These studies have focused on the regulatory properties of tyrosine hydroxylase, tryptophan hydroxylase and their cofactor tetrahydrobiopterin (BH4) in the biosynthesis and release of catecholamine and serotonin in the brain. We have identified two transcripts of human tryptophan hydroxylase (TPH) resulting from an alternative splicing at the 3' open reading frame. The two transcripts would give two isoforms of human TPH with different C termini, one of which contains a putative cAMP-dependent protein kinase (PKA) site, whereas the other one, which is 22-amino acid longer does not. The splicing site was found to exist in all the human tissues we tested by using RT-PCR techniques, but the ratio of the two transcripts varies. We have also identified the two isoforms at the protein level by analyzing protein extracts from human pineal gland and brainstem with anti-TPH antibody. These results provide knowledge about the regulatory mechanism of serotonin production by regulating the expression of TPH. It has been shown that TPH can be phosphorylated by PKA and calmodulin- dependent protein kinase II (CamPKII) and be activated in the presence of 14-3-3 protein. To identify the phosphorylation sites in TPH, we expressed both isoforms in E.coli and insect cells. Several mutants were constructed at three potential phosphorylatable serine sites: Ser58, Ser260 and Ser443. We found that Ser58 and Ser443 were phosphorylated by PKA to the extent of 0.35 mol/subunit of TPH and 0.45 mol/subunit of TPH, respectively, while Ser260 was not phosphorylated by PKA. CamPKII phosphorylated these serines poorly, to an extent of 0.08 mol/subunit TPH. Ser260 is the best site for CamPKII, it can be phosphorylated to an extent of 0.06 mol/subunit of TPH. The difference between the phosphorylation by PKA and CamPKII may reveal some clues to different regulatory mechanisms about which we have not known. In addition, Ser260 is the putative site for interaction with 14-3-3. However, due to the poor phosphorylation of this site, we were not able to show significant interaction between 14-3-3 and phosphorylated human TPH. With the technique of in vitro perfusion of striatal slices, we have shown that BH4 plays an important dual role in regulating dopamine release: through its cofactor activity with nitric oxide synthase, it stimulates the formation of nitric oxide (NO) which indirectly stimulates dopamine release. Through its coenzyme activity with tyrosine hydroxylase, it also stimulates directly dopamine synthesis and, secondarily, its release.
