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Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin.

Pomeraniec L, Hector-Greene M, Ehrlich M, Blobe GC, Henis YI - Mol. Biol. Cell (2015)

Bottom Line: However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8.ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex.Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

No MeSH data available.


Related in: MedlinePlus

Stable TβRII/endoglin heteromeric complexes form independently of ligand and of endoglin interaction with GIPC and β-arrestin2. COS7 or bEnd.3 cells were cotransfected with pairs of expression vectors encoding myc-TβRII and HA-endoglin-WT, HA-endoglin-Del, or HA-endoglin-T650A. In control experiments with singly expressed myc-TβRII, HA-endoglin-WT or the mutants endoglin-Del and endoglin-T650A were replaced for empty vector. Labeling and patch/FRAP measurements (measuring the lateral diffusion of myc-TβRII) were carried out 24 h after transfection as described in Figure 1. (A, B) Representative FRAP curves of the lateral diffusion of myc-TβRII in a COS7 cell coexpressing HA-endoglin-WT without (A) or with (B) cross-linking of the HA-tagged proteins. (C, E, G) Average Rf and (D, F, H) average D values derived from multiple patch/FRAP measurements in COS7 cells (A–F) or bEnd.3 cells (G, H). Bars are mean ± SEM of 45–60 measurements. IgG cross-linking of HA-endoglin markedly reduced Rf of myc-TβRII, independent of TGF-β1, BMP-9 (whose addition yielded indistinguishable results from those obtained in the presence of TGF-β1), GIPC, or β-arrestin2 binding motifs (**p < 10–5; ***p < 10–6; Student's t test). No significant differences were found in the D values as a result of IgG-mediated cross-linking of HA-endoglin or after addition of TGF-β1 (or BMP-9) under similar experimental conditions.
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Figure 3: Stable TβRII/endoglin heteromeric complexes form independently of ligand and of endoglin interaction with GIPC and β-arrestin2. COS7 or bEnd.3 cells were cotransfected with pairs of expression vectors encoding myc-TβRII and HA-endoglin-WT, HA-endoglin-Del, or HA-endoglin-T650A. In control experiments with singly expressed myc-TβRII, HA-endoglin-WT or the mutants endoglin-Del and endoglin-T650A were replaced for empty vector. Labeling and patch/FRAP measurements (measuring the lateral diffusion of myc-TβRII) were carried out 24 h after transfection as described in Figure 1. (A, B) Representative FRAP curves of the lateral diffusion of myc-TβRII in a COS7 cell coexpressing HA-endoglin-WT without (A) or with (B) cross-linking of the HA-tagged proteins. (C, E, G) Average Rf and (D, F, H) average D values derived from multiple patch/FRAP measurements in COS7 cells (A–F) or bEnd.3 cells (G, H). Bars are mean ± SEM of 45–60 measurements. IgG cross-linking of HA-endoglin markedly reduced Rf of myc-TβRII, independent of TGF-β1, BMP-9 (whose addition yielded indistinguishable results from those obtained in the presence of TGF-β1), GIPC, or β-arrestin2 binding motifs (**p < 10–5; ***p < 10–6; Student's t test). No significant differences were found in the D values as a result of IgG-mediated cross-linking of HA-endoglin or after addition of TGF-β1 (or BMP-9) under similar experimental conditions.

Mentions: Next we used patch/FRAP to investigate heterocomplex formation between endoglin and TβRII. The studies were conducted on cells expressing HA-endoglin and myc-TβRII in the presence or absence of ligand (TGF-β1 or BMP-9), immobilizing (or not) HA-endoglin by IgG cross-linking, and measuring the lateral diffusion of Fab′-labeled myc-TβRII. In COS7 cells, cross-linking of HA-endoglin resulted in a 35% reduction in Rf (ΔRf) of myc-TβRII, whereas D was unaffected (Figure 3, A–D). Similar results were obtained in the presence or absence of ligands. Analogous experiments on bEnd.3 cells (Figure 3, G and H) yielded comparable results, with a slightly higher ΔRf (42%). Note that heterocomplex formation is directly proportional to the ΔRf value and does not require the statistical correction needed for homo-dimerization (Ehrlich et al., 2011). We conclude that a significant portion (0.3–0.4) of TβRII is in stable complexes with endoglin at the cell surface, independent of ligand stimuli. Moreover, analogous measurements on the interactions of TβRII with endoglin cytoplasmic-domain mutants (endoglin-Del or endoglin-T650A), yielded identical results to those obtained with endoglin-WT, suggesting that TβRII-endoglin interactions are also independent of GIPC or β-arrestin2 binding (Figure 3, E and F).


Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin.

Pomeraniec L, Hector-Greene M, Ehrlich M, Blobe GC, Henis YI - Mol. Biol. Cell (2015)

Stable TβRII/endoglin heteromeric complexes form independently of ligand and of endoglin interaction with GIPC and β-arrestin2. COS7 or bEnd.3 cells were cotransfected with pairs of expression vectors encoding myc-TβRII and HA-endoglin-WT, HA-endoglin-Del, or HA-endoglin-T650A. In control experiments with singly expressed myc-TβRII, HA-endoglin-WT or the mutants endoglin-Del and endoglin-T650A were replaced for empty vector. Labeling and patch/FRAP measurements (measuring the lateral diffusion of myc-TβRII) were carried out 24 h after transfection as described in Figure 1. (A, B) Representative FRAP curves of the lateral diffusion of myc-TβRII in a COS7 cell coexpressing HA-endoglin-WT without (A) or with (B) cross-linking of the HA-tagged proteins. (C, E, G) Average Rf and (D, F, H) average D values derived from multiple patch/FRAP measurements in COS7 cells (A–F) or bEnd.3 cells (G, H). Bars are mean ± SEM of 45–60 measurements. IgG cross-linking of HA-endoglin markedly reduced Rf of myc-TβRII, independent of TGF-β1, BMP-9 (whose addition yielded indistinguishable results from those obtained in the presence of TGF-β1), GIPC, or β-arrestin2 binding motifs (**p < 10–5; ***p < 10–6; Student's t test). No significant differences were found in the D values as a result of IgG-mediated cross-linking of HA-endoglin or after addition of TGF-β1 (or BMP-9) under similar experimental conditions.
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Figure 3: Stable TβRII/endoglin heteromeric complexes form independently of ligand and of endoglin interaction with GIPC and β-arrestin2. COS7 or bEnd.3 cells were cotransfected with pairs of expression vectors encoding myc-TβRII and HA-endoglin-WT, HA-endoglin-Del, or HA-endoglin-T650A. In control experiments with singly expressed myc-TβRII, HA-endoglin-WT or the mutants endoglin-Del and endoglin-T650A were replaced for empty vector. Labeling and patch/FRAP measurements (measuring the lateral diffusion of myc-TβRII) were carried out 24 h after transfection as described in Figure 1. (A, B) Representative FRAP curves of the lateral diffusion of myc-TβRII in a COS7 cell coexpressing HA-endoglin-WT without (A) or with (B) cross-linking of the HA-tagged proteins. (C, E, G) Average Rf and (D, F, H) average D values derived from multiple patch/FRAP measurements in COS7 cells (A–F) or bEnd.3 cells (G, H). Bars are mean ± SEM of 45–60 measurements. IgG cross-linking of HA-endoglin markedly reduced Rf of myc-TβRII, independent of TGF-β1, BMP-9 (whose addition yielded indistinguishable results from those obtained in the presence of TGF-β1), GIPC, or β-arrestin2 binding motifs (**p < 10–5; ***p < 10–6; Student's t test). No significant differences were found in the D values as a result of IgG-mediated cross-linking of HA-endoglin or after addition of TGF-β1 (or BMP-9) under similar experimental conditions.
Mentions: Next we used patch/FRAP to investigate heterocomplex formation between endoglin and TβRII. The studies were conducted on cells expressing HA-endoglin and myc-TβRII in the presence or absence of ligand (TGF-β1 or BMP-9), immobilizing (or not) HA-endoglin by IgG cross-linking, and measuring the lateral diffusion of Fab′-labeled myc-TβRII. In COS7 cells, cross-linking of HA-endoglin resulted in a 35% reduction in Rf (ΔRf) of myc-TβRII, whereas D was unaffected (Figure 3, A–D). Similar results were obtained in the presence or absence of ligands. Analogous experiments on bEnd.3 cells (Figure 3, G and H) yielded comparable results, with a slightly higher ΔRf (42%). Note that heterocomplex formation is directly proportional to the ΔRf value and does not require the statistical correction needed for homo-dimerization (Ehrlich et al., 2011). We conclude that a significant portion (0.3–0.4) of TβRII is in stable complexes with endoglin at the cell surface, independent of ligand stimuli. Moreover, analogous measurements on the interactions of TβRII with endoglin cytoplasmic-domain mutants (endoglin-Del or endoglin-T650A), yielded identical results to those obtained with endoglin-WT, suggesting that TβRII-endoglin interactions are also independent of GIPC or β-arrestin2 binding (Figure 3, E and F).

Bottom Line: However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8.ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex.Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

No MeSH data available.


Related in: MedlinePlus