Interest in the functions of proteins coded for by ras oncogenes has been given impetus by the recent discovery of mutational alterations which activate ras oncogenes in a number of human cancers (2,12,24,25). ras oncogenes were originally recognized as the oncogenes of two rat-derived viruses, the Harvey (Ha) and Kirsten (Ki) strains of murine sarcoma virus (MuSV) (15,16). By the application of molecular cloning techniques, it was recognized that the ras oncogenes of Ha-and Ki-MuSV were divergent members of a gene family present in a wide variety of vertebrates (5, 6). Recently, a third gene member of the ras gene family was recognized by identifying an activated oncogene in a human neuroblastoma (21). ras oncogenes, whether of viral or cellular origin, code for a protein designated p21 because its molecular weight is approximately 21,000 (19). Little is known about the function of the p21 coded for by any ras gene or ras oncogene. p21 molecules have a high affinity for guanine nucleotides and can be assayed by a guanine nucleotide binding assay (14). The p21 coded for by two ras genes, v-Ha-ras and v-Kiras, also can undergo autophosphorylation, using GTP as a substrate (18). Several investigators have studied the expression of ras genes in embryonic development in mice and in specialized mammalian cells in an attempt to gain clues about the normal function of the protein products of ras genes (9,13,17 (-600 Ci/mmol) was added directly to the culture to a final concentration of 25 ,uCi/ml. Cultures were labeled for 2.0 h at 31°C. Cell extracts were prepared as indicated in the legend to Fig. 1.Source of antisera. Three monoclonal antibodies were used (7). The three sera were prepared from rat myeloma cells grown in cell culture, YAG 172, YAG 238, and YAG 259. YAG 172 and YAG 238 detect the p21 coded for by Ha-rasrelated murine genes and YAG 259 detects the p21 coded for by Ha-ras genes, Ki-ras genes, and N-ras genes (7,22). To prepare purified YAG 259 antibody, the rat myeloma cells were grown in a serum-free medium, and the antibody molecules were purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography (7). Purified antibody 259 was iodinated by the chloramine-T method to a specific activity of 7 x 106 cpm/,ug. A polyclonal rat serum prepared from several rats carrying rat tumor cells induced by v-Ha-ras has been previously described (17,19

The v-ras oncogene of Harvey murine sarcoma virus encodes a 21,000-dalton protein, p21, which mediates transformationH produced by that virus. Previous work has shown that both p21V-/t6I. and the cellular homolog p2l"cr appear to bind guanine nucleotides. We report here the expression in Escherichia coli of v-rasH to produce a biochemically active p21 fusion protein which retains both guanine nucleotide binding and autophosphorylating activity. Furthermore, direct interaction of this protein with GTP is unequivocally demonstrated by photoaffinity labeling it with bo-32P]GTP.

No abstract

We have recently documented that phosphorylation of the GluR1 subunit of alpha-amino-3-hydroxy-5-methylisoxazole-propionate (AMPA) glutamate receptors is influenced by calcium-independent forms of phospholipase A(2) (iPLA(2)) activity in the brain. Given the importance of GluR1 subunit phosphorylation in the control of AMPA receptor delivery to synaptic membranes, we tested the influence of iPLA(2) activity on AMPA receptor distribution between neuronal compartments, using organotypic cultured hippocampal slices. In agreement with earlier reports, the iPLA(2) inhibitor bromoenol lactone (BEL) markedly enhanced the phosphorylation of the GluR1 subunit at both Ser831 and Ser845 residues. GluR1 subunit phosphorylation levels were selectively increased by (R)-BEL, an enantio-selective inhibitor of iPLA(2)gamma, but not by (S)-BEL, an iPLA(2)beta inhibitor. The iPLA(2)gamma inhibitor R-BEL also promoted the insertion of new GluR1 subunits into synaptic membranes and exacerbated AMPA-mediated cell death in the CA1 region of the hippocampus. The latter effect was selectively abolished by IEM 1460 and philanthotoxin-433, two antagonists specific for AMPA receptors lacking GluR2 subunits. These results provide evidence that iPLA(2)gamma-related regulation of AMPA receptor GluR1 subunit phosphorylation could represent an important mechanism modulating hippocampal cell death induced by AMPA receptor overstimulation.

18 Dopamine (DA) is the predominant catecholamine neurotransmitter in the mammalian brain, where it controls various functions: endocrine regulation, food intake, cognition, emotion, addiction and locomotion.1 This catecholamine also plays multiple roles in the periphery as a modulator of the cardiovascular system, catecholamine release, hormone secretion, vascular tone, renal function, and gastrointestinal motility. These physiological effects are all mediated through DA receptors (D1-D5) which are members of the large family of G protein-coupled receptors.1,2 DA receptors can be subdivided into D1 and D2 classes: the D1-like receptor subfamily which includes D1 and D5 receptors, and the D2-like subfamily, which includes D2, D3 and D4 receptors. D1 and D2 receptors constitute major DA receptor subtypes that are expressed differentially in various regions of the human brain. The intensity and duration of these effects are primarily regulated by the rapid reuptake of DA by the plasma membrane DA transporter (DAT) back into presynaptic DA neurons.3-8 DAT is a member of the NaCl 2 -coupled neurotransmitter transporter family, which constitutes plasma membrane transporters for serotonin and ABSTRACT: Dopamine (DA) receptors, which are heavily expressed in the caudate/putamen of the brain, represent the molecular target of several drugs used in the treatment of various neurological disorders, such as Parkinson's disease. Although most of the drugs are very effective in alleviating the symptoms associated with these conditions, their long-term utilization could lead to the development of severe side-effects. In addition to uncovering novel mediators of physiological DA receptor functions, recent research advances are suggesting a role of these receptors in toxic effects on neurons. For instance, accumulating evidence indicates that DA receptors, particularly D1 receptors, are central in the neuronal toxicity induced by elevated synaptic levels of DA. In this review, we will discuss recent findings on DA receptors as regulators of long term neuronal dysfunction and neurodegenerative processes. RÉSUMÉ:Les récepteurs dopaminergiques, exprimés notamment dans les noyaux caudé et putamen du cerveau, sont la cible pharmacologique de plusieurs médicaments employés dans le traitement de maladies neurologiques telle que la maladie de Parkinson. Bien que ces médicaments soient efficaces pour renverser les symptômes cliniques de la maladie, ils sont associés au développement d'effets secondaires incommodants majeurs lorsque utilisés à long terme. L'avancement de la recherche sur la fonction des récepteurs dopaminergiques a mis en lumière plusieurs nouvelles voies de signalisation, dont certaines sont reconnues pour initier la neurodégénérescence. Ainsi, des études récentes ont démontré l'implication des récepteurs dopaminergiques D1 dans la toxicité induite par des niveaux synaptiques élevés de dopamine. Cet article a pour but d'exposer le rôle potentiel d'un dérèglement de la signalisation des récepteurs dopaminergiques dans l'a...