Fig. 4: Structural analysis of key motifs in APJR for differential βarr1 and Gi signaling.

a Overlay of cmpd644-bound APJR structures in the Gi-coupled (green) and βarr1-coupled (blue) states. Key regions and residues involved in ligand binding and receptor activation are highlighted. The ligand is depicted in purple. b–e The conformational changes during βarr1 coupling in the key activation motifs of APJR: the toggle switch W261^6.48 and P^5.50I^3.40F^6.44 motif (b); the rearrangement of the sodium ion-binding pocket (c); the N^7.49P^7.50xxY^7.53 motif (d); and the D^3.29R^3.50Y^3.51 motif (e). Movements of APJR residues upon βarr1 coupling are indicated by red arrows. f Molecular interactions within the intracellular pocket surrounding residue Y309^7.53 of APJR in coupling to βarr1. Key interactions include a hydrophobic network formed by the L^3.43Y^5.58L^6.40 motif, along with contributions from residues L120^3.43, Y221^5.58, and L253^6.40. The left panel diagrammatically represents the spatial relationships among these interacting residues. g Functional assays were conducted to evaluate the impact of various substitutions within the L^3.43Y^5.58L^6.40 motif on βarr1 recruitment and Gi protein signaling, quantified as net BRET (% of WT Emax) and data are represented as mean ± SEM (n = 3 independent experiments). See also Supplementary Table 8 for details. h, Schematic representation of the βarr1-bound conformations of APJR. The key residues and motifs contributing to biased signaling are marked with different colors (purple for the Y271^6.58–F291^7.35 motif; orange for the sodium ion-binding pocket; green for the L^3.43Y^5.58L^6.40 motif and Y^7.53). Movements of residues are indicated by red arrows.