Adam G. Grieve,1,∗ Yi-Chun Yeh,2,5 Yu-Fen Chang,3 Hsin-Yi Huang,3 Lucrezia Zarcone,1 Johannes Breuning,1,6 Nicholas Johnson,4,7 Kvido Stříšovský,4 Marion H. Brown,1 Anant B. Parekh,2 and Matthew Freeman1,8,∗∗


"1Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK
2Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, UK
3LumiSTAR Biotechnology, Inc., National Biotechnology Research Park, Taipei City 115, Taiwan
4Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences (IOCB), Prague, 166 10, Czech Republic
Adam G. Grieve: [email protected]; Matthew Freeman: [email protected]
∗Corresponding author [email protected]
∗∗Corresponding author [email protected]
5Present address: International Center of Wound Repair and Regeneration, National Cheung Kung University, Tainan City 701, Taiwan
6Present address: GlaxoSmithKline (GSK), Stevenage, SG1 2NY, UK
7Present address: Beatson Institute for Cancer Research, Glasgow, G61 1BD, UK
8Lead contact"


Calcium Imaging, Molecular Biology


"Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2’s recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

Keywords: rhomboid protease, CRAC channel, Orai1, RHBDL2, transmembrane, calcium, T cell, signalling



subsequently imaged on an inverted microscope (DMi8, Leica) equipped with a 63x objective lens (N.A. 1.4) and a multiwavelength LED light source (pE-4000, CoolLED) in regular Ca2+ containing solution.

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The pE-4000 Universal Illumination System offers 16 selectable wavelengths from 365 - 770 nm, making it a highly flexible illuminator covering a wide variety of fluorophores

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Molecular cell

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