β-Arrestins Scaffold Cofilin with Chronophin to Direct Localized Actin Filament Severing and Membrane Protrusions Downstream of Protease-activated Receptor-2

Abstract

Protease-activated receptor-2 (PAR-2) mediates pro-inflammatory signals in a number of organs, including enhancing leukocyte recruitment to sites of injury and infection. At the cellular level, PAR-2 promotes activation of the actin filamentsevering protein cofilin, which is crucial for the reorganization of the actin cytoskeleton and chemotaxis. These responses require the scaffolding functions of ␤-arrestins; however, the mechanism by which ␤-arrestins spatially regulate cofilin activity and the role of this pathway in primary cells has not been investigated. Here, using size-exclusion chromatography and co-immunoprecipitation, we demonstrate that PAR-2 promotes the formation of a complex containing ␤-arrestins, cofilin, and chronophin (CIN) in primary leukocytes and cultured cells. Both association of cofilin with CIN and cell migration are inhibited in leukocytes from ␤-arrestin-2 ؊/؊ mice. We show that, in response to PAR-2 activation, ␤-arrestins scaffold cofilin with its upstream activator CIN, to facilitate the localized generation of free actin barbed ends, leading to membrane protrusion. These studies suggest that a major role of ␤-arrestins in chemotaxis is to spatially regulate cofilin activity to facilitate the formation of a leading edge, and that this pathway may be important for PAR-2-stimulated immune cell migration.Protease-activated-receptor-2 (PAR-2) 2 is a G-protein-coupled receptor that signals, through ␤-arrestin-promoted scaffolds, to promote reorganization of the actin cytoskeleton and chemotaxis (1, 2). In vivo, PAR-2 plays an important role in the recruitment of leukocytes to the sites of inflammation, because this is impaired in PAR-2 Ϫ/Ϫ mice and enhanced by administration of PAR-2 agonists (3-8). However, no studies have yet linked ␤-arrestin-dependent scaffolding of actin assembly proteins to PAR-2-stimulated chemotaxis under physiological conditions.␤-Arrestins are multifunctional proteins that mediate receptor desensitization and internalization and serve as signaling scaffolds. A role for ␤-arrestin scaffolds in signaling by PAR-2 and other receptors was first identified for the spatial regulation of ERK1/2 activity (9 -11). They are now known to scaffold numerous other signaling molecules (12-15), many of which are involved in actin reorganization and chemotaxis (1, 13, 16 -19). An attractive hypothesis is that ␤-arrestins exert spatial control over actin assembly events at the leading edge to promote membrane protrusion and cell migration. A recent advance in this field was the discovery that ␤-arrestins are required for PAR-2-dependent activation of the actin filamentsevering protein, cofilin (14), which binds to the sides of actin filaments, destabilizing them and promoting their severing. Filament severing has two functions: the reorganization of existing filaments and the creation of free actin barbed ends for monomer addition (20). Actin is a polar molecule containing a barbed and pointed end; addition of actin monomers to a growing filament occurs at the barbed end. Alt...

βArrestin1 Regulates the Guanine Nucleotide Exchange Factor RasGRF2 Expression and the Small GTPase Rac-mediated Formation of Membrane Protrusion and Cell Motility

“…This multiprotein complex formation facilitated cofilin dephosphorylation, actin polymerization, and protrusion formation. Depletion of ␤Arr2 resulted in cell migration defects (43). Here, we showed that ␤Arr1 regulates rasgrf2 expression at the transcription level with a consequent effect on Rac and cofilin activities and cell migration.…”

“…␤Arr2 was reported to function as a scaffold for cofilin and the phosphatases slingshot and chronophin in HEK293, leukocytes, and breast cancer cells (42)(43)(44). This multiprotein complex formation facilitated cofilin dephosphorylation, actin polymerization, and protrusion formation.…”

Transactivation of the PAR1-PAR2 Heterodimer by Thrombin Elicits β-Arrestin-mediated Endosomal Signaling

“…However, the role of ␤-arrestins in PAR1-PAR2-induced pathological responses is not known and is important to investigate. In addition, ␤-arrestins have been implicated in activated PAR2-induced cytoskeleton reorganization and chemotaxis through activation of cofilin (47). Whether PAR1-PAR2 heterodimer-mediated recruitment of ␤-arrestins also regulates cell migration and/or endothelial adherens junction and barrier maintenance is not known but is critical to understand.…”

Cofilin under control of β-arrestin-2 in NMDA-dependent dendritic spine plasticity, long-term depression (LTD), and learning

“…β-Arrestins were recently shown to control the spatial localization of cofilin and its regulating proteins that act downstream of protease-activated receptor-2 (PAR-2) in fibroblasts and primary leukocytes (34,44), and this process has been implicated in the formation of a leading edge and subsequent chemotaxis (34). Whereas β-arrestin-1 was shown to scaffold cofilin with LIMK, the kinase that phosphorylates and inactivates cofilin, β-arrestin-2 has been implicated in associating cofilin with the phosphatases CIN and SSH, leading to cofilin dephosphorylation and activation (33,34,44). The ability of β-arrestins to scaffold cofilin with its enzymes and to regulate dynamic changes in F-actin organization made it a prime candidate for the regulation of cofilin translocation into dendritic spines.…”

“…The scaffolding proteins β-arrestins, which were recently suggested to regulate cofilin localization in migrating cells (33,34), were prime candidates for the regulation of cofilin translocation in response to NMDAR activation. First, we examined the effects of NMDA on the localization of WT cofilin, constitutively active cofilin S3A , or inactive phospho-mimetic cofilin S3D in .…”