Figure 6
Influence of monovalent (1a,b), bivalent (2a,b) and bivalent control (3a,b) dopamine D2 receptor antagonists on receptor dimerization.
(a) Chemical structures of monovalent ligands (1a–c), bivalent ligands (2a–c), and control ligands (3a,b). (b) Monomer/dimer ratios calculated from fitted fluorescence intensity distributions of Alexa546-labeled SNAP-D2L receptors incubated with monovalent (1a,b), bivalent (2a,b), control (3a,b) ligands using a mixed Gaussian model (Supplementary Table S2, Supplementary Fig. S6a). Data represent mean ± s.d. of n analysed cells (n = 16 for 1a, 8 for 1b, 8 for 2a, 16 for 2b, 6 for 3a and 8 for 3b. (c) Average diffusion coefficients (Dlat) of the corresponding ligand-SNAP-D2L receptor complexes of the same analyzed cells in (b). Data in (b,c) represent mean ± s.d., Statistical analysis was performed by an unpaired t-test (**p-value < 0.01, ****p-value < 0.0001) and showed that the receptor mobility is negatively correlated with the size of the receptor complexes. (d–g) Comparison of the apparent lifetimes of particle colocalization of the monomeric SNAP-CD86 and dimeric SNAP-CD28 control proteins proteins (e and f respectively) and the SNAP-D2L receptor in the absence and presence of the monovalent ligand 1a or bivalent ligand 2a (g, h, and f respectively). (d) Representative intensity profile of one trajectory which showed intensity doubling from the beginning of the particle tracking followed by one step intensity change which was used to calculate the colocalization time of two particles. (e–i) The apparent lifetime of particle colocalizations (τ; 95 confidence interval) was calculated by fitting colocalization time data with a one-phase exponential decay function. 120 trajectories like those shown in a were analyzed from 8 different cells in (e), 4 in (f), 6 in (g), 6 in (h) and 4 in (i), respectively.
