CENTRAL HYPOTHESIS - We hypothesize that in accordance with the strict temporal and spatial control of NO production in vascular endothelial cells, argininosuccinate synthase, the rate-limiting step of the citrulline-NO cycle, is coordinately and tightly regulated via translational and posttranslational mechanisms, in addition to transcriptional regulation. This premise underlies the rationale for the specific aims our present research which focuses on posttranscriptional events that regulate AS function. First, we hypothesize that the determinants of AS subcellular localization are AS-interacting proteins which guide AS to distinct destinations within the cell. Methodology used to examine this hypothesis employs: 1) immunofluorescence microscopy; 2) pull-down analysis; 3) immunoprecipitation analysis; 4) tandem affinity purification analysis; 5) mass-spectrometry; and 6) NO determinations. Second, we hypothesize that a small protein encoded by an upstream open reading frame (uORF) represses AS mRNA translation under stress conditions by routing stalled AS mRNA particles into discrete cytoplasmic foci known as stress granules. To test this hypothesis we employ: 1) in vitro translation; 2) RNA immunoprecipitation; 3) RNA EMSA; 4) polysome profiling; 5) immunofluorescence microscopy; 6) western blotting; and 7) fluorescence in situ hybridization (FISH). Third, we hypothesize that endothelial AS protein is phosphorylated at serine/theronine residues which mediate an acute response, altering AS activity. To test this hypothesis the following methodology has been employed: 1) proteomics and mass-spectrometry; 2) mutational analysis; 3) NO determinations; 4) AS activity assays; and 5) western blotting. Fourth, to translate our cell culture work to a whole animal system, we are studying how changes in AS expression correlate with impaired vascular endothelial NO production in a diabetic rat model system. Experimental approaches include: 1) animal model characterization; 2) real time RT-PCR; 3) western blotting; 4) NO measurements; 5) immunofluorescence microscopy; and 6) FISH. Overall, results from this research have the potential to distinguish new therapeutic targets to be used to prevent or inhibit vascular endothelial dysfunction found in diabetes, obesity and other related vascular diseases.