Phosphorylation-induced conformation of β2-adrenoceptor related to arrestin recruitment revealed by NMR

Abstract

The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated β₂-adrenoceptor (β₂AR) and the phosphorylated β₂AR-β-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M215^5.54 and M279^6.41, located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the β-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables β-arrestin to recognize dozens of GPCRs.

Fig. 1

Distribution of the NMR probes on the structure of β₂AR. The crystal structure of β₂AR (PDB ID: 3SN6) is shown as a ribbon model. The N-terminal region, intracellular loop 3, and the C-terminal region, which are either substituted with T4 lysozyme, not observed, or truncated, are shown with dotted lines. The methionine residues observed for the analysis of the conformation of the TM region are shown as sticks. M82^2.53, M215^5.54, and M279^6.41, with resonances that reportedly exhibit remarkable chemical shift change upon activation, are shown as red sticks. The C-terminal region, which was segmentally labeled for the NMR observation, is shown by a red dotted line. The putative phosphorylated residues in the C-terminal region are shown. The structural model was prepared with Cuemol (http://www.cuemol.org/)

Fig. 2

NMR spectra of the C-terminal region of the segmentally labeled β₂AR. a Schematic diagram of the preparation of segmentally labeled β₂AR. b Overlay of the ¹H-¹⁵N HSQC spectra of the unphosphorylated (black) and phosphorylated (red) {Cterm- [²H, ¹³C, ¹⁵N]} β₂AR in rHDLs. Resonances that were not observed in the phosphorylated β₂AR are indicated with boxes, and resonances that exhibited different chemical shifts between the unphosphorylated and phosphorylated spectra are indicated with arrows. c Overlay of the ¹H-¹³C HMQC spectra of {Cterm- [²H, Thrγ2-, Ileδ1-[¹³C,¹H]} β₂AR in rHDLs. d ¹H and ¹³C linewidths of the T360 methyl signals of the unphosphorylated (gray bars) and phosphorylated (red bars) β₂AR in rHDL. The linewidths were calculated by fitting the Lorentzian function to the signals using Topspin 3.1. The error bars indicate the standard errors of the fit

Fig. 3

Conformation of the TM region of phosphorylated β₂AR. Overlay of the ¹H-¹³C HMQC spectra of [²H-9AA, αβγ-²H, methyl-¹³C-Met] β₂AR in rHDLs in the unphosphorylated state (black) and phosphorylated state (red). Cross-sections of the resonances from M215^5.54 and M279^6.41 are shown above the spectra

Fig. 4

Interaction between the C-terminal region of β₂AR and the membrane surface examined by the methyl-directed cross-saturation experiments. a Schematic diagram of the methyl-directed cross-saturation experiment, using segmentally-labeled β₂AR. b, c Methyl-directed cross-saturation spectra of the C-terminal region of unphosphorylated β₂AR (b) and phosphorylated β₂AR (c). Spectra with and without irradiation are shown in the right and left panels, respectively. d Intensity reduction ratio of each resonance. Gray and red bars indicate the results in the unphosphorylated state and the phosphorylated state, respectively. The error bars represent the experimental errors, calculated from the root sum square of (noise level/signal intensity) in the two spectra, with and without irradiation

Fig. 5

Conformation of the TM region of the phosphorylated β₂AR bound to β-arrestin. Overlay of the ¹H-¹³C HMQC spectra of [²H-9AA, αβγ-²H, methyl-¹³C-Met] β₂AR in rHDLs in the unphosphorylated state (black), phosphorylated state (red), and β-arrestin 1-bound state (blue). The centers of the resonances from M215^5.54 to M279^6.41 are indicated with dots. In the phosphorylated state, the resonance from M279^6.41 could not be observed

Fig. 6

Role of the phosphorylation of the C-terminal region in signal transduction. In the unphosphorylated state, the C-terminal region of β₂AR is unstructured. Upon phosphorylation by GRKs, the TM-proximal region of the C-terminal region of β₂AR adheres to the cytoplasmic face of the TM region, and the intracellular halves of the TM helices become spatially rearranged, which preferentially activates arrestin-mediated signal transduction. The adhesion of the phosphorylated C-terminal region to the TM-region and the spatial rearrangement of the TM helices would generate the structural motif for β-arrestin binding