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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 plays an important role in Orai1-mediated SOCE in T cells. (a) SOCE measurements in Jurkat and HEK293 cells depleted of CRACR2A and CRACR2B. Top panel, averaged responses from Jurkat T cells: scrambled (Scr, n = 75 cells), CRACR2A (R2A, n = 79), or CRACR2B (R2B, n = 70) siRNAs. Bottom, averaged responses from HEK293 cells: Scr (n = 41), R2A (n = 47) or R2B (n = 49). Bar graphs represent averaged peak [Ca2+]i ± s.e.m. from three independent experiments. (b) Measurement of IL-2 expression in Jurkat T cells transfected with siRNAs. A representative of three independent experiments is shown. (c) Real-time PCR analysis of human CRACR2A and CRACR2B transcripts from various tissues and cell lines. Normalized mRNA levels are plotted relative to those of brain tissue. Data represent average ± s.d. from 2 independent experiments performed in triplicate. * indicates tissues or cell-lines showing high expression of CRACR2A transcripts, distinct from CRACR2B. (d) Expression of CRACR2A and CRACR2B in murine primary cells. The mRNA levels of CRACR2A or CRACR2B were measured from mouse embryonic fibroblasts (MEFs), thymocytes (Thy), naïve CD4+ T cells (Naïve), effector CD4+ (ThN), or CD8+ (CTL) T cells. Normalized mRNA levels are plotted relative to those of MEFs. N.D., not detected. Data represent average ± s.d. from 2 independent experiments performed in triplicate. (e) Examination of functional redundancy between CRACR2 proteins. SOCE was measured in HEK293 cells stably expressing control (Scr, black trace, n = 46 cells) or CRACR2B shRNA (red, n = 49). CRACR2B-depleted cells with ectopic expression of CRACR2A (R2A, blue, n = 41) or CRACR2B (R2B, cyan, n = 43) were examined for SOCE. A representative of three independent experiments is shown. The plasmids contain IRES-GFP and GFP+ cells were selected for analysis. (f) Effect of CRACR2 protein expression on Orai1-mediated SOCE. SOCE was measured in Orai1- MEFs expressing CRACR2A or CRACR2B together with Orai1. Each trace shows averaged responses from 25 (vector), 30 (Orai1), 35 (Orai1+R2A) or 33 (Orai1+R2B) MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.
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Figure 3: CRACR2A plays an important role in Orai1-mediated SOCE in T cells. (a) SOCE measurements in Jurkat and HEK293 cells depleted of CRACR2A and CRACR2B. Top panel, averaged responses from Jurkat T cells: scrambled (Scr, n = 75 cells), CRACR2A (R2A, n = 79), or CRACR2B (R2B, n = 70) siRNAs. Bottom, averaged responses from HEK293 cells: Scr (n = 41), R2A (n = 47) or R2B (n = 49). Bar graphs represent averaged peak [Ca2+]i ± s.e.m. from three independent experiments. (b) Measurement of IL-2 expression in Jurkat T cells transfected with siRNAs. A representative of three independent experiments is shown. (c) Real-time PCR analysis of human CRACR2A and CRACR2B transcripts from various tissues and cell lines. Normalized mRNA levels are plotted relative to those of brain tissue. Data represent average ± s.d. from 2 independent experiments performed in triplicate. * indicates tissues or cell-lines showing high expression of CRACR2A transcripts, distinct from CRACR2B. (d) Expression of CRACR2A and CRACR2B in murine primary cells. The mRNA levels of CRACR2A or CRACR2B were measured from mouse embryonic fibroblasts (MEFs), thymocytes (Thy), naïve CD4+ T cells (Naïve), effector CD4+ (ThN), or CD8+ (CTL) T cells. Normalized mRNA levels are plotted relative to those of MEFs. N.D., not detected. Data represent average ± s.d. from 2 independent experiments performed in triplicate. (e) Examination of functional redundancy between CRACR2 proteins. SOCE was measured in HEK293 cells stably expressing control (Scr, black trace, n = 46 cells) or CRACR2B shRNA (red, n = 49). CRACR2B-depleted cells with ectopic expression of CRACR2A (R2A, blue, n = 41) or CRACR2B (R2B, cyan, n = 43) were examined for SOCE. A representative of three independent experiments is shown. The plasmids contain IRES-GFP and GFP+ cells were selected for analysis. (f) Effect of CRACR2 protein expression on Orai1-mediated SOCE. SOCE was measured in Orai1- MEFs expressing CRACR2A or CRACR2B together with Orai1. Each trace shows averaged responses from 25 (vector), 30 (Orai1), 35 (Orai1+R2A) or 33 (Orai1+R2B) MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.

Mentions: To examine the physiological role of CRACR2 proteins, we measured SOCE in Jurkat T cells upon siRNA-mediated depletion of CRACR2A or CRACR2B. As seen in Fig. 3a, depletion of CRACR2A decreased SOCE by 50% while that of CRACR2B had a mild effect. Consistent with a decrease in SOCE, depletion of CRACR2A resulted in a stronger impairment in IL-2 production by Jurkat T cells than CRACR2B knockdown (Fig. 3b). Surprisingly, in HEK293 cells, depletion of CRACR2B had a stronger effect on SOCE than that of CRACR2A (Fig. 3a, bottom). Specificity and efficiency of CRACR2 knockdown was examined by RT-PCR and immunoblotting (Supplementary Information, Fig. S6a–d). To determine the reason for the differences between Jurkat and HEK293 cells, we measured the expression levels of CRACR2 transcripts in various human tissues and cell lines. Quantitative RT-PCR analysis revealed abundant expression of CRACR2A transcripts in spleen and thymus distinct from CRACR2B (Fig. 3c). Among cell lines, CRACR2A transcripts were higher in Jurkat T cells when compared with HEK293 or HeLa cells, while those of CRACR2B were higher in HEK293 cells (Fig. 3c). Since CRACR2A transcripts were abundant in Jurkat T cells, we examined their expression in primary T cells. Murine thymocytes, naïve and effector CD4+ T cells showed high expression of CRACR2A transcripts when compared with murine embryonic fibroblasts (MEFs), while CRACR2B transcripts were very low (Fig. 3d). These data suggest that the relative contribution of CRACR2A and CRACR2B in SOCE in T cells versus HEK293 cells is partially due to differences in their expression levels.


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 plays an important role in Orai1-mediated SOCE in T cells. (a) SOCE measurements in Jurkat and HEK293 cells depleted of CRACR2A and CRACR2B. Top panel, averaged responses from Jurkat T cells: scrambled (Scr, n = 75 cells), CRACR2A (R2A, n = 79), or CRACR2B (R2B, n = 70) siRNAs. Bottom, averaged responses from HEK293 cells: Scr (n = 41), R2A (n = 47) or R2B (n = 49). Bar graphs represent averaged peak [Ca2+]i ± s.e.m. from three independent experiments. (b) Measurement of IL-2 expression in Jurkat T cells transfected with siRNAs. A representative of three independent experiments is shown. (c) Real-time PCR analysis of human CRACR2A and CRACR2B transcripts from various tissues and cell lines. Normalized mRNA levels are plotted relative to those of brain tissue. Data represent average ± s.d. from 2 independent experiments performed in triplicate. * indicates tissues or cell-lines showing high expression of CRACR2A transcripts, distinct from CRACR2B. (d) Expression of CRACR2A and CRACR2B in murine primary cells. The mRNA levels of CRACR2A or CRACR2B were measured from mouse embryonic fibroblasts (MEFs), thymocytes (Thy), naïve CD4+ T cells (Naïve), effector CD4+ (ThN), or CD8+ (CTL) T cells. Normalized mRNA levels are plotted relative to those of MEFs. N.D., not detected. Data represent average ± s.d. from 2 independent experiments performed in triplicate. (e) Examination of functional redundancy between CRACR2 proteins. SOCE was measured in HEK293 cells stably expressing control (Scr, black trace, n = 46 cells) or CRACR2B shRNA (red, n = 49). CRACR2B-depleted cells with ectopic expression of CRACR2A (R2A, blue, n = 41) or CRACR2B (R2B, cyan, n = 43) were examined for SOCE. A representative of three independent experiments is shown. The plasmids contain IRES-GFP and GFP+ cells were selected for analysis. (f) Effect of CRACR2 protein expression on Orai1-mediated SOCE. SOCE was measured in Orai1- MEFs expressing CRACR2A or CRACR2B together with Orai1. Each trace shows averaged responses from 25 (vector), 30 (Orai1), 35 (Orai1+R2A) or 33 (Orai1+R2B) MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.
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Figure 3: CRACR2A plays an important role in Orai1-mediated SOCE in T cells. (a) SOCE measurements in Jurkat and HEK293 cells depleted of CRACR2A and CRACR2B. Top panel, averaged responses from Jurkat T cells: scrambled (Scr, n = 75 cells), CRACR2A (R2A, n = 79), or CRACR2B (R2B, n = 70) siRNAs. Bottom, averaged responses from HEK293 cells: Scr (n = 41), R2A (n = 47) or R2B (n = 49). Bar graphs represent averaged peak [Ca2+]i ± s.e.m. from three independent experiments. (b) Measurement of IL-2 expression in Jurkat T cells transfected with siRNAs. A representative of three independent experiments is shown. (c) Real-time PCR analysis of human CRACR2A and CRACR2B transcripts from various tissues and cell lines. Normalized mRNA levels are plotted relative to those of brain tissue. Data represent average ± s.d. from 2 independent experiments performed in triplicate. * indicates tissues or cell-lines showing high expression of CRACR2A transcripts, distinct from CRACR2B. (d) Expression of CRACR2A and CRACR2B in murine primary cells. The mRNA levels of CRACR2A or CRACR2B were measured from mouse embryonic fibroblasts (MEFs), thymocytes (Thy), naïve CD4+ T cells (Naïve), effector CD4+ (ThN), or CD8+ (CTL) T cells. Normalized mRNA levels are plotted relative to those of MEFs. N.D., not detected. Data represent average ± s.d. from 2 independent experiments performed in triplicate. (e) Examination of functional redundancy between CRACR2 proteins. SOCE was measured in HEK293 cells stably expressing control (Scr, black trace, n = 46 cells) or CRACR2B shRNA (red, n = 49). CRACR2B-depleted cells with ectopic expression of CRACR2A (R2A, blue, n = 41) or CRACR2B (R2B, cyan, n = 43) were examined for SOCE. A representative of three independent experiments is shown. The plasmids contain IRES-GFP and GFP+ cells were selected for analysis. (f) Effect of CRACR2 protein expression on Orai1-mediated SOCE. SOCE was measured in Orai1- MEFs expressing CRACR2A or CRACR2B together with Orai1. Each trace shows averaged responses from 25 (vector), 30 (Orai1), 35 (Orai1+R2A) or 33 (Orai1+R2B) MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.
Mentions: To examine the physiological role of CRACR2 proteins, we measured SOCE in Jurkat T cells upon siRNA-mediated depletion of CRACR2A or CRACR2B. As seen in Fig. 3a, depletion of CRACR2A decreased SOCE by 50% while that of CRACR2B had a mild effect. Consistent with a decrease in SOCE, depletion of CRACR2A resulted in a stronger impairment in IL-2 production by Jurkat T cells than CRACR2B knockdown (Fig. 3b). Surprisingly, in HEK293 cells, depletion of CRACR2B had a stronger effect on SOCE than that of CRACR2A (Fig. 3a, bottom). Specificity and efficiency of CRACR2 knockdown was examined by RT-PCR and immunoblotting (Supplementary Information, Fig. S6a–d). To determine the reason for the differences between Jurkat and HEK293 cells, we measured the expression levels of CRACR2 transcripts in various human tissues and cell lines. Quantitative RT-PCR analysis revealed abundant expression of CRACR2A transcripts in spleen and thymus distinct from CRACR2B (Fig. 3c). Among cell lines, CRACR2A transcripts were higher in Jurkat T cells when compared with HEK293 or HeLa cells, while those of CRACR2B were higher in HEK293 cells (Fig. 3c). Since CRACR2A transcripts were abundant in Jurkat T cells, we examined their expression in primary T cells. Murine thymocytes, naïve and effector CD4+ T cells showed high expression of CRACR2A transcripts when compared with murine embryonic fibroblasts (MEFs), while CRACR2B transcripts were very low (Fig. 3d). These data suggest that the relative contribution of CRACR2A and CRACR2B in SOCE in T cells versus HEK293 cells is partially due to differences in their expression levels.

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