No abstract

SynGAP is a Ras GTPase activating protein present at the postsynaptic density (PSD) in quantities matching those of the core scaffold protein PSD-95. SynGAP is reported to inhibit synaptic accumulation of AMPA receptors. Here, we characterize by immunogold electron microscopy the distribution of SynGAP at the PSD under basal and depolarizing conditions in rat hippocampal neuronal cultures. The PSD core, extending up to 40 nm from the postsynaptic membrane, typically shows label for SynGAP, while half of the synapses exhibit additional labeling in a zone 40–120 nm from the postsynaptic membrane. Upon depolarization with high K+, labeling for SynGAP significantly decreases at the core of the PSD and concomitantly increases at the 40–120 nm zone. Under the same depolarization conditions, label for PSD-95, the presumed binding partner of SynGAP, does not change its localization at the PSD. Depolarization-induced redistribution of SynGAP is reversible and also occurs upon application of NMDA. Activity-induced movement of SynGAP could vacate sites in the PSD core allowing other elements to bind to these sites, such as transmembrane AMPA receptor regulatory proteins, and simultaneously facilitate access of SynGAP to CaMKII and Ras, elements of a regulatory cascade.

Dopamine receptor genes are under complex transcription control, determining their unique regional distribution in the brain. We describe here a zinc finger type transcription factor, designated dopamine receptor regulating factor (DRRF), which binds to GC and GT boxes in the D 1A and D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Consequently, DRRF can modulate the activity of these dopamine receptor promoters. Highest DRRF mRNA levels are found in brain with a specific regional distribution including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala, and frontal cortex. Many of these brain regions also express abundant levels of various dopamine receptors. In vivo, DRRF itself can be regulated by manipulations of dopaminergic transmission. Mice treated with drugs that increase extracellular striatal dopamine levels (cocaine), block dopamine receptors (haloperidol), or destroy dopamine terminals (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) show significant alterations in DRRF mRNA. The latter observations provide a basis for dopamine receptor regulation after these manipulations. We conclude that DRRF is important for modulating dopaminergic transmission in the brain.T ranscriptional regulation in eukaryotes is governed by the coordinated action of regulatory factors that bind to specific DNA elements. One class of these factors comprises zinc finger proteins of which Sp1 is a prototypical example, having three Cys-2-His-2 zinc finger motifs (1). Other family members, Sp2, Sp3, and Sp4, with similar structural and functional features also have been identified (2, 3). Sp1, Sp3, and Sp4 bind to the same recognition sequence (GC boxes) with similar affinities (3, 4). While Sp1 and Sp4 generally act as transcription activators, Sp3 can act as repressor or activator (5). Sp2, on the other hand, has a DNA-binding specificity different (2) from that of Sp1, Sp3, or Sp4. Several additional factors with the same zinc finger motif as Sp1 have been cloned and found to bind to the GC box sequence (6-8).Central dopaminergic neurotransmission is crucial for normal brain function, and its aberrations are intricately involved in several neuropsychiatric disorders. The specific biological effects of dopamine are determined at least in part by the complex spatial and temporal regulation of genes encoding its receptors. and D 2 genes have revealed a delicate balance among several nuclear factors that tightly regulate expression of these genes (10-12). For example, the D 2 gene promoter is under strong negative control (13). One of its silencing elements (nucleotides Ϫ116 to Ϫ76), which consists of an Sp1 consensus sequence (GC box) and three TGGG repeats (GT box), interacts with Sp1, Sp3 (10), and an unidentified factor (13). In the present investigation, we characterized the nature and function of this nuclear protein, which regulates the expression of dopamine receptor genes.

SynGAP, a protein abundant at the postsynaptic density (PSD) of glutamatergic neurons, is known to modulate synaptic strength by regulating the incorporation of AMPA receptors at the synapse. Two isoforms of SynGAP, α1 and α2, which differ in their C-termini, have opposing effects on synaptic strength. In the present study, antibodies specific for SynGAP-α1 and SynGAP-α2 are used to compare the distribution patterns of the two isoforms at the postsynaptic density (PSD) under basal and excitatory conditions. Western immunoblotting shows enrichment of both isoforms in PSD fractions isolated from adult rat brain. Immunogold electron microscopy of rat hippocampal neuronal cultures shows similar distribution of both isoforms at the PSD, with a high density of immunolabel within the PSD core under basal conditions. Application of NMDA promotes movement of SynGAP-α1 as well as SynGAP-α2 out of the PSD core. In isolated PSDs both isoforms of SynGAP can be phosphorylated upon activation of the endogenous CaMKII. Application of tatCN21, a cell-penetrating inhibitor of CaMKII, to hippocampal neuronal cultures blocks NMDA-induced redistribution of SynGAP-α1 and SynGAP-α2. Thus CaMKII activation promotes the removal of two distinct C-terminal SynGAP variants from the PSD.