Fig. 3: A domain swapped architecture of L-TGF-β1 supports αvβ6-mediated autocrine TGF-β signaling without release. | Nature Communications

Fig. 3: A domain swapped architecture of L-TGF-β1 supports αvβ6-mediated autocrine TGF-β signaling without release.

From: Latent-TGF-β has a domain swapped architecture

Fig. 3

a Cell-based system to measure autocrine TGF-β activation without release. Two lines are developed either expressing WT L-TGF-β1, or a furin cleavage site mutant L-TGF-β1 (R249A), where mature TGF-β1 remains covalently linked to LAP. Shown are scatterplots showing equal surface expression of both forms of L-TGF-β1/GARP in their respective lines. Below, a cartoon shows L-TGF-β1/GARP expressed on the cell surface of TMLC TGF-β reporter cells and applied to wells coated with immobilized αvβ6 ectodomain. b αvβ6 supports autocrine TGF-β signaling without release. TMLC cells either expressing L-TGF-β1/GARP or L-TGF-β1 (R249A)/GARP were allowed to bind to the immobilized αvβ6 ectodomain, or control substrates (all substrates coated at 1 μg/ml), and amount of TGF-β activation determined by normalization to a recombinant TGF-β standard curve, as described4. The control substrates used are αvβ3 as a low-affinity TGF-β binding integrin, a polyclonal antibody to LAP as a high-affinity non-integrin L-TGF-β binder, or BSA, which does not bind to L-TGF-β. Data shown as mean ± SEM, P-values were generated by one way ANOVA with Šídák’s multiple comparisons test (N = 3 biologic replicates). Source data are provided as a Source Data file. c Cartoon of TMLC assay to measure effect of constraint on αvβ6 on TGF-β activation. Assays use L-TGF-β1/GARP TMLC either cultured with 1, empty nanodisc; 2, soluble αvβ6 ectodomain; 3, αvβ6 full-length in nanodisc; 4, immobilized αvβ6 ectodomain. d Constraint correlates with αvβ6-mediated TGF-β1 activation. Soluble αvβ6 ectodomain, full-length αvβ6 in nanodisc, or empty nanodisc as a control, were added to individual wells of TMLC L-TGF-β1/GARP cells, and TGF-β1 activation compared to TMLC L-TGF-β1/GARP cells incubated with immobilized αvβ6 ectodomain. The concentration of αvβ6 is indicated. TGF-β1 activation is indicated by luciferase activity in arbitrary units (AU). Data shown as mean ± SEM, P-values were generated by one-way ANOVA with Šídák’s multiple comparisons test (N = 3 biologic replicates). Source data are provided as a Source Data file. Cartoons in (a, c) created in BioRender. Nishimura, S. (2025) https://BioRender.com/29k4zvb.

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