Transient and rapid activation of Akt/GSK-3ß and mTORC1 signaling by D3 dopamine receptor stimulation in dorsal striatum and nucleus accumbens.

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time-course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK-3ß in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein-S6 (Ser240/244), and eukaryotic initiation factor-4E binding protein-1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock-out mice lacking D3R. In drd1a-EGFP transgenic mice, quinelorane activated Akt/GSK-3ß in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK-3ß pathway in the two populations of medium-size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.

Signaling pathways leading to phosphorylation of Akt and GSK-3ß by activation of cloned human and rat cerebral D2and D3 receptors.

Although dopamine (DA) regulates the serine/threonine kinase Akt and its downstream substrate glycogen synthase kinase-3ß (GSK-3ß), the direct influence of dopaminergic receptors remains poorly characterized. Short-term incubation of Chinese hamster ovary (CHO)-expressed human (h)D(2L) and hD3) receptors with DA (maximal effect, 5-10 min) phosphorylated Akt (Thr308 and Ser473) and GSK-3ß (Ser9), actions blocked by the selective D2 and D3 antagonists, 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) and (3aR,9bS)-N[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl] (4-phenyl)benzamide (S33084), respectively. Similar findings were acquired with the specific D2/D3 receptor agonist quinelorane, which also enhanced (10 min after administration) levels of p-Akt and p-GSK-3ß in rat nucleus accumbens, an action blocked by the D2/D3 receptor antagonist raclopride. Akt and GSK-3ß phosphorylation mediated via CHO-expressed hD(2L) and hD3 receptors was prevented by pertussis toxin and by inhibitors of insulin-like growth factor-1 receptors as well as phosphatidylinositol 3-kinase and Src. Likewise, chelation of intracellular Ca²+ and interference with an "atypical" phorbol ester-insensitive protein kinase C (PKC) abolished recruitment of Akt and GSK-3ß. Inactivation of PKCµ blocked Akt and GSK-3ß phosphorylation at hD(2L) receptors. However, blockade of conventional PKC isoforms attenuated the actions of DA at hD3 receptors only. Furthermore, phospholipase C (PLC), calmodulin, and Akt inhibitors abolished DA-induced GSK-3ß phosphorylation by hD3 receptors, whereas phosphorylation by hD(2L) receptors partially involved calmodulin, Akt, and extracellular signal-regulated kinase (ERK) 1/2. In conclusion, at both hD(2L) and hD3 receptors, DA elicited a G(i/o)- and Ca²+/calmodulin-dependent phosphorylation of Akt and GSK-3ß via transactivation of insulin-like growth factor 1 receptor. However, significant differences were seen regarding the involvement of PLC, calmodulin, and ERK1/2.

Unclassified variants identified in BRCA1 exon 11: Consequences on splicing.

Numerous mutations identified in breast/ovarian cancer families occur in splice sites of the BRCA1 gene. Splicing can also be disrupted by mutations occurring in exonic splicing enhancer (ESE) sequences. It is important to identify those mutations among the large number of nontruncating sequence variants that are identified during molecular diagnosis, as this could help to classify some of them as cancer predisposing. Several software programs have been designed to identify ESEs and can therefore be used to predict the outcome of genetic variation. However, it is not known whether these predictions are relevant in the case of BRCA1 exon 11 (3.4 kb). In this study, we assessed the consequences on splicing of 108 exon 11 variants identified in French breast/ovarian cancer families, most of them predicted to alter putative ESEs, and of nine variants located in the exon 11 alternative donor splice site. We employed a BRCA1 minigene consisting of exon 10 to 12, into which we introduced separately each of the variants to be tested. RNA was analyzed by RT-PCR after transient transfection of the resulting minigenes. None of the tested variants was found to dramatically alter splicing through disruption of an ESE. However, we identified several variants in the alternative donor splice site that are likely to be of biological significance as they appear to favor the expression of BRCA1-Delta11b over that of the full-length transcript. The results of this study will be of value to classify BRCA1 exon 11 variants of unknown significance. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.