-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 ...
-arrestins (-arrs), two ubiquitous proteins involved in serpentine heptahelical receptor regulation and signaling, form constitutive homo-and heterooligomers stabilized by inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Monomeric -arrs are believed to interact with receptors after agonist activation, and therefore, -arr oligomers have been proposed to represent a resting biologically inactive state. In contrast to this, we report here that the interaction with and subsequent titration out of the nucleus of the protooncogene Mdm2 specifically require -arr2 oligomers together with the previously characterized nucleocytoplasmic shuttling of -arr2. Mutation of the IP6-binding sites impair oligomerization, reduce interaction with Mdm2, and inhibit p53-dependent antiproliferative effects of -arr2, whereas the competence for receptor regulation and signaling is maintained. These observations suggest that the intracellular concentration of -arr2 oligomers might control cell survival and proliferation. bioluminescence resonance energy transfer ͉ FRET ͉ nucleocytoplasmic shuttling ͉ ubiquitination
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