Fig. 2: D3R Binding behaviour of bivalent ligands.
Competition binding experiments were carried out with [³H]spiperone and membranes from HEK293T cells either monoexpressing D3R or coexpressing D3R and NTSR1. Bivalent ligands 1d (a) and 2d (b) comprising the long 88-atom spacer or 1a (c) and 2a (d) comprising the short 22-atom spacer bind to D3R with binding affinities in the high picomolar to low nanomolar range (n = 4 for 1d and n = 3 for 1a, 2a, 2d). Coexpression of an excess of NTSR1 (relative stoichiometry D3R to NTSR1 1:4) results in a 5.5- to 1460-fold increase in affinity (n = 4 for 1a; n = 5 for 1d, 2d and n = 3 for 2a). Biphasic displacement curves revealing two distinct affinities corresponding to the high- and low-affinity binding mode are resolved when experiments are carried out at a 1:1 D3R to NTSR1 stoichiometry (n = 3 for 1d and 2d). Data are presented as mean ± s.e.m. of n biologically independent experiments.
