Unraveling the molecular architecture of a G protein-coupled receptor/β-arrestin/Erk module complex

Abstract

β-arrestins serve as signaling scaffolds downstream of G protein-coupled receptors, and thus play a crucial role in a plethora of cellular processes. Although it is largely accepted that the ability of β-arrestins to interact simultaneously with many protein partners is key in G protein-independent signaling of GPCRs, only the precise knowledge of these multimeric arrangements will allow a full understanding of the dynamics of these interactions and their functional consequences. However, current experimental procedures for the determination of the three-dimensional structures of protein-protein complexes are not well adapted to analyze these short-lived, multi-component assemblies. We propose a model of the receptor/β-arrestin/Erk1 signaling module, which is consistent with most of the available experimental data. Moreover, for the β-arrestin/Raf1 and the β-arrestin/ERK interactions, we have used the model to design interfering peptides and shown that they compete with both partners, hereby demonstrating the validity of the predicted interaction regions.

Figure 1. Similarity of the top 20-ranked conformations.

Top: the 20 best ranked conformations of the β-arrestin 1/Raf1 complex have been aligned on the β-arrestin (left, β-arrestin shown in surface, Raf in ribbon) and Raf1 (right, Raf shown in surface, β-arrestin in ribbon) partners. Bottom: the residues of β-arrestin (left) and Raf (right) are colored as a function of their presence in the 20 best ranked conformations, from dark red (present in more than 15 out of 20 conformations) to yellow (present in 1 to 4 conformation), grey residues are never in the interface in these 20 conformations. These superimpositions clearly show that the interaction regions on both molecules are highly similar across these 20 conformations.

Figure 2. Docking models.

The initial step is the docking of each partner (receptor, c-Src, c-Raf, MEK1 and ERK1) on the β-arrestin. During this initial step, the solutions obtained for the β-arrestin/ERK complex were not satisfying since they were not compatible with MEK interaction. Since ERK and MEK need to interact within the complex, resulting in ERK activation, we docked ERK on the predicted β-arrestin/MEK complex. The different models were then reassembled, and it should be noted that no other docking was necessary at this step since all the heterodimer models were compatible with each other.

Figure 3. Model of the β-arrestin-1/ERK signaling module.

Color code: β-arrestin in grey, c-Src in blue, Raf1 in green, Mek1 in yellow, Erk1 in dark red; experimentally identified residues are in red, other potentially important residues are in orange, phosphorylation sites are in dark blue. A: c-Src/β-arrestin interaction, SH3 domain and its interacting peptide of PDB 1AD5 in violet. B: Raf1/β-arrestin interaction. C: Mek1/β-arrestin interaction. D: Erk1/β-arrestin interaction. E: Mek1/Erk1 interaction. F: Raf1/Mek1 interaction, the region 270-307 figured by a red dashed line is unfolded.

Figure 4. Complete complex model. The model can accommodate the presence of Clathrin and the SH2 domain of c-Src.

The color code for the main complex is the same as in Fig. 3. Possible geometry of the complex including the β-arrestin/Erk1 module, including SH2 domain of c-Src (in orange) and clathrin (in marine blue).

Figure 5. Experimental validation of the predicted β-arrestin-Raf1 and β-arrestin-ERK2 interaction sites.

A) Equal amounts of GST-Raf1-RBD fusion protein were incubated with 25 ng of β-arrestin 1 and with or without 0.5mM of the indicated peptide. B) Representative blot for β-arrestin-Raf1 interaction site sequentially probed with anti-β-arrestin and anti-GST antibodies. B) Equal amounts of GST-ERK2 fusion protein were incubated with 25 ng of β-arrestin 1 and with or without 0.5mM of the indicated peptide. Representative blot for β-arrestin-ERK2 interaction site sequentially probed with anti-β-arrestin and anti-ERK antibodies. Immunoblots were quantified using Odyssey software and data were expressed as the ratio of β-arrestin 1 over GST-Raf1-RBD or GST-ERK respectively. The maximum response measured with the Control peptide was chosen as 100%. Data represent means ± SEM from three independent experiments. *,**: p < 0.05 specific peptides versus control peptides.

Figure 6. Model of the receptor/β-arrestin-1/ERK signaling complex, focusing on the Erk active site.

Color code: β-arrestin in grey, c-Src in blue, Raf1 in green, Mek1 in yellow, Erk1 in dark red (including ATP shown as spheres), receptor in pink, V2R peptide in spheres.