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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 endocytic adaptor protein Eps15R interacts with Smad1. (a) Xenopus Eps15R and deletion constructs. The N-terminal portion contains three Eps15 homology (EH) domains (green, orange and purple), followed by a coiled coil domain (blue) and carboxy-terminal DPF tripeptide repeat domain (black). (b) Eps15R binds to Smad1 in the yeast two-hybrid assay, as detected by β-galactosidase activity. Interaction requires the DPF domain of Eps15R. (c) Eps15R and Smad1 interact in Xenopus embryos. Embryos were injected with Myc-tagged full-length Eps15R mRNA alone or with Flag-tagged Smad1 mRNA. Lysates were immunoprecipitated (IP) with anti-FLAG antibodies and then western blotted with anti-Myc antibodies. Whole embryo lysates were immunoblotted with anti-Myc and anti-FLAG antibodies. (d) GFP-Eps15R is enriched in bright punctate foci located both juxta-membraneously and deeper within the cytoplasm of Xenopus animal cap cells. Lower fluorescence levels are found in the nucleus. (e) Histone B4-RFP and mCherry-GPI expression in cells shown in (d). (f) Limited co-localization of GFP-Eps15R and mCherry-Hrs 1 h after stimulation with 100 ng ml–1 BMP4/7.
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RSOB120060F1: The endocytic adaptor protein Eps15R interacts with Smad1. (a) Xenopus Eps15R and deletion constructs. The N-terminal portion contains three Eps15 homology (EH) domains (green, orange and purple), followed by a coiled coil domain (blue) and carboxy-terminal DPF tripeptide repeat domain (black). (b) Eps15R binds to Smad1 in the yeast two-hybrid assay, as detected by β-galactosidase activity. Interaction requires the DPF domain of Eps15R. (c) Eps15R and Smad1 interact in Xenopus embryos. Embryos were injected with Myc-tagged full-length Eps15R mRNA alone or with Flag-tagged Smad1 mRNA. Lysates were immunoprecipitated (IP) with anti-FLAG antibodies and then western blotted with anti-Myc antibodies. Whole embryo lysates were immunoblotted with anti-Myc and anti-FLAG antibodies. (d) GFP-Eps15R is enriched in bright punctate foci located both juxta-membraneously and deeper within the cytoplasm of Xenopus animal cap cells. Lower fluorescence levels are found in the nucleus. (e) Histone B4-RFP and mCherry-GPI expression in cells shown in (d). (f) Limited co-localization of GFP-Eps15R and mCherry-Hrs 1 h after stimulation with 100 ng ml–1 BMP4/7.

Mentions: Xenopus Eps15R encodes a protein of 897 amino acids that shares 73 per cent amino acid identity with human and mouse Eps15R. Like its mammalian orthologues, Xenopus Eps15R contains three copies of an EH domain and a coiled-coil domain that mediates protein dimerization [14]. At their C-termini, Xenopus and mammalian Eps15Rs contain a series of aspartate-proline-phenylalanine (DPF) tripeptide repeats, known as the DPF domain [10] (figure 1a). We found that Eps15R interacts with the MH1 domain of Smad1 through the DPF domain both in yeast by two-hybrid assay (figure 1b) and in Xenopus embryos by co-immunoprecipitation (figure 1c).Figure 1.


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 endocytic adaptor protein Eps15R interacts with Smad1. (a) Xenopus Eps15R and deletion constructs. The N-terminal portion contains three Eps15 homology (EH) domains (green, orange and purple), followed by a coiled coil domain (blue) and carboxy-terminal DPF tripeptide repeat domain (black). (b) Eps15R binds to Smad1 in the yeast two-hybrid assay, as detected by β-galactosidase activity. Interaction requires the DPF domain of Eps15R. (c) Eps15R and Smad1 interact in Xenopus embryos. Embryos were injected with Myc-tagged full-length Eps15R mRNA alone or with Flag-tagged Smad1 mRNA. Lysates were immunoprecipitated (IP) with anti-FLAG antibodies and then western blotted with anti-Myc antibodies. Whole embryo lysates were immunoblotted with anti-Myc and anti-FLAG antibodies. (d) GFP-Eps15R is enriched in bright punctate foci located both juxta-membraneously and deeper within the cytoplasm of Xenopus animal cap cells. Lower fluorescence levels are found in the nucleus. (e) Histone B4-RFP and mCherry-GPI expression in cells shown in (d). (f) Limited co-localization of GFP-Eps15R and mCherry-Hrs 1 h after stimulation with 100 ng ml–1 BMP4/7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120060F1: The endocytic adaptor protein Eps15R interacts with Smad1. (a) Xenopus Eps15R and deletion constructs. The N-terminal portion contains three Eps15 homology (EH) domains (green, orange and purple), followed by a coiled coil domain (blue) and carboxy-terminal DPF tripeptide repeat domain (black). (b) Eps15R binds to Smad1 in the yeast two-hybrid assay, as detected by β-galactosidase activity. Interaction requires the DPF domain of Eps15R. (c) Eps15R and Smad1 interact in Xenopus embryos. Embryos were injected with Myc-tagged full-length Eps15R mRNA alone or with Flag-tagged Smad1 mRNA. Lysates were immunoprecipitated (IP) with anti-FLAG antibodies and then western blotted with anti-Myc antibodies. Whole embryo lysates were immunoblotted with anti-Myc and anti-FLAG antibodies. (d) GFP-Eps15R is enriched in bright punctate foci located both juxta-membraneously and deeper within the cytoplasm of Xenopus animal cap cells. Lower fluorescence levels are found in the nucleus. (e) Histone B4-RFP and mCherry-GPI expression in cells shown in (d). (f) Limited co-localization of GFP-Eps15R and mCherry-Hrs 1 h after stimulation with 100 ng ml–1 BMP4/7.
Mentions: Xenopus Eps15R encodes a protein of 897 amino acids that shares 73 per cent amino acid identity with human and mouse Eps15R. Like its mammalian orthologues, Xenopus Eps15R contains three copies of an EH domain and a coiled-coil domain that mediates protein dimerization [14]. At their C-termini, Xenopus and mammalian Eps15Rs contain a series of aspartate-proline-phenylalanine (DPF) tripeptide repeats, known as the DPF domain [10] (figure 1a). We found that Eps15R interacts with the MH1 domain of Smad1 through the DPF domain both in yeast by two-hybrid assay (figure 1b) and in Xenopus embryos by co-immunoprecipitation (figure 1c).Figure 1.

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