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Eps15R is required for bone morphogenetic protein signalling and differentially compartmentalizes with Smad proteins.

Callery EM, Park CY, Xu X, Zhu H, Smith JC, Thomsen GH - Open Biol (2012)

Bottom Line: The endocytic adaptor protein Eps15R, or 'epidermal growth factor (EGF) receptor pathway substrate 15-related protein' is a component of EGF signal transduction, mediating internalization of the EGF receptor.This function resides in the Asp-Pro-Phe motif-enriched 'DPF domain' of Eps15R, which activates transcription and antagonizes Smad2 signalling.In living cells, Eps15R segregates into spatially distinct regions with different Smads, indicating an unrecognized level of Smad compartmentalization.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, PO Box 157, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. emc13@cam.ac.uk

ABSTRACT
Transforming growth factor β superfamily members signal through Smad transcription factors. Bone morphogenetic proteins (BMPs) act via Smads 1, 5 and 8 and TGF-βs signal through Smads 2 and 3. The endocytic adaptor protein Eps15R, or 'epidermal growth factor (EGF) receptor pathway substrate 15-related protein' is a component of EGF signal transduction, mediating internalization of the EGF receptor. We show that it interacts with Smad proteins, is required for BMP signalling in animal caps and stimulates Smad1 transcriptional activity. This function resides in the Asp-Pro-Phe motif-enriched 'DPF domain' of Eps15R, which activates transcription and antagonizes Smad2 signalling. In living cells, Eps15R segregates into spatially distinct regions with different Smads, indicating an unrecognized level of Smad compartmentalization.

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Related in: MedlinePlus

Eps15R enhances Smad1 signalling and is required for transcription in response to BMP signalling. (a,b) Over-expression of a control RNA encoding lactate dehydrogenase (LDH) does not impair development (a), whereas over-expression of Eps15R RNA causes defects in head and anterior mesoderm (b). (c–e) Phenotypes of embryos injected with an antisense MO oligonucleotide targeting Eps15R. (c) Controls. (d) Embryos injected with Eps15R MO exhibit shortened axes and ventrolateral defects. (e) The phenotype in (d) is significantly rescued by co-injection of MT-Eps15R RNA. (f) Morphometric analysis of PAT : AP length ratio in these embryos. Measurements were subjected to ANOVA and Tukey's test for least significant difference. (g) Diagrammatic depiction of the PAT and AP measurements. (h) Expression of BMP targets Xhox3 and Xbra is elevated when Eps15R is co-expressed with Flag-Smad1 in the animal cap assay; caps were harvested at NF11.5. (i) Eps15R MO inhibits transcription of BMP-responsive genes in animal caps treated with 100 ng ml–1 BMP4/7 heterodimers and harvested at NF11.5, yet there is no decrease in activation of these genes in response to 100 ng ml–1 FGF4 (FGF) or 10 ng ml–1 activin (act). Fold induction was calculated relative to control cap levels, and samples were normalized to the expression of ornithine decarboxylase.
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RSOB120060F2: Eps15R enhances Smad1 signalling and is required for transcription in response to BMP signalling. (a,b) Over-expression of a control RNA encoding lactate dehydrogenase (LDH) does not impair development (a), whereas over-expression of Eps15R RNA causes defects in head and anterior mesoderm (b). (c–e) Phenotypes of embryos injected with an antisense MO oligonucleotide targeting Eps15R. (c) Controls. (d) Embryos injected with Eps15R MO exhibit shortened axes and ventrolateral defects. (e) The phenotype in (d) is significantly rescued by co-injection of MT-Eps15R RNA. (f) Morphometric analysis of PAT : AP length ratio in these embryos. Measurements were subjected to ANOVA and Tukey's test for least significant difference. (g) Diagrammatic depiction of the PAT and AP measurements. (h) Expression of BMP targets Xhox3 and Xbra is elevated when Eps15R is co-expressed with Flag-Smad1 in the animal cap assay; caps were harvested at NF11.5. (i) Eps15R MO inhibits transcription of BMP-responsive genes in animal caps treated with 100 ng ml–1 BMP4/7 heterodimers and harvested at NF11.5, yet there is no decrease in activation of these genes in response to 100 ng ml–1 FGF4 (FGF) or 10 ng ml–1 activin (act). Fold induction was calculated relative to control cap levels, and samples were normalized to the expression of ornithine decarboxylase.

Mentions: Over-expression of Eps15R RNA caused defects of the eye and anterior mesendoderm (figure 2a,b). Loss of function was achieved using a translation-blocking antisense morpholino (MO). Morphant embryos had short axes, eye defects and swelling around the heart cavity (figure 2c,d). Co-injection of Myc-tagged (MT)-Eps15R RNA reduced the severity of defects (figure 2e). Whereas 81 per cent (n = 26) of Eps15R morphants had curved axes, such defects were only observed in 17 per cent (n = 24) of rescued embryos and 8 per cent (n = 24) of embryos injected with control MO. We quantitated the degree of axial rescue using the morphometric ratio of post-anal tail length : anteroposterior length (PAT/AP length ratio; figure 2f,g). Whereas this measure was decreased in Eps15R morphants (p < 0.01), there was no significant difference between the control and rescue groups, indicating a successful rescue of this phenotype (figure 2f).Figure 2.


Eps15R is required for bone morphogenetic protein signalling and differentially compartmentalizes with Smad proteins.

Callery EM, Park CY, Xu X, Zhu H, Smith JC, Thomsen GH - Open Biol (2012)

Eps15R enhances Smad1 signalling and is required for transcription in response to BMP signalling. (a,b) Over-expression of a control RNA encoding lactate dehydrogenase (LDH) does not impair development (a), whereas over-expression of Eps15R RNA causes defects in head and anterior mesoderm (b). (c–e) Phenotypes of embryos injected with an antisense MO oligonucleotide targeting Eps15R. (c) Controls. (d) Embryos injected with Eps15R MO exhibit shortened axes and ventrolateral defects. (e) The phenotype in (d) is significantly rescued by co-injection of MT-Eps15R RNA. (f) Morphometric analysis of PAT : AP length ratio in these embryos. Measurements were subjected to ANOVA and Tukey's test for least significant difference. (g) Diagrammatic depiction of the PAT and AP measurements. (h) Expression of BMP targets Xhox3 and Xbra is elevated when Eps15R is co-expressed with Flag-Smad1 in the animal cap assay; caps were harvested at NF11.5. (i) Eps15R MO inhibits transcription of BMP-responsive genes in animal caps treated with 100 ng ml–1 BMP4/7 heterodimers and harvested at NF11.5, yet there is no decrease in activation of these genes in response to 100 ng ml–1 FGF4 (FGF) or 10 ng ml–1 activin (act). Fold induction was calculated relative to control cap levels, and samples were normalized to the expression of ornithine decarboxylase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376731&req=5

RSOB120060F2: Eps15R enhances Smad1 signalling and is required for transcription in response to BMP signalling. (a,b) Over-expression of a control RNA encoding lactate dehydrogenase (LDH) does not impair development (a), whereas over-expression of Eps15R RNA causes defects in head and anterior mesoderm (b). (c–e) Phenotypes of embryos injected with an antisense MO oligonucleotide targeting Eps15R. (c) Controls. (d) Embryos injected with Eps15R MO exhibit shortened axes and ventrolateral defects. (e) The phenotype in (d) is significantly rescued by co-injection of MT-Eps15R RNA. (f) Morphometric analysis of PAT : AP length ratio in these embryos. Measurements were subjected to ANOVA and Tukey's test for least significant difference. (g) Diagrammatic depiction of the PAT and AP measurements. (h) Expression of BMP targets Xhox3 and Xbra is elevated when Eps15R is co-expressed with Flag-Smad1 in the animal cap assay; caps were harvested at NF11.5. (i) Eps15R MO inhibits transcription of BMP-responsive genes in animal caps treated with 100 ng ml–1 BMP4/7 heterodimers and harvested at NF11.5, yet there is no decrease in activation of these genes in response to 100 ng ml–1 FGF4 (FGF) or 10 ng ml–1 activin (act). Fold induction was calculated relative to control cap levels, and samples were normalized to the expression of ornithine decarboxylase.
Mentions: Over-expression of Eps15R RNA caused defects of the eye and anterior mesendoderm (figure 2a,b). Loss of function was achieved using a translation-blocking antisense morpholino (MO). Morphant embryos had short axes, eye defects and swelling around the heart cavity (figure 2c,d). Co-injection of Myc-tagged (MT)-Eps15R RNA reduced the severity of defects (figure 2e). Whereas 81 per cent (n = 26) of Eps15R morphants had curved axes, such defects were only observed in 17 per cent (n = 24) of rescued embryos and 8 per cent (n = 24) of embryos injected with control MO. We quantitated the degree of axial rescue using the morphometric ratio of post-anal tail length : anteroposterior length (PAT/AP length ratio; figure 2f,g). Whereas this measure was decreased in Eps15R morphants (p < 0.01), there was no significant difference between the control and rescue groups, indicating a successful rescue of this phenotype (figure 2f).Figure 2.

Bottom Line: The endocytic adaptor protein Eps15R, or 'epidermal growth factor (EGF) receptor pathway substrate 15-related protein' is a component of EGF signal transduction, mediating internalization of the EGF receptor.This function resides in the Asp-Pro-Phe motif-enriched 'DPF domain' of Eps15R, which activates transcription and antagonizes Smad2 signalling.In living cells, Eps15R segregates into spatially distinct regions with different Smads, indicating an unrecognized level of Smad compartmentalization.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, PO Box 157, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. emc13@cam.ac.uk

ABSTRACT
Transforming growth factor β superfamily members signal through Smad transcription factors. Bone morphogenetic proteins (BMPs) act via Smads 1, 5 and 8 and TGF-βs signal through Smads 2 and 3. The endocytic adaptor protein Eps15R, or 'epidermal growth factor (EGF) receptor pathway substrate 15-related protein' is a component of EGF signal transduction, mediating internalization of the EGF receptor. We show that it interacts with Smad proteins, is required for BMP signalling in animal caps and stimulates Smad1 transcriptional activity. This function resides in the Asp-Pro-Phe motif-enriched 'DPF domain' of Eps15R, which activates transcription and antagonizes Smad2 signalling. In living cells, Eps15R segregates into spatially distinct regions with different Smads, indicating an unrecognized level of Smad compartmentalization.

Show MeSH
Related in: MedlinePlus