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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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Identification of CRACR2A as a binding partner of Orai1 by large-scale affinity purification. (a) CRAC currents measured in HeLa cells stably expressing Orai1 and STIM1 (HeLa O+S cells). Left panel shows inwardly rectifying CRAC currents (red trace) obtained by subtracting 2-APB inhibited currents (blue trace) from whole-cell currents (black trace). The pipette solution contained 12 mM EGTA to deplete the intracellular Ca2+ stores and the external solution contained 6 mM CaCl2. Right panel shows current-voltage relationships of the currents in divalent free (DVF, red trace) or 6 mM CaCl2 (black) containing solution. (b) Glycerol gradient fractionation of DSP cross-linked HeLa O+S cells in store-filled (top two panels) and store-depleted (bottom two panels) conditions. Different fractions were separated on SDS-PAGE and immunoblotted for detection of Orai1 (left) and STIM1 (right). * represents fractions enriched in Orai1 or STIM1 proteins. Arrowheads denote the fractions in which MW markers were detected. For full scans see Supplementary Information, Fig. S12. (c) Affinity purification of Orai1 protein complex. The glycerol gradient fractions enriched in FLAG-Orai1 were pooled and immunoprecipitated with anti-FLAG resin. After elution with the FLAG peptide, fractions were separated on SDS-PAGE and visualized by silver staining. * indicates protein bands enriched in store-filled or store-depleted conditions. (d) Schematic showing the predicted domain structure of human CRACR2A protein. Human CRACR2A contains 395 amino acids with two predicted EF-hand motifs (SMART program) in its N terminus and a predicted coiled-coil domain with leucine rich (LR) sequence in its C terminus (Human Protein Reference Database and COILS). The mutants used in the current study are indicated.
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Figure 1: Identification of CRACR2A as a binding partner of Orai1 by large-scale affinity purification. (a) CRAC currents measured in HeLa cells stably expressing Orai1 and STIM1 (HeLa O+S cells). Left panel shows inwardly rectifying CRAC currents (red trace) obtained by subtracting 2-APB inhibited currents (blue trace) from whole-cell currents (black trace). The pipette solution contained 12 mM EGTA to deplete the intracellular Ca2+ stores and the external solution contained 6 mM CaCl2. Right panel shows current-voltage relationships of the currents in divalent free (DVF, red trace) or 6 mM CaCl2 (black) containing solution. (b) Glycerol gradient fractionation of DSP cross-linked HeLa O+S cells in store-filled (top two panels) and store-depleted (bottom two panels) conditions. Different fractions were separated on SDS-PAGE and immunoblotted for detection of Orai1 (left) and STIM1 (right). * represents fractions enriched in Orai1 or STIM1 proteins. Arrowheads denote the fractions in which MW markers were detected. For full scans see Supplementary Information, Fig. S12. (c) Affinity purification of Orai1 protein complex. The glycerol gradient fractions enriched in FLAG-Orai1 were pooled and immunoprecipitated with anti-FLAG resin. After elution with the FLAG peptide, fractions were separated on SDS-PAGE and visualized by silver staining. * indicates protein bands enriched in store-filled or store-depleted conditions. (d) Schematic showing the predicted domain structure of human CRACR2A protein. Human CRACR2A contains 395 amino acids with two predicted EF-hand motifs (SMART program) in its N terminus and a predicted coiled-coil domain with leucine rich (LR) sequence in its C terminus (Human Protein Reference Database and COILS). The mutants used in the current study are indicated.

Mentions: To identify novel regulators of the CRAC channel using Orai1 for affinity purification, we generated HeLa cells stably expressing Orai1 and STIM1 (HeLa O+S cells). Presence of an active CRAC channel complex was verified by detection of amplified CRAC currents (Fig. 1a, Supplementary Information, Fig. S1a)19–21. To capture Orai1 in its native complex, cells were treated with different concentrations of a membrane-permeable cross-linker, dithiobis succinimidyl propionate (DSP) and immunoblotted for Orai1. Upon treatment with 0.5 mM DSP, Orai1 and STIM1 were detected in high molecular weight complexes in non-reducing SDS-PAGE (Supplementary Information, Fig. S1b). These complexes were applied onto a 20–50% glycerol gradient to determine their size. The size of the Orai1 protein complex was estimated to be ~700 kDa under resting conditions and ~670 kDa after store depletion (Fig. 1b). Under resting conditions, STIM1 was primarily detected in a ~200 kDa protein complex (Fig. 1b, right), possibly as a dimer, whereas it co-migrated with Orai1 upon store depletion. These results are consistent with the observation that STIM1 self-associates at rest and forms a high molecular weight (MW) protein complex upon stimulation9, 30, 31.


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)

Identification of CRACR2A as a binding partner of Orai1 by large-scale affinity purification. (a) CRAC currents measured in HeLa cells stably expressing Orai1 and STIM1 (HeLa O+S cells). Left panel shows inwardly rectifying CRAC currents (red trace) obtained by subtracting 2-APB inhibited currents (blue trace) from whole-cell currents (black trace). The pipette solution contained 12 mM EGTA to deplete the intracellular Ca2+ stores and the external solution contained 6 mM CaCl2. Right panel shows current-voltage relationships of the currents in divalent free (DVF, red trace) or 6 mM CaCl2 (black) containing solution. (b) Glycerol gradient fractionation of DSP cross-linked HeLa O+S cells in store-filled (top two panels) and store-depleted (bottom two panels) conditions. Different fractions were separated on SDS-PAGE and immunoblotted for detection of Orai1 (left) and STIM1 (right). * represents fractions enriched in Orai1 or STIM1 proteins. Arrowheads denote the fractions in which MW markers were detected. For full scans see Supplementary Information, Fig. S12. (c) Affinity purification of Orai1 protein complex. The glycerol gradient fractions enriched in FLAG-Orai1 were pooled and immunoprecipitated with anti-FLAG resin. After elution with the FLAG peptide, fractions were separated on SDS-PAGE and visualized by silver staining. * indicates protein bands enriched in store-filled or store-depleted conditions. (d) Schematic showing the predicted domain structure of human CRACR2A protein. Human CRACR2A contains 395 amino acids with two predicted EF-hand motifs (SMART program) in its N terminus and a predicted coiled-coil domain with leucine rich (LR) sequence in its C terminus (Human Protein Reference Database and COILS). The mutants used in the current study are indicated.
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Related In: Results  -  Collection

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Figure 1: Identification of CRACR2A as a binding partner of Orai1 by large-scale affinity purification. (a) CRAC currents measured in HeLa cells stably expressing Orai1 and STIM1 (HeLa O+S cells). Left panel shows inwardly rectifying CRAC currents (red trace) obtained by subtracting 2-APB inhibited currents (blue trace) from whole-cell currents (black trace). The pipette solution contained 12 mM EGTA to deplete the intracellular Ca2+ stores and the external solution contained 6 mM CaCl2. Right panel shows current-voltage relationships of the currents in divalent free (DVF, red trace) or 6 mM CaCl2 (black) containing solution. (b) Glycerol gradient fractionation of DSP cross-linked HeLa O+S cells in store-filled (top two panels) and store-depleted (bottom two panels) conditions. Different fractions were separated on SDS-PAGE and immunoblotted for detection of Orai1 (left) and STIM1 (right). * represents fractions enriched in Orai1 or STIM1 proteins. Arrowheads denote the fractions in which MW markers were detected. For full scans see Supplementary Information, Fig. S12. (c) Affinity purification of Orai1 protein complex. The glycerol gradient fractions enriched in FLAG-Orai1 were pooled and immunoprecipitated with anti-FLAG resin. After elution with the FLAG peptide, fractions were separated on SDS-PAGE and visualized by silver staining. * indicates protein bands enriched in store-filled or store-depleted conditions. (d) Schematic showing the predicted domain structure of human CRACR2A protein. Human CRACR2A contains 395 amino acids with two predicted EF-hand motifs (SMART program) in its N terminus and a predicted coiled-coil domain with leucine rich (LR) sequence in its C terminus (Human Protein Reference Database and COILS). The mutants used in the current study are indicated.
Mentions: To identify novel regulators of the CRAC channel using Orai1 for affinity purification, we generated HeLa cells stably expressing Orai1 and STIM1 (HeLa O+S cells). Presence of an active CRAC channel complex was verified by detection of amplified CRAC currents (Fig. 1a, Supplementary Information, Fig. S1a)19–21. To capture Orai1 in its native complex, cells were treated with different concentrations of a membrane-permeable cross-linker, dithiobis succinimidyl propionate (DSP) and immunoblotted for Orai1. Upon treatment with 0.5 mM DSP, Orai1 and STIM1 were detected in high molecular weight complexes in non-reducing SDS-PAGE (Supplementary Information, Fig. S1b). These complexes were applied onto a 20–50% glycerol gradient to determine their size. The size of the Orai1 protein complex was estimated to be ~700 kDa under resting conditions and ~670 kDa after store depletion (Fig. 1b). Under resting conditions, STIM1 was primarily detected in a ~200 kDa protein complex (Fig. 1b, right), possibly as a dimer, whereas it co-migrated with Orai1 upon store depletion. These results are consistent with the observation that STIM1 self-associates at rest and forms a high molecular weight (MW) protein complex upon stimulation9, 30, 31.

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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