Fig. 5: Binding site mutations on TEN2 abolish LPHN3 binding in both trans and cis-like.

a Diagram for WT TEN2 −SS and TEN2 DHR −SS constructs. DHR mutation (D1737N, H1738T, R1739T) is on the TEN2 β-barrel located at the LPHN3-binding interface (black dots). Results for TEN2 and LPHN3 binding in different experimental setups (Fig. 4a–c) are summarized in the table. The DHR mutation breaks the interaction of TEN2 with LPHN3 in all experimental setups. b Representative images for cell-aggregation assays with TEN2 constructs and full-length LPHN3. WT TEN2 −SS induces cell aggregation with LPHN3, while TEN2 DHR −SS abolishes cell aggregation. HEK293 cells were co-transfected with TEN2 or LPHN3 and either tdTomato or EGFP as indicated. Scale bar indicates 100 µm. Quantification of aggregation index (%) is shown on the right (***p < 0.001 by one-way ANOVA). c TEN2 constructs expressed in mammalian cells were tested for their ability to bind soluble biotinylated LPHN3 or LPHN1 Lec domain using flow cytometry experiments (left) or cell-surface staining assays (right). The DHR mutation abolishes the cis-like interaction between TEN2 and LPHN. TEN2 construct expression was determined by HA tag fluorescence (Y axis) and purified Lec binding to TEN2-expressing cells was measured by fluorescence of DyLight attached to neutravidin (X axis). Dot plots represent the correlation between TEN2 expression and LPHN3 binding. Black cross indicates “high TEN2 expression and high LPHN3 binding” gate. Scale bar indicates 20 µm. Quantification of cell-surface-binding assays are shown next to the image (***p < 0.001 by one-way ANOVA). d Size-exclusion chromatograms showing the formation of a binary complex between soluble TEN2 −SS ECRΔ1 and full LPHN3 ECR (left, black line). Individual proteins are shown as control (dotted lines). LPHN binding mutant does not bind LPHN3 ECR (right, green line), as observed by the lack of co-elution in fractions ran on SDS-PAGE. Colors of the chromatograms and the boxes around the gels match. Data in b and c are presented as mean ± SEM, n = 3, and are representative of at least three independent experiments. Source data are provided as a Source Data file.