RESUMO
beta-Arrestins (betaarr) 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 betaarr-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 betaarr-binding site involves tandem repeat 22 in the carboxyl terminus. betaarr binds FLNA through both its N- and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that betaarr 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 betaarr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, betaarr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of betaarr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for betaarr, FLNA, and active ERK in this process. Our results indicate that betaarr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.
Assuntos
Arrestinas/metabolismo , Forma Celular , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/análise , Arrestinas/genética , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Proteínas Contráteis/análise , Proteínas Contráteis/genética , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática , Filaminas , Humanos , Imunoprecipitação , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Proteína Quinase 1 Ativada por Mitógeno/análise , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Muscarínico M1/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Técnicas do Sistema de Duplo-Híbrido , beta-ArrestinasRESUMO
beta-arrestins (betaarrs) are two highly homologous proteins that uncouple G protein-coupled receptors from their cognate G proteins, serve as adaptor molecules linking G protein-coupled receptors to clathrin-coat components (AP-2 complex and clathrin), and act as scaffolding proteins for ERK1/2 and JNK3 cascades. A striking difference between the two betaarrs (betaarr1 and betaarr2) is that betaarr1 is evenly distributed throughout the cell, whereas betaarr2 shows an apparent cytoplasmic localization at steady state. Here, we investigate the molecular determinants underlying this differential distribution. betaarr2 is constitutively excluded from the nucleus by a leptomycin B-sensitive pathway because of the presence of a classical leucine-rich nuclear export signal in its C terminus (L395/L397) that is absent in betaarr1. In addition, using a nuclear import assay in yeast we showed that betaarr2 is actively imported into the nucleus, suggesting that betaarr2 undergoes constitutive nucleocytoplasmic shuttling. In cells expressing betaarr2, JNK3 is mostly cytosolic. A point mutation of the nuclear export signal (L395A) in betaarr2, which was sufficient to redistribute betaarr2 from the cytosol to the nucleus, also caused the nuclear relocalization of JNK3. These data indicate that the nucleocytoplasmic shuttling of betaarr2 controls the subcellular distribution of JNK3.