The metabotropic GABAB receptor directly interacts with the activating transcription factor 4.

G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. In the central nervous system, heteromeric metabotropic gamma-aminobutyric acid type B (GABA(B)) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element. Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABA(B) receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family. As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABA(B)R and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo. In oocyte coexpression assays short term signaling of GABA(B)Rs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivo reporter assays. Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.

Turnover rates of the AMPA-type glutamate receptor GluR1 measured by transient gene expression.

Protein turnover rates have until now been measured by pulse chasing of the target protein after labeling it with radioactive amino acids. This procedure, however, requires severe amino acid depletion followed by specific immunoprecipitation of the target protein. In the present study, we assessed the turnover rates of an AMPA-type glutamate receptor, GluR1 (or GluRA), with the conventional method and a novel one using gene transfer, and compared both of them. GluR1 cDNA was introduced into PC12 cells and cultured rat hippocampal neurons by electroporation and lipofection, respectively. Expression of its mRNA was transient and had almost ceased 2 days in PC12 cells after transfection, while the receptor protein continued to be detectable by Western blotting for a week. When the levels of the receptor protein in PC12 cells were plotted on a semi-logarithmic scale, the decay curve appeared linear after 2 days: Its decay half time (tau 1/2) was calculated as 41 h. In contrast, the pulse chase experiment revealed that the decay half time was 2-4 h in PC12 cells although cell damage was seen during this procedure. The receptor decay speed was also measured in cultured hippocampal neurons using GluR1 cDNA attached to a tag sequence. Decay of the receptor protein was monitored by Western blotting probed by an anti-tag antibody: tau 1/2 was 52 h in hippocampal neurons, similar to that in PC12. These observations suggest that the transfection procedure is more sensitive and beneficial than the conventional pulse chasing method when measuring protein turnover rates in fragile neural cells.

The dopamine D2 receptor antagonist sulpiride causes long-lasting serotonin release.

The effects of the dopamine D2 receptor antagonist sulpiride on extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) were examined by using in vivo voltammetry. Sulpiride (1 or 3 mM, 2 microl over 24 min) was administered to freely moving rats via a cannula implanted in the striatum and 5-hydroxyindole levels were measured by using a carbon fiber voltammetry electrode implanted in the ipsilateral striatum. Six to 8 h after injection, 5-hydroxyindole levels increased 3-fold, peaked 1 to 2 days post-injection, and returned to normal levels within 2 to 4 days. These effects were suppressed by pretreatment with p-chlorophenylalanine. Two days after sulpiride injection, high-performance liquid chromatography of striatal homogenates revealed that although the 5-HT concentration was unchanged, the 5-HIAA concentration was increased significantly. These results suggest that the long-lasting elevation of 5-hydroxyindole concentrations was primarily due to increased 5-HT release.

Duration of catalepsy correlates with increased intrastriatal sulpiride.

To investigate the mechanism underlying sulpiride-induced catalepsy, we simultaneously examined cataleptic behavior and the kinetics of the dopamine receptor antagonist, sulpiride of dopamine, and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), using in vivo voltammetry. After intrastriatal administration of sulpiride to freely moving rats, the levels increased, peaked at 20 min, and remained elevated for more than 3 h. Sulpiride-induced cataleptic behavior also continued for 3 h. Levels of DOPAC peaked 180 min after the injection and did not return to baseline within the experimental period. Thus, the time-course of cataleptic behavior correlated better with elevated extracellular levels of sulpiride than with that of DOPAC. These findings suggest that sulpiride induces catalepsy via a direct action.

Identification of four different forms of syntaxin 3.

cDNAs for four different forms of syntaxin 3 were cloned from a mouse brain cDNA library, and the proteins encoded by these clones were named syntaxin 3A (previous syntaxin 3), 3B, 3C and 3D. Syntaxin 3B contained a different sequence in the carboxyl terminal region from that of syntaxin 3A. The amino terminal region of syntaxin 3C contained an 18 amino acid sequence instead of a 34 amino acid sequence present in syntaxins 3A and 3B. Syntaxin 3D consisted of only 86 amino acids and lacked any putative transmembrane segments. These forms of syntaxin 3 are probably generated by alternative splicing of the primary transcript of syntaxin 3 gene. Cytoplasmic portions of syntaxins 3A and 3B but not of syntaxin 3C or 3D bound to Munc-18/n-sec1.

A complex of rab3A, SNAP-25, VAMP/synaptobrevin-2 and syntaxins in brain presynaptic terminals.

Two monoclonal antibodies (SPM-1 and SPM-2) immunoprecipitate brain N-type calcium channels. On immunoaffinity chromatography of digitonin extracts of bovine brain membranes on SPM-1- and SPM-2-Sepharose, proteins of 36 (syntaxins A and B), 28 and 19 kDa are specifically retained by both columns. Here we show that the 19 and 28 kDa bands contain VAMP/synaptobrevin-2, and rab3A/smg25A and SNAP-25, respectively. Since SPM-1 and SPM-2 recognize only syntaxins and the 28 kDa band (rab3A/sm25A and SNAP-25), respectively, the results indicate that all these proteins form a complex. Our results suggest tight linkage between the components involved in neurotransmitter release.