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Structure of active ?-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

ABSTRACT: 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 arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ?-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ?-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ?-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ?-arrestin-1. The structure of the ?-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ?-arrestin-1 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 ?-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ?-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

SUBMITTER: Shukla AK

PROVIDER: S-EPMC3654799 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

Shukla Arun K AK Manglik Aashish A Kruse Andrew C AC Xiao Kunhong K Reis Rosana I RI Tseng Wei-Chou WC Staus Dean P DP Hilger Daniel D Uysal Serdar S Huang Li-Yin LY Paduch Marcin M Tripathi-Shukla Prachi P Koide Akiko A Koide Shohei S Weis William I WI Kossiakoff Anthony A AA Kobilka Brian K BK Lefkowitz Robert J RJ

Nature 20130421 7447

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 arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated recep ...[more]

PMID: 23604254

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Project description:G-protein-coupled receptors (GPCRs) are critically regulated by ?-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ?2 adrenergic receptor (?2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ?-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-?-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ?2AR-?-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ?2AR and ?-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ?-arrestin 1 to the ?2AR, 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 crosslinked residues suggest engagement of the finger loop of ?-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ?-arrestin 1 when coupled to the ?2AR. A molecular model of the ?2AR-?-arrestin signalling complex was made by docking activated ?-arrestin 1 and ?2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Dataset Information

Structure of active ?-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

Publications

Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

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