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.