Abstract
SummaryCalcium 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.
Highlights
Signaling controls most cellular functions and must be precisely regulated in time and space
We focused on three characteristics of known rhomboid substrates: their transmembrane domains (TMDs) have a type I orientation (NH2-out, COOH-in), many contain extracellular EGF-like domains, and they often contain helix-destabilizing amino acids (Freeman, 2014)
The top bioinformatic TMD hits (Table S1) were inserted into a reporter that was co-expressed with RHBDL2, so that RHBDL2dependent cleavage led to accumulation of extracellular alkaline phosphatase (AP) (Figure 1A, left), which was detected using a colorimetric phosphatase assay
Summary
Signaling controls most cellular functions and must be precisely regulated in time and space. Much signaling is triggered by integral membrane receptors, including, for example, growth factor and cytokine receptors, ion channels, and G protein-coupled receptors (GPCRs). Little is known about the regulation of the abundance and activity of most cell surface signaling proteins. A good example of switch-like signaling is the control of calcium ion (Ca2+) flux across the eukaryotic plasma membrane (PM), which acts as a barrier between high extracellular and low cytoplasmic Ca2+ concentrations. Opening of CRAC channels at the cell surface causes a rapid increase of cytosolic Ca2+, which activates important signaling pathways, the most studied being in the adaptive immune system. The founding and most ubiquitously expressed member of the family, was originally identified as a genetic cause of severe combined immunodeficiency in humans and plays essential roles in T cell immunity (Feske et al, 2006)
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