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.
Scott
Mark G H
MG
Institut Cochin, Dept. of Cell Biology, 27 rue du Faubourg St Jacques, 75014 Paris, France. scott@cochin.inserm.fr
Pierotti
Vincenzo
V
Storez
Hélène
H
Lindberg
Erika
E
Thuret
Alain
A
Muntaner
Olivier
O
Labbé-Jullié
Catherine
C
Pitcher
Julie A
JA
Marullo
Stefano
S
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D048430:Cell Shape; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D064448:Filamins; D006801:Humans; D047468:Immunoprecipitation; D008840:Microfilament Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000235:genetics; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D020798:Two-Hybrid System Techniques; D000071557:beta-Arrestins
D000199:Actins; D019390:Arrestins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D064448:Filamins; D008840:Microfilament Proteins; D044140:Receptor, Angiotensin, Type 1; D043643:Receptor, Muscarinic M1; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1