Abstract
Orai1 and STIM1 are critical components of Ca2+ release-activated Ca2+ (CRAC) channels that mediate store-operated Ca2+ 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 Ca2+ 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 Ca2+ and induced cell death, suggesting its active interaction with CRAC channels. These observations implicate CRACR2A, a novel Ca2+ 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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Acknowledgements
We thank Kenneth Philipson for critical reading of this manuscript and kind suggestions on Ca2+ overlay experiments. We thank Bernard Ribalet for help with patch clamp experiments, Baljit Khakh for sharing equipments and Earl Homsher for helpful discussions. This work was supported by National Institute of Health grant AI-083432, Stein Oppenheimer Endowment Award, Cancer Research Coordinating Committee Award (Y.G.) and a fellowship from the American Heart Association (S.S.).
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S.S. and Y.G. designed the research. S.S. and Y.G. carried out the experiments with technical help from H.J.J. K.D.K. performed and analysed Ca2+ binding assays and P.S. and J.W. carried out mass spectrometry. S.S. and Y.G. analysed the data and wrote the manuscript. Y.G. supervised the project.
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Srikanth, S., Jung, HJ., Kim, KD. et al. A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells. Nat Cell Biol 12, 436–446 (2010). https://doi.org/10.1038/ncb2045
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DOI: https://doi.org/10.1038/ncb2045
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