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Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.

Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY - Elife (2015)

Bottom Line: These results explain numerous disease-associated mutations.Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition.The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

No MeSH data available.


Related in: MedlinePlus

Distinct dimeric assembly of Fz4CRD and mouse Fz8CRD observed from crystal structures.(A) The superimposition of Fz4CRD structures from two crystal forms, coloured as pink and magenta for crystal form I and green and gray for crystal form II. (B) The structure of dimeric mouse Fz8CRD (PDB: 1IJA) coloured as cyan and blue. The encircled N and C denote the N- and C-termini.DOI:http://dx.doi.org/10.7554/eLife.06554.012
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fig3s2: Distinct dimeric assembly of Fz4CRD and mouse Fz8CRD observed from crystal structures.(A) The superimposition of Fz4CRD structures from two crystal forms, coloured as pink and magenta for crystal form I and green and gray for crystal form II. (B) The structure of dimeric mouse Fz8CRD (PDB: 1IJA) coloured as cyan and blue. The encircled N and C denote the N- and C-termini.DOI:http://dx.doi.org/10.7554/eLife.06554.012

Mentions: Fz receptors are members of the GPCR family (Nichols et al., 2013), known for formation of receptor dimers, although it is unclear whether dimerization is mediated by the CRD, transmembrane helices or intracellular domain. In the case of Fz4, β-galactosidase complementation in combination with bioluminescence resonance energy transfer and split-yellow fluorescence protein assays suggest that Fz4 exists as dimer on the cell membrane in the absence of Norrin or Wnts (Kaykas et al., 2004; Ke et al., 2013). However, ligand-independent receptor dimerization of Fz4 is not sufficient to activate signalling (Xu et al., 2004; Ke et al., 2013). Interestingly, we found that our Fz4CRD structures form the same dimeric assembly in two crystal lattices (r.m.s. deviation of 0.7 Å over 238 equivalent Cα atoms from two crystal forms; Figure 3—figure supplement 2A). The dimer interface has an average 1330 Å2 buried surface area, in agreement with the characteristics of known protein–protein interfaces (Lawrence and Colman, 1993). However, this Fz4CRD dimer (front-to-front) is distinct from the previously reported crystal structure of mouse Fz8CRD dimer (back-to-back; Figure 3—figure supplement 2B) (Dann et al., 2001). We were therefore curious to assess the dimerization characteristics of the CRDs of Fz receptors. Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) results (Figure 3B and Table 3) showed Fz4CRD, Fz5CRD and Fz8CRD exist as monomers in solution at 50 μM concentration, in agreement with previously reported SEC studies of Fz8CRD and SEC-MALS analyses of MuSKCRD and SmoCRD (Stiegler et al., 2009; Nachtergaele et al., 2013). SAXS measurements further support the conclusion that Fz4CRD is monomeric in solution at 290 μM concentration (Figure 3C,D). Taken together, our results suggest that the CRDs of Fz receptors exist as monomers and may not be involved in receptor dimerization; multiple GPCRs dimerize through their hepta-helical transmembrane domains (Rios et al., 2001). However, we cannot exclude the possibility that in the environment of the cellular membrane the weak interaction propensities of the CRDs, in combination with the transmembrane domains, are important for the dimerization of Fz receptors.10.7554/eLife.06554.013Table 3.


Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.

Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY - Elife (2015)

Distinct dimeric assembly of Fz4CRD and mouse Fz8CRD observed from crystal structures.(A) The superimposition of Fz4CRD structures from two crystal forms, coloured as pink and magenta for crystal form I and green and gray for crystal form II. (B) The structure of dimeric mouse Fz8CRD (PDB: 1IJA) coloured as cyan and blue. The encircled N and C denote the N- and C-termini.DOI:http://dx.doi.org/10.7554/eLife.06554.012
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4497409&req=5

fig3s2: Distinct dimeric assembly of Fz4CRD and mouse Fz8CRD observed from crystal structures.(A) The superimposition of Fz4CRD structures from two crystal forms, coloured as pink and magenta for crystal form I and green and gray for crystal form II. (B) The structure of dimeric mouse Fz8CRD (PDB: 1IJA) coloured as cyan and blue. The encircled N and C denote the N- and C-termini.DOI:http://dx.doi.org/10.7554/eLife.06554.012
Mentions: Fz receptors are members of the GPCR family (Nichols et al., 2013), known for formation of receptor dimers, although it is unclear whether dimerization is mediated by the CRD, transmembrane helices or intracellular domain. In the case of Fz4, β-galactosidase complementation in combination with bioluminescence resonance energy transfer and split-yellow fluorescence protein assays suggest that Fz4 exists as dimer on the cell membrane in the absence of Norrin or Wnts (Kaykas et al., 2004; Ke et al., 2013). However, ligand-independent receptor dimerization of Fz4 is not sufficient to activate signalling (Xu et al., 2004; Ke et al., 2013). Interestingly, we found that our Fz4CRD structures form the same dimeric assembly in two crystal lattices (r.m.s. deviation of 0.7 Å over 238 equivalent Cα atoms from two crystal forms; Figure 3—figure supplement 2A). The dimer interface has an average 1330 Å2 buried surface area, in agreement with the characteristics of known protein–protein interfaces (Lawrence and Colman, 1993). However, this Fz4CRD dimer (front-to-front) is distinct from the previously reported crystal structure of mouse Fz8CRD dimer (back-to-back; Figure 3—figure supplement 2B) (Dann et al., 2001). We were therefore curious to assess the dimerization characteristics of the CRDs of Fz receptors. Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) results (Figure 3B and Table 3) showed Fz4CRD, Fz5CRD and Fz8CRD exist as monomers in solution at 50 μM concentration, in agreement with previously reported SEC studies of Fz8CRD and SEC-MALS analyses of MuSKCRD and SmoCRD (Stiegler et al., 2009; Nachtergaele et al., 2013). SAXS measurements further support the conclusion that Fz4CRD is monomeric in solution at 290 μM concentration (Figure 3C,D). Taken together, our results suggest that the CRDs of Fz receptors exist as monomers and may not be involved in receptor dimerization; multiple GPCRs dimerize through their hepta-helical transmembrane domains (Rios et al., 2001). However, we cannot exclude the possibility that in the environment of the cellular membrane the weak interaction propensities of the CRDs, in combination with the transmembrane domains, are important for the dimerization of Fz receptors.10.7554/eLife.06554.013Table 3.

Bottom Line: These results explain numerous disease-associated mutations.Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition.The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

No MeSH data available.


Related in: MedlinePlus