Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs
Introduction
Cell migration and chemotaxis are essential processes in embryonic development, the inflammatory response, and play a key role in metastatic cancers [1], [2], [3]. The signaling mechanisms cells use to sense chemical gradients in their environment are complex and include multiple functional steps involving activation of chemokine G protein-coupled receptors (GPCRs), as well as other GPCRs [4], [5] and a network of actin regulatory signaling pathways. To ensure correct navigation of different cells to distinct destinations, the availability of the guiding cues and the cell's responsiveness to them must be tightly controlled. Thus, as the cell migrates, signaling must be quenched at the trailing edge. Arrestins, together with their partners in the GPCR desensitization process, G protein-coupled receptor kinases, are known to play the key role in regulating the sensitivity to chemokines and the signaling of other GPCRs involved in migration [6], [7]. Migration requires the coordinated activation of hundreds of proteins in distinct compartments of the cell [8]. Because arrestins are multi-functional regulators capable of orchestrating signaling and localizing proteins to distinct subcellular compartments [9], [10], they are also likely to affect the activity of various signaling proteins involved in generating the forces that promote movement. Indeed, over the last few years, arrestins have emerged as important regulators of the actin cytoskeleton [11], [12], [13].
Rho family GTPases are small G proteins that act as molecular switches that regulate the signal transduction pathways connecting plasma membrane receptors to the cytoskeleton [14], [15]. GTPases of the Rho family, which includes 20 proteins from three distinct types, Rho, Rac and Cdc42, control separate signal transduction pathways regulating the remodeling of actin cytoskeleton [15]. Rac activation induces the formation of protrusions known as lamellipodia that drive the cell migration. Cdc42 activity produces filopodia, a different type of cell protrusions involving actin polymerization [16]. Cdc42 activity may be involved in the control of the movement direction in response to external cues [17]. Rho proteins also regulate the actin-myosin contractility required to propel the cell forward [15], [18]. The functional information about other members of the Rho family is limited.
There is growing evidence for a role of the non-visual arrestins in facilitating small GTPase-mediated events. First, in was shown that arrestin-22 activates the small GTPase RhoA coordinately with Gαq following the activation of the angitotensin II 1A receptor (ATII1AR) [11]. Arrestin-2 also regulates RhoA activity by binding and inhibiting ARHGAP21, a RhoA GTPase activating protein, in response to ATII1AR stimulation [19]. Arrestin-3 interacts with the actin treadmilling protein cofilin upon activation of another GPCR, PAR2 [13], and both arrestins inhibit PAR-2-stimulated Cdk2 activity [20]. In contrast, the transforming growth factor beta (TGF-beta) superfamily co-receptor, the type III TGF receptor, activates Cdk2 via direct interaction with arrestin-3, which leads to inhibition of directed cell migration [21]. Both arrestin-2 and -3 regulate small GTPase guanyl nucleotide dissociation stimulator ralGDS upon activation of the fMLP receptor [22], and activates the ELMO-ARF cascade upon stimulation of the calcium-sensing receptor [12]. Furthermore, arrestins interact with tumor suppressor PTEN, and this interaction is enhanced by stimulation of the G12-coupled lysophosphatidic acid receptor and subsequent activation of RhoA [23]. In the context of 3-D culture, PTEN regulates the arrestin-2 interaction with ARHGAP21/Cdk2 and the activity of Cdk2, which is essential for the multicellular morphogenesis [24]. Thus, collectively the data suggests that arrestins could act both upstream as RhoA regulators as well as downstream as RhoA effectors.
We were interested in determining whether ubiquitous non-visual arrestins [10] regulate the activity of these GTPases. Arrestins have been shown to regulate a variety of proteins independently of G-protein coupled receptor (GPCR) activation [25], [26], [27], [28], [29], [30], but the effect of arrestins on the small GTPases under basal conditions has not been explored. Recently we found that arrestins promote focal adhesion disassembly, likely by recruiting clathrin to microtubules targeting focal adhesions to facilitate intergrin internalization [31]. Here we show that arrestins regulate the actin cytoskeleton to limit cell spreading by affecting the activity of the small GTPases RhoA and Rac1 in a receptor-independent manner. We also show that, in addition to microtubule dependent FA disassembly, arrestin-mediated regulation of the small GTPase RhoA likely contributes to the FA phenotype in arrestin null cells.
Section snippets
Materials
Restriction endonucleases and other DNA modifying enzymes were from New England Biolabs (Ipswich, MA). Cell culture reagents and media were from Mediatech-Corning (Manassas, VA) or Life-technologies (Carlsbad, CA). DNA purification kits were from Zymo Research (Irvine, CA). All other reagents were from Amresco (Solon, OH) or Sigma-Aldrich (St Louis, MO).
Activity of the small GTPases is altered in cells lacking arrestin-2 or arrestin-3
To test whether non-visual arrestins play a direct role in regulating cell shape under basal conditions, arrestin-2/3 double knock-out (DKO) mouse embryonic fibroblasts (MEFs) [33], [34] were plated on fibronectin (FN), and compared to wild type (WT) MEFs (Fig. 1A) in serum-free media. DKO MEFs are twice as large as WT cells, and show a dramatically different arrangement of the actin cytoskeleton [31]. DKO cells spread similarly on poly-d-lysine (PDL), which binds integrins but does not promote
Discussion
Small GTPases control cell shape by interacting with a variety of effectors that regulate the cytoskeleton [15], [36]. The dramatically altered morphology of DKO cells suggested that small GTPases are dysregulated. Indeed, we found that basal activity of RhoA and Rac1 was significantly reduced by the deletion of both non-visual arrestins. In contrast to the previous report, which detected enhanced Rac activity in MEFs lacking arrestin-2 [47], we found no changes in the Rac activity in MEFs with
Conclusions
Both non-visual arrestin subtypes regulate the activity of small GTPases RhoA and Rac1, thereby affecting cell spreading and motility. Non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins act independently of GPCRs. Our data reveal a completely novel function of arrestins. Arrestin-2 and arrestin-3 individually regulate RhoA independently of GPCR stimulation to
Acknowledgements
The authors thank Dr. Ian Macara for small GTPase mutants, Dr. Christopher Turner for GFP-paxillin construct, and Dr. L.A. Donoso for F4C1 pan-arrestin mouse monoclonal antibody. This work was supported by NIH RO1 Grants GM077561, GM109955 (these two were merged into R35 GM122491), EY011500 (VVG), NS065868 and DA030103 (EVG), DK069921 and VA grant I01BX002196 (RZ), and training grants GM007628, EY007135 (WMC). Confocal images were obtained using VUMC Cell Imaging Shared Resource (supported by
References (66)
- et al.
Role of G protein-coupled receptor kinases in cell migration
Curr. Opin. Cell Biol.
(2014) - et al.
Attractive guidance: how the chemokine SDF1/CXCL12 guides different cells to different locations
Semin. Cell Dev. Biol.
(2012) - et al.
G protein-coupled receptors stimulation and the control of cell migration
Cell. Signal.
(2009) - et al.
Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics
Exp. Cell Res.
(2000) - et al.
b-Arrestin-dependent regulation of the cofilin pathway downstream of protease-activated Receptor-2
J. Biol. Chem.
(2007) - et al.
Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia
Cell
(1995) - et al.
Visual and both non-visual arrestins in their "inactive" conformation bind JNK3 and Mdm2 and relocalize them from the nucleus to the cytoplasm
J. Biol. Chem.
(2006) - et al.
Identification of a motif in the carboxyl terminus of beta -arrestin2 responsible for activation of JNK3
J. Biol. Chem.
(2001) - et al.
JNK3 enzyme binding to arrestin-3 differentially affects the recruitment of upstream mitogen-activated protein (MAP) kinase kinases
J. Biol. Chem.
(2013) - et al.
Arrestin-3 binds c-Jun N-terminal kinase 1 (JNK1) and JNK2 and facilitates the activation of these ubiquitous JNK isoforms in cells via scaffolding
J. Biol. Chem.
(2013)
Silent scaffolds: inhibition of JNK3 activity in the cell by a dominant-negative arrestin-3 mutant
J. Biol. Chem.
Assay of Cdc42, Rac, and rho GTPase activation by affinity methods
Methods Enzymol.
The small GTP-binding protein Rac regulates growth-factor induced membrane ruffling
Cell
Arrestin mobilizes signaling proteins to the cytoskeleton and redirects their activity
J. Mol. Biol.
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia
Cell
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors
Cell
βArrestin1 regulates the guanine nucleotide exchange factor RasGRF2 expression and the small GTPase Rac-mediated formation of membrane protrusion and cell motility
J. Biol. Chem.
Acute activation of β2-adrenergic receptor regulates focal adhesions through βArrestin2- and p115RhoGEF protein-mediated activation of RhoA
J. Biol. Chem.
The structural basis of arrestin-mediated regulation of G protein-coupled receptors
Pharmacol. Ther.
Rho protein crosstalk: another social network?
Trends Cell Biol.
Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism
Curr. Biol.
Integrin engagement, the actin cytoskeleton, and c-Src are required for the calcitonin-induced tyrosine phosphorylation of paxillin and HEF1, but not for calcitonin-induced Erk1/2 phosphorylation
J. Biol. Chem.
Integrin-regulated FAK-Src signaling in normal and cancer cells
Curr. Opin. Cell Biol.
Gastrulation: making and shaping germ layers
Annu. Rev. Cell Dev. Biol.
Cell migration
Comp. Physiol. Ecol.
Chemokine signaling in development and disease
Development (Cambridge, England)
GRKs and arrestins: regulators of migration and inflammation
J. Leukoc. Biol.
Beta-arrestins and cell signaling
Annu. Rev. Physiol.
Arrestins are ubiquitous regulators of cellular signaling pathways
Genome Biol.
b-Arrestin 1 and Gaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation
J. Biol. Chem.
The calcium-sensing receptor changes cell shape via a b-arrestin-1-ARNO-ARF6-ELMO protein network
J. Cell Sci.
Rho GTPases and the actin cytoskeleton
Science
Rho GTPases in cell biology
Nature
Cited by (12)
Targeting arrestin interactions with its partners for therapeutic purposes
2020, Advances in Protein Chemistry and Structural BiologyCitation Excerpt :Non-visual arrestins were shown to perform several functions independently of GPCRs. These include regulation of cell spreading and motility via direct regulation of focal adhesions (Cleghorn et al., 2015) or the activity of small GTPases (Cleghorn et al., 2018). In addition, both arrestin-2 (Kook et al., 2014) and arrestin-3 (Kook, Vishnivetskiy, Gurevich, & Gurevich, 2019) were shown to be cleaved by caspases during apoptosis, with caspase-generated fragments facilitating (Kook et al., 2014) or suppressing (Kook et al., 2019) cell death.
Arrestins: Introducing Signaling Bias Into Multifunctional Proteins
2018, Progress in Molecular Biology and Translational ScienceCitation Excerpt :In fact, each nonvisual arrestin was found to interact with more than a hundred diverse proteins20 and the list of their binding partners keeps growing. Among other things, arrestins were implicated in recruiting signaling proteins to microtubules,21 in cytochrome C release during apoptosis,22 focal adhesion dynamics,23 activation of small GTPases,24–27 glucose maintenance,28 and many other cellular functions,29,30 including even heterologous desensitization of GPCRs phosphorylated by second messenger-activated kinases.31,32 Structurally, all four vertebrate arrestins are quite similar: elongated two-domain molecules, with the long axis of no more than 90 Å and the short axis of ~ 30–35 Å33–38 (Fig. 1).
Arrestin-3 binds parkin and enhances parkin-dependent mitophagy
2024, Journal of Neurochemistryβ Arrestins: Structure, Function, Physiology, and Pharmacological Perspectives
2023, Pharmacological ReviewsEffect of Yishen Daluo Prescription on Rho/ROCK Signaling Pathway in EAE Mice Based on Silencing of β-arrestin1
2023, Chinese Journal of Experimental Traditional Medical Formulae
- 1
Current address: Department of Biochemistry, University of Washington, Seattle, WA 98109, United States.