Frizzled Receptors Facilitate GPI-anchored Protein TIKI Inhibition Of Wnt Signaling At The Cell Surface

Wnt proteins constitute an evolutionarily conserved family of secreted glycosylated and lipidated ligands that play essential roles in embryogenesis,adult tissue homeostasis,and regeneration.Frizzled(FZD)proteins function as prime receptors and participate in multiple downstream signaling pathways,such as β-catenin-dependent canonical Wnt signaling and β-catenin-independent noncanonical Wnt signaling.Wnt signaling pathways are tightly regulated by multiple Wnt inhibitors,such as Dickkopf and Sclerostin proteins,which bind to LRP5/6,and the Wnt enzymatic inhibitors TIKI and Notum.TIKI proteins represent a new family of metalloproteases that can specifically cleave and inactivate Wnt proteins in Wnt-producing cells.Mechanistically,TIKI cleaves the amino terminus of Wnt3 a,and cleaved Wnt3 a forms oxidized oligomers,which lose the receptor binding ability.TIKI proteins also function in Wnt-receiving cells to antagonize Wnt signaling in early Xenopus embryonic development by an unknown mechanism.Besides Wnt3 a which participates in canonical Wnt signaling,TIKI proteins also cleave Wnt5 a,which is a non-canonical Wnt ligand.However,whether and how TIKI proteins regulate non-canonical Wnt signaling remains unclear.This study characterizes the properties of TIKI proteins and reveals the mechanism by which TIKI proteins antagonize Wnt signaling on the cell surface of Wnt-receiving cells by utilizing biochemical and cell biological approaches combined with biotin proximity labeling techniques,cell overexpression and gene knockout methods.It has the following key findings:(1)TIKI proteins are glycosylphosphatidylinositol(GPI)-anchored proteins,which can be regulated by GPI-specific lipases GDE3 and GDE6.Meanwhile,TIKI proteins function in Wnt-receiving cells in mammalian cell culture.(2)The potential association between TIKI2 protein and FZD receptors was identified by the biotin proximity labeling technique.TIKI interacts with the Wnt-FZD complex and cleaves FZD-bound Wnt3 a and Wnt5 a on the cell surface and in vitro.TIKI cleavage of Wnt proteins does not affect Wnt-FZD complex stability.(3)TIKI inhibits Wnt3a-or Wnt5a-induced phosphorylation of coreceptors LRP6 or ROR1/2.Mechanistically,TIKI cleavage of Wnt3 a prevents the Wnt3a-FZD complex from recruiting and activating the coreceptor LRP6.(4)In U2 OS cells,TIKI2 knockout renders cells hypersensitive to Wnt3 a and Wnt5 a,further revealing that endogenous TIKI2 controls Wnt signaling on the cell surface.This study characterizes TIKI proteins as GPI-anchored proteins and reveals a negative role of FZD receptors by acting as TIKI cofactors on the cell surface.This study also reveals an unexpected role of the Wnt3 a amino terminus in the engagement of the LRP6 coreceptor,which provides a new clue to explain the activation mechanism of the cell surface receptor complex of the Wnt signaling pathway.