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/β-catenin pathway by triggering heterodimerization of Frizzled-4 (FZD4) with Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to downstream β-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, with a contact interface between the lower hinge of FZD4 and the extracellular loops of Tspan12 that enables FZD4 to respond with a more gradual response to a wider range of Norrin concentrations. 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, indicating that Tspan12 is a core component of the FZD4-Norrin-LRP5/6 signaling complex. Together, these studies reveal the molecular mechanism underlying Tspan12-FZD4 complex assembly and enhancement of Norrin signaling and suggest new ways to target the FZD4-Tspan12 complex for ocular diseases of hypo- or hyper-vascularization.