Mutational Analysis of Norrin-Frizzled4 Recognition

Abstract

Norrin and Frizzled4 (Fz4) function as a ligand-receptor pair to control vascular development in the retina and inner ear. In mice and humans, mutations in either of the corresponding genes lead to defects in vascular development. The present work is aimed at defining the sequence determinants of binding specificity between Norrin and the Fz4 amino-terminal ligand-binding domain (the "cysteine-rich domain" (CRD)). The principal conclusions are as follows: 1) Norrin binds to the Fz4 CRD and does not detectably bind to the 14 other mammalian Frizzled and secreted Frizzled-related protein CRDs; 2) Norrin and Xenopus Wnt8 recognize largely overlapping regions of the Fz4 CRD; 3) surface determinants on the Fz4 and Fz8 CRDs that allow Norrin to distinguish between these two CRDs reside within several small regions on one face of the CRD; 4) Norrin function depends critically on three pairs of cysteines that form the highly conserved trio of disulfide bonds shared among all cystine knot proteins, but the remaining two putative disulfide bonds are less important; 5) Norrin-CRD binding depends on a largely contiguous group of amino acids in the extended ␤-sheet domain of Norrin that are predicted to face away from the interface between the two monomers in the Norrin homodimer; 6) Norrin-CRD binding is strongly modulated by interactions involving charged amino acid side chains; and 7) Norrin-CRD binding is enhanced ϳ10-fold by the addition of heparin. These observations are discussed in the context of Frizzled signaling and the structure and function of other cystine knot proteins.

An Essential Role for Frizzled5 in Neuronal Survival in the Parafascicular Nucleus of the Thalamus

“…However, as an indirect test of the specificity of this hypothesized interaction, 293/STF cells were cotransfected with Fz5 and Wnt9b together with the N-terminal ligand-binding domain [also referred to as the cysteine-rich domain (CRD)] from each of the 10 Frizzleds in the form of myc-epitope-tagged GPI-anchored derivatives. Previous work demonstrated efficient plasma membrane localization of these Frizzled CRD-myc-GPI derivatives and their accessibility to extracellular ligands, including Wnts and antibodies (Hsieh et al, 1999;Xu et al, 2004;Smallwood et al, 2007). In the cotransfection experiment shown in Figure 9C, the Fz5 and Fz8 CRDs efficiently inhibited signaling by coexpressed Wnt9b and Fz5, whereas the remaining eight Frizzled CRDs had little or no effect.…”

“…Wnt1, which strongly activates signaling in 293/STF cells, presumably by binding to one or more endogenous Frizzled receptors, is inhibited more than 10-fold by Fz5 CRD-myc-GPI. Signaling by Lrp5, Fz4, and Norrin, a nonWnt ligand specific for Fz4 (Xu et al, 2004;Smallwood et al, 2007) exhibits ϳ15% inhibition by Fz5 CRD-myc-GPI and ϳ50% inhibition by Fz4 CRD-myc-GPI, whereas signaling by Wnt9b and Fz5 is inhibited Ͼ10-fold by Fz5 CRD-myc-GPI and not at all by Fz4 CRD-myc-GPI.…”

An Essential Role of the Cysteine-rich Domain of FZD4 in Norrin/Wnt Signaling and Familial Exudative Vitreoretinopathy

“…FZD4 binding to Norrin is disrupted by the C204R mutation, suggesting that the CRD may be beyond the previously predicted region (i.e. the 114-amino acid region extending from the first to the tenth conserved CRD cysteine) (19) or that Norrin binding to FZD4 requires the CRD plus additional residues C-terminal to the CRD.…”

Secreted and Transmembrane Wnt Inhibitors and Activators

“…Thus, although structurally unrelated to Wnts, Norrin functions like a Wnt. Norrin function requires three pairs of cysteines that form the conserved trio of disulfide bonds shared among all cystine knot proteins (Smallwood et al 2007).…”

“…Among the 10 mammalian Fzs, Fz4 is the only Norrin receptor (Smallwood et al 2007). Yet Fz4 can also transduce the signal of Wnts, raising the question of how Fz4/LRP5 can respond to different types of ligands to activate b-catenin-dependent transcription.…”