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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 is essential for cluster formation of Orai1 and STIM1 in T cells. (a) TIRF microscopy images of STIM1-YFP in Jurkat T cells transfected with either control (top panel) or CRACR2A siRNA (bottom panel). Cells were imaged in Ca2+ free Ringer solution and ER Ca2+ was depleted with 1 µM of thapsigargin at the initial time point (T = 0). The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 10 cells transfected with control siRNA (black) and 15 cells transfected with CRACR2A siRNA (red). Bar, 5 µm. (b) Clustering of Orai1-GFP in Jurkat T cells co-expressing STIM1-mCherry. Cells were transfected with control siRNA (top panel) or CRACR2A siRNA (bottom panel). Jurkat cells expressing both Orai1 and STIM1 proteins were imaged by TIRF microscopy. The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 8 cells transfected with control siRNA and 10 cells transfected with CRACR2A siRNA. Bar, 5 µm. (c) TIRF microscopy analysis of Orai1K85A/K87A-GFP in Jurkat T cells. Jurkat T cells expressing STIM1-mCherry and wild type Orai1-GFP or Orai1K85A/K87A-GFP were imaged. The graph represents an average of normalized fluorescence intensity ± s.e.m. from 9 cells expressing Orai1-GFP and 12 cells expressing Orai1K85A/K87A-GFP. Bar, 5 µm. (d) Reconstitution of SOCE in Orai1- CD4+ T cells by expression of WT Orai1 or Orai1K85A/K87A. T cells transduced with retroviral vectors expressing WT Orai1 or Orai1K85A/K87A together with GFP from an IRES site were examined for SOCE. Each trace shows average ± s.e.m. from 70 (vector), 77 (WT Orai1), or 79 (K85A, K87A) GFP+ cells. A representative of three independent experiments is shown here. (e) Recovery of defect in SOCE of Orai1K85A/K87A mutant by co-expression of CRACR2 proteins. Orai1- MEFs were transduced with retroviruses encoding CRACR2A or CRACR2B together with Orai1K85A/K87A for measurement of SOCE. Each trace shows averaged responses from 35 (Orai1K85A/K87A), 32 (WT Orai1), 30 (Orai1K85A/K87A + R2A) or 39 (Orai1K85A/K87A + R2B) GFP+ MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.
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Figure 4: CRACR2A is essential for cluster formation of Orai1 and STIM1 in T cells. (a) TIRF microscopy images of STIM1-YFP in Jurkat T cells transfected with either control (top panel) or CRACR2A siRNA (bottom panel). Cells were imaged in Ca2+ free Ringer solution and ER Ca2+ was depleted with 1 µM of thapsigargin at the initial time point (T = 0). The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 10 cells transfected with control siRNA (black) and 15 cells transfected with CRACR2A siRNA (red). Bar, 5 µm. (b) Clustering of Orai1-GFP in Jurkat T cells co-expressing STIM1-mCherry. Cells were transfected with control siRNA (top panel) or CRACR2A siRNA (bottom panel). Jurkat cells expressing both Orai1 and STIM1 proteins were imaged by TIRF microscopy. The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 8 cells transfected with control siRNA and 10 cells transfected with CRACR2A siRNA. Bar, 5 µm. (c) TIRF microscopy analysis of Orai1K85A/K87A-GFP in Jurkat T cells. Jurkat T cells expressing STIM1-mCherry and wild type Orai1-GFP or Orai1K85A/K87A-GFP were imaged. The graph represents an average of normalized fluorescence intensity ± s.e.m. from 9 cells expressing Orai1-GFP and 12 cells expressing Orai1K85A/K87A-GFP. Bar, 5 µm. (d) Reconstitution of SOCE in Orai1- CD4+ T cells by expression of WT Orai1 or Orai1K85A/K87A. T cells transduced with retroviral vectors expressing WT Orai1 or Orai1K85A/K87A together with GFP from an IRES site were examined for SOCE. Each trace shows average ± s.e.m. from 70 (vector), 77 (WT Orai1), or 79 (K85A, K87A) GFP+ cells. A representative of three independent experiments is shown here. (e) Recovery of defect in SOCE of Orai1K85A/K87A mutant by co-expression of CRACR2 proteins. Orai1- MEFs were transduced with retroviruses encoding CRACR2A or CRACR2B together with Orai1K85A/K87A for measurement of SOCE. Each trace shows averaged responses from 35 (Orai1K85A/K87A), 32 (WT Orai1), 30 (Orai1K85A/K87A + R2A) or 39 (Orai1K85A/K87A + R2B) GFP+ MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.

Mentions: STIM1 is involved in clustering and gating of Orai1 and evokes large CRAC currents when co-expressed with Orai135–38. In contrast, CRACR2A did not induce large CRAC currents when co-expressed with Orai1 (data not shown), indicating that CRACR2A cannot substitute for STIM1. We hypothesized that binding of CRACR2A to Orai1 and STIM1 may stabilize their interaction in PM-proximal area, and consequently its depletion may impair clustering of Orai1 and STIM1. To examine the role of CRACR2A in STIM1 clustering, we transfected control or CRACR2A-depleted Jurkat T cells with STIM1-YFP and measured its accumulation into PM-proximal regions using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, STIM1-YFP clustered within ten minutes in control cells (Fig. 4a, top), but in CRACR2A-depleted cells clustering was severely reduced (Fig. 4a, bottom). To investigate the effects of CRACR2A on clustering of Orai1, we transfected control and CRACR2A-depleted Jurkat T cells with plasmids encoding Orai1-GFP and STIM1-mCherry in an equimolar ratio. Orai1 formed clusters upon thapsigargin treatment in control cells while CRACR2A depletion impaired this process (Fig. 4b). These data suggest that CRACR2A is important for clustering of Orai1 and STIM1.


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 is essential for cluster formation of Orai1 and STIM1 in T cells. (a) TIRF microscopy images of STIM1-YFP in Jurkat T cells transfected with either control (top panel) or CRACR2A siRNA (bottom panel). Cells were imaged in Ca2+ free Ringer solution and ER Ca2+ was depleted with 1 µM of thapsigargin at the initial time point (T = 0). The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 10 cells transfected with control siRNA (black) and 15 cells transfected with CRACR2A siRNA (red). Bar, 5 µm. (b) Clustering of Orai1-GFP in Jurkat T cells co-expressing STIM1-mCherry. Cells were transfected with control siRNA (top panel) or CRACR2A siRNA (bottom panel). Jurkat cells expressing both Orai1 and STIM1 proteins were imaged by TIRF microscopy. The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 8 cells transfected with control siRNA and 10 cells transfected with CRACR2A siRNA. Bar, 5 µm. (c) TIRF microscopy analysis of Orai1K85A/K87A-GFP in Jurkat T cells. Jurkat T cells expressing STIM1-mCherry and wild type Orai1-GFP or Orai1K85A/K87A-GFP were imaged. The graph represents an average of normalized fluorescence intensity ± s.e.m. from 9 cells expressing Orai1-GFP and 12 cells expressing Orai1K85A/K87A-GFP. Bar, 5 µm. (d) Reconstitution of SOCE in Orai1- CD4+ T cells by expression of WT Orai1 or Orai1K85A/K87A. T cells transduced with retroviral vectors expressing WT Orai1 or Orai1K85A/K87A together with GFP from an IRES site were examined for SOCE. Each trace shows average ± s.e.m. from 70 (vector), 77 (WT Orai1), or 79 (K85A, K87A) GFP+ cells. A representative of three independent experiments is shown here. (e) Recovery of defect in SOCE of Orai1K85A/K87A mutant by co-expression of CRACR2 proteins. Orai1- MEFs were transduced with retroviruses encoding CRACR2A or CRACR2B together with Orai1K85A/K87A for measurement of SOCE. Each trace shows averaged responses from 35 (Orai1K85A/K87A), 32 (WT Orai1), 30 (Orai1K85A/K87A + R2A) or 39 (Orai1K85A/K87A + R2B) GFP+ MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.
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Figure 4: CRACR2A is essential for cluster formation of Orai1 and STIM1 in T cells. (a) TIRF microscopy images of STIM1-YFP in Jurkat T cells transfected with either control (top panel) or CRACR2A siRNA (bottom panel). Cells were imaged in Ca2+ free Ringer solution and ER Ca2+ was depleted with 1 µM of thapsigargin at the initial time point (T = 0). The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 10 cells transfected with control siRNA (black) and 15 cells transfected with CRACR2A siRNA (red). Bar, 5 µm. (b) Clustering of Orai1-GFP in Jurkat T cells co-expressing STIM1-mCherry. Cells were transfected with control siRNA (top panel) or CRACR2A siRNA (bottom panel). Jurkat cells expressing both Orai1 and STIM1 proteins were imaged by TIRF microscopy. The graph below represents an average of normalized fluorescence intensity ± s.e.m. from measurements of 8 cells transfected with control siRNA and 10 cells transfected with CRACR2A siRNA. Bar, 5 µm. (c) TIRF microscopy analysis of Orai1K85A/K87A-GFP in Jurkat T cells. Jurkat T cells expressing STIM1-mCherry and wild type Orai1-GFP or Orai1K85A/K87A-GFP were imaged. The graph represents an average of normalized fluorescence intensity ± s.e.m. from 9 cells expressing Orai1-GFP and 12 cells expressing Orai1K85A/K87A-GFP. Bar, 5 µm. (d) Reconstitution of SOCE in Orai1- CD4+ T cells by expression of WT Orai1 or Orai1K85A/K87A. T cells transduced with retroviral vectors expressing WT Orai1 or Orai1K85A/K87A together with GFP from an IRES site were examined for SOCE. Each trace shows average ± s.e.m. from 70 (vector), 77 (WT Orai1), or 79 (K85A, K87A) GFP+ cells. A representative of three independent experiments is shown here. (e) Recovery of defect in SOCE of Orai1K85A/K87A mutant by co-expression of CRACR2 proteins. Orai1- MEFs were transduced with retroviruses encoding CRACR2A or CRACR2B together with Orai1K85A/K87A for measurement of SOCE. Each trace shows averaged responses from 35 (Orai1K85A/K87A), 32 (WT Orai1), 30 (Orai1K85A/K87A + R2A) or 39 (Orai1K85A/K87A + R2B) GFP+ MEFs. The bar graph shows averaged peak [Ca2+]i ± s.e.m. from three independent experiments.
Mentions: STIM1 is involved in clustering and gating of Orai1 and evokes large CRAC currents when co-expressed with Orai135–38. In contrast, CRACR2A did not induce large CRAC currents when co-expressed with Orai1 (data not shown), indicating that CRACR2A cannot substitute for STIM1. We hypothesized that binding of CRACR2A to Orai1 and STIM1 may stabilize their interaction in PM-proximal area, and consequently its depletion may impair clustering of Orai1 and STIM1. To examine the role of CRACR2A in STIM1 clustering, we transfected control or CRACR2A-depleted Jurkat T cells with STIM1-YFP and measured its accumulation into PM-proximal regions using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, STIM1-YFP clustered within ten minutes in control cells (Fig. 4a, top), but in CRACR2A-depleted cells clustering was severely reduced (Fig. 4a, bottom). To investigate the effects of CRACR2A on clustering of Orai1, we transfected control and CRACR2A-depleted Jurkat T cells with plasmids encoding Orai1-GFP and STIM1-mCherry in an equimolar ratio. Orai1 formed clusters upon thapsigargin treatment in control cells while CRACR2A depletion impaired this process (Fig. 4b). These data suggest that CRACR2A is important for clustering of Orai1 and STIM1.

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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