Fig. 6: Membrane anchoring restricts alternative splice-dependent interaction of TEN2 to LPHN3.

Same three experimental setups as in Fig. 4a–c were used to test the effect of alternative splicing on TEN2/LPHN3 interaction. Figure outline is identical in principle to that of in Fig. 5. a Diagram for WT TEN2 −SS and WT TEN2 +SS constructs that were used in the below experiments. The seven amino acid splice site on the TEN2 β-propeller is indicated by empty or filled red stars. Results for the interaction of TEN2 and LPHN3 in different experimental setups (as in Figs. 4a–c and 5b–d) are summarized in the table. The insertion of the splice site breaks the interaction of TEN2 with LPHN3 only in the cell-aggregation assays, but not in the other experimental setups. b Representative images for cell-aggregation assays with TEN2 −SS or TEN2 +SS and full-length LPHN3. TEN2 −SS induces cell aggregation with LPHN3, while TEN2 +SS abolishes cell aggregation. Scale bar indicates 100 µm. Figure modified from ref. ². c TEN2 −SS and TEN2 +SS expressed in mammalian cells were tested for their ability to bind soluble biotinylated LPHN3 or LPHN1 Lec domain using flow cytometry experiments (left) and using cell-surface staining assays (right). Both −SS and +SS mediate the interaction between TEN2 and LPHN in cis-like. Quantification of cell-surface-binding assays are shown next to the image. The cell-surface staining assays in c was performed in the same experiment as in Fig. 5c, and thus the control images are identical. Scale bar indicates 20 µm. (***p < 0.001 by one-way ANOVA.) d Size-exclusion chromatograms showing the formation of binary complexes between soluble full TEN2 ECR and full LPHN3 ECR (left, blue and green lines). Elution profile for individual TEN2 −SS ECR, TEN2 +SS ECR and LPHN3 ECR are shown for reference (gray lines). Both TEN2 −SS ECR and TEN2 +SS ECR bind to LPHN3 ECR (green and blue lines, respectively), as also observed by co-elution in the fractions ran on SDS-PAGE gel. Colors of the chromatograms match the colors of box around the SDS-PAGE gel. 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.