Elucidating the mechanisms involved in the regulation of biosynthesis of tryosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) is central in understanding factors controlling the development and expression of the catecholaminergic neuronal system. The proposed research will: I. Study the regulation of multiple forms of dopamine beta-hydroxylase. Ia. Isolate and sequence a cDNA clone for rat DBH, to deduce, for the first time, the amino acid sequence of this enzyme. A Lambdagt11 expression library of cDNA prepared from PC12 pheochromocytoma cell mRNA is being screened with antibodies to DBH to isolate the cDNA for rat DBH. Ib. Determine the differences between multiple subunit forms of DBH. The conversion of the 77K to the 73K subunit form of DBH will be examined as to whether it derives from proteolysis or variations in glycosylation. Ic. Determine the subcellular site of the post-translational processing of DBH and examine its relationship to the processing of neuropeptides. Relatively specific inhibitors such as monensin, chloroquine, and tunicamycin will be used. Id. Study the regulation of biosynthesis of multiple subunit forms of DBH in the locus coeruleus, a major center of noradrenergic neurons in the brain. The effect of factors which alter noradrenealine levels in the brain will be examined. II. Study the mechanisms of in vivo regulation of expression of the genes for catecholamine biosynthesis. IIa. Study the regulation of specific mRNA levels for TH and DBH under conditions which have been suggested to lead to long-term regulation of activity. 1. Regulation by glucocorticoids; 2. Ontogenetic changes; 3. Effect of reserpine treatment; 4. Effect of cell density; 5. Effect in schizophrenia. IIb. Determine the organization of DBH genes, relative to other genes of the pathway and attempt to identify regulatory sequences such as glucocorticoid receptor binding sites.
Showing the most recent 10 out of 11 publications
