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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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The DPF domain transactivates gene expression and differentially modulates Smad signalling. (a–c) Effects of over-expression of full-length Eps15R, or the DPF domain alone, on Xenopus development. (a) Uninjected embryos. (b) Embryos injected with 3 ng RNA encoding Eps15R. (c) Embryos injected with 3 ng RNA encoding the Eps15R DPF domain. (d) RT-PCR showing globin induction by the DPF domain in NF21 animal caps. (e) The DPF domain mimics the activity of Smad1 in the induction of Xbra and Xhox3 in NF11 gastrula caps but the full-length Eps15R lacks this ability. (f) Both full length Eps15R and the DPF domain can transactivate transcription in the yeast assay. (g) The cytoplasmic localization of GFP-Eps15R-ΔDPF is disrupted, displaying a diffuse, reticulated cytoplasmic distribution, in contrast to the punctate cytoplasmic localization of the full-length version shown in figure 1d. Nuclear enrichment is retained in the absence of the DPF domain. (h) The DPF domain antagonizes the ability of Smad2 to induce expression of genes such as chordin, goosecoid and Frzb in NF11 gastrula animal caps, which instead activates the ventral marker Xhox3.
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RSOB120060F3: The DPF domain transactivates gene expression and differentially modulates Smad signalling. (a–c) Effects of over-expression of full-length Eps15R, or the DPF domain alone, on Xenopus development. (a) Uninjected embryos. (b) Embryos injected with 3 ng RNA encoding Eps15R. (c) Embryos injected with 3 ng RNA encoding the Eps15R DPF domain. (d) RT-PCR showing globin induction by the DPF domain in NF21 animal caps. (e) The DPF domain mimics the activity of Smad1 in the induction of Xbra and Xhox3 in NF11 gastrula caps but the full-length Eps15R lacks this ability. (f) Both full length Eps15R and the DPF domain can transactivate transcription in the yeast assay. (g) The cytoplasmic localization of GFP-Eps15R-ΔDPF is disrupted, displaying a diffuse, reticulated cytoplasmic distribution, in contrast to the punctate cytoplasmic localization of the full-length version shown in figure 1d. Nuclear enrichment is retained in the absence of the DPF domain. (h) The DPF domain antagonizes the ability of Smad2 to induce expression of genes such as chordin, goosecoid and Frzb in NF11 gastrula animal caps, which instead activates the ventral marker Xhox3.

Mentions: The DPF domain is required for the interaction of Eps15R with Smad1 (figure 1b) and it also binds AP2, which recruits clathrin to the cell surface [24]. We tested the activity of the DPF domain by injecting embryos with RNA encoding this protein region. Development appeared significantly perturbed by injection of 3 ng mRNA encoding the DPF domain, relative to an equivalent amount of full-length RNA (figure 3a–c), although a precise quantitative comparison of the effects of the RNAs would require equivalent molar amounts to be injected. The embryos appeared ventralized, consistent with the ability of the DPF domain to induce expression of the ventral marker α-globin in animal caps (figure 3d). The DPF domain of Esp15R mimics Smad1-mediated gene induction, upregulating the expression of Xbra and Xhox3 in animal caps (figure 3e), whereas neither the full-length protein nor the N-terminal 597 domain possesses this ability. It is possible, in a manner analogous to Smad proteins [25], that intramolecular interactions between the N-terminal EH domains and the DPF repeats inhibit the function of the full-length protein, and that these do not occur when the DPF domain is expressed alone.Figure 3.


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)

The DPF domain transactivates gene expression and differentially modulates Smad signalling. (a–c) Effects of over-expression of full-length Eps15R, or the DPF domain alone, on Xenopus development. (a) Uninjected embryos. (b) Embryos injected with 3 ng RNA encoding Eps15R. (c) Embryos injected with 3 ng RNA encoding the Eps15R DPF domain. (d) RT-PCR showing globin induction by the DPF domain in NF21 animal caps. (e) The DPF domain mimics the activity of Smad1 in the induction of Xbra and Xhox3 in NF11 gastrula caps but the full-length Eps15R lacks this ability. (f) Both full length Eps15R and the DPF domain can transactivate transcription in the yeast assay. (g) The cytoplasmic localization of GFP-Eps15R-ΔDPF is disrupted, displaying a diffuse, reticulated cytoplasmic distribution, in contrast to the punctate cytoplasmic localization of the full-length version shown in figure 1d. Nuclear enrichment is retained in the absence of the DPF domain. (h) The DPF domain antagonizes the ability of Smad2 to induce expression of genes such as chordin, goosecoid and Frzb in NF11 gastrula animal caps, which instead activates the ventral marker Xhox3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120060F3: The DPF domain transactivates gene expression and differentially modulates Smad signalling. (a–c) Effects of over-expression of full-length Eps15R, or the DPF domain alone, on Xenopus development. (a) Uninjected embryos. (b) Embryos injected with 3 ng RNA encoding Eps15R. (c) Embryos injected with 3 ng RNA encoding the Eps15R DPF domain. (d) RT-PCR showing globin induction by the DPF domain in NF21 animal caps. (e) The DPF domain mimics the activity of Smad1 in the induction of Xbra and Xhox3 in NF11 gastrula caps but the full-length Eps15R lacks this ability. (f) Both full length Eps15R and the DPF domain can transactivate transcription in the yeast assay. (g) The cytoplasmic localization of GFP-Eps15R-ΔDPF is disrupted, displaying a diffuse, reticulated cytoplasmic distribution, in contrast to the punctate cytoplasmic localization of the full-length version shown in figure 1d. Nuclear enrichment is retained in the absence of the DPF domain. (h) The DPF domain antagonizes the ability of Smad2 to induce expression of genes such as chordin, goosecoid and Frzb in NF11 gastrula animal caps, which instead activates the ventral marker Xhox3.
Mentions: The DPF domain is required for the interaction of Eps15R with Smad1 (figure 1b) and it also binds AP2, which recruits clathrin to the cell surface [24]. We tested the activity of the DPF domain by injecting embryos with RNA encoding this protein region. Development appeared significantly perturbed by injection of 3 ng mRNA encoding the DPF domain, relative to an equivalent amount of full-length RNA (figure 3a–c), although a precise quantitative comparison of the effects of the RNAs would require equivalent molar amounts to be injected. The embryos appeared ventralized, consistent with the ability of the DPF domain to induce expression of the ventral marker α-globin in animal caps (figure 3d). The DPF domain of Esp15R mimics Smad1-mediated gene induction, upregulating the expression of Xbra and Xhox3 in animal caps (figure 3e), whereas neither the full-length protein nor the N-terminal 597 domain possesses this ability. It is possible, in a manner analogous to Smad proteins [25], that intramolecular interactions between the N-terminal EH domains and the DPF repeats inhibit the function of the full-length protein, and that these do not occur when the DPF domain is expressed alone.Figure 3.

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