Structural basis for regulation of Frizzled-4 signaling by the co-receptor Tetraspanin-12

Norrin is an atypical ligand that regulates retinal angiogenesis through the Wnt/{beta}-catenin pathway. Norrin triggers heterodimerization of Frizzled-4 (FZD4) with Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to downstream {beta}-catenin stabilization. Unlike Wnt ligands, Norrin requires the tetraspanin Tspan12 for signaling amplification, but it is not understood why. Here, we report a 3.4-angstrom structure of Tspan12 in complex with FZD4 determined by cryo-electron microscopy. The structure reveals that FZD4 and Tspan12 form a direct complex in the absence of Norrin. The transmembrane (TM) domain of Tspan12 is oriented in a tightly packed four-helix bundle and interacts with TM2 of FZD4 to promote trafficking of Tspan12 to the cell surface. The C-D helices of Tspan12, which mediate binding to Norrin, remain exposed while Tspan12 is in complex with FZD4, facilitating higher-affinity Norrin binding. Cell-based assays reveal that Tspan12 and FZD4 remain associated after Norrin recognition, suggesting that Tspan12 is a core component of the FZD4-Norrin-LRP5/6 signaling complex. Together, these studies reveal the molecular mechanism underlying the assembly of the Tspan12-FZD4 complex and the enhancement of Norrin signaling, suggesting new ways to target the FZD4-Tspan12 complex for ocular diseases characterized by hypo- or hyper-vascularization. SIGNIFICANCE STATEMENTBlood vessel formation in the retina is essential for vision, and its disruption leads to ocular diseases. Norrin regulates retinal angiogenesis by triggering the heterodimerization of Frizzled-4 with co-receptor LRP5/6, mimicking the function of Wnt ligands and leading to downstream {beta}-catenin signaling. Unlike Wnts, Norrin requires a tetraspanin family member, Tspan12, to amplify signaling. We report the structure of the FZD4-Tspan12 co-receptor complex and identify key interacting regions that enable Tspan12 trafficking to the cell surface and higher affinity capture of Norrin. Cell-based assays indicate Tspan12 remains a component of the active Norrin-FZD4-LRP5/6 signaling complex. Our results highlight an underappreciated role of tetraspanins in direct regulation of receptor signaling and suggest new therapeutic avenues for targeting abnormalities in vascular growth.