Figure 8. Influence of antagonists (4a,b) and agonists (5a,b) on D_2L, D_2S and D₃ receptor dimerization.

(a) Chemical structures of monovalent fluorescent antagonists (4a,b) and agonists (5a,b). (b) Representative images a single CHO cell stably expressing the D_2L receptor, labeled with 4a (38 nM), visualized by TIRF-M. Scale bar, 10 μm. Insert correspond to a higher magnification image of the area in the white box. (c) Plot of average mean square displacement (MSD ± s.d.) versus the time interval (δt) of the receptor ligand complexes that were tracked (r² = 0.99 – linear fit (blue)). (d) Intensity distribution of fluorescent spots identified over the first 10-frame time window of ligand receptor complex D_2L-4a. (e,f) Monomer/dimer ratios of D_2L, D_2S and D₃ receptors labeled with the fluorescent antagonists 4a,b and agonists 5a,b, calculated from fitted fluorescence intensity distributions of fluorescent ligand receptor complexes using a mixed Gaussian model (compare to (c) and Supplementary Table S5). (g) Average diffusion coefficients (D_lat) of the receptor complexes of the same cells analyzed in (e,f). Data in (e–g) represent mean ± s.d. n analysed cells (D_2L: n = 18 for 4a, 19 for 5a, 14 for 4b and 17 for 5b, D_2S: 13 for 4a, 12 for 5a, 19 for 4b and 12 for 5b, D₃: 12 for 4a, 13 for 5a, 15 for 4b and 10 for 5b). (e) Representative effects of the size of D_2L receptor-ligand complexes on their rates of lateral diffusion. The diffusion coefficient of D_2L-4a monomers (0.116 ± 0.030 μm² s⁻¹, n = 838 from 3 cells) is greater than that of the dimers (0.087 ± 0.037 μm² s⁻¹, n = 185 from 3 cells). This is also found for D_2L-5a monomers 0.134 ± 0.023 μm² s⁻¹ (n = 1396 from 3 cells) and dimers 0.106 ± 0.027 μm² s⁻¹ (n = 558 from 3 cells). Data represent means ± s.d. Statistical analysis was performed by an unpaired t-test (**p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001, n.s. - not significant).