Abstract
β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.
Highlights
Cancer cell metastasis involves multistep cellular processes, commencing with cell migration and invasion [1, 2]
We report that 2AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. 2ARs induce activation of RhoA, which is dependent on Arrestin2 but not Gs. Arrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G12/13-coupled receptors
Upon G␣i knockdown, the basal RhoA activity is significantly increased, and ISO stimulation failed to further activate RhoA (Fig. 4, H and I). These results suggest that Gi proteins regulate focal adhesions through RhoA. Arrestin2 Regulates p115RhoGEF—To indentify intermediates involved in the regulation of RhoA activity by 2AR and
Summary
Reagents—Anti-FLAG, anti-vinculin, rabbit anti-HA, and anti-actin antibodies were purchased from Sigma; antiGAPDH from Millipore; mouse anti-HA from Roche Applied Science; anti-p115RhoGEF from Cellular Signaling; anti-RhoA from Cytoskeleton; anti-paxillin from BD Biosciences. Transfection was performed using Lipofectamine 2000 (for cDNAs) or Lipofectamine RNAiMax (for siRNAs) for HEK293 and RCC7 cells, or using GenJet for MEFs. For immunofluorescence staining, cells were seeded on fibronectin-coated coverslips and stained using anti-vinculin, or anti-paxillin (for focal adhesions), anti-HA (for overexpressed Arrestin2), or anti-GFP (for overexpressed GFPp115RhoGEF) antibodies. Immunoprecipitation and GST Pulldown—Cells were washed with PBS and lysed in the lysis buffer (25 mM Tris, pH 8.0, 100 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM EDTA, 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin, and 2 g/ml pepstatin A). Anti-FLAG M2 beads were washed three times with lysis buffer, and immunoprecipitated proteins were boiled into SDS-PAGE sample buffer. Arrestin Regulates RhoA and Focal Adhesion teins bound to them were incubated with freshly prepared cell lysates overnight at 4 °C, washed three times with lysis buffer, and boiled into SDS-PAGE sample buffer. Graphs were generated using Prism software (GraphPad), and axis labels were generated using Adobe Illustrator
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