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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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Expression of EF-hand mutant of CRACR2A induces cell death in Jurkat T cells. (a) Cell death induced by CRACR2A overexpression in T cells. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were examined for cell death by Annexin V staining at 24, 48, and 72 h after transfection. One representative from three independent experiments is shown. (b) Quantification of the data shown in (a). Bar graphs represent average ± s.e.m. from three independent experiments. (c) Live cell population is reduced in Jurkat cells expressing CRACR2AEF2MUT-GFP. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were assessed for percent of live, GFP-positive populations at 24, 48 and 72 h after transfection. Cells with high expression of GFP (GFPhigh) were analyzed by flow cytometry. Data represents average ± s.e.m. from three independent experiments. (d) A proposed model showing possible role(s) of CRACR2A in CRAC channel function. Under resting conditions, Orai1 and STIM1 are distributed at the PM and ER membranes, respectively (1. Resting state) while CRACR2A (green) localizes in the cytoplasm. Upon store depletion, Orai1 and STIM1 translocate to form clusters at the junctional regions between PM and ER (State 2). CRACR2A may either be actively involved in translocation of Orai1 (i), STIM1 (ii) or both (iii). It is also possible that CRACR2A passively interacts with Orai1 and STIM1 at sites of clustering (iv). Based on our data, we propose that CRACR2A is important for stabilization of Orai1- STIM1 complex via direct protein interaction under physiological conditions where amounts of Orai1 and STIM1 proteins are limiting (State 3). Upon increase of cytoplasmic [Ca2+] via opening of CRAC channels, the EF hands of CRACR2A bind Ca2+ ions, resulting in its dissociation from Orai1 and STIM1 (State 4), thereby destabilizing the Orai1-STIM1 complex. The EF-hands of STIM1 and CRACR2A are indicated and their Ca2+-bound status is colored in red. The schematic does not represent molecular stoichiometry of Orai1, STIM, and CRACR2A proteins.
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Figure 7: Expression of EF-hand mutant of CRACR2A induces cell death in Jurkat T cells. (a) Cell death induced by CRACR2A overexpression in T cells. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were examined for cell death by Annexin V staining at 24, 48, and 72 h after transfection. One representative from three independent experiments is shown. (b) Quantification of the data shown in (a). Bar graphs represent average ± s.e.m. from three independent experiments. (c) Live cell population is reduced in Jurkat cells expressing CRACR2AEF2MUT-GFP. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were assessed for percent of live, GFP-positive populations at 24, 48 and 72 h after transfection. Cells with high expression of GFP (GFPhigh) were analyzed by flow cytometry. Data represents average ± s.e.m. from three independent experiments. (d) A proposed model showing possible role(s) of CRACR2A in CRAC channel function. Under resting conditions, Orai1 and STIM1 are distributed at the PM and ER membranes, respectively (1. Resting state) while CRACR2A (green) localizes in the cytoplasm. Upon store depletion, Orai1 and STIM1 translocate to form clusters at the junctional regions between PM and ER (State 2). CRACR2A may either be actively involved in translocation of Orai1 (i), STIM1 (ii) or both (iii). It is also possible that CRACR2A passively interacts with Orai1 and STIM1 at sites of clustering (iv). Based on our data, we propose that CRACR2A is important for stabilization of Orai1- STIM1 complex via direct protein interaction under physiological conditions where amounts of Orai1 and STIM1 proteins are limiting (State 3). Upon increase of cytoplasmic [Ca2+] via opening of CRAC channels, the EF hands of CRACR2A bind Ca2+ ions, resulting in its dissociation from Orai1 and STIM1 (State 4), thereby destabilizing the Orai1-STIM1 complex. The EF-hands of STIM1 and CRACR2A are indicated and their Ca2+-bound status is colored in red. The schematic does not represent molecular stoichiometry of Orai1, STIM, and CRACR2A proteins.

Mentions: Since overexpression of CRACR2AEF2MUT increased basal [Ca2+]i, we measured its effect on cell death in T cells. Jurkat T cells expressing CRACR2A-GFP or CRACR2AEF2MUT-GFP showed twofold and fivefold increase in apoptotic cell population respectively, when compared with GFP-expressing cells at 72 h post transfection (Fig. 7a, b). As a result, population of cells expressing CRACR2A-GFP or CRACR2AEF2MUT-GFP drastically decreased by 72 h while that of control cells remained unchanged (Fig. 7c). These results suggest that CRACR2A plays a pivotal role in regulation of [Ca2+]i and an uncontrolled increase in [Ca2+]i upon expression of CRACR2AEF2MUT can result in cell death.


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)

Expression of EF-hand mutant of CRACR2A induces cell death in Jurkat T cells. (a) Cell death induced by CRACR2A overexpression in T cells. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were examined for cell death by Annexin V staining at 24, 48, and 72 h after transfection. One representative from three independent experiments is shown. (b) Quantification of the data shown in (a). Bar graphs represent average ± s.e.m. from three independent experiments. (c) Live cell population is reduced in Jurkat cells expressing CRACR2AEF2MUT-GFP. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were assessed for percent of live, GFP-positive populations at 24, 48 and 72 h after transfection. Cells with high expression of GFP (GFPhigh) were analyzed by flow cytometry. Data represents average ± s.e.m. from three independent experiments. (d) A proposed model showing possible role(s) of CRACR2A in CRAC channel function. Under resting conditions, Orai1 and STIM1 are distributed at the PM and ER membranes, respectively (1. Resting state) while CRACR2A (green) localizes in the cytoplasm. Upon store depletion, Orai1 and STIM1 translocate to form clusters at the junctional regions between PM and ER (State 2). CRACR2A may either be actively involved in translocation of Orai1 (i), STIM1 (ii) or both (iii). It is also possible that CRACR2A passively interacts with Orai1 and STIM1 at sites of clustering (iv). Based on our data, we propose that CRACR2A is important for stabilization of Orai1- STIM1 complex via direct protein interaction under physiological conditions where amounts of Orai1 and STIM1 proteins are limiting (State 3). Upon increase of cytoplasmic [Ca2+] via opening of CRAC channels, the EF hands of CRACR2A bind Ca2+ ions, resulting in its dissociation from Orai1 and STIM1 (State 4), thereby destabilizing the Orai1-STIM1 complex. The EF-hands of STIM1 and CRACR2A are indicated and their Ca2+-bound status is colored in red. The schematic does not represent molecular stoichiometry of Orai1, STIM, and CRACR2A proteins.
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Figure 7: Expression of EF-hand mutant of CRACR2A induces cell death in Jurkat T cells. (a) Cell death induced by CRACR2A overexpression in T cells. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were examined for cell death by Annexin V staining at 24, 48, and 72 h after transfection. One representative from three independent experiments is shown. (b) Quantification of the data shown in (a). Bar graphs represent average ± s.e.m. from three independent experiments. (c) Live cell population is reduced in Jurkat cells expressing CRACR2AEF2MUT-GFP. Jurkat T cells expressing GFP, CRACR2A-GFP or CRACR2AEF2MUT-GFP were assessed for percent of live, GFP-positive populations at 24, 48 and 72 h after transfection. Cells with high expression of GFP (GFPhigh) were analyzed by flow cytometry. Data represents average ± s.e.m. from three independent experiments. (d) A proposed model showing possible role(s) of CRACR2A in CRAC channel function. Under resting conditions, Orai1 and STIM1 are distributed at the PM and ER membranes, respectively (1. Resting state) while CRACR2A (green) localizes in the cytoplasm. Upon store depletion, Orai1 and STIM1 translocate to form clusters at the junctional regions between PM and ER (State 2). CRACR2A may either be actively involved in translocation of Orai1 (i), STIM1 (ii) or both (iii). It is also possible that CRACR2A passively interacts with Orai1 and STIM1 at sites of clustering (iv). Based on our data, we propose that CRACR2A is important for stabilization of Orai1- STIM1 complex via direct protein interaction under physiological conditions where amounts of Orai1 and STIM1 proteins are limiting (State 3). Upon increase of cytoplasmic [Ca2+] via opening of CRAC channels, the EF hands of CRACR2A bind Ca2+ ions, resulting in its dissociation from Orai1 and STIM1 (State 4), thereby destabilizing the Orai1-STIM1 complex. The EF-hands of STIM1 and CRACR2A are indicated and their Ca2+-bound status is colored in red. The schematic does not represent molecular stoichiometry of Orai1, STIM, and CRACR2A proteins.
Mentions: Since overexpression of CRACR2AEF2MUT increased basal [Ca2+]i, we measured its effect on cell death in T cells. Jurkat T cells expressing CRACR2A-GFP or CRACR2AEF2MUT-GFP showed twofold and fivefold increase in apoptotic cell population respectively, when compared with GFP-expressing cells at 72 h post transfection (Fig. 7a, b). As a result, population of cells expressing CRACR2A-GFP or CRACR2AEF2MUT-GFP drastically decreased by 72 h while that of control cells remained unchanged (Fig. 7c). These results suggest that CRACR2A plays a pivotal role in regulation of [Ca2+]i and an uncontrolled increase in [Ca2+]i upon expression of CRACR2AEF2MUT can result in cell death.

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