{"doi":"10.1101/2025.09.25.678640","title":"Structural basis for regulation of Frizzled-4 signaling by the co-receptor Tetraspanin-12","abstract":"ABSTRACT Norrin is an atypical ligand that regulates retinal angiogenesis through the Wnt/β-catenin pathway. Norrin triggers 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. 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 STATEMENT Blood 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 β-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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":575923,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9436,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":695284,"name":"Payal Pratap","orcid":"0000-0002-7170-6866","position":1,"is_corresponding":false},{"id":1133383,"name":"Kevin Alexander Estrada Alamo","orcid":"0000-0002-3829-357X","position":2,"is_corresponding":false},{"id":314020,"name":"Katherine J. Susa","orcid":"0000-0003-0077-667X","position":3,"is_corresponding":false},{"id":1484499,"name":"Alyana J. Granados","orcid":null,"position":0,"is_corresponding":true}],"reference_count":81,"raw_metadata":null,"created_at":"2026-07-19T02:57:52.712371Z","pmid":"41040411","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}