Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins.: FLNA AND ßarr COOPERATE TO REGULATE ERK AND CELL SHAPE

14 pagesbeta-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

Recruitment of Activated G Protein-coupled Receptors to Pre-existing Clathrin-coated Pits in Living Cells

International audienceThe process of clathrin-mediated endocytosis tightly regulates signaling of the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). A fundamental question in the cell biology of membrane receptor endocytosis is whether activated receptors can initiate the formation of clathrin-coated pits (CPs) or whether they are simply mobilized to pre-existing CPs. Here, using various approaches, including a dynamic assay to monitor the distribution of CPs and GPCR-beta-arrestin complexes in live HeLa cells, we demonstrate for the first time that activated GPCRs do not initiate the de novo formation of CPs but instead are targeted to pre-existing CPs

Homo- and Hetero-oligomerization of β-Arrestins in Living Cells

International audienceArrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous β-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of β-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged β-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, β-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of β-arrestin2 and β-arrestin1 prevented β-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that β-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions