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J. Biol. Chem., Vol. 282, Issue 6, 4057-4068, February 9, 2007

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Mutational Analysis of Norrin-Frizzled4 Recognition^*

Philip M. Smallwood, John Williams, Qiang Xu, Daniel J. Leahy^¶, and Jeremy Nathans^||**1

From the Department of Molecular Biology and Genetics, ^||Department of Neuroscience, ^**Department of Ophthalmology, ^¶Department of Biophysics and Biophysical Chemistry, and the Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

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 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.

Received for publication, October 12, 2006 , and in revised form, December 4, 2006.

^* This work was supported by the Howard Hughes Medical Institute and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ To whom correspondence should be addressed: 805 PCTB, 725 N. Wolfe St., The Johns Hopkins University School of Medicine, Baltimore, MD 21205. Tel.: 410-955-4679; Fax: 410-614-0827; E-mail: jnathans{at}jhmi.edu.

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