The functions of G-protein coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein coupled receptor kinases (GRKs), and the arrestins1. G proteins mediate activation of second messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors2, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization3. Arrestins activated by interaction with phosphorylated receptors can also mediate G protein-independent signaling by serving as adaptors to link receptors to numerous signaling pathways4. Despite their central role in regulation and signaling of GPCRs, a structural understanding of β-arrestin activation and interaction with GPCRs is still lacking. Here, we report the crystal structure of β-arrestin1 in complex with a fully phosphorylated 29 amino acid carboxy-terminal peptide derived from the V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate β-arrestin15. To capture this active conformation, we utilized a conformationally-selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of β-arrestin1. The structure of the β-arrestin1:V2Rpp:Fab30 complex shows striking conformational differences in β-arrestin1 compared to its inactive conformation. These include rotation of the amino and carboxy-terminal domains relative to each other, and a major reorientation of the “lariat loop” implicated in maintaining the inactive state of β-arrestin1. These results reveal, for the first time at high resolution, a receptor-interacting interface on β-arrestin, and they suggest a potentially general molecular mechanism for activation of these multifunctional signaling and regulatory proteins.

G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins.

β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-βarr complexes: the "tail" conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-βarr conformations can carry out distinct functions.O ver the past decade, significant efforts have been made to understand the molecular properties and regulatory mechanisms that control the function of β-arrestin (βarr) interactions with G protein-coupled receptors (GPCRs) (1, 2). Once activated, GPCRs initiate a highly conserved signaling and regulatory cascade marked by interactions with: (i) heterotrimeric G proteins, which mediate their actions largely by promoting second-messenger generation (3); (ii) GPCR kinases (GRKs), which phosphorylate activated conformations of receptors (4); and (iii) βarrs, which bind to the phosphorylated receptors to mediate desensitization of G protein signaling and receptor internalization (5, 6). In addition to their canonical function of desensitization and internalization, βarrs have been appreciated as independent signaling units by virtue of their crucial role as both adaptors and scaffolds for an increasing number of signaling pathways (7-11).There are two driving forces that mediate βarr interactions with an activated GPCR: phosphorylation of the C-terminal tail of the receptor by GRKs and/or binding to the transmembrane core of the receptor. How each of these interactions contributes to βarr functionality remains unclear. Moreover, GPCRs tend to either interact with βarr transiently, termed "class A" GPCRs [e.g., β 2 -adrenergic receptor (β 2 AR)], or tightly, known as "class B" GPCRs [e.g., vasopressin type 2 receptor (V 2 R)]. For the current study, we use a previously described chimeric β 2 V 2 R construct, which comprises the β 2 AR with its C-terminal tail exchanged with the V 2 R C-terminal tail (12-14). The β 2 V 2 R construct provides an ideal system for studying a GPCR-βarr complex in vitro, because it maintains identical pharmacological properties to the WT β 2 AR and has a robustly increased class B affinity for βarr1, which allows stable β 2 V 2 R-βarr complexes to be formed and purified.Structural insights have shed some light onto the complexity of the interaction between GPCRs and βarrs. A recent struc...

Classically, G protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (βarr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-βarr 'megaplex'. Nevertheless, the megaplex's molecular architecture remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine βarr to the core and phosphorylated tail, respectively, of a single active human chimeric β 2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β 2 V 2 R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and βarr. Our findings provide a structural basis for GPCR-mediated sustained, internalized G protein signaling. Nguyen et al.