Cooperative Regulation of Extracellular Signal-Regulated Kinase Activation and Cell Shape Change by Filamin A and β-Arrestins

Abstract

␤-Arrestins (␤arr) are multifunctional adaptor proteins that can act as scaffolds for G protein-coupled receptor activation of mitogen-activated protein kinases (MAPK). Here, we identify the actin-binding and scaffolding protein filamin A (FLNA) as a ␤arr-binding partner using Son of sevenless recruitment system screening, a classical yeast two-hybrid system, coimmunoprecipitation analyses, and direct binding in vitro. In FLNA, the ␤arr-binding site involves tandem repeat 22 in the carboxyl terminus. ␤arr binds FLNA through both its N-and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that ␤arr and FLNA act cooperatively to activate the MAPK extracellular signal-regulated kinase (ERK) downstream of activated muscarinic M1 (M1MR) and angiotensin II type 1a (AT1AR) receptors and provide experimental evidence indicating that this phenomenon is due to the facilitation of ␤arr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, ␤arr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of ␤arr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for ␤arr, FLNA, and active ERK in this process. Our results indicate that ␤arr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.Cytoskeletal reorganization is fundamental for cell shape change, signaling, locomotion, and many other important dynamic cellular processes. The MAPK JNK, p38, and ERK play key roles in the regulation of cytoskeletal dynamics (21), as many extracellular signals that promote a change in cell shape converge at MAPK, which in turn phosphorylate downstream targets involved in actin cytoskeleton regulation. For example, ERK can phosphorylate myosin light chain kinase (27, 37), calpain (18), focal adhesion kinase (22), and ribosomal S6 kinase (15), which all play important roles in cytoskeletal reorganization and cell migration.Initially appreciated for their roles in G protein-coupled receptor desensitization and endocytosis, ␤-arrestins are now considered multifunctional adaptor molecules, with over 20 binding partners, including trafficking proteins, nonreceptor tyrosine kinases, guanine nucleotide exchange factors, and MAPK components, identified to date (28, 29). There exist two isoforms of ␤arr, ␤arr1 and ␤arr2. These share a high degree of homology (ϳ80%) and several biological functions, such as their abilities to bind to agonist-occupied GPCRs, preventing G protein-mediated signal transduction, and to bridge receptors with components of the clathrin-dependent endocytic machinery (clathrin and AP-2). With regard to MAPK, ␤arr can act as scaffolds for GPCR-mediated activation of ERK1/2 (12, 33), JNK3 (35), and p38 (44). ␤arr bind multiple components of MAPK cascades to promote efficient activation of MAPK, additionally redirecting them to extranuclear compartments (12,33,35). The ...

Emerging Roles of β-Arrestins

“…Recently, β-arrestin was also shown to act in concert with filamin A in the activation of ERK and play an important role in actin reorganization in membrane ruffles. 15 β-arrestin has also been shown to act as a scaffold which influences the activation and subcellular localization of JNK3 and p38. 16,17 Furthermore, β-arrestin can also affect the signaling of receptor tyrosine kinases such as the EGFR by mediating the GPCR-induced internalization of this receptor.…”

Neuroprotective roles of the P2Y2 receptor

“…GPCR internalization often requires arrestin binding to the desensitized receptor that provides a scaffold for multiple protein-protein interactions [113,114]. A role for arrestin-2 in cell migration has been reported [115], and arrestin has been shown to associate with LIM kinase/cofilin [116] and FLNa [117] providing a mechanism for activation of arrestins by a GPCR, such as the P2Y 2 R. The β1-adrenergic receptor-mediated transactivation of the EGFR is mediated by arrestin 1 and 2 following G protein receptor kinase 5/6-dependent phosphorylation of the β1 receptor [118]. These interactions lead to Src-dependent activation of MMPs and consequent release of the HB-EGF ligand to enable autocrine activation of the EGFR [119].…”

A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells

“…CK2 plasmid was obtained from David Litchfield (University of Western Ontario) via Addgene and has been described previously 53 . AT1AR and M1MR have been described previously 55 . The antibodies pAKT, total AKT, phospho PTEN (Ser380/Thr382/Thr383), PTEN (138G6), Myc (71D10), b-arr1/2 (D24H9), HA (C29F4) and GAPDH were from Cell Signalling; PTEN mAb clone 6H2.1 was from Millipore; green fluorescent protein and Myc (9E10) were from Roche; anti-c-erbB2 (clone e2-4001) was from Thermo Scientific; tubulin and actin were from Santa Cruz; and FLAG was from Sigma.…”

“…For co-immunoprecipitations of b-arr with Rluc-PTEN-YFP, cells were lysed in buffer containing 50 mM Hepes (pH 7.4), 250 mM NaCl, 2 mM EDTA, 0.5% NP-40, 10% glycerol supplemented with protease and phosphatase inhibitors (Roche) as used for previous b-arr interaction studies 21,54,55 . For co-immunoprecipitations of PTEN-tail fusions with PTEN-Dtail, HEK293T cells expressing relevant plasmids were lysed in buffer containing 10 mM Tris-HCl (pH 7.5), 140 mM NaCl, 5 mM EDTA and 0.1% Nonidet P-40 supplemented with protease and phosphatase inhibitors (Roche).…”