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...