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Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders.

ABSTRACT: Catecholamine-regulated proteins (CRPs) have been shown to bind dopamine and other structurally related catecholamines; in particular, the 40-kDa CRP (CRP40) protein has been previously cloned and functionally characterized. To determine putative human homologs, BLAST analysis using the bovine CRP40 sequence identified a human established sequence tag (EST) with significant homology (accession #BQ224193). Using this EST, we cloned a recombinant human brain CRP40-like protein, which possessed chaperone activity. Radiolabeled dopamine binding studies with recombinant human CRP40 protein demonstrated the ability of this protein to bind dopamine with low affinity and high capacity. The full-length human CRP40 nucleotide sequence was elucidated (accession #DQ480334) with RNA ligase-mediated rapid amplification of complementary DNA ends polymerase chain reaction, while Northern blot hybridization suggested that human CRP40 is an alternative splice variant of the 70-kDa mitochondrial heat shock protein, mortalin. Human SH-SY5Y neuroblastoma cells treated with the antipsychotic drug, haloperidol, exhibited a significant increase in CRP40 messenger RNA expression compared to untreated control cells, while other dopamine agonists/antagonists also altered CRP40 expression and immunolocalization. In conclusion, these results show that we have cloned a splice variant of mortalin with a novel catecholamine binding function and that this chaperone-like protein may be neuroprotective in dopamine-related central nervous system disorders.

SUBMITTER: Gabriele J

PROVIDER: S-EPMC2866950 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders.

Gabriele Joseph J Pontoriero Giuseppe F GF Thomas Nancy N Thomson Christy A CA Skoblenick Kevin K Pristupa Zdenek B ZB Mishra Ram K RK

Cell stress & chaperones 20090312 6

Catecholamine-regulated proteins (CRPs) have been shown to bind dopamine and other structurally related catecholamines; in particular, the 40-kDa CRP (CRP40) protein has been previously cloned and functionally characterized. To determine putative human homologs, BLAST analysis using the bovine CRP40 sequence identified a human established sequence tag (EST) with significant homology (accession #BQ224193). Using this EST, we cloned a recombinant human brain CRP40-like protein, which possessed cha ...[more]

PMID: 19280369

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Project description:BACKGROUND AND PURPOSE: This study investigates the role of alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), on catecholamine synthesis and catabolism in the central nervous system of mice. EXPERIMENTAL APPROACH: Activities of the main catecholamine synthetic and catabolic enzymes were determined in whole brains obtained from alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptor knockout (KO) and C56Bl\7 wild-type (WT) mice. KEY RESULTS: Although no significant differences were found in tyrosine hydroxylase activity and expression, brain tissue levels of 3,4-dihydroxyphenylalanine were threefold higher in alpha(2A)- and alpha(2C)-adrenoceptor KO mice. Brain tissue levels of dopamine and noradrenaline were significantly higher in alpha(2A) and alpha(2C)KOs compared with WT [WT: 2.8 +/- 0.5, 1.1 +/- 0.1; alpha(2A)KO: 6.9 +/- 0.7, 1.9 +/- 0.1; alpha(2B)KO: 2.3 +/- 0.2, 1.0 +/- 0.1; alpha(2C)KO: 4.6 +/- 0.8, 1.5 +/- 0.2 nmol.(g tissue)(-1), for dopamine and noradrenaline respectively]. Aromatic L-amino acid decarboxylase activity was significantly higher in alpha(2A) and alpha(2C)KO [WT: 40 +/- 1; alpha(2A): 77 +/- 2; alpha(2B): 40 +/- 1; alpha(2C): 50 +/- 1, maximum velocity (V(max)) in nmol.(mg protein)(-1).h(-1)], but no significant differences were found in dopamine beta-hydroxylase. Of the catabolic enzymes, catechol-O-methyltransferase enzyme activity was significantly higher in all three alpha(2)KO mice [WT: 2.0 +/- 0.0; alpha(2A): 2.4 +/- 0.1; alpha(2B): 2.2 +/- 0.0; alpha(2C): 2.2 +/- 0.0 nmol.(mg protein)(-1).h(-1)], but no significant differences were found in monoamine oxidase activity between all alpha(2)KOs and WT mice. CONCLUSIONS AND IMPLICATIONS: In mouse brain, deletion of alpha(2A)- or alpha(2C)-adrenoceptors increased cerebral aromatic L-amino acid decarboxylase activity and catecholamine tissue levels. Deletion of any alpha(2)-adrenoceptor subtypes resulted in increased activity of catechol-O-methyltransferase. Higher 3,4-dihydroxyphenylalanine tissue levels in alpha(2A) and alpha(2C)KO mice could be explained by increased 3,4-dihydroxyphenylalanine transport.

Dataset Information

Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders.

Publications

Cloning, characterization, and functional studies of a human 40-kDa catecholamine-regulated protein: implications in central nervous system disorders.

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