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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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An EF-hand mutant of CRACR2A causes spontaneous clustering of STIM1. (a) CRACR2A binds Ca2+ via its EF-hands. 45Ca2+ overlay experiments were performed with purified full-length CRACR2A, CRACR2A truncated in its N terminus (ΔN, amino acids 119–395), C terminus (ΔC, amino acids 1–154), or mutated in its EF-hand2 (EF2MUT). 2 or 10 µg of each protein were used to examine Ca2+ binding. Similar amount of purified calmodulin (CaM) was run (left panel) to compare Ca2+ binding affinity. Compare Ca2+ binding of CaM with ΔC mutant of CRACR2A for similar molar concentrations of each protein. Panels on the right show Ponceau S staining of the same blots to compare protein amounts. (b) Live cell epifluorescence and TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP and CRACR2AEF2MUT-mCherry in the absence or presence of store depletion. Left panels show epifluorescence images from the middle of the cell and right panels show TIRF images from the footprint of the cell. Arrowheads in the top panel show clustering of STIM1-YFP in cells co-expressing CRACR2AEF2MUT. An arrow in the same panel depicts minimal clustering in a cell expressing only STIM1-YFP. Arrowheads in the bottom TIRF panels show co-accumulation of CRACR2AEF2MUT-mCherry with STIM1-YFP at sites of newly formed clusters after store depletion (+TG). Bar, 10 µm. (c) TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP in the absence (top) or presence (bottom) of CRACR2AEF2MUT. Cells co-expressing mCherry (top) or CRACR2AEF2MUT-mCherry (bottom) were selected. CRACR2AEF2MUT induced clustering of STIM1-YFP without ER Ca2+ depletion (compare images at T = 0 s in the top and bottom panels). Thapsigargin was added at T = 0 s and images of STIM1 clustering are shown at 200 and 500 s. Arrowheads represent preformed clusters of STIM1 that expand upon store depletion. Bar graph depicts averaged number of HEK293 cells (± s.d.) showing STIM1 clustering in the presence (n = 112 cells) or absence (n = 106) of CRACR2AEF2MUT. Cells moderately expressing STIM1-YFP were examined. Bar, 10 µm.
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Figure 5: An EF-hand mutant of CRACR2A causes spontaneous clustering of STIM1. (a) CRACR2A binds Ca2+ via its EF-hands. 45Ca2+ overlay experiments were performed with purified full-length CRACR2A, CRACR2A truncated in its N terminus (ΔN, amino acids 119–395), C terminus (ΔC, amino acids 1–154), or mutated in its EF-hand2 (EF2MUT). 2 or 10 µg of each protein were used to examine Ca2+ binding. Similar amount of purified calmodulin (CaM) was run (left panel) to compare Ca2+ binding affinity. Compare Ca2+ binding of CaM with ΔC mutant of CRACR2A for similar molar concentrations of each protein. Panels on the right show Ponceau S staining of the same blots to compare protein amounts. (b) Live cell epifluorescence and TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP and CRACR2AEF2MUT-mCherry in the absence or presence of store depletion. Left panels show epifluorescence images from the middle of the cell and right panels show TIRF images from the footprint of the cell. Arrowheads in the top panel show clustering of STIM1-YFP in cells co-expressing CRACR2AEF2MUT. An arrow in the same panel depicts minimal clustering in a cell expressing only STIM1-YFP. Arrowheads in the bottom TIRF panels show co-accumulation of CRACR2AEF2MUT-mCherry with STIM1-YFP at sites of newly formed clusters after store depletion (+TG). Bar, 10 µm. (c) TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP in the absence (top) or presence (bottom) of CRACR2AEF2MUT. Cells co-expressing mCherry (top) or CRACR2AEF2MUT-mCherry (bottom) were selected. CRACR2AEF2MUT induced clustering of STIM1-YFP without ER Ca2+ depletion (compare images at T = 0 s in the top and bottom panels). Thapsigargin was added at T = 0 s and images of STIM1 clustering are shown at 200 and 500 s. Arrowheads represent preformed clusters of STIM1 that expand upon store depletion. Bar graph depicts averaged number of HEK293 cells (± s.d.) showing STIM1 clustering in the presence (n = 112 cells) or absence (n = 106) of CRACR2AEF2MUT. Cells moderately expressing STIM1-YFP were examined. Bar, 10 µm.

Mentions: The EF-hands are highly conserved among CRACR2 family (Supplementary Information, Fig. S2 and S5). To test whether these predicted EF-hands bind Ca2+, 45Ca2+ overlay analysis was performed using recombinant full-length and various mutants of CRACR2A together with calmodulin (CaM) as a positive control. WT CRACR2A and ΔC mutant showed binding to Ca2+ while truncation of the N terminus or mutations in the second EF-hand abolished Ca2+ binding (Fig. 5a). These results together with those in Fig. 2 suggest that the EF-hand motifs of CRACR2A bind to Ca2+ and Ca2+ binding reduces its interaction with 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)

An EF-hand mutant of CRACR2A causes spontaneous clustering of STIM1. (a) CRACR2A binds Ca2+ via its EF-hands. 45Ca2+ overlay experiments were performed with purified full-length CRACR2A, CRACR2A truncated in its N terminus (ΔN, amino acids 119–395), C terminus (ΔC, amino acids 1–154), or mutated in its EF-hand2 (EF2MUT). 2 or 10 µg of each protein were used to examine Ca2+ binding. Similar amount of purified calmodulin (CaM) was run (left panel) to compare Ca2+ binding affinity. Compare Ca2+ binding of CaM with ΔC mutant of CRACR2A for similar molar concentrations of each protein. Panels on the right show Ponceau S staining of the same blots to compare protein amounts. (b) Live cell epifluorescence and TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP and CRACR2AEF2MUT-mCherry in the absence or presence of store depletion. Left panels show epifluorescence images from the middle of the cell and right panels show TIRF images from the footprint of the cell. Arrowheads in the top panel show clustering of STIM1-YFP in cells co-expressing CRACR2AEF2MUT. An arrow in the same panel depicts minimal clustering in a cell expressing only STIM1-YFP. Arrowheads in the bottom TIRF panels show co-accumulation of CRACR2AEF2MUT-mCherry with STIM1-YFP at sites of newly formed clusters after store depletion (+TG). Bar, 10 µm. (c) TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP in the absence (top) or presence (bottom) of CRACR2AEF2MUT. Cells co-expressing mCherry (top) or CRACR2AEF2MUT-mCherry (bottom) were selected. CRACR2AEF2MUT induced clustering of STIM1-YFP without ER Ca2+ depletion (compare images at T = 0 s in the top and bottom panels). Thapsigargin was added at T = 0 s and images of STIM1 clustering are shown at 200 and 500 s. Arrowheads represent preformed clusters of STIM1 that expand upon store depletion. Bar graph depicts averaged number of HEK293 cells (± s.d.) showing STIM1 clustering in the presence (n = 112 cells) or absence (n = 106) of CRACR2AEF2MUT. Cells moderately expressing STIM1-YFP were examined. Bar, 10 µm.
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Figure 5: An EF-hand mutant of CRACR2A causes spontaneous clustering of STIM1. (a) CRACR2A binds Ca2+ via its EF-hands. 45Ca2+ overlay experiments were performed with purified full-length CRACR2A, CRACR2A truncated in its N terminus (ΔN, amino acids 119–395), C terminus (ΔC, amino acids 1–154), or mutated in its EF-hand2 (EF2MUT). 2 or 10 µg of each protein were used to examine Ca2+ binding. Similar amount of purified calmodulin (CaM) was run (left panel) to compare Ca2+ binding affinity. Compare Ca2+ binding of CaM with ΔC mutant of CRACR2A for similar molar concentrations of each protein. Panels on the right show Ponceau S staining of the same blots to compare protein amounts. (b) Live cell epifluorescence and TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP and CRACR2AEF2MUT-mCherry in the absence or presence of store depletion. Left panels show epifluorescence images from the middle of the cell and right panels show TIRF images from the footprint of the cell. Arrowheads in the top panel show clustering of STIM1-YFP in cells co-expressing CRACR2AEF2MUT. An arrow in the same panel depicts minimal clustering in a cell expressing only STIM1-YFP. Arrowheads in the bottom TIRF panels show co-accumulation of CRACR2AEF2MUT-mCherry with STIM1-YFP at sites of newly formed clusters after store depletion (+TG). Bar, 10 µm. (c) TIRF microscopy analysis of HEK293 cells expressing STIM1-YFP in the absence (top) or presence (bottom) of CRACR2AEF2MUT. Cells co-expressing mCherry (top) or CRACR2AEF2MUT-mCherry (bottom) were selected. CRACR2AEF2MUT induced clustering of STIM1-YFP without ER Ca2+ depletion (compare images at T = 0 s in the top and bottom panels). Thapsigargin was added at T = 0 s and images of STIM1 clustering are shown at 200 and 500 s. Arrowheads represent preformed clusters of STIM1 that expand upon store depletion. Bar graph depicts averaged number of HEK293 cells (± s.d.) showing STIM1 clustering in the presence (n = 112 cells) or absence (n = 106) of CRACR2AEF2MUT. Cells moderately expressing STIM1-YFP were examined. Bar, 10 µm.
Mentions: The EF-hands are highly conserved among CRACR2 family (Supplementary Information, Fig. S2 and S5). To test whether these predicted EF-hands bind Ca2+, 45Ca2+ overlay analysis was performed using recombinant full-length and various mutants of CRACR2A together with calmodulin (CaM) as a positive control. WT CRACR2A and ΔC mutant showed binding to Ca2+ while truncation of the N terminus or mutations in the second EF-hand abolished Ca2+ binding (Fig. 5a). These results together with those in Fig. 2 suggest that the EF-hand motifs of CRACR2A bind to Ca2+ and Ca2+ binding reduces its interaction with 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.

Show MeSH
Related in: MedlinePlus