Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors

Abstract

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.

Keywords: GPCR; GRK; arrestin; biased signaling; drug discovery; membrane proteins; phosphorylation codes; rhodopsin.

Figure 1

Crystal structure of rhodopsin-arrestin complex with rhodopsin C-terminal tail interacting with arrestin N-terminal domain. See also Figure S1. (A–B) Two views of T4L-rhodopsin-arrestin fusion complex with rhodopsin in green and arrestin in brown (T4L not shown). (C–D) Two views of rhodopsin C-terminal tails of overlayed four rhodopsin-arrestin complexes in an asymmetric unit. Complex A is shown in green, B in magenta, C in cyan, and D in yellow.

Figure 2

Interface between rhodopsin C-terminal tail and arrestin N-terminal domain. See also Figures S1–2 and Table S3. (A) The interface between rhodopsin C-tail and arrestin N-terminal domain with the rhodopsin C-tail covered with a 2mFo-DFc density map contoured at 1 σ. Rhodopsin is in green, and arrestin in brown. (B) Interface residues between rhodopsin C-tail and arrestin N-terminal domain, with rhodopsin residues colored green and arrestin residues brown. (C) A schematic diagram of the interactions between rhodopsin C-tail residues (green) and arrestin residues (brown). Solid lines and arrows indicate hydrogen bonds and salt bridges, respectively. (D) Charge-distribution surface of the arrestin N-terminal domain in the rhodopsin-arrestin complex with an electrostatic scale from −3 to +3 eV corresponding to red to blue colors. Labeled are the rhodopsin C-tail residues involved in the charge interaction network.

Figure 3

Validation of the interface between rhodopsin C-tail and arrestin N-terminal domain by DEER spectroscopy. (A) Interface between the rhodopsin C-tail and arrestin showing the intermolecular distances between pairs of R1 nitroxide spin-labeled side chains at arrestin position 107 and rhodopsin position 335 or 337. (B) Experimental distance distributions between the R1 nitroxide pairs shown in (A). (C) Interface between rhodopsin C-tail and arrestin showing the intermolecular distances between pairs of R1 nitroxide spin-labeled side chains at rhodopsin position 342 and arrestin position 106 or 107. (D) Experimental distance distributions between the R1 nitroxide pairs shown in (C).

Figure 4

Validation of the interface between rhodopsin C-tail and arrestin by disulfide cross-linking. Crystal structures showing interface residues (left), and Western blots showing disulfide cross-linking data (right). The black asterisk indicates arrestin, and the arrowhead the rhodopsin-arrestin crosslinking product. See also Figure S3. (A) The disulfide cross-linking between D330 of rhodopsin and K167 of arrestin, D331 of rhodopsin and R19 of arrestin, and E332 of rhodopsin and R19, K167 and K168 of arrestin. (B) The disulfide cross-linking between A333, S334 and A335 of rhodopsin and R19 of arrestin. (C) The disulfide cross-linking between T336 of rhodopsin and K16 and R19 of arrestin, and E341 of rhodopsin and K111 of arrestin.

Figure 5

Molecular dynamics simulations suggest a key interface between rhodopsin C-tail and arrestin. See also Figure S5. (A–B) Interaction frequencies of pT336, pS338 and E341 to key residues within arrestin’s A, B, and C phosphate binding pockets for simulation 1 (A) and simulation 2 (B). (C) Displacement of selected residues within the C-tail region away from their crystallographic position measured by RMSDs of their heavy atoms. RMSD traces were smoothed over a 10 ns window. Residues comprising the phosphorylation code region remain stable within 3 Å of their initial positions (pT336: 2.72 ± 0.5 Å, pS338: 2.01 ± 0.78 Å, E341: 2.98 ± 0.66 Å), while residues out of the code region (D330 and S334) exhibit a number of metastable states reaching values upwards of 19 Å away. (D) Root mean square fluctuation (RMSF) of C-tail alpha carbons around their average position between 1 and 3 µs suggests a key interaction region comprising residues 335 to 341 in agreement with previous RMSD observations. (E) Average RMSF values for each residue converted into B-factor values (B-factor=[8*π²]/3*(RMSF)²) and mapped onto the initial simulation model for C-tail simulations.

Figure 6

Phosphorylation codes derived from the interfaces of the rhodopsin C-tail with visual arrestin and the V2R C-tail peptide with β-arrestin-1. See also Figures S5–7 and Table S4. (A) A sequence alignment of rhodopsin C-tail with the V2R peptide based on the phospho-residues that bind to the positively charged pockets of arrestin. (B) An overlay of the rhodopsin C-tail (green) with the V2R C-tail (4JQI, magenta) binding at arrestin N-terminal surfaces. Only visual arrestin surface is shown. Blue indicates positive charges, and red negative charges at arrestin surfaces. (C) A model depiction of the conserved arrestin N-terminal surface “reading” the phosphorylation code of rhodopsin or V2R. (D) Mutations of phosphorylation code residues on rhodopsin C-tail (left panel), and the binding (normalized luminescence) of the mutant rhodopsin to visual arrestin determined by Tango assay (right panel). (E) Mutations that abolish the phosphorylation codes on the V2R C-tail of the β₂AR-V2R C-tail chimera (left panel), and the binding (normalized luminescence) of the mutant chimeric receptors to β-arrestin-1 determined by Tango assay (right panel) in the presence or absence of 1 µM salmeterol xinafoate (SX). In (D) and (E), phosphorylation codes are highlighted in red boxes. The relative activity values obtained from three experiments are represented as mean ± s.d. (n=3, *P<0.05, **P<0.01, and ***P<0.001).

Figure 7

Mutations turning partial phosphorylation codes at the β₂AR C-tail to full codes increase the binding affinity of the receptor to β-arrestin-1. See also Figure S7. (A) Amino acid sequence of β₂AR C-tail. Partial phosphorylation codes are highlighted with red lines (same as in panel (B) and (C)), and serine, threonine or glutamic acid residues that form the partial codes are colored in red. Two clusters of phosphorylation sites are noted. (B) Mutations that turn partial phosphorylation codes to full codes (in red box) or replace the partial code serine or threonine residues with alanine at the β₂AR C-tail (left panel), and the binding affinity (normalized luminescence) of the mutant receptor to β-arrestin-1 determined by Tango assay in the presence or absence of 1 µM SX (right panel). (C) Mutations that turn partial phosphorylation codes to full codes (in red box) on the β₂AR C-tail (left panel), and the binding affinity (normalized luminescence) of the mutant receptor to β-arrestin-1 determined by Tango assay in the presence or absence of 1 µM SX (right panel). The relative activity values obtained from three experiments are represented as mean ± s.d. (n=3, *P<0.05, **P<0.01, and ***P<0.001).