Lipids modulate the dynamics of GPCR:β-arrestin interaction

Abstract

β-arrestins are key molecular partners of G Protein-Coupled Receptors (GPCRs), triggering not only their desensitization but also intracellular signaling. Existing structural data point to high conformational plasticity of GPCR:β-arrestin interaction, with two completely different orientations between receptor and β-arrestin. Combining molecular dynamics simulations and fluorescence spectroscopy, we show that β-arrestin 1 interacts with membranes even in the absence of a receptor, an interaction that is enhanced by PI(4,5)P2, presumably holding the β-arrestin 1 C-edge loop into the lipid bilayer. This key interaction helps β-arrestin 1 to adopt a "receptor-ready" orientation and consequently favors its coupling to the ghrelin receptor (GHSR). In addition, we show that the GHSR:β-arrestin 1 assembly is a dynamic complex where β-arrestin can adopt several orientations. PI(4,5)P2 decreases the dynamics of the complex and shifts the equilibrium between the different arrangements, favoring one of them. Taken together, our results highlight how PI(4,5)P2 plays a true third-player role in the GPCR:β-arrestin interaction, not only by preparing β-arrestin for its further interaction with receptors but also by modulating its orientation once the protein:protein complex is formed.

Fig. 1. Conformational plasticity of the GPCR:β-arrestin interaction in existing structural data.

A Summary of β-arrestin contacts with the receptor and with the membrane as observed in the NTS1R structure (PDB: 6UP7). B Conformations adopted by the finger-loop of β-arrestin in the different receptors. C Orientations of β-arrestin in the different known structures of GPCR:β-arrestin complexes showing two main sets of orientations with NTS1R structures aside. D Position of the C-edge loop of β-arrestin with respect to the membrane among the same set of structures; black points correspond to the position of the membrane predicted by OPM (Orientations of Proteins in Membrane) for the complex with the beta-1 adrenergic receptor (B1AR) (PDB: 6TKO).

Fig. 2. Structural aspects of β-arrestin 1 and its interaction with membranes.

A Side view of the β-arrestin 1 model used CGMD simulations. The protein backbone beads are shown as pink surface representations with the finger loop and the C-edge loop highlighted in orange and green, respectively. Residues chosen to insert MB are depicted as orange (L68C) and green spheres (L191C). B Changes in the MB normalized emission intensity for the two positions of wild-type β-arrestin 1 labeling in the absence of nanodiscs (no ND), in the presence of POPC/POPG nanodiscs (ND, no PIP2), or in the presence of POPC/POPG nanodiscs containing 2.5% PI(4,5)P2 (ND, PIP2). Data were inferred from the emission spectra (see Supplementary Fig. 2) and are the mean value ± SD of three experiments. C Percentages of CGMD simulation frames where β-arrestin 1, the finger loop, or the C-edge loop are in contact with the membrane are shown as a function of the simulated system (β-arrestin 1 in the presence of a PI(4,5)P2-free, PI(4,5)P2-enriched, or POPS-enriched membrane). Bars correspond to the means and standard deviations calculated over five independent simulations. All statistical values in (B) and (C) were determined using one-way ANOVA tests with Bonferroni correction between all groups (*p ≤ 0.05, **p ≤ 0.01).

Fig. 3. β-arrestin 1 interaction with PI(4,5)P2 and predicted orientations to the cell membrane.

A PI(4,5)P2 binding site encompassing positively charged residues (K250 and K324) and the membrane insertion of C-edge through a hydrophobic residue (L335) are shown. B Statistical distributions of PI(4,5)P2 (orange meshes) along the obtained CGMD trajectories. It confirmed the specific binding of PI(4,5)P2 in the same site as that described in the literature. Secondary sites distributed around the C-edge loop and the N-domain of β-arrestin 1 were also found. C The definition of “tilt” and “roll” angles aimed to report the position of the protein with respect to the membrane surface. D β-arrestin 1 assumes a specific orientation at the membrane surface (see line contours), compatible with further interaction with receptors. Black dots correspond to “tilt” and “roll” angles extrapolated from existing GPCR:β-arrestin structures in the PDB after receptor alignment to B1AR (PDB: 6TKO).

Fig. 4. Role of the PI(4,5)P2 in the interaction of β-arrestin 1 with GHSR.

A Changes in the emission intensity of MB attached to either C68 or C191 of ΔC β-arrestin 1 free in solution (no ND), in the presence of POPC/POPG nanodiscs containing unphosphorylated GHSR and 10 µM MK0677 (ND, no PIP2) or in the presence of POPC/POPG nanodiscs containing 2.5% PI(4,5)P2, unphosphorylated GHSR and 10 µM MK0677 (ND, PIP2). The scheme was created in BioRender. BANERES, J. (2025) https://BioRender.com/ ngj6am4. B Same experiment with wild-type β-arrestin 1 instead of ΔC β-arrestin 1. The scheme was created in BioRender. BANERES, J. (2025) https://BioRender.com/aha8piq. In all cases, data are the mean value ± SD of three experiments. Statistical values were obtained by means a one-way ANOVA test with Bonferroni post-test groups (*p ≤ 0.05, **p ≤ 0.01).

Fig. 5. Distinct orientations of β-arrestin 1 coupled to the GHSR.

Models of the GHSR:β-arrestin 1 complex built from NTS1R (green, PDB: 6UP7) or B1AR (blue, PDB: 6TKO) structures. The location of the residues used to attach the fluorescent probes in β-arrestin 1 (V167 or L191) and in GHSR (F71^1.60) are depicted in orange sticks.

Fig. 6. Relative orientation of the protein components in the GHSR:β-arrestin 1 complex.

Sensitized-emission decays from phosphorylated GHSR labeled with Alexa Fluor 488 (A) or Bodipy-TMR (B) on C711.60 and assembled in nanodiscs containing or not 2.5% PI(4,5)P2, in the presence of wild-type β-arrestin 1 labeled either on C167 (A) or on C191 (B) with Lumi-4 Tb, and of 10 µM MK0677. Data are presented as the log of normalized fluorescence intensity as a function of time and are the mean ± SD of three independent measurements. (C, D) Corresponding residual values representing the goodness of the multi-exponential fits. (E, F) Corresponding mean distances and molecular fractions (populations) between the fluorescent probes calculated from the sensitized-emission lifetimes of the two dominant exponential components of the mean sensitized-emission decays (see Supplementary Table 1). The distances between the two probes calculated from the cryo-EM structures of the two representative orientations, i.e. NTS1R and B1AR (Table 1), are indicated in dotted lines.

Fig. 7. Results from CGMD simulations of the GHSR:β-arrestin 1 complex starting from the NTS1R-like or B1AR-like orientations.

A Top views of snapshots from a CGMD simulation replica of the GHSR:β-arrestin 1 complex with the protein backbone beads being shown as surface representations. The NTS1R-like starting conformation is highlighted in green, intermediate conformations are shown in gray, and the fully B1AR-like transitioned complex is depicted in blue. The residues used for the rotation angle and the F71^1.60:L191 distance measurement are shown as yellow discs. B Distribution of the rotation angle of β-arrestin 1 for all CGMD simulation replicas starting from the NTS1R-like (green) and the B1AR-like orientation (blue) performed with (solid line) or without PI(4,5)P2 (dashed line). The rotation angles of the respective experimental structures are highlighted by vertical lines. C 2D histogram corresponding to data obtained starting from the NTS1R-like orientation and showing the correlation between the β-arrestin 1 rotation angle and the F71^1.60:L191 distance that was measured experimentally. The rotation angles of the NTS1R-like (green) and B1AR-like (blue) experimental structures are indicated by horizontal lines. D The difference in the percentage of frames where PI(4,5)P2 interacts with the protein complex between the simulation replica starting from the B1AR-like or the NTS1R-like orientation is mapped onto the backbone beads. Residues colored in red indicate a more frequent interaction of the respective beads with PI(4,5)P2 in simulations starting from the B1AR-like conformation whereas residues colored in blue show no differences between both conformations.

Fig. 8. Influence of the C-edge loop-membrane interaction on the conformational equilibrium of the GHSR:β-arrestin 1 complex.

A Boxplot showing the distribution of the C-edge loop z-position from NTS1R-like (green), B1AR-like (blue), and intermediate snapshots (gray) in the presence (dark) or absence of PI(4,5)P2 (light). Only CG metadynamics trajectories starting from the NTS1R-like orientation were considered in this analysis. The box represents the interquartile range (IQR), with the lower and upper hinges indicating the first (Q1) and third (Q3) quartiles, respectively. The line inside the box denotes the median. The whiskers extend to the smallest and largest values within 1.5 times the IQR from the hinges. The dotted line corresponds to the position of the intracellular membrane surface. Statistical values were determined using unpaired Student’s t-tests with Bonferroni correction on the mean values of n = 20 independent simulation replicas, which are presented as black scatter (*p ≤ 0.05, **p ≤ 0.01). B Side views of representative snapshots from a CG metadynamics trajectory starting from the NTS1R orientation in the presence (dark) and absence of PI(4,5)P2 (light). The protein backbone beads are shown as surface representations with the same color scheme as in A. The intra- and extracellular membrane surfaces are depicted as black lines.