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...

First identified as mediators of G-protein-coupled receptor desensitization and internalization and later as signaling platforms, ␤-arrestins play a requisite role in chemotaxis and reorganization of the actin cytoskeleton, downstream of multiple receptors. However, the precise molecular mechanisms underlying their involvement have remained elusive. Initial interest in ␤-arrestins as facilitators of cell migration and actin reorganization stemmed from the known interplay between receptor endocytosis and actin filament formation, because disruption of the actin cytoskeleton inhibits these ␤-arrestin-dependent events. With growing interest in the mechanisms by which cells can sense a gradient of agonist during cell migration, investigators began to hypothesize that ␤-arrestins may contribute to directed migration by controlling chemotactic receptor turnover at the plasma membrane. Finally, increasing evidence emerged that ␤-arrestins are more than just clathrin adaptor proteins involved in turning off receptor signals; they are actually capable of generating their own signals by scaffolding signaling molecules and controlling the activity of multiple cellular enzymes. This new role of ␤-arrestins as signaling scaffolds has led to the hypothesis that they can facilitate cell migration by sequestering actin assembly activities and upstream regulators of actin assembly at the leading edge. This Minireview discusses recent advances in our understanding of how ␤-arrestin scaffolds contribute to cell migration, focusing on recently identified ␤-arrestin interacting proteins and phosphorylation targets that have known roles in actin reorganization.

F 1 F 0 ATP synthase was prepared from iron-overloaded heart mitochondria to study the effect of iron on mitochondria. As F 1 F 0 ATP synthase was able to transport iron, proteoliposomes containing F 1 F 0 ATP synthase were prepared to compare iron uptake between control and iron-overloaded mitochondria. A threefold increase in V max (nmol/min/mg) (6.35 ± 0.17 vs. 2.08 ± 0.06) and an eightfold increase in K m (µM) (7.5 ± 0.7 vs. 0.85 ± 0.5) by F 1 F 0 ATP synthase for iron uptake were observed in iron-overloaded mitochondria. Mitochondrial ATP synthase prepared from iron-overloaded heart has a canonical subunit composition in an altered stoichiometry compared to the control enzyme: the α, β, γ, OSCP, d, a, δ, and c subunits increased, but the b, e, A6L, and F6 subunits decreased significantly. In addition, the pattern of β subunit isomers with different pIs changed. These isomers appeared to be associated with augmented phosphorylation by excess iron.

β‐arrestin serves as a multifunctional scaffold protein that brings together MAPK cascade modules and spatially restricts them within the cell. Downstream of protease‐activated‐receptor‐2 (PAR‐2), ERK activity is prolonged by β‐arrestins at the plasma membrane and we hypothesize that this is dependent upon precise molecular interactions that are facilitated by interaction of the ERK module with β‐arrestins. We have shown that Raf binds directly to β‐arrestin 1 in vitro, but MEK1 and ERK2 bind indirectly to via Raf. Using bioluminescence resonance energy transfer (BRET), we have shown direct interaction of Raf with β‐arrestin‐1 in cells upon PAR‐2 activation. Furthermore, using recombinant proteins, we have determined that the C‐terminus of β‐arrestin‐1 directly binds Raf, while the N‐terminus of β‐arrestin binds Raf upon PAR‐2 activation, suggesting that the composition of the β‐arrestin/ERK scaffold is dependent upon the activating receptor. Expression of either N or C‐terminal β‐arrestin truncations partially rescued PAR‐2 stimulated ERK1/2 activation in β‐arrestin knockout cells; expression of both N and C‐terminal truncations together fully rescues PAR‐2 stimulated ERK1/2 activation. Taken together, β‐arrestin 1 recruits the ERK cascade modules via direct binding to Raf and subsequent recruitment of MEK1/2 and ERK1/2 through interactions with sites on both its N and C‐terminus.