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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 regulates SOCE and CRAC channel-mediated Ca2+ oscillations in T cells. (a) Measurement of SOCE in HeLa O+S cells expressing CRACR2A. Averaged responses from HeLa O+S cells expressing mCherry (n = 33 cells), CRACR2A-mCherry (n = 36), or CRACR2AEF2MUT-mCherry (n = 39) are shown. Bar graph shows peak [Ca2+]i values immediately upon addition of 2 mM Ca2+ (Left). A decrease in sustained [Ca2+]i is plotted as 1-Ca2+ss / Ca2+peak, where Ca2+ss represents steady state [Ca2+]i at 800 sec and Ca2+peak represents the peak [Ca2+]i (right). Bar graphs show average ± s.e.m. from three independent experiments. (b) Measurement of SOCE in Jurkat T cells expressing CRACR2A. Averaged responses from cells expressing mCherry (n = 55 cells), CRACR2A-mCherry (n = 50), or CRACR2AEF2MUT-mCherry (n = 60) are shown. Bar graph depicts average ± s.e.m. of [Ca2+]i before (basal, gray bars) and after (peak, open bars) store depletion from three independent experiments. * represents statistically significant differences in resting and stimulated [Ca2+]i (P < 0.001 by t-test). (c) Expression of CRACR2AEF2MUT in Jurkat T cells disrupts normal Ca2+ oscillations induced by thapsigargin. Jurkat T cells expressing mCherry or CRACR2AEF2MUT-mCherry were treated with 10 nM thapsigargin in 2 mM Ca2+ containing Ringer solution to induce asynchronous [Ca2+]i oscillations. Left panel shows pseudocolored images of transfected cells for [Ca2+]i (>70% transfection efficiency in each case) and right panel shows the traces of [Ca2+]i oscillations averaged from 50 cells. Data are representative of three independent experiments. Bar, 50 µm.
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Figure 6: CRACR2A regulates SOCE and CRAC channel-mediated Ca2+ oscillations in T cells. (a) Measurement of SOCE in HeLa O+S cells expressing CRACR2A. Averaged responses from HeLa O+S cells expressing mCherry (n = 33 cells), CRACR2A-mCherry (n = 36), or CRACR2AEF2MUT-mCherry (n = 39) are shown. Bar graph shows peak [Ca2+]i values immediately upon addition of 2 mM Ca2+ (Left). A decrease in sustained [Ca2+]i is plotted as 1-Ca2+ss / Ca2+peak, where Ca2+ss represents steady state [Ca2+]i at 800 sec and Ca2+peak represents the peak [Ca2+]i (right). Bar graphs show average ± s.e.m. from three independent experiments. (b) Measurement of SOCE in Jurkat T cells expressing CRACR2A. Averaged responses from cells expressing mCherry (n = 55 cells), CRACR2A-mCherry (n = 50), or CRACR2AEF2MUT-mCherry (n = 60) are shown. Bar graph depicts average ± s.e.m. of [Ca2+]i before (basal, gray bars) and after (peak, open bars) store depletion from three independent experiments. * represents statistically significant differences in resting and stimulated [Ca2+]i (P < 0.001 by t-test). (c) Expression of CRACR2AEF2MUT in Jurkat T cells disrupts normal Ca2+ oscillations induced by thapsigargin. Jurkat T cells expressing mCherry or CRACR2AEF2MUT-mCherry were treated with 10 nM thapsigargin in 2 mM Ca2+ containing Ringer solution to induce asynchronous [Ca2+]i oscillations. Left panel shows pseudocolored images of transfected cells for [Ca2+]i (>70% transfection efficiency in each case) and right panel shows the traces of [Ca2+]i oscillations averaged from 50 cells. Data are representative of three independent experiments. Bar, 50 µm.

Mentions: To examine how CRACR2A influences Orai1-mediated SOCE, we measured SOCE in HeLa O+S cells expressing mCherry, CRACR2A-mCherry, or CRACR2AEF2MUT-mCherry. CRACR2A expression significantly increased the peak [Ca2+]i, while CRACR2AEF2MUT-mCherry increased both peak and sustained [Ca2+]i (Fig. 6a). To examine its effect on endogenous CRAC channel function, we measured SOCE in Jurkat T cells expressing WT or mutant CRACR2A. Expression of CRACR2A as well as CRACR2AEF2MUT enhanced SOCE in Jurkat T cells, with the latter having a stronger effect (Fig. 6b). We also observed a significant increase in resting [Ca2+]i in Jurkat cells expressing CRACR2AEF2MUT (Fig. 6b, right panel and Fig. 6c), supporting our observations in HEK293 cells.


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 regulates SOCE and CRAC channel-mediated Ca2+ oscillations in T cells. (a) Measurement of SOCE in HeLa O+S cells expressing CRACR2A. Averaged responses from HeLa O+S cells expressing mCherry (n = 33 cells), CRACR2A-mCherry (n = 36), or CRACR2AEF2MUT-mCherry (n = 39) are shown. Bar graph shows peak [Ca2+]i values immediately upon addition of 2 mM Ca2+ (Left). A decrease in sustained [Ca2+]i is plotted as 1-Ca2+ss / Ca2+peak, where Ca2+ss represents steady state [Ca2+]i at 800 sec and Ca2+peak represents the peak [Ca2+]i (right). Bar graphs show average ± s.e.m. from three independent experiments. (b) Measurement of SOCE in Jurkat T cells expressing CRACR2A. Averaged responses from cells expressing mCherry (n = 55 cells), CRACR2A-mCherry (n = 50), or CRACR2AEF2MUT-mCherry (n = 60) are shown. Bar graph depicts average ± s.e.m. of [Ca2+]i before (basal, gray bars) and after (peak, open bars) store depletion from three independent experiments. * represents statistically significant differences in resting and stimulated [Ca2+]i (P < 0.001 by t-test). (c) Expression of CRACR2AEF2MUT in Jurkat T cells disrupts normal Ca2+ oscillations induced by thapsigargin. Jurkat T cells expressing mCherry or CRACR2AEF2MUT-mCherry were treated with 10 nM thapsigargin in 2 mM Ca2+ containing Ringer solution to induce asynchronous [Ca2+]i oscillations. Left panel shows pseudocolored images of transfected cells for [Ca2+]i (>70% transfection efficiency in each case) and right panel shows the traces of [Ca2+]i oscillations averaged from 50 cells. Data are representative of three independent experiments. Bar, 50 µm.
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Figure 6: CRACR2A regulates SOCE and CRAC channel-mediated Ca2+ oscillations in T cells. (a) Measurement of SOCE in HeLa O+S cells expressing CRACR2A. Averaged responses from HeLa O+S cells expressing mCherry (n = 33 cells), CRACR2A-mCherry (n = 36), or CRACR2AEF2MUT-mCherry (n = 39) are shown. Bar graph shows peak [Ca2+]i values immediately upon addition of 2 mM Ca2+ (Left). A decrease in sustained [Ca2+]i is plotted as 1-Ca2+ss / Ca2+peak, where Ca2+ss represents steady state [Ca2+]i at 800 sec and Ca2+peak represents the peak [Ca2+]i (right). Bar graphs show average ± s.e.m. from three independent experiments. (b) Measurement of SOCE in Jurkat T cells expressing CRACR2A. Averaged responses from cells expressing mCherry (n = 55 cells), CRACR2A-mCherry (n = 50), or CRACR2AEF2MUT-mCherry (n = 60) are shown. Bar graph depicts average ± s.e.m. of [Ca2+]i before (basal, gray bars) and after (peak, open bars) store depletion from three independent experiments. * represents statistically significant differences in resting and stimulated [Ca2+]i (P < 0.001 by t-test). (c) Expression of CRACR2AEF2MUT in Jurkat T cells disrupts normal Ca2+ oscillations induced by thapsigargin. Jurkat T cells expressing mCherry or CRACR2AEF2MUT-mCherry were treated with 10 nM thapsigargin in 2 mM Ca2+ containing Ringer solution to induce asynchronous [Ca2+]i oscillations. Left panel shows pseudocolored images of transfected cells for [Ca2+]i (>70% transfection efficiency in each case) and right panel shows the traces of [Ca2+]i oscillations averaged from 50 cells. Data are representative of three independent experiments. Bar, 50 µm.
Mentions: To examine how CRACR2A influences Orai1-mediated SOCE, we measured SOCE in HeLa O+S cells expressing mCherry, CRACR2A-mCherry, or CRACR2AEF2MUT-mCherry. CRACR2A expression significantly increased the peak [Ca2+]i, while CRACR2AEF2MUT-mCherry increased both peak and sustained [Ca2+]i (Fig. 6a). To examine its effect on endogenous CRAC channel function, we measured SOCE in Jurkat T cells expressing WT or mutant CRACR2A. Expression of CRACR2A as well as CRACR2AEF2MUT enhanced SOCE in Jurkat T cells, with the latter having a stronger effect (Fig. 6b). We also observed a significant increase in resting [Ca2+]i in Jurkat cells expressing CRACR2AEF2MUT (Fig. 6b, right panel and Fig. 6c), supporting our observations in HEK293 cells.

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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