Phosphorylated peptide of G protein-coupled receptor induces dimerization in activated arrestin

Andreas M Stadler,Anneliese Cousin,Joachim Granzin,Renu Batra-Safferling

doi:10.1038/s41598-020-67944-0

Andreas M Stadler, Anneliese Cousin + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-67944-0

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Abstract

Termination of the G-protein-coupled receptor signaling involves phosphorylation of its C-terminus and subsequent binding of the regulatory protein arrestin. In the visual system, arrestin-1 preferentially binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction.Here, we have investigated binding of a synthetic phosphopeptide of bovine rhodopsin (residues 323–348) to the active variants of visual arrestin-1: splice variant p44, and the mutant R175E. Unlike the wild type arrestin-1, both these arrestins are monomeric in solution. Solution structure analysis using small angle X-ray scattering supported by size exclusion chromatography results reveal dimerization in both the arrestins in the presence of phosphopeptide. Our results are the first report, to our knowledge, on receptor-induced oligomerization in arrestin, suggesting possible roles for the cellular function of arrestin oligomers. Given high structural homology and the similarities in their activation mechanism, these results are expected to have implications for all arrestin isoforms.

Highlights

Size exclusion chromatography on all three arrestin proteins were performed in the presence of unphosphorylated peptide which resulted in no change in the retention volumes and were similar to the control runs performed without any peptide (Supplementary Table S1). These results are consistent with the reports that binding of arrestin to the receptor requires phosphorylation of the C-terminus of the receptor[22,23]. It is highly intriguing how just four members of the arrestin family interact with hundreds of different G protein-coupled receptors (GPCRs) and other signaling proteins to regulate a multitude of physiological processes
Using rhodopsin and visual arr-1 as prototype models for GPCR-arrestin interaction, we have investigated the effect of rhodopsin C-terminal peptide on global conformation of constitutively active variants of arr-1
Using SAXS, we demonstrate that the phosphopeptide induces dimerization in both the pre-active arrestins

Summary

Introduction

Termination of the GPCR-mediated signaling involves a two-step arrestin-mediated desensitization, where activated receptor is first phosphorylated by a G protein-coupled receptor kinase (GRK) followed by high affinity binding of arrestin that sterically blocks the interaction between receptor and G-protein, leading to the signal termination. Activation of the receptor allows binding of its phosphorylated C-terminus to the N-domain of arrestin, disrupting the polar core network and releasing the C-tail of arrestin. Opening of the loop 139 (middle loop) and C-loop leads to accommodation of ICL2 helix of the receptor, forming the high-affinity activated state of arrestin, with the α-helical finger loop at the interface. P44 is the splice variant of visual arrestin (arr-1), which differs from arrestin only in the C-terminus where it lacks the last 35 amino acids that are replaced by an alanine residue[13].

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