Structural Basis of Lymphangiogenic Receptor VEGFR-3 Activation Mediated by Distinctive Clustering of the Ligand-Receptor Complex.

Vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 are critical for lymphangiogenesis, yet the structural mechanisms beyond simple dimerization that drive activation remain unknown. Here, we report the first cryo-EM structure of the human VEGFR-3 full ectodomain in complex with VEGF-C. Such a canonical ligand-induced 2:2 hetero-tetrameric complex further self-assembles into distinct higher-order assemblies-a lateral cis-cluster and an inverted trans-like-cluster. Further analysis of these canonical 2:2 hetero-tetramer in higher-order assemblies identifies that the specificity of VEGF-C for VEGFR-3 and VEGFR-2 over VEGFR-1 is mainly governed by its N-terminal α1 helix, which is structurally accommodated by the D2 domains of VEGFR-3 and VEGFR-2 but sterically excluded by the protruding D1-D2 connecting loop of VEGFR-1. Furthermore, we identify a unique interface within the membrane-proximal D5 domain occurring between neighboring canonical 2:2 complexes, mediated by a specific "WTP motif," as a key driver of cis-clustering. Structure-guided mutagenesis and real-time optogenetic assays support a critical role for cis-clustering in signal amplification and show that enforced receptor clustering can drive robust activation, whereas trans-like-clustering appears dispensable. Collectively, these findings transform the traditional "monomer-to-dimer" activation model of VEGFR-3/VEGF-C into a "dimer-to-cluster" paradigm, providing a blueprint for engineering next-generation therapeutics targeting lymphatic vascular diseases.