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A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells

Nature Cell Biology volume 12, pages 436–446 (2010)Cite this article

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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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Figure 1: Identification of CRACR2A as a binding partner of Orai1 by large-scale affinity purification.
Figure 2: CRACR2A interacts directly with Orai and STIM1.
Figure 3: CRACR2A has an important role in Orai1-mediated SOCE in T cells.
Figure 4: CRACR2A is essential for cluster formation of Orai1 and STIM1 in T cells.
Figure 5: An EF-hand mutant of CRACR2A causes spontaneous clustering of STIM1.
Figure 6: CRACR2A regulates SOCE and CRAC channel-mediated Ca2+ oscillations in T cells.
Figure 7: Expression of an EF-hand mutant of CRACR2A induces cell death in Jurkat T cells.

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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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Authors and Affiliations

  1. Department of Physiology, David Geffen School of Medicine at the University of California, Los Angeles, 90095, California, USA

    Sonal Srikanth, Hea-Jin Jung, Kyun-Do Kim & Yousang Gwack

  2. The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David Geffen School of Medicine at the University of California, Los Angeles, 90024, California, USA

    Puneet Souda & Julian Whitelegge

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Contributions

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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Correspondence to Yousang Gwack.

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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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