Figure 1.

Intercellular adhesion mediated by Lphn3 heterophilic contacts with its ligands Flrt3 and Ten2 is differentially altered by cancer-related GAIN domain mutations. (a) Schematic representation of Lphn3 domains organization, depicting cancer-related GAIN domain mutations (K561N, A760G, D798H, S810L and E811Q), Flag tag fused to the amino-terminal, and hemagglutinin tag (HA) introduced in the first extracellular loop. Domain legends: amino-terminal fragment (NTF), carboxyl-terminal fragment (CTF), Lectin (LEC), Olfacftomedin (OLF), hormone binding domain (HRM), GPCR proteolysis site (GPS), seven transmembrane domains (7TM), PDZ-binding domain (P.DZbd). Representative epifluorescence microscopy images of aggregation assays generated by mixing indicated cell populations separately transfected with (b,c) DsRed or EGFP alone, or (d–o) in combination with EGFP for Lphn3-WT and indicated Lphn3 variants alongside (d–i) cells co-expressing DsRed and Flrt3 or (j–o) Teneurin2. (p,q) Quantification of aggregation index calculated from assays conducted in between Lphn3-expressing cells and Flrt3- or Teneurin2-expressing cells, respectively. Scale bar: 50 µm. Subdomain A (GAIN Sub A) and B (GAIN Sub B) of GAIN domain. Data are represented as mean values of three independent experiments (n = 3). Dotted line represents the values obtained in control conditions (Ctrl). Statistical analysis was performed using one-way ANOVA. Error bars indicate S.E.M., p values between Lphn3-variants and control data are indicated by # inside histograms while p values between Lphn3-variants and Lphn3-WT are indicated by *: #### or **** p ≤ 0.0001, * p ≤ 0.05.