Molecular Cloning, Structure, and Expression of Dopamine‐β–Hydroxylase from Bovine Adrenal Medulla

Wu, Hongjiang; Parmer, Robert J.; Koop, Allen H.; Rozansky, David J.; O’Connor, Daniel T.

doi:10.1111/j.1471-4159.1990.tb08826.x

Cited by 18 publications

(8 citation statements)

References 51 publications

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“…Eighteen DBH SNPs had minor allele frequencies greater than 5%. In the coding region, we found 8 non-synonymous SNPs (cSNPs), but only one (Ala172Thr) was common (minor allele frequency >5%), and none were in likely functional domains, e.g., Cys residues (that mediate inter-subunit disulfide bonds) (47), or Cu 2+ cofactor-binding His-His or His-Xxx-His domains (48) (Suppl. Figure 2).…”

Section: Resultsmentioning

confidence: 99%

Human dopamine beta-hydroxylase (DBH) regulatory polymorphism that influences enzymatic activity, autonomic function, and blood pressure

Chen

Wen²,

Rao³

et al. 2010

Journal of Hypertension

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Rationale Dopamine beta-hydroxylase (DBH) plays an essential role in catecholamine synthesis by converting dopamine into norepinephrine. Here we systematically investigated DBH polymorphisms associated with enzymatic activity as well as autonomic and BP/disease phenotypes in vivo. Methods and Results 70 genetic variants were discovered at the locus; across ethnicities, much of the promoter was spanned by a 5’ haplotype block, with a larger block spanning the promoter in whites than blacks. DBH secretion was predicted by genetic variants in the DBH promoter, rather than the amino acid coding region. The C allele of common promoter variant C-970T increased plasma DBH activity, epinephrine excretion, the heritable change in BP during environmental stress in twin pairs, and also predicted higher basal BP in three independent populations. Mutagenesis and expression studies with isolated/transfected DBH promoter/luciferase reporters in chromaffin cells indicated that variant C-970T was functional. C-970T partially disrupted consensus transcriptional motifs for n-MYC and MEF-2, and this variant affected not only basal expression, but also the response to exogenous/co-transfected n-MYC or MEF-2; during ChIP, these two endogenous factors interacted with the motif. Conclusions These results suggest that common DBH promoter variant C-970T plays a role in the pathogenesis of human essential hypertension: common genetic variation in the DBH promoter region seems to initiate a cascade of biochemical and physiological changes eventuating in alterations of basal BP. These observations suggest new molecular strategies for probing the pathophysiology, risk, and rational treatment of systemic hypertension.

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Section: Resultsmentioning

confidence: 99%

Human dopamine beta-hydroxylase (DBH) regulatory polymorphism that influences enzymatic activity, autonomic function, and blood pressure

Chen

Wen²,

Rao³

et al. 2010

Journal of Hypertension

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“…DBM signal sequences from several species and construction of DBMsignal/PHMs chimera. A, comparison of the signal sequences of rat (27), mouse (54), human (16), and bovine DBM (11,21). The deduced amino acid sequences of the NH 2 -terminal region are shown.…”

Section: Construction Of Expressionmentioning

confidence: 99%

“…(1), noncovalent association with phosphatidylserine (3,55), and incorporation of myristate (54). The only region of rat (27), human (15,16), mouse (56), or bovine (11,19,21) DBM sufficiently hydrophobic to function as a membrane anchor is a 20-amino acid region near the NH 2 terminus (Fig. 1A).…”

Section: Fully Active Phm Is Formed Upon Expression Of the Dbmsignal/mentioning

confidence: 99%

Dopamine β-Monooxygenase Signal/Anchor Sequence Alters Trafficking of Peptidylglycine α-Hydroxylating Monooxygenase

Oyarce

Steveson

Jin

et al. 2001

Journal of Biological Chemistry

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Dopamine ␤-monooxygenase (DBM) and peptidylglycine ␣-hydroxylating monooxygenase (PHM) are essential for the biosynthesis of catecholamines and amidated peptides, respectively. The enzymes share a conserved catalytic core. We studied the role of the DBM signal sequence by appending it to soluble PHM (PHMs) and expressing the DBMsignal/PHMs chimera in AtT-20 and Chinese hamster ovary cells. PHMs produced as part of DBMsignal/PHMs was active. In vitro translated and cellular DBMsignal/PHMs had similar masses, indicating that the DBM signal was not removed. DBMsignal/PHMs was membrane-associated and had the properties of an intrinsic membrane protein. After in vitro translation in the presence of microsomal membranes, trypsin treatment removed 2 kDa from DBMsignal/PHMs while PHMs was entirely protected. In addition, a Cys residue in DBMsignal/PHMs was accessible to Cys-directed biotinylation. Thus the chimera adopts the topology of a type II membrane protein. Pulse-chase experiments indicate that DBMsignal/PHMs turns over rapidly after exiting the trans-Golgi network. Although PHMs is efficiently localized to secretory granules, DBMsignal/ PHMs is largely localized to the endoplasmic reticulum in AtT-20 cells. On the basis of stimulated secretion, the small amount of PHMs generated is stored in secretory granules. In contrast, the expression of DBMsignal/ PHMs in PC12 cells yields protein that is localized to secretory granules.Catecholamine synthesis from tyrosine involves several cytosolic enzymes and one secretory granule enzyme, dopamine ␤-monooxygenase (DBM) 1 (1). DBM from adrenal chromaffin cells and neurons occurs in soluble and membrane forms (1-3). Although soluble DBM is secreted along with its product catecholamines, membrane DBM undergoes endocytosis (4, 5). DBM monomers form tetramers composed of two noncovalently bound disulfide-linked dimers (6, 7). Soluble and membrane forms of DBM both are heavily glycosylated and derived from a single translation product (8). Phospholipids as well as an uncleaved signal sequence seem to play a role in the attachment of DBM to membranes (8 -10).Sequence analysis of membrane DBM from bovine adrenal medulla revealed an uncleaved signal sequence in 30% of the protein (11). Similarly, sequence analysis of rat DBM produced by in vitro transcription/translation in the presence of microsomal membranes indicated that the signal sequence was not removed (12). These studies suggest that DBM is a type II integral membrane protein. The NH 2 -terminal sequences of rat, mouse, human, and bovine DBM vary in length, but each contains a stretch of 20 hydrophobic amino acids, which is longer than the hydrophobic domains typically found in cleaved signal sequences (13, 14) (Fig. 1A). Although rat and mouse DBM also contain a lengthy stretch of amino acids preceding the hydrophobic domain, human and bovine DBM do not. However, analysis of the genomic sequence encoding human DBM (15, 16) identified an in-frame upstream Met codon that is likely to represent the actual translational sta...

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“…Cloning of DBH cDNA revealed a single open reading frame and no obvious membrane attachment site . The only extensive hydrophobic region is at the N-terminal putative signal sequence (Lamouroux et al ., 1987 ;Taljanidisz et al ., 1989 ;Lewis et al ., 1990 ;McMahon et al ., 1990 ;Wang et al ., 1990 ;Wu et al ., 1990) . Transfection of cells with a single cDNA generated both membrane-bound and soluble forms of DBH (Lewis and Asnani, 1992) .…”

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confidence: 99%

Mutagenesis of Rat Dopamine β‐Hydroxylase: Examination in Cell‐Free System

Feng

Sabban

1995

Journal of Neurochemistry

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Dopamine β‐hydroxylase (DBH; EC 1.14.17.1) exists as membrane‐bound and soluble forms in neurosecretory vesicles. The features of DBH required for glycosylation and incorporation into membranes were studied in a cell‐free system. Translation of full‐length DBH with microsomal membranes generated two glycosylated products (GH and GL) depending on the magnesium concentration. Carboxyl‐terminal, in contrast to amino‐terminal, truncations gave translation products that were glycosylated by microsomal membranes. Site‐directed mutants were generated with the second AUG codon and the region of a putative signal sequence cleavage site modified. Translation without membranes indicated that the second AUG is not used to initiate translation. The mutant with Glu41→ Leu41 and Ser43→ Thr43 yielded only the GH form with membranes, whereas mutation of Ser43→ Ala43 generated both GH and GL forms. Both glycosylated forms comigrated with the microsomal membranes on sucrose gradients. Endoglycosidase H digestion indicated that the differences between the GH and GL forms are not due to the sugar moiety. The results suggest a role for cleavage of a signal sequence in the formation of different forms of DBH. The possibility that these mutations change the secondary structure near the signal cleavage site, affecting processing, is discussed.

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Molecular Cloning, Structure, and Expression of Dopamine‐β–Hydroxylase from Bovine Adrenal Medulla

Cited by 18 publications

References 51 publications

Human dopamine beta-hydroxylase (DBH) regulatory polymorphism that influences enzymatic activity, autonomic function, and blood pressure

Human dopamine beta-hydroxylase (DBH) regulatory polymorphism that influences enzymatic activity, autonomic function, and blood pressure

Dopamine β-Monooxygenase Signal/Anchor Sequence Alters Trafficking of Peptidylglycine α-Hydroxylating Monooxygenase

Mutagenesis of Rat Dopamine β‐Hydroxylase: Examination in Cell‐Free System

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