Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 3 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in v i m at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 moVmol subunit and 2 mol/ mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-3 ) and Serl9 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-11, which phosphorylates Serl9 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser4O about twice as fast as Serl9. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-I1 correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3=4 > 1 + 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/ mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.Tyrosine hydroxylase (TH) is a homotetrameric enzyme which catalyses the rate-limiting step in catecholamine synthesis. It is found predominantly in the adrenal medulla and central and sympathetic nervous systems, where its activity is increased by agonists that stimulate catecholamine secretion, providing a mechanism for replenishing the stores of catecholamine hormones and neurotransmitters that have been lost via secretion.

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.

ABSTRACT␥-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the ␤1 subunit. Within the rat striatum, this subunit coassembles with ␣2, ␤1, and ␥1, suggesting that ␥-aminobutyric acid type A receptors consisting of arrangements other than ␣␤ ؉ ␥, ␦, or do exist. Expression of ␣2␤1␥1 in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for ␥-aminobutyric acid compared with ␣2␤1␥1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.In the mammalian central nervous system, inhibitory neurotransmission is mediated primarily by the ␥-aminobutyric acid (GABA), which acts on GABA type A (GABA-A) receptors, ligand-gated ion channels acting over a rapid time frame. Over the past 10 years, it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from ␣1-␣6, ␤1-␤3, ␥1-␥3, ␦, , and to form what is most likely a pentameric macromolecule (1-6). The subunits show distinct patterns of temporal and spatial expression, so that GABA-A receptor subtypes have a defined localization presumably reflecting their physiological role (7)(8)(9)(10)(11).In this article, we report the identification and characterization of a further member of the GABA-A receptor gene family that we have termed theta (). MATERIALS AND METHODSCloning of Subunit cDNA. Full-length cDNA was cloned starting from sequence information in GenBank entry U47334. PCR was performed under standard conditions, on human whole-brain cDNA (CLONTECH) with oligonucleotide primers specific to the 5Ј and 3Ј ends of the U47334 sequence. A single PCR product of approximately 1,600 bp was obtained. The 5Ј and 3Ј ends of the coding region corresponding to full-length U47334 sequence were obtained by 5Ј and 3Ј anchored PCR using human brain Marathon cDNA (CLON-TECH). Full-length cDNA (GenBank accession no. AF144648) was generated by PCR using a primer derived from sequences surrounding the initiating methionine incorporating a consensus Kozak sequence (12), and a primer based on the 3Ј untranslated region of the anchored PCR product. The PCR product (1,958 bp) was sequenced completely on both strands by primer walking by using dye terminator chemistry and an Applied Biosystems model 373A sequencer.Epitope-tagged subunit was constructed that contained nucleotides Ϫ224 to ϩ99 (i.e., the 5Ј untranslated region, the signal peptide, 6 amino acids of the mature protein) of bovine ...

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichiu coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (1 3 -40-fold) by incubation with Fe(I1) salts (concentration of iron at half-maximal activation = 6-14 pM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(I1) and Ni(I1). They all bind stoichiometric amounts of Fe(I1) and Zn(I1) with high affinity (Kd = 0.2-3 pM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(I1) and enzyme activation. In the absence of any free Fe(I1) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a tetrahydropteridine-dependent enzyme which catalyses the rate-limiting step in the biosynthesis of catecholamines [ 11. Early studies showed that the enzyme activity is stimulated by ferrous iron and inhibited by certain iron chelators [2 -51.Subsequently, it was shown that the enzyme isolated from various tissues contained significant amounts of iron [6 -81, which seems to be involved in oxygen activation, an essential step in the catalytic reaction [6-81. However, partly due to the difficulties involved in obtaining pure tyrosine hydroxylase, no systematic study has been performed in order to determine the metal-binding properties of this enzyme.The recent expression of cloned tyrosine hydroxylase in high yield in eukaryotic cells using the baculovirus system [9, 101 and in prokaryotic cells [ 10 a] has, however, changed this situation. Human tyrosine hydroxylase exists as four different isozymes (TH1-TH4), generated through alternative splicing of pre-mRNA [ l l -131. Since these isozymes have different amino acid sequences in their regulatory N-terminal region and different distribution in the nervous system [14], it has been postulated that the occurrence of multiple molecular forms of the enzyme has important functional consequences. Previous studies, in which the isozymes have been expressed in low quantities in eukaryotic systems, have also demonstrated different kinetic properties of the various isozymes [15, 161. Correspondence to J. Haavik, Department of Biochemistry, UniAbbreviations. TH 1 -TH4, tyrosine hydroxylase isozymes 1 -4. Enzyme. Tyrosine 3-monooxygenase or tyrosine hydroxylase versity of Bergen, N-5009 Berge...

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia Cali and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTHZ and hTH2 with a stoichiometry of about 1 .O mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for CAMP-dependent protein kinase ( K , = 5 ~L M , V,,, = 9.5 pmol . min-' . mg kinase-'). The incorporation of about 1 .O mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(I1) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by CAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.Tyrosine hydroxylase (TH) catalyses the rate-limiting step in the biosynthesis of catecholamines [I], and is regulated in vivo by long-term and short-term mechanisms. The long-term mechanism involves a modulation of TH gene expression[2], while short-term regulation involves activation of the enzyme by phosphorylation [2 -41 and feedback inhibition by catecholamines [5, 61.Human TH (hTH) exists as four different isozyme forms (hTH1-4), generated by alternative splicing of pre-mRNA [7-91. All four isozymes have been detected in the human adrenal medulla [lo], although hTHl and hTH2 seem to be the most abundant species [7-lo]. Three of the isozymes (hTH1, hTH2 and hTH4) have recently been expressed in Escherichia roli as metal-free apoenzymes, and found to be activated by Fe(I1) on binding of 1 Fe/subunit [ll]. Little is known about their regulatory properties in vitro, except that both hTHl and hTH2 are inhibited by catecholamines [12]. This inhibition seems to be partially reversed by phosphorylation [I 21. Furthermore, the isozymes have different phosphorylation sites and may be regulated by different secondmessenger systems [12].