title: Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.
abstract: BACKGROUND
  Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed.
  
  
  RESULTS
  Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner.
  
  
  CONCLUSIONS
  We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.
authors.forename: Sumit
authors.initials: S
authors.lastname: Kumar
authors.forename: Mihaela
authors.initials: M
authors.lastname: Žigman
authors.forename: Trushar R
authors.initials: TR
authors.lastname: Patel
authors.forename: Benjamin
authors.initials: B
authors.lastname: Trageser
authors.forename: Julia Christina
authors.initials: JC
authors.lastname: Gross
authors.forename: Karolin
authors.initials: K
authors.lastname: Rahm
authors.forename: Michael
authors.initials: M
authors.lastname: Boutros
authors.forename: Dietmar
authors.initials: D
authors.lastname: Gradl
authors.forename: Herbert
authors.initials: H
authors.lastname: Steinbeisser
authors.forename: Thomas
authors.initials: T
authors.lastname: Holstein
authors.forename: Jörg
authors.initials: J
authors.lastname: Stetefeld
authors.affiliation: Centre for Organismal Studies, Department of Molecular Evolution and Genomics, University of Heidelberg, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany. suat.oezbek@cos.uni-heidelberg.de.
authors.forename: Suat
authors.initials: S
authors.lastname: Özbek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D013329:Structure-Activity Relationship; D060449:Wnt Signaling Pathway; D060509:Wnt3A Protein / Q000737:chemistry* / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology
chemical_list: D050505:Mutant Proteins; D043562:Receptors, G-Protein-Coupled; D060509:Wnt3A Protein; C511610:Fzd8 protein, mouse