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

Abstract

ABSTRACT β-Arrestins (βarr) 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 βarr-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 βarr-binding site involves tandem repeat 22 in the carboxyl terminus. βarr binds FLNA through both its N- and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that βarr 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 βarr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, βarr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of βarr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for βarr, FLNA, and active ERK in this process. Our results indicate that βarr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.