Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites

Abstract

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [(35)S]GTPgammaS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.

Figure 1

D2S receptors are specifically involved in the regulation of the state of phosphorylation of TH. Striatal slices from wild-type mice (A), D2 KO mice (B), and 2DL KO mice (C) were incubated for 10 min in the presence of quinpirole (10 nM to 1 μM). The levels of phospho[Ser-40]-TH were determined as described in Materials and Methods. (Upper) Representative autoradiograms. (Lower) Summary of data expressed as means ± SEM (n = 6). The amount of phosphorylated TH is expressed as a percentage of that determined in the absence of quinpirole (control). *, P < 0.01 and **, P < 0.001 vs. respective control group; one-way ANOVA followed by Dunnett's test.

Figure 2

D2S receptors are specifically involved in the regulation of TH activity. Striatal slices from wild-type mice (A), D2 KO mice (B), and D2L KO mice (C) were incubated for 5 min in the presence of quinpirole (1 μM) and for 15 min in the presence of quinpirole plus m-hydroxybenzylhydrazine (100 μM). The samples were then sonicated in 0.1 M perchloric acid and centrifuged. The levels of DOPA recovered in the supernatant were determined by HPLC and normalized according to protein content (see Materials and Methods). Data are expressed as means ± SEM (n = 16). The amount of dopa is expressed as a percentage of that determined in the absence of quinpirole, and 100% corresponds to 25.4 pmol/min per mg of protein. *, P < 0.05 vs. respective control group (Student's t test).

Figure 3

D2L receptors are specifically involved in the regulation of the state of phosphorylation of DARPP-32. Striatal slices from wild-type mice (A), D2 KO mice (B), and D2L KO mice (C) were incubated for 2 min in the presence of quinpirole (1 μM) and then for 8 min in the presence of quinpirole plus SKF81297 (10 μM). The levels of phospho[Thr-34]-DARPP-32 were determined as described in Materials and Methods. (Upper) Representative autoradiograms. (Lower) Summary of data expressed as means ± SEM (n = 6). The amount of phosphorylated DARPP-32 is expressed as a percentage of that determined in the absence of drugs (control). **, P < 0.01 and ***, P < 0.001 vs. control; one-way ANOVA followed by Newman-Keuls test. †, The effect of SKF81297 was significantly reduced by quinpirole (P < 0.01; two-way ANOVA).