Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

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 ...