Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation

Abstract

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].

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

“…Activity was decreased at 167 M iron to 71 and 93% of the maximal value for DTH I and DTH II, respectively (not shown). Dopamine, one of the end products of the catecholamine biosynthesis pathway, has been shown to inhibit vertebrate TH activity (37)(38)(39)(40). We found that this regulation also occurs in Drosophila, although DTH seems to be less sensitive to dopamine inactivation than vertebrate TH.…”

“…Dopamine and other catecholamines inhibit and stabilize vertebrate TH activity (37)(38)(39)(40), and iron stimulates dopamine binding (31). We have shown here that this regulation is conserved in Drosophila TH and that the level of inhibition by dopamine depends on ferrous iron concentration.…”

“…For vertebrate TH, it has been demonstrated that catecholamine binds to the active site iron oxidized in a ferric redox state (47,49), thus stabilizing the enzyme in an inactivated form (32). Phosphorylation by PKA decreases the binding affinity of human TH for dopamine (40,52), and conformation studies suggest that the catalytic site lays in close proximity or interacts with the PKA phosphorylation site of the regulatory domain (53). Deletion of the NH 2 -terminal regulatory domain of human TH1 up to the PKA phosphorylation site (Ser 40 ) abolishes the inhibitory effect of dopamine (54).…”

“…Phosphorylation by PKA decreases the binding affinity of human TH for dopamine (40,52), and conformation studies suggest that the catalytic site lays in close proximity or interacts with the PKA phosphorylation site of the regulatory domain (53). Deletion of the NH 2 -terminal regulatory domain of human TH1 up to the PKA phosphorylation site (Ser 40 ) abolishes the inhibitory effect of dopamine (54). All these data strongly suggest that: 1) there is a direct interaction between the region surrounding the PKA site in the regulatory domain, and the iron binding site occurring close to the active site in the catalytic domain; 2) this interaction is required for dopamine binding and inhibition of TH activity; and 3) this interaction is relieved by phosphorylation of Ser 40 .…”

Regulation of tyrosine hydroxylase by stress‐activated protein kinases

“…However, these studies employed CaM-KII, which also phosphorylates Ser40 (Graham et al 2000). We have previously shown that phosphorylation of Ser40 in hTH1 shows a slight positive cooperativity with Hill coefficients in the range of 1.4-1.6, suggesting a kinetic interaction between phosphorylation sites on different subunits (Almas et al 1992). Similarly, the interaction between Ser19 and Ser40 phosphorylation may also occur on the same or different subunits of the TH tetramer.…”