Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins.

• 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.

Nyckelord

Actins

metabolism

Amino Acid Sequence

Animals

Arrestins

analysis

genetics

metabolism

Binding Sites

Cell Membrane

chemistry

metabolism

Cell Shape

Cells

Cultured

Contractile Proteins

analysis

genetics

metabolism

Cytoskeletal Proteins

metabolism

Enzyme Activation

Humans

Immunoprecipitation

Microfilament Proteins

analysis

genetics

metabolism

Mitogen-Activated Protein Kinase 1

analysis

metabolism

Molecular Sequence Data

Protein Interaction Mapping

Protein Structure

Tertiary

genetics

Receptor

Angiotensin

Type 1

metabolism

Receptor

Muscarinic M1

metabolism

Repetitive Sequences

Amino Acid

genetics

Two-Hybrid System Techniques

Publikations- och innehållstyp

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