A Regulatory Domain in the N Terminus of Tryptophan Hydroxylase 2 Controls Enzyme Expression

Abstract

Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 (TPH2) is expressed predominantly in the central nervous system, whereas tryptophan hydroxylase 1 (TPH1) is expressed mostly in peripheral tissues. Although the two enzymes share considerable sequence homology, the regulatory domain of TPH2 contains an additional 41 amino acids at the N terminus that TPH1 lacks. Here we show that the extended TPH2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, TPH2 is synthesized less efficiently and is also less stable than TPH1. Removal of the unique portion of the N terminus of TPH2 results in expression of the enzyme at a level similar to that of TPH1, whereas protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10 -20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased TPH2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain serotonin synthesis. Serotonin (5-hydroxytryptamine (5HT)5 ) is involved in a wide range of functions throughout the body, including the regulation of vascular tone, appetite, wakefulness, and mood. Its role in mood regulation is supported by the successful treatment of a number of psychiatric disorders with drugs that regulate extracellular 5HT. Currently, this is accomplished by inhibiting either the serotonin transporter or monoamine oxidases, both of which increase the extracellular 5HT by blocking clearance of released transmitter (1-4). As the rate-limiting enzymes in 5HT synthesis, tryptophan hydroxylases (TPHs) provide another target for the regulation of 5HT levels. Two TPH isoforms, encoded by separate genes, have been identified in mammals (5). In adults, TPH1 is mostly expressed in nonneuronal cells and plays an essential role in peripheral 5HT synthesis (5-7). In contrast, TPH2 is expressed in neurons (6, 7) and controls brain 5HT synthesis (8). Since its discovery, genetic variants in the TPH2 gene have been identified in cohorts of patients with depression, suicidality, and bipolar disorder (9 -18). Thus, mechanisms that specifically regulate TPH2 activity may be important for the etiology or management of psychiatric disorders.TPH2 is part of a family of amino acid hydroxylases that includes TPH1, tyrosine hydroxylase (TH), and phenylalanine hydroxylase. All members of this family share a similar structure composed of an N-terminal regulatory domain, a central catalytic domain, and a C-termi...

Brainstem Deficiency of the 14-3-3 Regulator of Serotonin Synthesis: A Proteomics Analysis in the Sudden Infant Death Syndrome

“…serine-/threonine-phosphorylated motifs that lead in turn to activity changes in bound ligands, altered associations of bound ligands with other cellular components, and changes in intracellular localization of 14-3-3-bound cargo (37). Of major importance to our finding of significantly reduced 14-3-3 isoforms in the medullary 5-HT system in SIDS is their key role in TPH2 modulation (35).…”

“…The remarkable stability of 14-3-3 proteins after death is likely a result of their highly compact well-folded structure (37). Moreover, a study of postmortem changes in proteins in a rat model indicate that 14-3-3 levels increase after 48 h (52), rather than decrease, i.e.…”

Tryptophan Hydroxylase as Novel Target for the Treatment of Depressive Disorders

“…We have shown that this domain is associated with an inhibitory effect on TPH2 activity [63] . Other studies reported a negative effect of this domain on translational efficiency, stability and solubility of TPH2 compared to tyrosine hydroxylase and PAH [180][181][182][183] .…”

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

“…The additional sequences in TH and TPH2 (Fig. 1), which include the phosphorylation-targeted Ser 19 (17,19), most probably constitute functional domains in the neuronal hydroxylases, as recently suggested for the N-terminal sequence in TPH2 (59). The 43-residue N-terminal region of TH was used in this work to investigate determinants for binding of this functional domain to membranes and 14-3-3 proteins.…”