Regulation of Akt signaling by D2 and D3 dopamine receptors in vivo

Abstract

The serine/threonine kinase Akt is a downstream target of dopamine receptor signaling that is inhibited/dephosphorylated in response to direct and indirect dopamine receptor agonists. Although pharmacological studies uncovered the involvement of D2-class dopamine receptors in Akt regulation, they did not identify the role of individual receptor subtypes in this process. Here we used knock-out mice lacking the D1, D2, D2 long, or D3 dopamine receptors as well as a D4 receptor-selective antagonist to address the function of each of these receptors in the regulation of Akt in vivo. Under basal conditions, D2, D2 long, and D3 knock-out mice display enhanced striatal Akt activation, whereas D1 knock-out mice and mice treated with the D4 receptor antagonist L745870 (3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]pyridine trihydrochloride) have phospho-Akt levels comparable with those of normal control animals. Furthermore, both amphetamine and apomorphine lose their ability to inhibit Akt in D2 knock-out mice but retain their normal effect on this signaling molecule in D1 knock-out animals. Finally, D3 knock-out mice show a reduced sensitivity of Akt-mediated signaling to dopaminergic drugs but retain the action of these drugs on Akt at high dose regimens. These results indicate that D2 receptors are essential for the inhibition of Akt by dopamine and that D3 receptors also participate in this signaling potentially by enhancing D2 receptor response. Identification of the functions of individual dopamine receptor subtypes in Akt regulation may help the development of new pharmaceutical approaches for mental disorders related to abnormal dopamine transmission such as bipolar disorder and schizophrenia.

Figure 1.

D₂ and D₃ dopamine receptors regulate Akt phosphorylation under basal conditions. A–E, Western blots (A–D) and densitometric (E–G) analysis of phospho-Thr-308 Akt levels in extracts prepared from the striatum of different drug-naive DA receptor knock-out mice (HO) (A, D₁; B, D₂; C, D₂L; D, D₃) and WT littermates. E–G, Phospho-Thr 308 Akt (E), Phospho-Ser-9 GSK3β (F) or phospho-Ser-473 Akt (G) levels in extracts prepared from the striatum of drug-naive DA receptor knock-out mice and WT littermates. Results are presented in arbitrary units normalized to phospho-protein levels observed in WT littermates. Phospho-independent antibodies directed against respective kinases were used as loading controls. n = 5–10 mice per group; data are average ± SEM. *p ≤ 0.05, ***p ≤ 0.005.

Figure 2.

D₄ receptor blockade does not affect striatal Akt phosphorylation. Western blots (A) and densitometric analysis (B) of phospho-Akt (Thr-308) levels in striatal extracts from WT or DAT-KO mice 30 min after injection of 5 mg/kg of the D₄ receptor blocker L745870. Results are presented in arbitrary units normalized to phospho-Akt levels observed in vehicle-treated mice of the same genotype. Phospho-independent antibodies directed against Akt were used as loading controls. n = 5 mice per group; data are average ± SEM.

Figure 3.

Regulation of Akt by DA drugs in D₁ and D₂ receptor knock-out mice. Phospho-Thr-308 Akt levels in extracts prepared from the striatum of WT, D₁, and D₂ DA receptor knock-out mice injected with apomorphine (3 mg/kg) or amphetamine (3 mg/kg). Representative Western blots (A, C) show results obtained from two separate striatal extracts prepared from different mice. Analyses were conducted at 60 min after injection. Results of densitometric analysis (B, C) are presented in arbitrary units normalized to vehicle-treated mice of the same genotype. n = 5–10 mice per group; data are average ± SEM. *p ≤ 0.05, **p ≤ 0.01.

Figure 4.

Regulation of Akt by DA drugs in D₃ receptor knock-out mice. A, B, Relative phospho-Akt (Thr-308) levels in extracts prepared from the striatum of WT or D₃ DA receptor knock-out mice 60 min after injection of amphetamine, 3 mg/kg (A, B) or 1 mg/kg (C, D), or of apomorphine (3 or 6 mg/kg) (E, F). Representative Western blots (A, C, E) show results obtained from two separate striatal extracts prepared from different mice. Results of densitometric analysis (B, D, F) are presented in arbitrary units normalized to vehicle-treated mice of the same genotype. n = 5–10 mice per group; data are average ± SEM. *p ≤ 0.05, **p ≤ 0.01.