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Optogenetics-based localization of talin to the plasma membrane promotes activation of β3 integrins.

ABSTRACT: Interaction of talin with the cytoplasmic tails of integrin β triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbβ3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVβ3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbβ3 or αVβ3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2-talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]-decorated plasma membrane. This resulted in β3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated β3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in β3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP.

SUBMITTER: Liao Z

PROVIDER: S-EPMC8131925 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transforming growth factor β (TGFβ) induces hepatic stellate cell (HSC) differentiation into tumor-promoting myofibroblast, although underlying mechanism remains incompletely understood. Focal adhesion kinase (FAK) is activated in response to TGFβ stimulation, so it transmits TGFβ stimulus to extracellular signal-regulated kinase and P38 mitogen-activated protein kinase signaling. However, it is unknown whether FAK can, in return, modulate TGFβ receptors. In this study, we tested whether FAK phosphorylated TGFβ receptor 2 (TGFβR2) and regulated TGFβR2 intracellular trafficking in HSCs. The FAKY397F mutant and PF-573,228 were used to inhibit the kinase activity of FAK, the TGFβR2 protein level was quantitated by immunoblotting, and HSC differentiation into myofibroblast was assessed by expression of HSC activation markers, alpha-smooth muscle actin, fibronectin, or connective tissue growth factor. We found that targeting FAK kinase activity suppressed the TGFβR2 protein level, TGFβ1-induced mothers against decapentaplegic homolog phosphorylation, and myofibroblastic activation of HSCs. At the molecular and cellular level, active FAK (phosphorylated FAK at tyrosine 397) bound to TGFβR2 and kept TGFβR2 at the peripheral plasma membrane of HSCs, and it induced TGFβR2 phosphorylation at tyrosine 336. In contrast, targeting FAK or mutating Y336 to F on TGFβR2 led to lysosomal sorting and degradation of TGFβR2. Using RNA sequencing, we identified that the transcripts of 764 TGFβ target genes were influenced by FAK inhibition, and that through FAK, TGFβ1 stimulated HSCs to produce a panel of tumor-promoting factors, including extracellular matrix remodeling proteins, growth factors and cytokines, and immune checkpoint molecule PD-L1. Functionally, targeting FAK inhibited tumor-promoting effects of HSCs in vitro and in a tumor implantation mouse model. Conclusion: FAK targets TGFβR2 to the plasma membrane and protects TGFβR2 from lysosome-mediated degradation, thereby promoting TGFβ-mediated HSC activation. FAK is a target for suppressing HSC activation and the hepatic tumor microenvironment.

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Optogenetics-based localization of talin to the plasma membrane promotes activation of β3 integrins.

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