Mutational Analysis of Norrin-Frizzled4 Recognition

Philip M Smallwood,John Williams,Qiang Xu,Daniel J Leahy,Jeremy Nathans

doi:10.1074/jbc.m609618200

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 beta-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 approximately 10-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.

Highlights

“canonical” signaling pathway in conjunction with the coreceptors Lrp5 or Lrp6 [8]
How selective is the binding of Norrin to the Fz4 cysteine-rich domain (CRD) as compared with other Frizzled and secreted Frizzled-related proteins (sFRPs) CRDs? Second, which regions of the Fz4 CRD are responsible for Norrin binding, and what is the relationship of these regions to those responsible for Wnt binding? which regions of Norrin are involved in Fz4 binding and canonical pathway activation?
Norrin Binds Only to the Fz4 CRD—To assess Norrin and Wnt binding to various CRD targets, we employed alkaline phosphatase (AP) fusion proteins as probes

Summary

Introduction

“canonical” signaling pathway in conjunction with the coreceptors Lrp or Lrp6 [8]. Wnts bind to a compact domain at the Frizzled NH2 terminus referred to as the cysteine-rich domain (CRD)2 [9, 10], for which a high resolution crystallographic structure has been obtained [11]. Norrin, a protein that is not a member of the Wnt family, has been identified as a ligand that binds the Fz4 CRD with high affinity and potently activates the canonical signaling pathway [7]. Norrin has the ability to discriminate between the Fz4 CRD and the 14 other mammalian Frizzled and sFRP CRDs, and by site-directed mutagenesis we delimit the regions on both Norrin and Fz4 responsible for this recognition. These results have implications for other ligand-receptor families in which closely related proteins exhibit differential recognition of potential binding partners

Methods

Results

Conclusion