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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Modified Research Career Development Award (K04)
Project #
5K04NS001121-02
Application #
3074954
Study Section
Biochemistry Study Section (BIO)
Project Start
1987-04-01
Project End
1992-03-31
Budget Start
1988-04-01
Budget End
1989-03-31
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
New York Medical College
Department
Type
Schools of Medicine
DUNS #
City
Valhalla
State
NY
Country
United States
Zip Code
10595
McMahon, A; Kvetnansky, R; Fukuhara, K et al. (1992) Regulation of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA levels in rat adrenals by a single and repeated immobilization stress. J Neurochem 58:2124-30
McMahon, A; Sabban, E L (1992) Regulation of expression of dopamine beta-hydroxylase in PC12 cells by glucocorticoids and cyclic AMP analogues. J Neurochem 59:2040-7
Kilbourne, E J; Nankova, B B; Lewis, E J et al. (1992) Regulated expression of the tyrosine hydroxylase gene by membrane depolarization. Identification of the responsive element and possible second messengers. J Biol Chem 267:7563-9
Feng, Z; Angeletti, R H; Levin, B E et al. (1992) Glycosylation and membrane insertion of newly synthesized rat dopamine beta-hydroxylase in a cell-free system without signal cleavage. J Biol Chem 267:21808-15
Kilbourne, E J; McMahon, A; Sabban, E L (1991) Membrane depolarization by isotonic or hypertonic KCl: differential effects on mRNA levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA in PC12 cells. J Neurosci Methods 40:193-202
Kilbourne, E J; Osaka, H; Sabban, E L (1991) Hypomethylation of the rat tyrosine hydroxylase gene correlates with its expression in several cell types. Brain Res Dev Brain Res 58:143-6
Badoyannis, H C; Sharma, S C; Sabban, E L (1991) The differential effects of cell density and NGF on the expression of tyrosine hydroxylase and dopamine beta-hydroxylase in PC12 cells. Brain Res Mol Brain Res 11:79-87
McMahon, A; Geertman, R; Sabban, E L (1990) Rat dopamine beta-hydroxylase: molecular cloning and characterization of the cDNA and regulation of the mRNA by reserpine. J Neurosci Res 25:395-404
Kilbourne, E J; Sabban, E L (1990) Differential effect of membrane depolarization on levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNAs in PC12 pheochromocytoma cells. Brain Res Mol Brain Res 8:121-7
Sabban, E L; Schwartz, J; McMahon, A (1990) Effect of compounds which disrupt proton gradients on secretion of neurosecretory proteins from PC12 pheochromocytoma cells. Neuroscience 38:561-70

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