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The Netrin-related domain of Sfrp1 interacts with Wnt ligands and antagonizes their activity in the anterior neural plate.

Lopez-Rios J, Esteve P, Ruiz JM, Bovolenta P - Neural Dev (2008)

Bottom Line: In contrast, the functional relevance of the SfrpNTR has been barely addressed.In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Neurobiología Molecular Celular y del Desarrollo, Instituto Cajal, CSIC, Dr. Arce 37, Madrid, 28002, Spain. Javier.Lopez-Rios@unibas.ch

ABSTRACT

Background: Secreted frizzled related proteins (SFRPs) are multifunctional modulators of Wnt and BMP (Bone Morphogenetic Protein) signalling necessary for the development of most organs and the homeostasis of different adult tissues. SFRPs fold in two independent domains: the cysteine rich domain (SfrpCRD) related to the extracellular portion of Frizzled (Fz, Wnt receptors) and the Netrin module (SfrpNTR) defined by homologies with molecules such as Netrin-1, inhibitors of metalloproteinases and complement proteins. Due to its structural relationship with Fz, it is believed that SfrpCRD interferes with Wnt signalling by binding and sequestering the ligand. In contrast, the functional relevance of the SfrpNTR has been barely addressed.

Results: Here, we combine biochemical studies, mutational analysis and functional assays in cell culture and medaka-fish embryos to show that the Sfrp1NTR mimics the function of the entire molecule, binds to Wnt8 and antagonizes Wnt canonical signalling. This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR. In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.

Conclusion: On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

Show MeSH
Distantly related NTR domains mimic the activity of Sfrp1NTR with different efficiencies. (a-j) Brightfield views of embryos injected with different full-length or chimerical mRNAs as indicated. Insets in (c,d,f,g) correspond to embryos processed for double in situ hybridization for rx3 (red) and foxA2 (blue). Note that injections of Sfrp1 (b), Sfrp2 (c, and inset) lead to similar expansion of anterior structures compared to control embryos (a), while Sfrp3 has a very weak anteriorizing effect (d) observed only in 4% of the injected embryos (Table 1; inset in (d) shows an embryo injected with a high dose (500 ng/μl) of Sfrp3 mRNA). Similarly, Sfrp3NTR induces a weak anteriorisation at a low frequency (embryo shown in (g); Table 1), whereas the distantly related NTR motif from Netrin-1 (i) induces an expansion of the forebrain as observed with Sfrp1NTR. Note that the functionality of the NTR domain depends on an intact tertiary structure, since cysteine to serine mutations in Sfrp1NTR-C177S;C180S and Netrin-1NTR-C471S;C475S constructs (h,j) induce a total or partial loss of the effect. See Table 1. Scale bar: 0.1 mm.
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Figure 7: Distantly related NTR domains mimic the activity of Sfrp1NTR with different efficiencies. (a-j) Brightfield views of embryos injected with different full-length or chimerical mRNAs as indicated. Insets in (c,d,f,g) correspond to embryos processed for double in situ hybridization for rx3 (red) and foxA2 (blue). Note that injections of Sfrp1 (b), Sfrp2 (c, and inset) lead to similar expansion of anterior structures compared to control embryos (a), while Sfrp3 has a very weak anteriorizing effect (d) observed only in 4% of the injected embryos (Table 1; inset in (d) shows an embryo injected with a high dose (500 ng/μl) of Sfrp3 mRNA). Similarly, Sfrp3NTR induces a weak anteriorisation at a low frequency (embryo shown in (g); Table 1), whereas the distantly related NTR motif from Netrin-1 (i) induces an expansion of the forebrain as observed with Sfrp1NTR. Note that the functionality of the NTR domain depends on an intact tertiary structure, since cysteine to serine mutations in Sfrp1NTR-C177S;C180S and Netrin-1NTR-C471S;C475S constructs (h,j) induce a total or partial loss of the effect. See Table 1. Scale bar: 0.1 mm.

Mentions: To explore this possibility further, we next investigated whether the relevance of the NTR domain in antagonizing Wnt ligands could be extended to other SFRP family members or even to distantly related NTR domains [11]. According to phylogenetic analysis, the SFRP family is composed of three subfamilies: Sfrp1/2/5, Tlc/Sizzled and the very divergent Sfrp3/4 [13]. We thus compared the activity of Sfrp1 and Sfrp1NTR with equivalent constructs from Sfrp2 and Sfrp3 (Figure 1), close and a divergent members of the SFRP family, respectively. Furthermore, we also chose to analyze the NTR domain of Netrin-1 (Figure 1), a secreted protein involved in axon guidance where the NTR domain was first identified [11,20] as a distantly related module. When assayed for their ability to reproduce the Sfrp1 over-expression phenotype (Figures 2b and 7b), Sfrp2 and Sfrp2NTR displayed a significant anteriorising activity almost identical to that of Sfrp1 and Sfrp1NTR, respectively (Figure 7c,f; Table 1), while Sfrp3 and Sfrp3NTR had a much weaker activity and expansion of anterior markers was only observed upon injection of high mRNA concentrations (Figure 7d(inset),g; Table 1). Intriguingly, Netrin-1NTR mRNA injections led to a mild expansion of the forebrain at lower frequency than those of Sfrp1NTR (Figure 7i; Table 1). These results indicate that, despite the evolutionary distance, this module can mimic SFRP function, presumably by binding to endogenous Wnt.


The Netrin-related domain of Sfrp1 interacts with Wnt ligands and antagonizes their activity in the anterior neural plate.

Lopez-Rios J, Esteve P, Ruiz JM, Bovolenta P - Neural Dev (2008)

Distantly related NTR domains mimic the activity of Sfrp1NTR with different efficiencies. (a-j) Brightfield views of embryos injected with different full-length or chimerical mRNAs as indicated. Insets in (c,d,f,g) correspond to embryos processed for double in situ hybridization for rx3 (red) and foxA2 (blue). Note that injections of Sfrp1 (b), Sfrp2 (c, and inset) lead to similar expansion of anterior structures compared to control embryos (a), while Sfrp3 has a very weak anteriorizing effect (d) observed only in 4% of the injected embryos (Table 1; inset in (d) shows an embryo injected with a high dose (500 ng/μl) of Sfrp3 mRNA). Similarly, Sfrp3NTR induces a weak anteriorisation at a low frequency (embryo shown in (g); Table 1), whereas the distantly related NTR motif from Netrin-1 (i) induces an expansion of the forebrain as observed with Sfrp1NTR. Note that the functionality of the NTR domain depends on an intact tertiary structure, since cysteine to serine mutations in Sfrp1NTR-C177S;C180S and Netrin-1NTR-C471S;C475S constructs (h,j) induce a total or partial loss of the effect. See Table 1. Scale bar: 0.1 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 7: Distantly related NTR domains mimic the activity of Sfrp1NTR with different efficiencies. (a-j) Brightfield views of embryos injected with different full-length or chimerical mRNAs as indicated. Insets in (c,d,f,g) correspond to embryos processed for double in situ hybridization for rx3 (red) and foxA2 (blue). Note that injections of Sfrp1 (b), Sfrp2 (c, and inset) lead to similar expansion of anterior structures compared to control embryos (a), while Sfrp3 has a very weak anteriorizing effect (d) observed only in 4% of the injected embryos (Table 1; inset in (d) shows an embryo injected with a high dose (500 ng/μl) of Sfrp3 mRNA). Similarly, Sfrp3NTR induces a weak anteriorisation at a low frequency (embryo shown in (g); Table 1), whereas the distantly related NTR motif from Netrin-1 (i) induces an expansion of the forebrain as observed with Sfrp1NTR. Note that the functionality of the NTR domain depends on an intact tertiary structure, since cysteine to serine mutations in Sfrp1NTR-C177S;C180S and Netrin-1NTR-C471S;C475S constructs (h,j) induce a total or partial loss of the effect. See Table 1. Scale bar: 0.1 mm.
Mentions: To explore this possibility further, we next investigated whether the relevance of the NTR domain in antagonizing Wnt ligands could be extended to other SFRP family members or even to distantly related NTR domains [11]. According to phylogenetic analysis, the SFRP family is composed of three subfamilies: Sfrp1/2/5, Tlc/Sizzled and the very divergent Sfrp3/4 [13]. We thus compared the activity of Sfrp1 and Sfrp1NTR with equivalent constructs from Sfrp2 and Sfrp3 (Figure 1), close and a divergent members of the SFRP family, respectively. Furthermore, we also chose to analyze the NTR domain of Netrin-1 (Figure 1), a secreted protein involved in axon guidance where the NTR domain was first identified [11,20] as a distantly related module. When assayed for their ability to reproduce the Sfrp1 over-expression phenotype (Figures 2b and 7b), Sfrp2 and Sfrp2NTR displayed a significant anteriorising activity almost identical to that of Sfrp1 and Sfrp1NTR, respectively (Figure 7c,f; Table 1), while Sfrp3 and Sfrp3NTR had a much weaker activity and expansion of anterior markers was only observed upon injection of high mRNA concentrations (Figure 7d(inset),g; Table 1). Intriguingly, Netrin-1NTR mRNA injections led to a mild expansion of the forebrain at lower frequency than those of Sfrp1NTR (Figure 7i; Table 1). These results indicate that, despite the evolutionary distance, this module can mimic SFRP function, presumably by binding to endogenous Wnt.

Bottom Line: In contrast, the functional relevance of the SfrpNTR has been barely addressed.In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Neurobiología Molecular Celular y del Desarrollo, Instituto Cajal, CSIC, Dr. Arce 37, Madrid, 28002, Spain. Javier.Lopez-Rios@unibas.ch

ABSTRACT

Background: Secreted frizzled related proteins (SFRPs) are multifunctional modulators of Wnt and BMP (Bone Morphogenetic Protein) signalling necessary for the development of most organs and the homeostasis of different adult tissues. SFRPs fold in two independent domains: the cysteine rich domain (SfrpCRD) related to the extracellular portion of Frizzled (Fz, Wnt receptors) and the Netrin module (SfrpNTR) defined by homologies with molecules such as Netrin-1, inhibitors of metalloproteinases and complement proteins. Due to its structural relationship with Fz, it is believed that SfrpCRD interferes with Wnt signalling by binding and sequestering the ligand. In contrast, the functional relevance of the SfrpNTR has been barely addressed.

Results: Here, we combine biochemical studies, mutational analysis and functional assays in cell culture and medaka-fish embryos to show that the Sfrp1NTR mimics the function of the entire molecule, binds to Wnt8 and antagonizes Wnt canonical signalling. This activity requires intact tertiary structure and is shared by the distantly related Netrin-1NTR. In contrast, the Sfrp1CRD cannot mirror the function of the entire molecule in vivo but interacts with Fz receptors and antagonizes Wnt8-mediated beta-catenin transcriptional activity.

Conclusion: On the basis of these results, we propose that SFRP modulation of Wnt signalling may involve multiple and differential interactions among Wnt, Fz and SFRPs.

Show MeSH