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Beta-arrestins operate an on/off control switch for focal adhesion kinase activity

Abstract

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins β-arrestins and G proteins. Depletion of β-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that β-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK–β-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK–β-arrestin complex to the plasma membrane, β-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient β-arrestin and by a specific inhibitor of β-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of β-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

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Abbreviations

AP-2:

Adaptor protein 2

AT1AR:

Angiotensin II type 1 receptor

β-arr:

β-Arrestin

BRET:

Bioluminescence resonance energy transfer

FA:

Focal adhesion

FERM:

4.1, Ezrin, radixin, moesin

GFP:

Green fluorescent protein

MEF:

Mouse embryonic fibroblast

SiRNA:

Small interfering RNA

WT:

Wild type

YFP:

Yellow fluorescent protein

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Acknowledgements

The authors are grateful to Dr. R. J. Lefkowitz (Duke University, USA) for providing the β-arr mouse embryonic fibroblasts, to Dr. R. Leduc for the HEK-AT1AR cell line and to Dr. E. Escher (Université de Sherbrook, Canada) for the gift of the biased ligand, to Dr A. Benmerah (Imagine-INSERM-U1163, France), Dr. J.-A. Girault (Institut Fer-à-Moulin, INSERM-839, France), Dr. S. Laporte (McGill University, Canada), and Dr. A. N. Shrivastava (IBENS CNRS-UMR8197, Inserm-U1024, France), for providing reagents, to M. Sierks for help with the MEFs work and to J. Paradis (IRIC, Université de Montréal, Canada) for helpful discussion.

Funding

This work was supported by La Ligue Contre le Cancer, Comité de l’Oise (Nos. 2017-6751150, 2015-4756069) to H. Enslen; the Fondation pour la Recherche Médicale (“Team FRM”) (No. DEQ20120323720) to S. Marullo. The S. Marullo team is supported by the Inserm, the CNRS, the Université de Paris, and is a member of the “Who am I?” LABoratory of EXcellence (Grant ANR-11-LABX-0071) funded by the “Investments for the Future” program operated by The French National Research Agency (Grant ANR-11-IDEX-0005-01). RA Alexander was supported by FRM and the Who am I? LABoratory of EXcellence. M. Bouvier is supported by a Foundation grant from CIHR and holds the Canada Research Chair in Signal Transduction and Molecular Pharmacology.

Author information

RAA, IL, KS, MGHS, MB, SM, and HE designed research. RAA, IL, KS, GA, ML, ABo, HK, ED, and HE, performed research. ABe contributed new reagents. RAA, IL, KS, MGHS, HK, AA, MB, SM, and HE analysed data. HE supervised the project. RAA and HE wrote the manuscript, which was subsequently reviewed by the other authors.

Correspondence to Hervé Enslen.

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The experiments comply with the current laws of France, the country in which they were performed.

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Alexander, R.A., Lot, I., Saha, K. et al. Beta-arrestins operate an on/off control switch for focal adhesion kinase activity. Cell. Mol. Life Sci. (2020). https://doi.org/10.1007/s00018-020-03471-5

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Keywords

  • G-protein-coupled receptors
  • Beta-arrestin
  • β-Arrestin
  • AP-2
  • FAK
  • G proteins