Fig. 1: Interaction between different activation statuses of β-arrestin1 (βarr1) and Gα.

a Schematic diagram of G protein-coupled receptor (GPCR) signal transduction. Agonist-induced receptor activation initiates two major signaling pathways. On one hand, the activated receptor recruits and activates heterotrimeric G proteins, triggering guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange on the Gα subunit, followed by Gα and Gβγ dissociation, leading to distinct G protein-mediated downstream signaling. On the other hand, the activated receptor recruits GPCR kinase (GRK), which phosphorylates the receptor. Subsequently, the phosphorylated receptor promotes βarr recruitment and activation, resulting in receptor desensitization, internalization, or βarr-mediated signaling. b Structural features of basal βarr1 (gray, PDB: 1G4M). The auto-inhibitory C-terminal tail is highlighted in red. The polar core is indicated by an orange circle. The three-element interaction comprising β-strand XX (red), the αI, and β-strand I (blue) is marked in a blue circle, with hydrophobic side chains shown in yellow. c Structural superimposition of basal βarr1 (gray, PDB: 1G4M) and V2Rpp-bound active βarr1 (wheat, PDB: 4JQI). V2Rpp is shown as a green ribbon. Structural rearrangement upon activation, including loop rearrangement and C-terminal tail release, is highlighted in dark orange. Activation-induced interdomain rotation is shown as a black curved arrow. d–o Microscale thermophoresis (MST)-based affinity measurement between βarr1 and Gαs or Gαi1 in different activation states. The difference in normalized fluorescence ΔFnorm[‰] of labeled βarr1 is plotted as a function of the concentration of Gαs or Gαi1. Data points represent the mean values ± SEM from 3–4 independent experiments. Curve fitting was performed using GraphPad Prism 10. The dissociation constant is obtained using the Kd mode of MO.Affinity Analysis software (v2.3) and presented as the best-fit Kd ± Kd confidence, corresponding to a 68% confidence level (~1 standard deviation). MST experiments were performed at 20% excitation power and medium MST power.