Figure 2. Differential effects of PG01042, PG01037, and VK4-116 on β-arrestin recruitment in HEK-293T cells transfected with D₃R and D₁R.

a-c. Results from β-arrestin recruitment experiments in HEK-293T cells transfected with β-arrestin-1-Rluc cDNA (0.5 μg), D₃R-YFP cDNA (1 μg cDNA) with or without D₁R cDNA (1.5 μg cDNA) (D₁R-D₃R cells and D₃R cells, respectively). In a-b, cells are treated for 10 min with the D₂-like receptor agonist pramipexole (D3Rago; 30 nM) or the D₃R ligands PG01042, PG01037 and VK4-116 (10 or 100 nM). In c, cells are pre-treated or not with D₁R TM6 or TM7 peptides (4 μM for 4 h) and with VK4-116 (10 or100 nM) for 15 min before the D₁R agonist SKF81297 (30 nM; D1Rago). Coelenterazine H (5 μM) was added before pramipexole or the selective D₃R ligands for 7 minutes and β-arrestin-1 recruitment was measured by BRET (see Material and Methods). Values are mean ± S.D. (n = 8). *, *** and ****: p < 0.05, p < 0.001 and p < 0.0001 versus basal, respectively (one-way ANOVA followed by Dunnett’s post hoc comparisons). d. Evolution of the C_α atoms (spheres) of Y32^1.35 in TM 1, L89^2.64 in TM 2, I101^3.23 in TM 3, F170^4.62 in TM 4, F188^5.38 in TM 5, H354^6.60 in TM 6, and P362^7.32 in TM 7 during three replicas of unbiased 1 μs MD simulations of the D₃R monomer (gray) and the D₁R-D₃R heteromer (blue) with no ligand bound. Contour plots of the distances between Y32^1.35 in TM 1 and F188^5.38 in TM 5 (distance TM1-TM5) and between L89^2.64 in TM 2 and H354^6.60 in TM 6 (distance TM2-TM6) during the MD simulations. Distributions of these distances are shown in the axes. e. Evolution of I183^ECL2 (spheres), and contour plots and distributions of X,Z coordinates corresponding to the C_b atom of I183^ECL2 during MD simulations. Black arrows represent the movements of D₃R in the D₁R-D₃R heteromer (blue) relative to the D₃R protomer (gray). f. Detailed view of I183^ECL2 of D₃R in the D₁R-D₃R heteromer during MD simulations with no ligand bound (blue) and PG01042 (green), PG01037 (orange) and VK4-116 (red) bound to D₃R. Distributions of the Z coordinate corresponding to the C_α atom of I183^ECL2 during MD simulations. The ethyl group of VK4-116 that triggers the upward movement of I183^ECL2 is highlighted. g. Detailed views of I183^ECL2, F188^5.38, S192^5.42, and T115^3.37 of D₃R in the D₁R-D₃R heteromer during MD simulations with no ligand bound (blue) and PG01042 (green), PG01037 (orange) and VK4-116 (red) bound to D₃R. Frequency contacts (%) between side-chain residues, color-coded according to the ligand bound to D₃R, as calculated with the GetContacts software. Black arrows represent the movements of these side chains of D₃R relative to the unliganded D₃R (blue). The xy plane is as defined by the Orientations of Proteins in Membranes (OPM) [73].