Ptic plasticity. The dopamine- and cAMPregulated phosphoprotein of 32 kDa (DARPP32) is usually a key component inside the integration of dopaminergic and glutamatergic signaling and is very enriched in striatal tissue [33?5]. To evaluate differences in RNA expression data from microarray (no regulation) and qPCR (down-regulation in RGS9-deficient mice) DARPP32 protein levels were determined in Western blot analysis revealing no important difference between each mouse strains (Fig. 2a). DARPP32 activity is regulated by phosphorylation and possesses four functional relevant phosphorylation internet sites: Thr34, Thr75, Ser102 and Ser137 [36]. Applying phosphorylation-specific antibodies, we quantified relative phosphorylation state levels from the most important phosphorylated types of DARPP32, pDARPP32-Thr34 and pDARPP32-Thr75. Interestingly, phosphorylation of DARPP32 was substantially elevated at Thr34, but not at Thr75 (Fig. 2b). The extracellular signal-regulated kinases 1 and two (ERK1 and ERK2) are downstream effectors of dopamine signaling thatFigure 1. Intracellular localization and functional interaction of chosen signaling components expressed in striatum. Transcripts of signaling elements which might be drastically downregulated in striata of RGS9-deficient mice are shown in red. The complete notations of the transcripts are offered in Table 1 and Table S4 (in File S1). doi:ten.1371/journal.pone.0092605.gPLOS 1 | plosone.orgAdaptive Gene Regulation in RGS9-Deficient MiceFigure two. Striatal DARPP32-Thr34 phosphorylation is enhanced in RGS9-deficient mice. (a) Total DARPP32 from striatal homogenates of 3-month-old wt and RGS9-deficient mice was measured by Western blot quantification and normalized to actin. (b) DARPP32 phosphorylation at Thr34 and Thr75 is provided relative to total DARPP32. Representative Western blots are shown, quantification was performed with n = 6? per genotype. doi:10.1371/journal.pone.0092605.gregulate excitability and glutamatergic transmission in striatal sMSN [37]. The ERK activities are tightly regulated by phosphorylation.Price of 1250731-69-1 Despite the fact that ERK1 and ERK2 mRNA levels (Table 1, Table S4 in File S1) and protein concentrations (Fig.3-Formyl-1H-indazole-5-carboxylic acid supplier 3a) have been reduced in RGS9-deficient mice, ERK1/2 phosphorylation was strongly improved (Fig.PMID:33556578 3b) indicating sustained activation. Control of glutamatergic transmission is also dependent on Ca2+ signaling. Hence, we investigated concentration of Ca2+-dependent proteins, e.g. CaMK IIb and CaMK IIc (Fig. 4a, b). Moreover, protein concentration of GluR2, one of the most abundant striatal AMPA receptor subunit that accounts for Ca2+ impermeability of AMPA receptors [38], was determined (Fig. 4c). In agreement with gene expression information, protein concentration of CaMK IIc and GluR2 were drastically decreased in RGS9-deficient mice. Western blot analysis also revealed a lowered concentration of CaMK IIb and phospho-CaMK IIb in RGS9-deficient mice but tendencies were not substantial.Functional Evaluation in Acute Striatal SamplesThe hyperphosphorylation of DARPP32-Thr34, that was observed in RGS9-deficient mice (Fig. 2b), implies enhanced basal PKA activity or an imbalance within the activities with the opposing D1R and D2R signaling [36] (Fig. 1). To dissect pathways involved hyperphosphorylation of DARPP32 we assessed basal and stimulated adenylyl cyclase activity in acute striatal samples of wt and RGS9-deficient mice by a label-free AlphaScreen-based cAMP accumulation assay (Fig. 5). Below basal conditions and forskolin stimulation.