Inactive β1-integrin acts as a junctional scaffold for angiopoietin/TIE2/FOXO1 signaling
Tuomas Sipilä, Srinivas Kumar Ponna, Abhinandan Venkatesha Murthy, Anne Pink, Giray Enkavi, Shraman Kumar Bohra, Klaudia Lewna, Keerthana Ganesh, Qina Liu, Mirka Korhonen, Tommi Kajander, Michael Potente, Johanna Ivaska, Ilpo Vattulainen, Veli-Matti Leppänen, Pipsa Saharinen
Tuomas Sipilä, Srinivas Kumar Ponna, Abhinandan Venkatesha Murthy, Anne Pink, Giray Enkavi, Shraman Kumar Bohra, Klaudia Lewna, Keerthana Ganesh, Qina Liu, Mirka Korhonen, Tommi Kajander, Michael Potente, Johanna Ivaska, Ilpo Vattulainen, Veli-Matti Leppänen, Pipsa Saharinen
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Research Article Angiogenesis Cell biology Vascular biology

Inactive β1-integrin acts as a junctional scaffold for angiopoietin/TIE2/FOXO1 signaling

Abstract

The blood and lymphatic vascular systems are regulated by angiopoietin (ANGPT) growth factors, which signal via endothelial TIE receptor tyrosine kinases and integrins. However, mechanistic understanding of how these receptors crosstalk is limited. Here, we show how β1-integrin inactivation regulates endothelial ANGPT/TIE2 signaling. By integrating biophysical analyses, X-ray crystallography, size-exclusion chromatography–small-angle X-ray scattering and atomistic molecular dynamics simulations, we show that ANGPT2 binds through its asymmetrically positioned C-terminal fibrinogen-like domains to both TIE2 and α5β1-integrin, forming a trimeric complex compatible with the inactive α5β1-integrin conformation. Inactive β1-integrin colocalizes with ANGPT-induced TIE2 in cell-cell junctions and stabilizing β1-integrin in its inactive state enhances junctional TIE2 accumulation and promotes nuclear exclusion of the TIE2 transcriptional effector FOXO1 in cultured endothelial cells. Endothelial-specific β1-integrin deletion in adult mice reduces venous TIE2 phosphorylation, whereas endotoxemia diminishes junctional β1-integrin along with decreased phosphorylated TIE2. In contrast, without TIE2, ANGPT2 uniquely engages active β1-integrin, via its N-terminal superclustering domain. Altogether, our results provide structural and mechanistic evidence of ANGPT signaling via α5β1-integrin and support a model in which inactive α5β1-integrin acts as a junctional scaffold for ANGPT/TIE2/FOXO1 signaling, explaining how integrin conformational switching spatially organizes growth factor signaling in the endothelium.

Authors

Tuomas Sipilä, Srinivas Kumar Ponna, Abhinandan Venkatesha Murthy, Anne Pink, Giray Enkavi, Shraman Kumar Bohra, Klaudia Lewna, Keerthana Ganesh, Qina Liu, Mirka Korhonen, Tommi Kajander, Michael Potente, Johanna Ivaska, Ilpo Vattulainen, Veli-Matti Leppänen, Pipsa Saharinen

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Figure 5

Direct interactions of α5β1-integrin with N- and C-terminal ANGPT2 domains.

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Direct interactions of α5β1-integrin with N- and C-terminal ANGPT2 domai...
(A and B) Schematic representation of ANGPT2 (A) and α5β1-ECD (B) recombinant proteins used. (C) Binding of ANGPT2-FLD-Fc to immobilized α5β1-ECD in SPR. Two-state reaction kinetics were fitted to the original data. (D) Binding of α5β1-ECD (1.3 μM) to immobilized ANGPT2-N-Fc in SPR in the presence and absence of Mn2+ and Mg2+. (E) Binding of ANGPT2-FLD-Fc to immobilized α5Calf1-2 in SPR. (F) Binding of α5Calf1 to immobilized ANGPT2-FLD-Fc in SPR. (G) Schematic representation of N-terminal ANGPT2 and β1-ECD constructs. (H) Binding of β1-ECD to immobilized ANGPT2-N-Fc in SPR. (I) Binding of β1-head and -leg piece (β1-leg) to immobilized ANGPT2-N-Fc in SPR. (J and K) Binding of ANGPT219–202–SUMO (J) and ANGPT219–76-SUMO (K) to fluorescently labelled β1-ECD (2 nM) in MST. (L) Binding of ANGPT2-N-Fc to fluorescently labelled β1-PSI (20 nM) in MST. (M) Schematic representation of ANGPT2 binding through FLD and SCD domains to Calf1 and PSI domains of α5 and β1-integrins, respectively. Representative experiments with average KD are shown. n = 3 independent experiments (C–F and H–L). Nd, not determined.