STIM1 were transfected with plasmids encoding Myc-Orai1 alone (Vec) or together with FLAG-CRACR2A (CRACR2A). After immunoprecipitation with anti-FLAG resin (CRACR2A), precipitates were immunoblotted with anti-STIM1, anti-Myc (Orai1), and anti-FLAG (CRACR2A) antibodies. (b) CRACR2A directly binds to the N terminus of Orai1. Left panels: GST pulldown analysis was performed between full-length GST-Orai1 and 6× His-tagged CRACR2A or CRACR2AEF2MUT proteins in the presence or absence of 2 mM CaCl2 (left panels). Lower panel shows input levels of GST-Orai1. Right panels: pulldown analysis with GST-fused fragments of the N terminus (N-, amino acids 64–91), intracellular loop (intra, amino acids 137–173) and the C terminus (C-, amino acids 254–301) of Orai1 and full-length 6× His-tagged CRACR2A. Lower panel shows input levels of Orai1 fragments. All the recombinant proteins were purified from E. coli. (c) CRACR2A interacts with Orai2 and Orai3 proteins. HeLa cells stably expressing STIM1 were transfected with plasmids encoding FLAG-Orai2 (FO2, top) or FLAG-Orai3 (FO3, bottom) together with Myc-CRACR2A. Lysates were immunprecipitated with anti-FLAG resin and immunoblotted for detection of CRACR2A (anti-Myc, left, indicated with *) and Orai2 or Orai3 (anti-FLAG, right). NS, non-specific band. (d) Interaction between the cytoplasmic fragments of STIM1 and CRACR2A. A schematic of STIM1 with cytoplasmic domains of coiled-coil (CC, amino-acid residues 250–400), serine/threonine rich sequence (S/T, residues 400–600), and PEST sequence (residues 600–685) is shown. The CAD/SOAR fragment is indicated. Purified GST-fused STIM1 fragments were immunoblotted with anti-GST antibodies (left). Their interactions with purified full-length Orai1, CRACR2A, or STIM1 proteins were assessed by pulldown analysis in the absence (−Ca2+) or presence (+Ca2+) of 2 mM CaCl2. STIM1 fragments and CRACR2A were purified from E. coli, while full-length Orai1 and STIM1 proteins were purified from baculovirus infected insect cells. For full scans see Supplementary Information, Fig. S12.

Mentions: To examine whether CRACR2A binds Orai1, we carried out immunoprecipitation and pulldown analysis. For immunoprecipitation experiments, HeLa cells stably expressing either STIM1 alone or STIM1 and Orai1 proteins were transiently transfected with a plasmid encoding CRACR2A. As seen in Fig. 2a, both STIM1 and CRACR2A co-immunoprecipitated with Orai1 and showed enhanced binding upon store depletion. These results were further validated by reverse immunoprecipitation using FLAG-tagged CRACR2A (Fig. 2a, right). To test for a direct interaction between CRACR2A and Orai1, recombinant proteins purified from E. coli (Supplementary Information, Fig. S4) were used for in vitro binding assays. GST pulldown analysis showed a strong interaction between CRACR2A and Orai1 in the absence of Ca2+ while the binding was minimal in the presence of 2 mM Ca2+ (Fig. 2b, first panel). To confirm an inhibitory role of Ca2+, we used an EF-hand mutant of CRACR2A that abolishes Ca2+ binding (CRACR2AEF2MUT, Fig. 1d and see below). As shown in Fig. 2b, Ca2+ had no effect on Orai1-CRACR2AEF2MUT interaction, supporting its inhibitory role.


A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells.

Srikanth S, Jung HJ, Kim KD, Souda P, Whitelegge J, Gwack Y - Nat. Cell Biol. (2010)

CRACR2A directly interacts with Orai and STIM1. (a) CRACR2A co-immunoprecipitates with Orai1 and STIM1. Left two panels: HeLa cells stably expressing either STIM1 (HeLa), or STIM1 and Orai1 (Orai1) were transfected with a plasmid encoding CRACR2A. After immunoprecipitation with anti-FLAG resin (Orai1) and elution with the FLAG peptide, eluted fractions were immunoblotted using anti-STIM1, anti-CRACR2A, and anti-FLAG (Orai1) antibodies. Right two panels: HeLa cells stably expressing STIM1 were transfected with plasmids encoding Myc-Orai1 alone (Vec) or together with FLAG-CRACR2A (CRACR2A). After immunoprecipitation with anti-FLAG resin (CRACR2A), precipitates were immunoblotted with anti-STIM1, anti-Myc (Orai1), and anti-FLAG (CRACR2A) antibodies. (b) CRACR2A directly binds to the N terminus of Orai1. Left panels: GST pulldown analysis was performed between full-length GST-Orai1 and 6× His-tagged CRACR2A or CRACR2AEF2MUT proteins in the presence or absence of 2 mM CaCl2 (left panels). Lower panel shows input levels of GST-Orai1. Right panels: pulldown analysis with GST-fused fragments of the N terminus (N-, amino acids 64–91), intracellular loop (intra, amino acids 137–173) and the C terminus (C-, amino acids 254–301) of Orai1 and full-length 6× His-tagged CRACR2A. Lower panel shows input levels of Orai1 fragments. All the recombinant proteins were purified from E. coli. (c) CRACR2A interacts with Orai2 and Orai3 proteins. HeLa cells stably expressing STIM1 were transfected with plasmids encoding FLAG-Orai2 (FO2, top) or FLAG-Orai3 (FO3, bottom) together with Myc-CRACR2A. Lysates were immunprecipitated with anti-FLAG resin and immunoblotted for detection of CRACR2A (anti-Myc, left, indicated with *) and Orai2 or Orai3 (anti-FLAG, right). NS, non-specific band. (d) Interaction between the cytoplasmic fragments of STIM1 and CRACR2A. A schematic of STIM1 with cytoplasmic domains of coiled-coil (CC, amino-acid residues 250–400), serine/threonine rich sequence (S/T, residues 400–600), and PEST sequence (residues 600–685) is shown. The CAD/SOAR fragment is indicated. Purified GST-fused STIM1 fragments were immunoblotted with anti-GST antibodies (left). Their interactions with purified full-length Orai1, CRACR2A, or STIM1 proteins were assessed by pulldown analysis in the absence (−Ca2+) or presence (+Ca2+) of 2 mM CaCl2. STIM1 fragments and CRACR2A were purified from E. coli, while full-length Orai1 and STIM1 proteins were purified from baculovirus infected insect cells. For full scans see Supplementary Information, Fig. S12.
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Figure 2: CRACR2A directly interacts with Orai and STIM1. (a) CRACR2A co-immunoprecipitates with Orai1 and STIM1. Left two panels: HeLa cells stably expressing either STIM1 (HeLa), or STIM1 and Orai1 (Orai1) were transfected with a plasmid encoding CRACR2A. After immunoprecipitation with anti-FLAG resin (Orai1) and elution with the FLAG peptide, eluted fractions were immunoblotted using anti-STIM1, anti-CRACR2A, and anti-FLAG (Orai1) antibodies. Right two panels: HeLa cells stably expressing STIM1 were transfected with plasmids encoding Myc-Orai1 alone (Vec) or together with FLAG-CRACR2A (CRACR2A). After immunoprecipitation with anti-FLAG resin (CRACR2A), precipitates were immunoblotted with anti-STIM1, anti-Myc (Orai1), and anti-FLAG (CRACR2A) antibodies. (b) CRACR2A directly binds to the N terminus of Orai1. Left panels: GST pulldown analysis was performed between full-length GST-Orai1 and 6× His-tagged CRACR2A or CRACR2AEF2MUT proteins in the presence or absence of 2 mM CaCl2 (left panels). Lower panel shows input levels of GST-Orai1. Right panels: pulldown analysis with GST-fused fragments of the N terminus (N-, amino acids 64–91), intracellular loop (intra, amino acids 137–173) and the C terminus (C-, amino acids 254–301) of Orai1 and full-length 6× His-tagged CRACR2A. Lower panel shows input levels of Orai1 fragments. All the recombinant proteins were purified from E. coli. (c) CRACR2A interacts with Orai2 and Orai3 proteins. HeLa cells stably expressing STIM1 were transfected with plasmids encoding FLAG-Orai2 (FO2, top) or FLAG-Orai3 (FO3, bottom) together with Myc-CRACR2A. Lysates were immunprecipitated with anti-FLAG resin and immunoblotted for detection of CRACR2A (anti-Myc, left, indicated with *) and Orai2 or Orai3 (anti-FLAG, right). NS, non-specific band. (d) Interaction between the cytoplasmic fragments of STIM1 and CRACR2A. A schematic of STIM1 with cytoplasmic domains of coiled-coil (CC, amino-acid residues 250–400), serine/threonine rich sequence (S/T, residues 400–600), and PEST sequence (residues 600–685) is shown. The CAD/SOAR fragment is indicated. Purified GST-fused STIM1 fragments were immunoblotted with anti-GST antibodies (left). Their interactions with purified full-length Orai1, CRACR2A, or STIM1 proteins were assessed by pulldown analysis in the absence (−Ca2+) or presence (+Ca2+) of 2 mM CaCl2. STIM1 fragments and CRACR2A were purified from E. coli, while full-length Orai1 and STIM1 proteins were purified from baculovirus infected insect cells. For full scans see Supplementary Information, Fig. S12.
Mentions: To examine whether CRACR2A binds Orai1, we carried out immunoprecipitation and pulldown analysis. For immunoprecipitation experiments, HeLa cells stably expressing either STIM1 alone or STIM1 and Orai1 proteins were transiently transfected with a plasmid encoding CRACR2A. As seen in Fig. 2a, both STIM1 and CRACR2A co-immunoprecipitated with Orai1 and showed enhanced binding upon store depletion. These results were further validated by reverse immunoprecipitation using FLAG-tagged CRACR2A (Fig. 2a, right). To test for a direct interaction between CRACR2A and Orai1, recombinant proteins purified from E. coli (Supplementary Information, Fig. S4) were used for in vitro binding assays. GST pulldown analysis showed a strong interaction between CRACR2A and Orai1 in the absence of Ca2+ while the binding was minimal in the presence of 2 mM Ca2+ (Fig. 2b, first panel). To confirm an inhibitory role of Ca2+, we used an EF-hand mutant of CRACR2A that abolishes Ca2+ binding (CRACR2AEF2MUT, Fig. 1d and see below). As shown in Fig. 2b, Ca2+ had no effect on Orai1-CRACR2AEF2MUT interaction, supporting its inhibitory role.

Bottom Line: Studies using knockdown mediated by small interfering RNA (siRNA) and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion.Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca(2+) and induced cell death, suggesting its active interaction with CRAC channels.These observations implicate CRACR2A, a novel Ca(2+) binding protein that is highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, David Geffen School of Medicine at the University of California, Los Angeles, California 90095, USA.

ABSTRACT
Orai1 and STIM1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate store-operated Ca(2+) entry (SOCE) in immune cells. Although it is known that Orai1 and STIM1 co-cluster and physically interact to mediate SOCE, the cytoplasmic machinery modulating these functions remains poorly understood. We sought to find modulators of Orai1 and STIM1 using affinity protein purification and identified a novel EF-hand protein, CRACR2A (also called CRAC regulator 2A, EFCAB4B or FLJ33805). We show that CRACR2A interacts directly with Orai1 and STIM1, forming a ternary complex that dissociates at elevated Ca(2+) concentrations. Studies using knockdown mediated by small interfering RNA (siRNA) and mutagenesis show that CRACR2A is important for clustering of Orai1 and STIM1 upon store depletion. Expression of an EF-hand mutant of CRACR2A enhanced STIM1 clustering, elevated cytoplasmic Ca(2+) and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca(2+) binding protein that is highly expressed in T cells and conserved in vertebrates, as a key regulator of CRAC channel-mediated SOCE.

Show MeSH
Related in: MedlinePlus