Mutational Analysis of Norrin-Frizzled4 Recognition
2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês
10.1074/jbc.m609618200
ISSN1083-351X
AutoresPhilip M. Smallwood, John C. Williams, Qiang Xu, Daniel J. Leahy, Jeremy Nathans,
Tópico(s)Genetic Syndromes and Imprinting
ResumoNorrin 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 β-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. 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 β-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. The Frizzled family of cell surface receptors is present throughout the animal kingdom, with 10, four, and three family members encoded in the genomes of mammals, Drosophila, and Caenorhabditis elegans, respectively. The Frizzleds play an essential role in processes as diverse as embryonic segment polarity, midgut development, and bristle orientation in Drosophila, and axon guidance, retinal vascular development, and hair follicle orientation in mice (1Gubb D. Garcia-Bellido A. J. Embryol. Exp. Morphol. 1982; 68: 37-57PubMed Google Scholar, 2Bhanot P. Fish M. Jemison J.A. Nusse R. Nathans J. Cadigan K.M. Development. 1999; 126: 4175-4186PubMed Google Scholar, 3Chen C.M. Struhl G. Development. 1999; 126: 5441-5452PubMed Google Scholar, 4Wang Y. Thekdi N. Smallwood P.M. Macke J.P. Nathans J. J. 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Berger W. Invest. Ophthalmol. Vis. Sci. 2005; 46: 3372-3382Crossref PubMed Scopus (109) Google Scholar). Norrin binds with nanomolar affinity to the Fz4 CRD but not to several other Frizzled CRDs. Like Wnts, Norrin associates with the extracellular matrix, which limits its range of action to those target cells immediately surrounding its site of synthesis. Thus, Norrin appears to function in many respects like a Wnt despite a complete absence of primary sequence homology with the Wnt family. In this paper, we have addressed several questions raised by the discovery that Norrin and Fz4 constitute a ligand-receptor pair. First, how selective is the binding of Norrin to the Fz4 CRD as compared with other Frizzled and sFRP 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? Finally, which regions of Norrin are involved in Fz4 binding and canonical pathway activation? As described below, 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. Site-directed Mutagenesis—Mutations were constructed by tandem PCR. All DNA segments derived from PCR were sequenced to confirm the presence of the desired mutation and to rule out spurious mutations. Production of Xenopus Wnt8 (Xwnt8), Norrin, and Fz4 CRD Fusion Proteins—AP-3Myc-Norrin was secreted from transiently transfected 293 cells using a vector with a cytomegalovirus immediate early gene enhancer and promotor; the AP-3Myc-Norrin was collected in Dulbecco's modified Eagle's medium/F-12 medium containing penicillin/streptomycin and 10% calf serum and stored at 4 °C. Xwnt8-Myc-AP was produced under the control of a metallothionein promotor in stably transfected hygromycin-resistant Drosophila S2 cells. The secreted fusion protein was collected in Schneider Drosophila medium (Invitrogen) supplemented with penicillin/streptomycin, 10% calf serum, 50 μg/ml hygromycin, and 0.5 mm CuSO4 and stored at 4 °C. To produce Fz CRD-IgG fusion proteins, the CRD (i.e. the 114-amino acid region extending from the first to the tenth conserved CRD cysteine) was inserted between MluI and ApaI sites in a vector that contains a cytomegalovirus immediate early gene enhancer and promotor, followed by DNA coding for the mouse Fz8 signal peptide, the site for CRD insertion, 25 amino acids of Fz8 “linker” sequence immediately COOH-terminal to the Fz8 CRD, and the constant region of human IgG. CRD-IgG fusion proteins were secreted from transiently transfected 293 cells, collected in serum-free Dulbecco's modified Eagle's medium/F-12 containing penicillin/streptomycin, and stored in aliquots at -80 °C. Binding to Cell Surface CRD-Myc-GPI—To display CRDs at the cell surface, each CRD was inserted between MluI and ApaI sites in a vector that contains a cytomegalovirus immediate early gene enhancer and promotor followed by DNA coding for the mouse Fz8 signal peptide, the site of CRD insertion, 25 amino acids of Fz8 “linker” sequence immediately COOH-terminal to the Fz8 CRD, a Myc epitope, and the COOH-terminal GPI-anchoring peptide from decay activating factor (10Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3546-3551Crossref PubMed Scopus (286) Google Scholar). The CRD-Myc-GPI vectors were transiently transfected with Fugene 6 into COS cells that were grown on uncoated circular glass coverslips in 24-well trays in 293 cell medium as described above. Two days after transfection, the coverslips were incubated in fresh growth medium lacking bicarbonate and containing Xwnt8-Myc-AP conditioned medium (diluted 1:2) or AP-3Myc-Norrin conditioned medium (diluted 1:5) or in fresh growth medium lacking bicarbonate and containing 0.1% calf serum with anti-Myc monoclonal antibody (ascites diluted 1:1,000). After gentle rocking at 4 °C for 2 h, the coverslips were washed four times with PBS supplemented with 1 mm calcium and 1 mm magnesium (PBS/Ca/Mg), fixed in 0.5% gluteraldehyde in PBS/Ca/Mg at room temperature for 15 min, washed twice with PBS/Ca/Mg, and heated in a water bath at 65 °C for 90 min to inactivate endogenous alkaline phosphatase (AP). Immobilized AP was visualized with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Quantitative Binding to CRD-IgG Fusion Proteins—The wells of a 96-well plate carrying immobilized protein A (Reacti-Bind plates; Pierce) were coated with CRD-IgG fusion proteins by incubating overnight at 4 °C with 50 μl of a 1:5 dilution of CRD-IgG in serum-free conditioned medium, an amount of CRD-IgG that appears to saturate the binding capacity of the wells. The wells were then incubated with 1% bovine serum albumin in PBS for 3 h at room temperature; washed three times with PBS/Ca/Mg, 0.1% bovine serum albumin; and incubated with serial 2-fold dilutions of Xwnt8-Myc-AP, AP-3Myc-Norrin, or AP-conjugated anti-human IgG. After a 3-h incubation at room temperature, the wells were washed three times in PBS/Ca/Mg, 0.1% bovine serum albumin and once in PBS/Ca/Mg, and the bound AP activity was measured using a soluble 5-bromo-4-chloro-3-indolyl phosphate/tetrazolium enzyme assay (Blue-Phos; Kirkegaard and Perry Laboratories). For comparisons of Fz4 CRD-IgG binding between wild type (WT) AP-3Myc-Norrin and the various AP-3Myc-Norrin mutants, the relative concentration of AP in the conditioned medium was determined for each construct using the BluePhos substrate. The relative efficiency of binding was determined by comparing the binding signal at the interpolated midpoint of the WT AP-3Myc-Norrin dilution series (i.e. between dilutions of 1:2 and 1:4; geometric mean, 1:2.8) with the binding signal at the interpolated point along the dilution series of each mutant AP-3Myc-Norrin, corresponding to an AP concentration in the conditioned medium equivalent to the 1:2.8 dilution of the WT AP-3Myc-Norrin. By performing all of the binding assays with a dilution series of the AP fusion protein, quantitative comparisons with the WT could be made despite ∼3-fold variations in AP yield between the different AP-3Myc-Norrin conditioned media. Two independent preparations of AP-3Myc-Norrin conditioned medium were tested for each Norrin mutant. Heparin Binding—For heparin affinity purification of AP-3Myc-Norrin, 0.25 ml of heparin-Sepharose (Sigma) was washed three times with PBS/calcium/magnesium and then incubated with 5 ml of AP-3Myc-Norrin in complete conditioned medium (described above) at room temperature for 3 h with gentle end-over-end rotation. The resin was transferred to a column and washed with PBS/calcium/magnesium, and the AP-3Myc-Norrin was eluted with PBS/calcium/magnesium containing 1 m NaCl. The partially purified AP-3Myc-Norrin was diluted with 7 volumes 10 mm NaPO4, pH 7.2, calcium/magnesium supplemented with 0.01% bovine serum albumin (to reduce the final NaCl concentration to 150 mm) and stored at 4 °C. Binding to Fz4 CRD-IgG that had been captured onto protein A microwells was performed in the presence of various concentrations of porcine intestinal heparin (Sigma). Luciferase Assays—For a typical luciferase assay, a G418-resistant stable 293 cell line (STF cells) (7Xu Q. Wang Y. Dabdoub A. Smallwood P.M. Williams J. Woods C. Kelley M.W. Jiang L. Tasman W. Zhang K. Nathans J. Cell. 2004; 116: 883-895Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar) carrying the Super Top Flash firefly luciferase reporter of canonical Wnt signaling (a construct with seven tandem LEF/TCF binding sites) was transfected in triplicate in a 24-well tray using Fugene 6 with the following quantities of expression plasmid DNA per well: Norrin, 50 ng; Fz4, 50 ng; Lrp6, 50 ng; Renilla luciferase, 1 ng. Two days after transfection, cells were washed with PBS and assayed using the Promega dual luciferase assay reagents. The firefly luciferase activity was normalized to the co-expressed Renilla luciferase activity, and the average of the triplicate samples was determined. Modeling Fz4 CRD and Norrin Structures—The locations of different amino acid side chains on the Fz4 CRD surface were modeled by highlighting the corresponding residues in the high resolution Fz8 CRD crystal structure (11Dann C.E. Hsieh J.C. Rattner A. Sharma D. Nathans J. Leahy D.J. Nature. 2001; 412: 86-90Crossref PubMed Scopus (365) Google Scholar) using the program PyMol (available on the World Wide Web at pymol.sourceforge.net/). The amino acid sequences of the Fz4 and Fz8 CRDs align without recourse to insertion or deletion. To model the three-dimensional structure of Norrin, we first superimposed the structures of TGF-β family members TGF-β2 (Protein Data Bank code 2TGI) (27Daopin S. Piez K.A. Ogawa Y. Davies D.R. Science. 1992; 257: 369-373Crossref PubMed Scopus (374) Google Scholar), TGF-β3 (Protein Data Bank code 1TGJ) (28Mittl P.R. Priestle J.P. Cox D.A. McMaster G. Cerletti N. Grutter M.G. Protein Sci. 1996; 5: 1261-1271Crossref PubMed Scopus (127) Google Scholar), BMP7 (Protein Data Bank code 1BMP) (29Griffith D.L. Keck P.C. Sampath T.K. Rueger D.C. Carlson W.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 878-883Crossref PubMed Scopus (242) Google Scholar), and BMP2 (Protein Data Bank code 2GOO) (30Allendorph G.P. Vale W.W. Choe S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7643-7648Crossref PubMed Scopus (203) Google Scholar) using the program CCP4mg (31Potterton E. McNicholas S. Krissinel E. Cowtan K. Noble M. Acta Crystallogr. D. 2002; 58: 1955-1957Crossref PubMed Scopus (203) Google Scholar). This superposition was then used to align the amino acid sequences of these proteins. The subunits of TGF-β family dimers consist of two extended β-hairpins separated by an α-helical region. Each of the four β strands that make up the two hairpins contains cysteines that are conserved between TGF-β family members and Norrin and anchor alignment of the β-strand regions of Norrin with the TGF-β/BMP sequences. Branching out from these cysteines, a conserved pattern of hydrophilic and hydrophobic residues is evident between Norrin and the TGF-β family members in conserved secondary structure elements, which enabled reliable alignment of these regions of Norrin and TGF-β sequences (see Fig. 6). This sequence alignment was then used to identify sites on the BMP2 structure homologous to positions in the Norrin amino acid sequence. Since no TGF-β family member stands out as more related to Norrin, BMP2 was chosen for modeling because there is a relatively high resolution structure (2.2 Å), and a structure of BMP2 complexed with both types of TGF-β receptor allows comparison of these sites with interaction sites identified on Norrin. Norrin Binds Only to the Fz4 CRD—To assess Norrin and Wnt binding to various CRD targets, we employed AP fusion proteins as probes. For Xwnt8, a fusion with a Myc epitope and AP at the COOH terminus was produced in soluble form from stably transfected Drosophila S2 cells (10Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3546-3551Crossref PubMed Scopus (286) Google Scholar); for human Norrin, a fusion with AP and three Myc epitopes at the NH2 terminus was produced in soluble form from transiently transfected 293 cells (Fig. 1A). Both AP fusion proteins were collected in medium containing 10% serum and, unless otherwise noted, were used for binding assays without further purification. Signaling assays were performed with a β-catenin-responsive luciferase reporter in stably transfected 293 cells transiently expressing Fz4, Lrp6, and human Norrin carrying a COOH-terminal rhodopsin tag (Fig. 1A). For binding assays, the CRD targets were presented in two formats (Fig. 1). In the first format, the CRD was displayed as a Myc-tagged and GPI-anchored protein on the surface of transfected COS cells, and binding was performed with live cells. Cell surface localization and accessibility of the CRD was confirmed for each CRD construct by incubating live cells with an anti-Myc monoclonal antibody. In this format, the assay measures binding in the context of plasma membrane lipids, glycoconjugates, and cell-associated extracellular matrix molecules. Cell surface binding was scored qualitatively based on the average AP intensity per cell (Fig. 1B). In the second format, the CRD was expressed as a fusion to the constant region of human IgG, which was then immobilized in protein A-coated microwells. Binding in this context occurs free of cell-associated molecules and was scored quantitatively (Fig. 1, C and D). To systematically assess the specificity of Norrin-Fz4 binding, each of the 10 Frizzled and five sFRP CRDs encoded in the mouse genome was displayed on the surface of transfected COS cells and probed with anti-Myc, AP-3Myc-Norrin, or Xwnt8-Myc-AP. As seen in Fig. 2 and supplemental Table 1, all 15 CRDs accumulate at the cell surface, and four of them (from Fz4, Fz5, Fz7, and Fz8) efficiently bind Xwnt8-Myc-AP. In previous work, we had assayed the CRDs of Fz2-Fz8 for AP-Myc-Norrin binding and observed binding only to the Fz4 CRD (7Xu Q. Wang Y. Dabdoub A. Smallwood P.M. Williams J. Woods C. Kelley M.W. Jiang L. Tasman W. Zhang K. Nathans J. Cell. 2004; 116: 883-895Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). Here we extend this analysis to the complete set of mammalian CRDs and observe that only the Fz4 CRD shows detectable AP-3Myc-Norrin binding. A dendrogram showing the related-ness of the 15 mouse CRD sequences does not place the Fz4 CRD sequence on an outlying branch within this family, implying that relatively modest sequence differences between Fz4 and the other 14 CRDs are responsible for the ability of Norrin to uniquely recognize Fz4. Identifying CRD Sequences Involved in Norrin Binding—To define sequences within the Fz4 CRD responsible for its selective recognition by Norrin, we first used the high resolution x-ray structure of the Fz8 CRD to predict which Fz4 CRD amino acids are likely to be exposed at the protein surface. Since the Fz4 and Fz8 CRD sequences align without insertion or deletion and are identical at 46 of the 114 positions, including the 10 conserved cysteines, it is likely that the α-carbon positions of the Fz4 CRD closely match those of the Fz8 CRD (Fig. 3B). By way of comparison, the Fz8 and sFRP3 CRDs are identical at 52 of 114 positions with two insertions/deletions, and their x-ray structures show nearly identical tertiary structures with a root mean square deviation of 0.97 Å for 101 of 122 α-carbons (11Dann C.E. Hsieh J.C. Rattner A. Sharma D. Nathans J. Leahy D.J. Nature. 2001; 412: 86-90Crossref PubMed Scopus (365) Google Scholar). Three strategies were used to create Fz4 CRD sequence variants for binding assays. In the first, amino acids predicted to reside on the surface of the Fz4 CRD were mutated to alanine in 12 groups of 2-5 residues/group (Fz4 alanine scanning; Fig. 3, A, D, and E). In the second strategy, analogous block substitutions were generated, but in this case only those amino acids that differed between Fz4 and Fz8 and were predicted to reside on the surface were mutated; at each position, the Fz8 amino acid was inserted in place of its counterpart in Fz4 (Fz8 blocks into Fz4; Fig. 3, A and C). In the third strategy, nine chimeras between Fz4 and Fz8 CRDs were generated, such that each had only a single junction between Fz4 and Fz8 sequences (i.e. the NH2-terminal region was derived from Fz4 and the COOH-terminal region from Fz8 or the reverse; Fz4/Fz8 chimeras or Fz8/Fz4 chimeras, respectively; Fig. 3A). In all of these strategies, the cysteines were left unaltered. Because both Fz4 and Fz8 CRDs bind to Xwnt8-Myc-AP but only the Fz4 CRD binds to AP-3Myc-Norrin, we would predict that all correctly folded CRD constructs generated with the second and third strategies would retain Xwnt8-Myc-AP binding, and, among these, differences in AP-3Myc-Norrin binding would reflect sequence differences between Fz4 and Fz8 that are relevant to the specific recognition of Fz4 by Norrin. CRDs generated with each of the three strategies were expressed as Myc-GPI-anchored proteins and tested for binding to anti-Myc, Xwnt8-Myc-AP, and AP-3Myc-Norrin (Table 1 and Fig. 3E). All of the CRD constructs accumulate at the cell surface, and approximately half show binding to both Xwnt8-Myc-AP and AP-3Myc-Norrin that is comparable with the WT Fz4 CRD. For this subset of mutants, we can conclude that the CRD tertiary structure is largely unperturbed and that the mutated surface region is unlikely to be critically involved in AP-3Myc-Norrin or Xwnt8-Myc-AP binding. For the remaining CRD mutants that are variably defective in binding, the defects in Xwnt8-Myc-AP and AP-3Myc-Norrin binding are closely correlated (Table 1 and Fig. 3E). Although these data cannot distinguish between binding defects due to a distortion of tertiary structure and those due to defects in residues that have direct contact with the ligand, the surface locations of the alanine scanning and Fz8 block substitutions argue for the latter as the more likely explanation. We infer, therefore, that AP-3Myc-Norrin and Xwnt8-Myc-AP bind to largely overlapping sites on the CRD. The surface regions defined here for Xwnt8-Myc-AP binding are in good agreement with the regions defined by earlier mutagenesis studies using the Fz8 and Drosophila Fz2 CRDs (10Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3546-3551Crossref PubMed Scopus (286) Google Scholar, 11Dann C.E. Hsieh J.C. Rattner A. Sharma D. Nathans J. Leahy D.J. Nature. 2001; 412: 86-90Crossref PubMed Scopus (365) Google Scholar).TABLE 1Semiquantitative binding of Norrin and Xwnt8 AP fusion proteins to COS cells expressing Fz4 CRD-Myc-GPI constructsProbeTargetAP-3Myc-NorrinXwnt8-APAnti-MycFz4 WT+++++++++Fz4 alanine scanning A++++++++Fz4 alanine scanning B−−+++Fz4 alanine scanning C++/++/−+++Fz4 alanine scanning D+++++++++Fz4 alanine scanning E+++++++++Fz4 alanine scanning F+/−−+++Fz4 alanine scanning G++++/++++Fz4 alanine scanning H++++++++Fz4 alanine scanning I+/−+/−+++Fz4 alanine scanning J++++
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