Interaction of nuclear factors from young and old rat brain regions with regulatory sequences of the D2 dopamine receptor gene promoter.

Alterations in the number or functional state of D2 dopamine receptors have been implicated in the decreased motor abilities associated with normal aging, Parkinson's disease and other neurodegenerative diseases. Previous work has demonstrated a substantial decrease in D2 receptor-containing neurons, receptor proteins, steady-state mRNA levels, and the rate of mRNA synthesis with age in the rat striatum in particular and in mammalian brains in general. These observations suggest that one key area of regulatory control is at the level of transcriptional initiation and/or elongation. In the present study gel mobility shift experiments were used to assess the interaction of nuclear proteins from different rat brain regions with DNA containing putative DNA regulatory sites of the transcriptionally active rat D2 receptor gene promoter. Oligonucleotides containing either of the two SP1 binding sites immediately upstream of the primary transcriptional start site were bound by proteins found in nuclear extracts obtained from rat striatum, hippocampus, cortex, and cerebellum. Extracts from striatum and hippocampus formed predominantly low molecular weight complexes which do not contain SP1, as well as a small amount of high molecular weight complexes which may contain SP1 or an SP1-related protein. Cerebellar extracts formed two similar sets of complexes, but they were formed in roughly equal amounts. Extracts from cortex produced a more involved pattern of complexes, but still formed both high molecular weight complexes which contain SP1 and low molecular weight complexes which do not contain SP1. There were differences in the gel mobility as well as the relative amounts of complexes formed with the two SP1-specific oligonucleotides among different brain regions. With respect to possible age-related changes in transcription of the D2 dopamine receptor gene, there appeared to be no statistically significant difference in the DNA-protein complexes formed with striatal nuclear proteins from a population of young rats versus a population of old rats.

The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters.

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.

The mobile group I intron 3 alpha of the yeast mitochondrial COXI gene encodes a 35-kDa processed protein that is an endonuclease but not a maturase.

Three mitochondrial mutants were characterized that block the splicing of aI3 alpha, a mobile group I intron of the COXI gene of yeast mtDNA. Mutant C1085 alters helical structures known to be important for splicing of group I introns. M44 and C1072 are point mutants in exon 3 that block correct splicing but allow some splicing at cryptic 5'-splice sites. M44 alters the P1 helix needed for 5'-splice site definition, while the mutation in C1072 is a new kind of mutation because it is located upstream of the exon sequence involved in the P1 helix. All three mutants accumulate novel proteins of 35 and 44 kDa (p35 and p44, respectively) detected both by labeling of mitochondrial translation products and by Western blotting. Partial protease digestions indicate that p44 and p35 are closely related, probably as precursor and processed protein. The level of the intron-encoded endonuclease activity, I-SceIII, is elevated approximately 10-fold in the mutants. Partial purification of I-SceIII from the mutants showed that most, if not all, of the activity is associated with p35. Finally, because aI3 alpha splices accurately in a petite mutant, we conclude that aI3 alpha splicing does not depend on a mtDNA-encoded maturase.

DNA binding and regulatory effects of transcription factors SP1 and USF at the rat amyloid precursor protein gene promoter.

Two DNA elements which we have termed SAA and GAG have been shown to control expression of the rat amyloid precursor protein (APP) gene, and the region containing the SAA element has been shown to interact with nuclear proteins [Hoffman and Chernak (1994) Biochem. Biophys. Res. Commun. 201, 610-617]. In this report we study DNA sequences and proteins which influence the activity of the SAA element. An oligonucleotide containing the SAA element is specifically bound by nuclear proteins derived from rat PC12 cells, consistently forming four complexes designated C25, C30, C35 and C40 in electrophoretic mobility shift assays (EMSAs). We demonstrate that the C25, C30 and C40 complexes involve the binding of nuclear proteins to an SP1 consensus sequence located within the SAA element and that the C25 complex contains a protein antigenically related to the human SP1 protein. We establish further that the C35 complex requires a USF recognition site located within the SAA element and contains a protein antigenically related to the human upstream stimulatory factor (USF) protein. Using APP promoter/luciferase reporter gene constructs, we demonstrate that both the SP1 and the USF sites can play a role in the transcriptional activity of the SAA element. Finally, we show that complexes similar to the C25, C30 and C35 complexes are formed by rat cortex nuclear extracts and the SAA element in EMSA experiments, suggesting the relevance of our in vitro observations to the in vivo functioning of the rat APP promoter.

Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes.

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.

The rat amyloid precursor protein promoter contains two DNA regulatory elements which influence high level gene expression.

We have investigated the transcriptional activity and regulatory elements of the rat amyloid precursor protein promoter. A DNA fragment containing 375 base pairs upstream of the start codon drives transcription in rat PC12 cells at a level greater than five-fold that of the SV40 promoter. This fragment contains a predominant transcription start point and several additional start points which are similar to those found in the human promoter. The strong promoter activity appears to be dependent upon two small DNA regulatory elements. Deletion of one element at positions -260 through -248 reduces activity by 85%. This is the first report of a positive regulatory element at this location. Deletion of a second element at positions -223 through -192 reduces activity by 30%. Gel mobility shift assays with nuclear extracts from whole rat brain suggest that nuclear proteins interact directly with the second element but not with the first one.