AGR2 dimerization with ERMIT assay, principles and validation of the method.Figure 1The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. A) Upper panel: NMR structure of the non-covalent dimer of AGR2 (PDB ID: 2LNS). The dimer domain is highlighted with a yellow circle (residue 54-70 of each monomer). Monomer A is colored green and monomer B in pink. The figure was generated in Chimera. Lower panel: a close-up illustration of the dimer domain, showing that the dimer is stabilized through two salt-bridges between E60 and K64 of each monomer. The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. B) Molecular modeling showing root-mean-square deviation (RMSD) plot for the MD simulation of the AGR2 WT and E60A mutant. The interaction-energies are defined as the sum of the short-range Coulomb interactions and the short-range Lennard-Jones potential between monomer A and B. The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or a siRNA targeting AGR2 (siAGR2) for 24 h (C) Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2, anti-ERK1 antibodies (for loading control). The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. Western blot showing the expression of AGR2 dimers (D) and monomers (M) in HEK293T subjected to DSP-mediated cross-linking and that were previously transfected with either a control siRNA (siCTL) or treated or not with tunicamycin (Tun) prior to cross-linking (D). Reduced or non-reduced samples were resolved by SDS-PAGE and immunoblotted using anti-AGR2 anti-calnexin (CANX) antibodies (for loading control). The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. E) Principles of the ERMIT assay. A wild type IRE1 bait is used to report for dimerization, whereas a kinase catalytic mutant (K599A) is used as a control to prove that signal observed with the wild type form is due to IRE1 activation. The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. F) AGR2 dimerization was monitored with ERMIT (wild-type (WT)). The luminescence signal was abrogated when using the kinase dead (KD) constructs and by the constructs exhibiting mutations in the dimerization domain (E60A or C81S or double mutant (DM)). The graph represents average signal normalized on reporter protein expression ± SD (n=5; **: WT/KD (p=0.002), E60/WT (p=0.0034), E60/KD (p=0.001), C81/KD (p=0.004), DM/WT (p=0.0021) and DM/KD (p=0.0017), respectively; *: C81/WT (p=0.0098)). The ERMIT assay relies on the signaling properties of IRE1, one of the three ER stress sensors and reports for a dimerization event occurring in the lumen of the ER. The assay can be applied to heterodimerization or homodimerization events. G) Cells expressing various bait and prey constructs and the XBP1 splicing reporter or the XBP1 splicing reporter alone were exposed to increasing concentrations of DTT. The ERMIT signals obtained with both baits were then normalized to that of XBP1s to obtain results independent of the activation of endogenous IRE1 by the use of chemical ER stressors (n = 4).