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 filament-severing protein cofilin, which is crucial for the reorganization of the actin cytoskeleton and chemotaxis. These responses require the scaffolding functions of beta-arrestins; however, the mechanism by which beta-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 beta-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 beta-arrestin-2(-/-) mice. We show that, in response to PAR-2 activation, beta-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 beta-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.
Highlights
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]
We previously showed that PAR-2 promotes rapid cofilin dephosphorylation that is decreased in the absence of -arrestins or by expression of a dominant negative CIN mutant [14]
We hypothesize that PAR-2 activation results in recruitment of CIN and cofilin to -arrestins into a scaffolding complex to promote localized generation of free actin barbed ends and membrane protrusion
Summary
Materials and Cell Lines—All chemicals were from Sigma or Fisher Scientific unless stated otherwise. Cells were washed in 0.1 M glycine/PBS for 10 min, fixed, stained, and imaged as described in microscopy section. For cells stained with Alexa595 phalloidin, the laser intensity was set at 35% and amplifier gain was 850. Protein Analysis—Cleared lysates were prepared as follows: Cells (either cultured cells or bone marrow leukocytes) were treated with or without 100 nM to 1 M 2fAP for indicated times and lysed in 0.5 ml of lysis buffer, as described previously [10]. Protein was precipitated with methanol/chloroform from every other fraction within the included volume and analyzed by SDS-PAGE, followed by Western analysis with antibodies to -arrestin-1 and -2, total cofilin, and CIN. Bone marrow leukocytes were lysed, and total lysates were analyzed by SDS-PAGE followed by Western blotting with anti-phospho and anti-total cofilin. Integrated intensities were determined, and the average ratio of phospho-cofilin to total cofilin levels was calculated
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