Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation

Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-s pecific alternative splicing of the TH primary transcript generates two dis tinct 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), Expres sion of DTH I is restricted to the central nervous system, whereas DTH II i s expressed in non-nervous tissues like the epidermis, The two enzymes pres ent a single structural difference; DTH II specifically contains a very aci dic segment of 71 amino acids inserted in the regulatory domain. We show he re that the enzymatic and regulatory properties of vertebrate TH are genera lly conserved in insect TH and that the isoform DTH II presents unique char acteristics. The two DTH isoforms were expressed as apoenzymes in Escherich ia 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 t o be activated, The two isoforms also have a different pH profile. As for m ammalian TH, enzymatic activity of the Drosophila enzymes is decreased by d opamine binding, and this effect is dependent on ferrous iron levels. Howev er, DTH II appears comparatively less sensitive than DTH I to dopamine inhi bition. The central nervous system isoform DTH I is activated through phosp horylation by cAMP-dependent protein kinase (PKA) in the absence of dopamin e. In contrast, activation of DTH II by PKA is only manifest in the presenc e of dopamine. Site-directed mutagenesis of Ser(32), a serine residue occur ring in a PKA site conserved in all known TH proteins, abolishes phosphoryl ation of both isoforms and activation by PKA. We propose that tissue-specif ic alternative splicing of TH has a functional role for differential regula tion of dopamine biosynthesis in the nervous and non-nervous tissues of ins ects.