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

Abstract

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous ␤-arrestins are believed to function exclusively as monomers, although selfassociation is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of ␤-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged ␤-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, ␤-arrestins constitutively form both homo-and hetero-oligomers. Co-expression of ␤-arrestin2 and ␤-arrestin1 prevented ␤-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that ␤-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.Arrestins play a central role in the regulation and signaling of serpentine heptahelical G protein-coupled receptors (GPCRs).5 Arrestin 1 and 4 are restricted to retinal rods and cones where they regulate rhodopsin (1). In contrast, arrestin 2 and 3, also referred to as ␤-arrestin 1 (␤arr1) and 2 (␤arr2), respectively, are ubiquitous and translocate to a large variety of ligand-activated GPCRs. Originally identified as negative regulators of GPCR function, promoting desensitization (2), ␤arrs were subsequently shown to be adaptor proteins connecting GPCRs to the endocytic machinery (3, 4). ␤arrs also serve as signaling scaffolds linking receptors to a growing number of effector pathways (5). For example, ␤arrs act as scaffolds for the activation of ERK and JNK3 (5). In addition, ␤arr2 redistributes the ubiquitin ligase Mdm2 and the kinase JNK3 from the nucleus to the cytoplasm, a property related to the presence of a leucine-rich nuclear export signal (NES) in ␤arr2 (6, 7). This signal is absent from ␤arr1, determining some differences in both subcellular distribution and functional roles between the two isoforms (6 -8).Crystal structures of visual arrestin (9, 10) revealed that this molecule contains two globular domains and an extended COOH-terminal tail locking the molecule into an inactive state. Upon binding to receptors, the arrestin C-tail is released, leading to an open active conformation (11). In crystals, visual arrestin is a tetramer composed of two asymmetric dimers (9, 10). In vitro experiments showed that, in solution, tetramers are in equilibrium with monomers at physiological concentrations (12, 13), and it was proposed that self-association might regulate arrestin activity by limiting availability of active monomeric species (13). The crystal structure of ␤arr1 is very similar to that of visual arrestin, but unlike visual arrestin, full-length ␤arr1 was found to be monomeric (14). In addition, because of their lower ...

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

“…6). An alternative explanation of the findings would be that high concentration of arrestin-3 promotes its oligomerization (69,70) and that oligomers are unable to serve as scaffold leading to reduced efficacy of arrestin-3-dependent JNK activation. However, two lines of evidence argue against this explanation.…”

Using BRET to study chemical compound‐induced disruptions of the p53‐HDM2 interactions in live cells

“…Filter sets were 485±10 nm for luciferase emission and 530±12.5 nm for YFP emission. BRET ratios were calculated as described [8]. The data show the average values of triplicates from three independent experiments.…”

Distinct conformational changes in β-arrestin report biased agonism at seven-transmembrane receptors

“…To rule out the possibility that changes in BRET ratio are due to intermolecular interaction between ␤-arrestin molecules brought together through dimerization (14) or aggregation at the plasma membrane, we coexpressed Luc-␤-arr and ␤-arr-YFP under conditions that lead to comparable levels of fluorescence and luminescence as obtained in Luc-␤-arr-YFP-expressing cells. As shown in Fig.…”