Abstract
Accessory proteins in Frizzled (FZD) receptor complexes are thought to determine ligand selectivity and signaling amplitude. Genetic evidence indicates that specific combinations of accessory proteins and ligands mediate vascular β-catenin signaling in different CNS structures. In the retina, the tetraspanin TSPAN12 and the ligand norrin (NDP) mediate angiogenesis, and both genes are linked to familial exudative vitreoretinopathy (FEVR), yet the molecular function of TSPAN12 remains poorly understood. Here, we report that TSPAN12 is an essential component of the NDP receptor complex and interacts with FZD4 and NDP via its extracellular loops, consistent with an action as co-receptor that enhances FZD4 ligand selectivity for NDP. FEVR-linked mutations in TSPAN12 prevent the incorporation of TSPAN12 into the NDP receptor complex. Invitro and in Xenopus embryos, TSPAN12 alleviates defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction. This study sheds light on the poorly understood function of accessory proteins in FZD signaling.
Highlights
A multitude of metazoan developmental processes are controlled by wnt/b-catenin signaling, a central signaling pathway that is perturbed in human diseases, including cancer, bone disease, and vascular disease (Clevers and Nusse, 2012)
TSPAN12 Missense Mutations Impair FZD4 Signaling in a Ligand-Specific Manner We introduced ten previously identified familial exudative vitreoretinopathy (FEVR)-linked missense mutations (Kondo et al, 2011; Nikopoulos et al, 2010a; Poulter et al, 2010, 2012; Yang et al, 2011) individually into hemagglutinin (HA)-tagged TSPAN12 (Figures 1A and S1)
When we compared the TOPFlash reporter activity (Veeman et al, 2003) of 293T cells transfected with FZD4, LRP5, and wild-type TSPAN12 versus mutated TSPAN12, we found that the ability of TSPAN12 to enhance NDP/FZD4 signaling was impaired to various degrees by the FEVR-linked mutations
Summary
A multitude of metazoan developmental processes are controlled by wnt/b-catenin signaling, a central signaling pathway that is perturbed in human diseases, including cancer, bone disease, and vascular disease (Clevers and Nusse, 2012). The combinatorial assembly of receptor complexes from multiple additional families of membrane proteins is thought to provide ligand selectivity and contribute to context-specific signaling outcomes (van Amerongen and Nusse, 2009). Despite their important roles, the function of accessory proteins in determining ligand-receptor selectivity and the mechanisms of receptor complex assembly are poorly understood (Schulte, 2015). The critical in vivo role of distinct ligands and specific accessory membrane proteins in b-catenin signaling is evident in CNS blood vessel development and blood-CNS barrier formation, processes that require canonical b-catenin signaling in endothelial cells (Liebner et al, 2008; Wang et al, 2012; Ye et al, 2009; Zhou et al, 2014). NDP has been studied in several disease models for its vascular and neuroprotective roles (Chen et al, 2015; Ohlmann and Tamm, 2012)
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