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. 2013 Mar 11;4(6):940–951. doi: 10.1021/cn3002202

Figure 6.

Figure 6

PD128907- and quinpirole-induced tolerance is not elicited in the D3 D187A mutant receptor. Tolerance property defined as the ratio of second/first agonist-induced GIRK response was determined for quinpirole (A) and PD128907 (B) in AtT-20 cells transiently transfected with either wild type D2, D3, D3 C147K, D3 D187A, or D3 C147K+D187A mutant receptors. The tolerance induced by quinpirole (A) in wild type D3 receptor is significantly less than in the D2 receptor and D3 C147K, D3 D187A and D3C147K+D187A mutant receptors; *P < 0.01, ANOVA (Holm’s-Sidak post hoc test). However, these D3 mutant receptors exhibit quinpirole-induced tolerance when compared to wild type D2 receptor; **P < 0.05, ANOVA (Holm’s-Sidak post hoc test). The tolerance induced by PD128907 (B) in wild type D3 receptor is significantly attenuated only in the D3 D187A mutant receptors; *P < 0.01, ANOVA (Holm’s-Sidak post hoc test). However, the D3 D187A mutant receptor exhibits PD128907-induced tolerance when compared to wild type D2 receptor; **P < 0.05, ANOVA (Holm’s-Sidak post hoc test). The bars represent the mean values ± SEM (n = 10–12 cells). (C) Comparison of PD128907-induced GIRK dose response curves obtained from whole cell voltage clamp recording of AtT-20 cells expressing either wild type D3 receptor (open circle) or D3 D187A mutant receptor (filled square). The PD128907-induced GIRK currents (pA) were normalized for cell size (cell capacitance, pF). The data points were fit with a four-parameter Hill equation. The bars represent the mean values ± SEM (n = 6–8 cells). (D) Cumulative data comparing the mean tolerance of MAPK activation in AtT-20 cells transfected with either wild type human D3 or D3 D187A mutant receptors. Cells were pretreated for 1 min with either vehicle (SES) or 200 nM PD128907, washed 6 times with SES (3 min per wash), and subsequently treated with vehicle (SES) or 200 nM PD128907 for 5 min. The levels of PD128907-induced phosphorylated MAPK proteins were normalized to the levels of total MAPK proteins. Tolerance was determined by dividing the 200 nM PD128907-induced phosphorylation of ERK1/2 at 5 min in cells pretreated with 200 nM quinpirole by the 200 nM PD128907-induced phosphorylation of ERK1/2 at 5 min in cells pretreated with control SES. The wild type D3 receptor expressing cells exhibits significantly reduced response following PD128907 pretreatment compared to D3 D187A mutant expressing cells (*P < 0.05, Student’s t test). The experiments were repeated three independent times.