Elsevier

Cellular Signalling

Volume 42, January 2018, Pages 259-269
Cellular Signalling

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs

https://doi.org/10.1016/j.cellsig.2017.11.003Get rights and content

Highlights

  • Arrestin-2 and arrestin-3 differentially regulate small GTPases RhoA, Rac1, and Cdc42.

  • The absence of arrestin-2 and arrestin-3 enhances cell spreading.

  • Arrestin-2 and arrestin-3 regulate small GTPases and cytoskeleton independently of GPCRs.

  • Inhibition of RhoA and loss of arrestins improves spreading independently of integrin signaling.

Abstract

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that 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 regulate these small GTPases independently of G-protein-coupled receptor activation.

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

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    Current address: Department of Biochemistry, University of Washington, Seattle, WA 98109, United States.

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