The Olfactomedin domain of LPHN3 is required for FLRT3 binding while the Olfactomedin and Lectin domains both contribute to TEN1 binding. (A) Schematics of epitope-tagged wild-type (WT) LPHN3 and protein-protein interaction domain mutants. The GPCR domain spans the plasma membrane (dotted line), the N-terminus of the extracellular domain has an HA tag, and the C-terminus of the intracellular domain has a FLAG tag. (B) Immunostaining for surface (HA live-labeling) and total (FLAG permeablized) WT and mutant LPHN3 proteins in transfected HEK cells. (C) Binding of ecto-FLRT3-Fc to HEK cells expressing GFP, WT LPHN3, or mutant LPHN3. (D) Binding of Fc-ecto-TEN1 conditioned media to HEK cells expressing GFP, WT LPHN3, or mutant LPHN3. Binding was categorized as strong (+++), weak (+), or absent (-). Scale bars = 10 μm.