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 ß-arrestins and G proteins. Depletion of ß-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that ß-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-ß-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-ß-arrestin complex to the plasma membrane, ß-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 ß-arrestin and by a specific inhibitor of ß-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 ß-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.
Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/genetics , Multiprotein Complexes/genetics , Neoplasms/genetics , beta-Arrestins/genetics , Adaptor Protein Complex 2/genetics , Animals , Cell Membrane/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , GTP-Binding Proteins/genetics , HEK293 Cells , Humans , Mice , Multiprotein Complexes/metabolism , Neoplasms/drug therapy , Phosphorylation/drug effects , Protein Binding/genetics , Protein Domains/genetics , Receptor, Angiotensin, Type 1/genetics , Receptors, G-Protein-Coupled/genetics , Vasopressins/pharmacologyABSTRACT
ß-Arrestins 1 and 2 (ß-arr1 and ß-arr2) are ubiquitous proteins with common and distinct functions. They were initially identified as proteins recruited to stimulated G protein-coupled receptors (GPCRs), regulating their desensitization and internalization. The discovery that ß-arrs could also interact with more than 400 non-GPCR protein partners brought to light their central roles as multifunctional scaffold proteins regulating multiple signalling pathways from the plasma membrane to the nucleus, downstream of GPCRs or independently from these receptors. Through the regulation of the activities and subcellular localization of their binding partners, ß-arrs control various cell processes such as proliferation, cytoskeletal rearrangement, cell motility, and apoptosis. Thus, the identification of ß-arrs binding partners and the characterization of their mode of interaction in cells are central to the understanding of their function. Here we provide methods to explore the molecular interaction of ß-arrs with other proteins in cellulo.
Subject(s)
Protein Interaction Mapping/methods , beta-Arrestins/metabolism , Bioluminescence Resonance Energy Transfer Techniques , HEK293 Cells , Humans , Immunoprecipitation , Protein Binding , Saccharomyces cerevisiae/metabolism , Two-Hybrid System TechniquesABSTRACT
AIMS: Vascular endothelial growth factor (VEGF)-initiated angiogenesis requires coordinated proteolytic degradation of extracellular matrix provided by the urokinase plasminogen activator/urokinase receptor (uPA/uPAR) system and regulation of cell migration provided by integrin-matrix interaction. In this study, we investigated the mechanisms underlying the uPAR-dependent modulation of VEGF-induced endothelial migration. METHODS AND RESULTS: We used flow cytometry to quantify integrins at the cell surface. Stimulation of human and murine endothelial cells with VEGF resulted in internalization of α5ß1-integrins. Micropatterning and immunocytochemistry revealed co-clustering of uPAR and α5ß1-integrins and retrieval via clathrin-coated vesicles. It was also contingent on receptors of the low-density lipoprotein receptor (LDL-R) family. VEGF-induced integrin redistribution was inhibited by elimination of uPAR from the endothelial cell surface or by inhibitory peptides that block the uPAR-integrin interaction. Under these conditions, the migratory response of endothelial cells upon VEGF stimulation was impaired both in vitro and in vivo. CONCLUSIONS: The observations indicate that uPAR is an essential component of the network through which VEGF controls endothelial cell migration. uPAR is a bottleneck through which the VEGF-induced signal must be funnelled for both focused proteolytic activity at the leading edge and for redistribution of integrins.
