Extended Data Fig. 3: In vitro characterization of NRS activation.
From: Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation
a, Characterization of hybrid transmembrane sensors containing the ECR of the human CD4 receptor fused to the NotchJTS-LexA module (CD4-NRS-LexA) in Drosophila Schneider-2 cells using a luciferase-based assay. Addition of the CD4-ECR to the NRS basis (CD4-NRS-LexA) suppresses NRS activity. When the CD4-ECR is severed by secTEVp at cognate TEVp at TEVs interposed between CD4 and NRS-LexA components of the sensor (CD4-3TEVs-NRS-LexA, CD4-6TEVs-NRS-LexA), it becomes activated (magenta). Co-expression of cleavable sensors and intraTEVp does not result in sensor activation (grey). NΔEGF-LexA/NΔECN-LexA set, CD4-3TEVs-NRS-LexA set and CD4-6TEVs-NRS-LexA set were tested in separate assays but are displayed in the same graph. Data (n = 10 biological replicates from three independent experiments for all groups, except CD4-3TEVs-NRS-LexA group n = 3 from one experiment) were normalized and presented as multiples of control dataset in box-whisker plots (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). NΔEGF-LexA/NΔECN-LexA groups were compared with two-tailed Mann–Whitney U test, CD4-3TEVs-NRS-LexA dataset by ordinary one-way ANOVA with Tukey’s test, CD4-6TEVs-NRS-LexA dataset with Kruskal–Wallis one-way ANOVA with Dunn’s test (confidence interval = 95 % for all comparisons). P values are displayed above data. See also Source Data. b, NRS-LexA activity of the same sensor set as in a, visualized through expression of a lexAop-DsRed reporter (CD4-3TEVs-NRS-LexA not shown). Representative confocal images of Schneider-2 cell cultures with NRS-LexA signals (magenta, arrows) counterstained with Hoechst (blue). Scale bar = 100 µm. Experiment was independently repeated 3x with similar results. c, Protein sequence alignment of the JTS of Drosophila (Uniprot: P07207) and human Notch1 receptors (Uniprot: P46531). Positions of the TM helix (grey box) and S2, S3 and S4 protease cleavage sites are indicated. For control sensors in this study the critical valine residue at the S3 cleavage site (light brown box) was point mutated (V1763K). Black boxes delineate highly conserved residues. d, Function of NΔECN-LexA and CD4-6TEVs-NRS-LexA variants (grey circles) requires γ-secretase activity as application of 10 µM DAPT suppresses their activation (white circles). Data (n = 3 biological replicates from one experiment for all groups) are presented as multiples of control dataset in box-whisker plots (all data points plotted; horizontal line represents median, whiskers minimum and maximum values). Data groups (-DAPT/+DAPT for each sensor) were compared with two-tailed unpaired t-test (confidence interval = 95 %). P values are displayed above data. See also Source Data. e, Surface and total expression quantified by ELISA shows that Cirl-NRS-LexA variants as shown in b are delivered to the cell surface. Surface (n = 24 biological replicates from six independent experiments for all groups, except Cirl-NRSΔS3-LexA group n = 12 biological replicates from three independent experiments) and total ELISA data (n = 28 biological replicates from seven independent experiments for all groups, except Cirl-NRSΔS3-LexA group n = 12 biological replicates from three independent experiments) were normalized and presented as multiples of control dataset in box-whisker plots (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). Data were analysed with Kruskal–Wallis one-way ANOVA with Dunn’s test (confidence interval = 95 % for all comparisons). P values are displayed above and below data. Surface/total expression ratio (right panel) normalized to Cirl-NRS-LexA ratio indicates degree of surface trafficking of each Cirl-NRS-LexA variant and Cirl. See also Source Data.
