Structure of active β-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 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.

Functional map of arrestin-1 at single amino acid resolution

“…Phosphosensing mechanism in arrestin-1. Binding of arrestin mutants (blue and ribbon width indicate residues which increase binding to P-ROS* upon mutation; red indicates residues which decrease binding to P-ROS* upon mutation) plotted on the crystal structures of inactive (8), preactivated p44 arrestin (9), and a homology model of arrestin-1 based on the crystal structure of arrestin-2 bound to a receptor phosphopetide (10). Several residues including three hydrophobic phenylalanines (Phe375, Phe377, and Phe380) and the charged Arg382 anchor the C tail of arrestin-1 into the three-element interaction and polar core regulatory sites (Left).…”

“…Detailed structural information on the inactive state of arrestin-1 has been available for some time (7,8). These inactive structures have recently been complemented with structures of a preactivated state of the arrestin-1 splice variant p44 (9) and of arrestin-2 bound to a receptor phosphopeptide (10). Analysis of these 3D structures provides many clues of how arrestins function.…”

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

“…A model of the receptor-bound state of non-visual arrestins is necessary to elucidate the structural basis of arrestin-mediated signaling. In addition, truncated arrestin-2 was recently co-crystallized with the multiphosphorylated C terminus of the vasopressin V2 receptor (36). Although this structure was not obtained by co-crystallization of arrestin-2 with a receptor, the crystal structure of arrestin-2 in complex with an antibody fragment (Fab30) and the phosphorylated peptide (V2Rpp) provided the first glimpse of the possible active form of arrestin-2.…”

Neuropeptide Receptors in Pain Circuitries: Useful Targets for CNS Imaging with Non-Peptide Ligands Suitable for PET?

“…Regarding development of pharmacophore models, also basic structures of NT3 (sortilin) and CGRP receptors with transmembrane domains distinct from the common GPCRs or with multimeric complex composition (see RAMP and CLR as receptor associated proteins) are a new challenge to the right choice of suitable lead 23 [96]. On the analgesic route of pain circuits, however, not only µ opioid receptors but also κ-receptors appear to be of great relevance not only in relays of the spinothalamic tract conveying signals to higher centers but also as potential modulators of dopamine release in cortical and subcortical brain areas [240].…”

“…Additionally, arrestin has been recognized during the last decades as a signal protein on its own which can associate with further regulatory proteins in multi protein complexes. Those can bias the conventional signaling pathways governed by G proteins in dependence on cell type and microenvironment [8] [20]- [23]. Ligand bias at 7-transmembrane receptors (7TMRs) responsible for a predominance either of signaling via G proteins or of β-arrestin regulated pathways has been described for instance for neurokinin 1 and opioid receptors [24]- [26].…”