Human Tyrosine Hydroxylase Isoforms

Alterio, Jeanine; Ravassard, Philippe; Haavik, Jan; Caër, Jean-Pierre Le; Biguet, Nicole Faucon; Waksman, Gilles; Mallet, Jacques

doi:10.1074/jbc.273.17.10196

Cited by 38 publications

(16 citation statements)

References 47 publications

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“…BH 4 has an inhibitory effect on DTH I and II activities but at higher levels (400 M and above) (not shown). The kinetic data obtained for the recombinant Drosophila enzymes are comparable to the values obtained for vertebrate TH expressed in E. coli (32,33).…”

Section: Expression and Purification Of Recombinant Dth Type I Andsupporting

confidence: 69%

“…Either the amino acid segment inserted in the regulatory region prevents a conformational change required for enzyme activation or alternatively the TH enzyme containing this segment spontaneously presents a conformation similar to that of the phosphorylated enzyme. A comparable lack of regulation by PKA has been recently reported for the human isoform HTH3 in the absence of dopamine (33). On the opposite side, we show that phosphorylation of DTH II by PKA activates the enzyme in the presence of dopamine (Fig.…”

Section: Fig 6 Inhibition Of Dth Activity By Dopaminesupporting

confidence: 46%

“…For recombinant rat TH expressed in E. coli, it has been shown that phosphorylation by PKA in the absence of dopamine results in a moderate decrease in the K m for BH 4 and no increase in V max (32). Such a decrease in K m was shown to induce only an apparent 2-fold activation of the human isoform HTH1 when assayed in standard initial velocity conditions (33). Therefore, a decrease in K m is probably not sufficient to explain the strong activation of DTH1 by PKA phosphorylation we observed in the bacterial extract in the absence of dopamine (Fig.…”

Section: Fig 6 Inhibition Of Dth Activity By Dopaminementioning

confidence: 76%

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Differential Regulation of Drosophila Tyrosine Hydroxylase Isoforms by Dopamine Binding and cAMP-dependent Phosphorylation

Vié

Cigna

Toci

et al. 1999

Journal of Biological Chemistry

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Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559 -26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower K m value for the cosubstrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMPdependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser 32 , a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.Tyrosine hydroxylase (TH 1 ) (tyrosine 3-monooxygenase, EC 1.14.16.2) is an eukaryotic enzyme catalyzing the first and rate-limiting step in dopamine and other catecholamine biosynthesis, i.e. the hydroxylation of the monophenol amino acid L-tyrosine to produce the ortho-diphenol L-dihydroxyphenylalanine (2, 3). The enzyme is active in the presence of ferrous iron, O 2 , and a tetrahydrobiopterin co-substrate. A single gene encodes TH, which is required for embryonic development and survival in mammals (4, 5). In vertebrates, TH activity is exquisitely regulated at each step of its expression: control of gene transcription, RNA alternative processing, mRNA stability, and direct modulation of the enzyme by catecholamine feedback inhibition and protein kinase activation (6 -8).In contrast, much less is known on the regulatory properties of tyrosine hydroxylase in insects. Muta...

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Section: Expression and Purification Of Recombinant Dth Type I Andsupporting

confidence: 69%

Section: Fig 6 Inhibition Of Dth Activity By Dopaminesupporting

confidence: 46%

Section: Fig 6 Inhibition Of Dth Activity By Dopaminementioning

confidence: 76%

See 1 more Smart Citation

Differential Regulation of Drosophila Tyrosine Hydroxylase Isoforms by Dopamine Binding and cAMP-dependent Phosphorylation

Vié

Cigna

Toci

et al. 1999

Journal of Biological Chemistry

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“…Construction of the Expression Vectors-The complete coding sequence of HTH3 was excised from PET3a (Stratagene) (10) by NdeIBamHI digestion. The fragment was blunted by Klenow polymerase and transferred into the SmaI restriction site of PGEX-2T (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, the amount of the species containing exon 2 (HTH-3 and HTH-4) is enhanced in pheochromocytoma, a tumor of adrenal medulla (4). The four mRNAs encode proteins that differ in the N-terminal regulatory region and present different enzymatic activities (1,10). These data suggest that alternative splicing of TH premessenger RNA is significant and prompt us to further study the diversity of HTH mRNAs.…”

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confidence: 99%

A Human Tyrosine Hydroxylase Isoform Associated with Progressive Supranuclear Palsy Shows Altered Enzymatic Activity

Bodeau-Péan¹,

Ravassard²,

Neuner-Jehle³

et al. 1999

Journal of Biological Chemistry

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A novel human tyrosine hydroxylase (HTH) messenger RNA subgroup generated by alternative splicing and characterized by the absence of the third exon was recently identified. The corresponding putative protein lacks 74 amino acids including Ser 31 and Ser 40 , two major phosphorylation sites implicated in the regulation of HTH activity. These mRNA species are detected in adrenal medulla and are overexpressed in patients suffering from progressive supranuclear palsy, a neurodegenerative disease mostly affecting catecholaminergic neurons of the basal ganglia.In the present work, an HTH protein isoform lacking exon 3 was identified in human adrenal medulla. For this purpose, an antibody was raised against the HTH exon 3. The effect of the removal of exon 3 on the enzymatic activity of HTH was studied in vitro by comparing a purified recombinant fusion protein without exon 3 (glutathione S-transferase (GST)-HTH⌬3) to the equivalent protein containing exon 3 (GST-HTH3). In initial velocity conditions, GST-HTH⌬3 has 30% of the maximal velocity of GST-HTH3. Moreover, the skipping of exon 3 results in the absence of activation of GST-HTH by heparin and increases by 10-fold the retroinhibition constant for dopamine, demonstrating the involvement of exon 3 in the regulation of HTH enzymatic activity. The identification of a variably expressed HTH isoform that lacks an exon implicated in activity regulation supports the view that HTH alternative splicing contributes to the functional diversity within the catecholaminergic system and may be implicated in some neurological diseases. Tyrosine hydroxylase (TH)1 has been the subject of extensive investigation, largely because it catalyzes the rate-limiting step in the biosynthesis of catecholamines. The regulation of the TH level and of its enzymatic activity is thus a major mechanism for controlling the amount of these important amines in blood and synapses. TH is regulated by almost all possible mechanisms of transcriptional and post-transcriptional control including regulation of the transcription rate, alternative splicing of the premessenger, variable stability of the mRNA, translational control, and modulation of the enzymatic activity (1). Recently, it has been shown that some human TH (HTH) gene sequence variants are associated with abnormal TH enzymatic activity and may be involved in some neurodegenerative diseases. Indeed, point mutations in the coding sequence of HTH have been found that decrease the activity of the enzyme in patients with L-DOPA-responsive dystonia (2) and inherited juvenile L-DOPA-responsive parkinsonism (3). There is also emerging evidence that alternative splicing of TH premessenger RNA may have physiological and even pathological consequences (4, 5). Initially, four HTH mRNA variants generated by alternative splicing were cloned and characterized by (i) the differential use of two splicing donor sites within exon 1 and (ii) the differential inclusion of exon 2 (4, 6, 7). All four HTH species are present in human catecholaminergic tissues (8, 9)....

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Distribution of the catecholaminergic neurons in the central nervous system of human embryos and fetuses

Verney

1999

Microsc. Res. Tech.

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Human Tyrosine Hydroxylase Isoforms

Cited by 38 publications

References 47 publications

Differential Regulation of Drosophila Tyrosine Hydroxylase Isoforms by Dopamine Binding and cAMP-dependent Phosphorylation

Differential Regulation of Drosophila Tyrosine Hydroxylase Isoforms by Dopamine Binding and cAMP-dependent Phosphorylation

A Human Tyrosine Hydroxylase Isoform Associated with Progressive Supranuclear Palsy Shows Altered Enzymatic Activity

Distribution of the catecholaminergic neurons in the central nervous system of human embryos and fetuses

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