Biphasic activation of \u03b2-arrestin 1 upon interaction with a GPCR revealed by methyl-TROSY NMR

Yutaro Shiraishi,Takumi Ueda,Shubhi Pandey,Ichio Shimada,Hemlata Dwivedi-Agnihotri,Yutaka Kofuku,Arun K Shukla

doi:10.1038/s41467-021-27482-3

Abstract

β-arrestins (βarrs) play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs. However, the effects of the C tail- and TM core-mediated interactions on the conformational activation of βarrs have remained elusive. Here, we show the conformational changes for βarr activation upon the C tail- and TM core-mediated interactions with a prototypical GPCR by nuclear magnetic resonance (NMR) spectroscopy. Our NMR analyses demonstrated that while the C tail-mediated interaction alone induces partial activation, in which βarr exists in equilibrium between basal and activated conformations, the TM core- and the C tail-mediated interactions together completely shift the equilibrium toward the activated conformation. The conformation-selective antibody, Fab30, promotes partially activated βarr into the activated-like conformation. This plasticity of βarr conformation in complex with GPCRs engaged in different binding modes may explain the multifunctionality of βarrs.

Highlights

Β-arrestins play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs
The N and C lobes are connected by a hinge region, and the finger loop is located near the interface between the N and C lobes6. βarrs bind to two distinct elements in GPCRs: the carboxyl terminal region phosphorylated by GPCR kinases (GRKs), and the cytoplasmic face of the agonist-activated transmembrane region[7]
Since arrestins reportedly interact with membrane lipids when bound to GPCRs9,10,21, purified β2V2R was embedded within a lipid bilayer of reconstituted high-density lipoproteins[22]

Summary

Introduction

Β-arrestins (βarrs) play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs. We show the conformational changes for βarr activation upon the C tail- and TM coremediated interactions with a prototypical GPCR by nuclear magnetic resonance (NMR) spectroscopy. Recent studies have indicated that the TM core can activate βarr without the C tail-mediated interaction[13,14] These findings suggest that the C tail- and TM core-mediated interactions could induce the conformational changes of βarrs. The effects of these interactions on the conformational changes for βarr activation in solution have remained elusive, because activated arrestin is inherently dynamic[15] and the available structures have been visualized with stabilization methods, including pre-activated mutants[8,10,12], fusion proteins with a GPCR8,12, and the conformation-selective antibody fragment, Fab[309,11,12]. This conformational plasticity of βarr[1] in complex with a GPCR may explain its ability to engage multiple scaffolding partners and in turn mediate distinct functional outcomes

Methods

Results

Conclusion