Limits...
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.

Show MeSH

Related in: MedlinePlus

Differential compartmentalization of Eps15R/Smad complexes. Live imaging of Eps15R/Smad complexes monitored by bimolecular fluorescence complementation (BiFC). (a,c,e,g) Greyscale images of BiFC fluorescence. (b,d,f,h) Merged images of BiFC fluorescence in green and CFP-histone H2B (to label nucleus) and CFP-GPI (to label membranes) in red. Total numbers of cells scored for each BiFC complex were: n = 78 (Eps15R/Smad1); n = 27 (Eps15R-ΔDPF /Smad1); n = 45 (Eps15R/Smad2); n = 24 (Eps15R-ΔDPF/Smad2). (a,b) Cells injected with VN-Eps15R and VC-Smad1 have nuclear BiFC fluorescence, with enrichment in localized regions. (c,d) VN-Eps15R-ΔDPF does not interact with VC-Smad1. (e,f) Complexes of VN-Eps15R and VC-Smad2 are distributed in punctate dots throughout the cell. Some complexes are closely associated with the membrane (arrows), consistent with the known association of Eps15R with coated pits. (g,h) VN-Eps15R-ΔDPF does not interact with VC-Smad2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3376731&req=5

RSOB120060F4: Differential compartmentalization of Eps15R/Smad complexes. Live imaging of Eps15R/Smad complexes monitored by bimolecular fluorescence complementation (BiFC). (a,c,e,g) Greyscale images of BiFC fluorescence. (b,d,f,h) Merged images of BiFC fluorescence in green and CFP-histone H2B (to label nucleus) and CFP-GPI (to label membranes) in red. Total numbers of cells scored for each BiFC complex were: n = 78 (Eps15R/Smad1); n = 27 (Eps15R-ΔDPF /Smad1); n = 45 (Eps15R/Smad2); n = 24 (Eps15R-ΔDPF/Smad2). (a,b) Cells injected with VN-Eps15R and VC-Smad1 have nuclear BiFC fluorescence, with enrichment in localized regions. (c,d) VN-Eps15R-ΔDPF does not interact with VC-Smad1. (e,f) Complexes of VN-Eps15R and VC-Smad2 are distributed in punctate dots throughout the cell. Some complexes are closely associated with the membrane (arrows), consistent with the known association of Eps15R with coated pits. (g,h) VN-Eps15R-ΔDPF does not interact with VC-Smad2.

Mentions: Nuclear fluorescence was seen in isolated Xenopus ectodermal cells after co-injection of VN-Eps15R and VC-Smad1 RNAs (figure 4a,b). Complexes were present in intense punctate foci, suggesting that the proteins function within a nuclear sub-compartment. This result, in combination with our finding that Eps15R enhances Smad1 signalling and transactivates gene expression, suggests that Eps15R acts as a component of the Smad1 transcriptional complex. No fluorescence was detected outside the nucleus, indicating that Eps15R does not localize with the cytoplasmic fraction of Smad1 and that Smad1 does not associate with Eps15R near the cell membrane. Deletion of the DPF domain prevented the interaction of Eps15R with Smad1 (figure 4c,d), confirming the importance of this domain in facilitating the interaction and serving as a specificity control for BiFC. As GFP-Eps15R-ΔDPF bears the identical R589* mutation yet is still expressed in cells (figure 3g), this point mutation does not render the truncated protein unstable.Figure 4.


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)

Differential compartmentalization of Eps15R/Smad complexes. Live imaging of Eps15R/Smad complexes monitored by bimolecular fluorescence complementation (BiFC). (a,c,e,g) Greyscale images of BiFC fluorescence. (b,d,f,h) Merged images of BiFC fluorescence in green and CFP-histone H2B (to label nucleus) and CFP-GPI (to label membranes) in red. Total numbers of cells scored for each BiFC complex were: n = 78 (Eps15R/Smad1); n = 27 (Eps15R-ΔDPF /Smad1); n = 45 (Eps15R/Smad2); n = 24 (Eps15R-ΔDPF/Smad2). (a,b) Cells injected with VN-Eps15R and VC-Smad1 have nuclear BiFC fluorescence, with enrichment in localized regions. (c,d) VN-Eps15R-ΔDPF does not interact with VC-Smad1. (e,f) Complexes of VN-Eps15R and VC-Smad2 are distributed in punctate dots throughout the cell. Some complexes are closely associated with the membrane (arrows), consistent with the known association of Eps15R with coated pits. (g,h) VN-Eps15R-ΔDPF does not interact with VC-Smad2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120060F4: Differential compartmentalization of Eps15R/Smad complexes. Live imaging of Eps15R/Smad complexes monitored by bimolecular fluorescence complementation (BiFC). (a,c,e,g) Greyscale images of BiFC fluorescence. (b,d,f,h) Merged images of BiFC fluorescence in green and CFP-histone H2B (to label nucleus) and CFP-GPI (to label membranes) in red. Total numbers of cells scored for each BiFC complex were: n = 78 (Eps15R/Smad1); n = 27 (Eps15R-ΔDPF /Smad1); n = 45 (Eps15R/Smad2); n = 24 (Eps15R-ΔDPF/Smad2). (a,b) Cells injected with VN-Eps15R and VC-Smad1 have nuclear BiFC fluorescence, with enrichment in localized regions. (c,d) VN-Eps15R-ΔDPF does not interact with VC-Smad1. (e,f) Complexes of VN-Eps15R and VC-Smad2 are distributed in punctate dots throughout the cell. Some complexes are closely associated with the membrane (arrows), consistent with the known association of Eps15R with coated pits. (g,h) VN-Eps15R-ΔDPF does not interact with VC-Smad2.
Mentions: Nuclear fluorescence was seen in isolated Xenopus ectodermal cells after co-injection of VN-Eps15R and VC-Smad1 RNAs (figure 4a,b). Complexes were present in intense punctate foci, suggesting that the proteins function within a nuclear sub-compartment. This result, in combination with our finding that Eps15R enhances Smad1 signalling and transactivates gene expression, suggests that Eps15R acts as a component of the Smad1 transcriptional complex. No fluorescence was detected outside the nucleus, indicating that Eps15R does not localize with the cytoplasmic fraction of Smad1 and that Smad1 does not associate with Eps15R near the cell membrane. Deletion of the DPF domain prevented the interaction of Eps15R with Smad1 (figure 4c,d), confirming the importance of this domain in facilitating the interaction and serving as a specificity control for BiFC. As GFP-Eps15R-ΔDPF bears the identical R589* mutation yet is still expressed in cells (figure 3g), this point mutation does not render the truncated protein unstable.Figure 4.

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