REGULATION OF BIOSYNTHESIS OF DOPAMINE BETA-HYDROXYLASE

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Elucidating the mechanisms involved in the regulation of biosynthesis of dopamine beta-hydroxylase (DBH) is central in understanding factors controlling the development and expression of the noradrenergic neuronal system. The proposed research will: 1. 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 will be screened with antibodies to DBH to isolate the cDNA for rat DBH. 2. Characterize the biochemical differences between the multiple subunit forms of DBH which have recently been shown by us and others to be present in PC12 pheochromocytoma cells, and in bovine adrenal medulla and rat brain. We will examine whether the alteration is due to cleavage of a C- or N-terminal polypeptide anchoring the 77K form in the membrane, and whether the subunit forms are differentially glycosylated. The first possibility will utilize specific peptide mapping, based on the amino acid sequence. The glycosylation will be studied by endoglycosidase digestions of radiolabeled DBH, and specific sugar labeling experiments. The possible presence of O-linked oligosaccharide also will be examined. 3. 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. 4. 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 noradrenaline levels in the brain will be examined. 5. Study the regulation of specific mRNA levels for DBH under conditions which have been suggested to lead to long-term regulation of DBH activity. a. Regulation by glucocorticoids in the adrenal medulla; b. Ontogenetic development of DBH in the adrenal medulla and locus coeruleus; c. Effect of reserpine on the locus coeruleus and the adrenal medulla; d. Alteration of DBH in schizophrenia. 6. Determination of the genomic organization of DBH genes. This should provide information regarding the possible existence of common exons for catcholamine biosynthetic enzymes.