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 7

SAXS models of N-terminal and C-terminal ANGPT2 complexes with α5 and β1-integrin.

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SAXS models of N-terminal and C-terminal ANGPT2 complexes with α5 and β1...
(A) Schematic representation of ANGPT2-SCD and β1-integrin recombinant proteins used for SAXS. (B–D) SEC-SAXS of ANGPT219–76–SUMO in complex with β1-head. The SEC elution profile (B); fit of ab initio using DAMMIF (green) and homology model, rigid body refined using SASREF (black), with SAXS data (blue) (C); and overlay of ab initio and homology models (D). (E) Schematic representation of the N-terminal truncated dimeric ANGPT2147–496, α5Calf1-2 and TIE2 (LBD, red) used for SPR and SAXS. (F) Binding of ANGPT2147–496 to α5Calf1-2 in SPR. (G–I) SEC-SAXS analysis of ANGPT2147–496 in complex with α5Calf1-2. The SEC elution profile (G), fit of ab initio (green) and homology model (black) with SAXS data (blue) (H), ab initio (DAMMIF) and homology models (SREFLEX) (I). (J–L) SEC-SAXS analysis of ANGPT2147–496 in complex with α5Calf1-2 and TIE2 LBD. The SEC elution profile (J), fit of ab initio using DAMMIF (green) and homology model rigid body refined using SREFLEX (black) with SAXS data (blue) (K), and overlay of ab initio and homology models (L). The χ² value is presented as a measure of the model’s goodness of fit and statistical confidence (C, H, and K). For homology model, ANGPT219–76 (Supplemental Figure 6A), ANGPT2147–496 (Supplemental Figure 7A), β1-head (PDB ID: 7NXD), TIE2 LBD (PDB ID: 2GY7), and our crystal structure of α5Calf1-2 were used.