12 Coleman and Neckameyer Archives of Insect Biochemistry and Physiology 59:12–31 (2005) Serotonin Synthesis by Two Distinct Enzymes in Drosophila melanogaster Chandra M. Coleman and Wendi S. Neckameyer* Annotation of the sequenced Drosophila genome suggested the presence of an additional enzyme with extensive homology to mammalian tryptophan hydroxylase, which we have termed DTRH. In this work, we show that enzymatic analyses of the putative DTRH enzyme expressed in Escherichia coli confirm that it acts as a tryptophan hydroxylase but can also hydroxylate phenylalanine, in vitro. Building upon the knowledge gained from the work in mice and zebrafish, it is possible to hypothesize that DTRH may be primarily neuronal in function and expression, and DTPH, which has been previously shown to have phenylalanine hydroxylation as its primary role, may be the peripheral tryptophan hydroxylase in Drosophila. The experiments presented in this report also show that DTRH is similar to DTPH in that it exhibits differential hydroxylase activity based on substrate. When DTRH uses tryptophan as a substrate, substrate inhibition, catecholamine inhibition, and decreased tryptophan hydroxylase activity in the presence of serotonin synthesis inhibitors are observed. When DTRH uses phenylalanine as a substrate, end product inhibition, increased phenylalanine hydroxylase activity after phosphorylation by cAMP-dependent protein kinase, and a decrease in phenylalanine hydroxylase activity in the presence of the serotonin synthesis inhibitor, αmethyl-DL-tryptophan are observed. These experiments suggest that the presence of distinct tryptophan hydroxylase enzymes may be evolutionarily conserved and serve as an ancient mechanism to appropriately regulate the production of serotonin in its target tissues. Arch. Insect Biochem. Physiol. 59:12–31, 2005. © 2005 Wiley-Liss, Inc. KEYWORDS: Drosophila; aromatic amino acid hydroxylase; serotonin; tryptophan hydroxylase INTRODUCTION Biogenic amines, which include dopamine and serotonin, are derivatives of amino acids. The biogenic amines are formed by the actions of the aromatic amino acid hydroxylase superfamily members, which include tryptophan hydroxylase (TRH) (EC 1.14.16.4), tyrosine hydroxylase (TH) (EC 1.14.16.2), and phenylalanine hydroxylase (PAH) (EC 1.14.16.1). TRH catalyzes the synthesis of 5-hydroxytryptamine from tryptophan, the first and rate-limiting step in serotonin (5-hydroxytryptophan, 5-HT) biosynthesis, and is thus a marker for 5-HT synthesis (Fig. 1). TH catalyzes the formation of hydroxy-tyrosine (L-DOPA), the first and rate-limiting step in dopamine biosynthe- sis. The third member of the aromatic amino acid hydroxylase family, PAH, is a metabolic enzyme that catalyzes the conversion of phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. In insects, the biogenic amines act as both neuromodulators and neurotransmitters (Brown and Nestler, 1985). In lower vertebrates and in invertebrates, 5-HT neurons are found not only in the brain but in the ventral spinal cord and thoracic ganglia. The number of these neurons is small (about 100 in Drosophila) but, as in higher vertebrates, these neurons send projections to most parts of the nervous system (Valles and White, 1988). In addition to being widely distributed, 5HT activates multiple receptor subtypes that are dif- Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, St. Louis, Missouri Contract grant sponsor: NIGMS; Contract grant number: T32 GM008306. *Correspondence to: Wendi S. Neckameyer, Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. E-mail neckamws@slu.edu Received 15 September 2004; Accepted 11 January 2005 © 2005 Wiley-Liss, Inc. DOI: 10.1002/arch.20050 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila 13 Fig. 1. Pathways for the production of dopamine and serotonin. Shown are the dopamine and serotonin synthesis pathways. The enzymes involved in these pathways are also highlighted. It has been shown that DTPH is involved in the dopamine and serotonin synthesis pathways. ferentially expressed and coupled to different intracellular signaling systems (Saudou et al., 1992). 5-HT has been implicated in the control of salivation in the adult female mosquito; since 5-HT innervation is absent in male salivary glands, it has been suggested that 5-HT is also involved in bloodfeeding in these animals (Novak and Rowley, 1994). In Drosophila melanogaster, the serotonergic neurons have been localized and have been implicated in salivary gland secretion, heart and oviduct contractions, circadian rhythms, and learning and memory (Brown and Nestler, 1985). 5-HT is one of the first transmitter systems to appear during development, and is involved in proliferation and differentiation of neuronal and other target areas (Ballion et al., 2000). 5-HT plays a role in the modulation of sleep, aggression, sexual activity, appetite, learning, and memory (Azmtitia and Whitaker-Azmtitia, 1991). Clinically, 5-HT has been implicated in depression, obsessive compulsive behavior, anorexia, bulimia, sudden infant death syndrome, Alzheimer’s disease, and schizophrenia (Azmitia and Segal, 1978; Peroutka, 1990). The broad range of functions for 5-HT complements the extensive anatomy of the serotonergic neurons; in mammals, the brain serotonin system is the single largest brain system known (Azmtitia and Whitaker-Azmtitia, 1991). We have previously shown that in Drosophila, one enzyme (Drosophila tryptophan-phenylalanine hydroxylase, DTPH) is capable of hydroxylating both tryptophan and phenylalanine. DTPH exhibits differential hydroxylase activity based solely on May 2005 substrate (Coleman and Neckameyer, 2004). When DTPH uses phenylalanine as a substrate, regulatory control is observed that is not seen when tryptophan is used as a substrate. These studies suggest that regulation of DTPH enzymatic activity occurs, at least in part, through the actions of its substrate. Recent studies in mice (Walther et al., 2003) suggest the presence of two distinct tryptophan hydroxylase isoforms, Tph 1 and Tph 2. Tph1 is expressed in the duodenum, and to a lesser extent, in the brain, and represents the nonneuronal tryptophan hydroxylase gene; Tph2 is expressed exclusively in the brain and is considered the neuronal form. It has been shown that disruption of Tph1 leads to a loss of peripheral 5-HT; mice lacking Tph1 exhibit abnormal cardiac activity, which ultimately leads to heart failure in these animals (Côté et al., 2003). Tph2 plays a fundamental role in 5HT synthesis in the CNS (Zhang et al., 2004). In zebrafish, two tryptophan hydroxylase genes have been found that are expressed in the diencephalon of the developing zebrafish brain. Surprisingly, neither gene is expressed in the raphe nuclei, suggesting that additional tryptophan hydroxylase genes may exist, at least in zebrafish (Bellipanni et al., 2003). Annotation of the sequenced Drosophila genome suggested the presence of an additional enzyme with extensive homology to mammalian tryptophan hydroxylase, which we have termed DTRH. In this work, we show that enzymatic analyses of the putative DTRH enzyme expressed in E. coli confirm that it acts as a tryptophan hydroxylase, but can also hydroxylate phenylalanine in vitro. Build- 14 Coleman and Neckameyer ing upon the knowledge gained from the work in mice and zebrafish, it is possible to hypothesize that DTRH may be primarily neuronal in function and expression, and acts as Tph2 does in mice. We have hypothesized that DTPH, which may have phenylalanine hydroxylation as its primary role, is the peripheral tryptophan hydroxylase in Drosophila and acts similarly to Tph1 in mice. The experiments presented in this report also show that DTRH is similar to DTPH in that it exhibits differential hydroxylase activity based on substrate. When DTRH uses tryptophan as a substrate, substrate inhibition, catecholamine inhibition, and decreased tryptophan hydroxylase activity in the presence of serotonin synthesis inhibitors are observed. When DTRH uses phenylalanine as a substrate, end product inhibition, increased PAH activity after phosphorylation by cAMP-dependent protein kinase (PKA), and a decrease in PAH activity in the presence of the 5-HT synthesis inhibitor, α-methyl-DL-tryptophan (AMTP) are observed. These experiments suggest that the presence of distinct tryptophan hydroxylase enzymes may be evolutionarily conserved, and serve as an ancient mechanism to appropriately regulate the production of 5-HT in its target tissues. EXPERIMENTAL PROCEDURES Generation of Constructs and Preparation of Extracts We identified and obtained a cDNA clone, GH12537, from the Berkeley Drosophila Genome Project, corresponding to the gene CG9122, which encodes the transcript CT9937. This transcript encodes a protein with significant homology to mammalian tryptophan hydroxylase (56.8% when compared to Tph1 and 57.2% when compared to Tph2; Genome Annotation Database of Drosophila). The sequencing of the cDNA was confirmed since the annotation cited the protein as having 555 amino acids; the other amino acid hydroxylase family members are between 450 and 500 amino acids in length. It has also been stated that there are numerous and significant discrepancies in the annotation of the Drosophila genome, which calls for caution in interpreting predicted Drosophila genes (Karlin et al., 2001). The cDNA encoding the presumptive full-length DTRH was subjected to Polymerase Chain Reaction (PCR) to generate a fragment containing only the coding region flanked by 5′-Eco RI-Nde I and Nde I-Eco RI-3′ sites. The Eco RI fragment was subcloned into the SK+ bacterial phagemid (Stratagene, La Jolla, CA), and the sequence was confirmed to ensure no errors had been introduced during the PCR process. This clone was then digested with Nde I and subcloned into the bacterial expression vector pET11a (Stratagene), and transformed into the E. coli strain BL21/DE3; the Nde I site creates an initiator methionine codon at the beginning of the DTPH coding region. Log-phase cells were induced with isopropylthio-β-D-galactoside (IPTG, Gibco BRL, Gaithersburg, MD) (0.6 mg/ml culture) and allowed to grow for an additional 2.5 h. The cultures were spun at 5,000g for 10 min and the cell pellet was resuspended in 1/ 10 volume Buffer A (50 mM Tris-HCl, pH 7.0, 1 mM dithiothreitol [DTT], and 4 µg/ml each of aprotonin, leupeptin, and phenymethylsulfonyl fluoride [PMSF]). An equal volume of this buffer containing 5 mg/ml lysozyme was added to the resuspended pellet. The samples were gently mixed and allowed to rotate for 30 min at 4°C, followed by the addition of 50 µg/ml DNAse I (Sigma, St. Louis, MO). The sample was rotated at room temperature for 10 min, followed by a centrifugation at 2,500g for 5 min. The final pellet was resuspended in buffer A (1/10 original culture volume); aliquots were quick-frozen in liquid nitrogen and stored at –80°C. The protein concentration of the prepared extract was determined using a modified Bradford Assay (Bio-Rad, Richmond, CA). Southern Analysis Four micrograms of wild-type Canton S genomic DNA was digested with various restriction enzymes, electrophoresed through a 0.7% agarose gel, transferred to nitrocellulose filter paper, and probed with either 32P-DTRH or 32P-DTPH cDNA containing the complete coding region for these Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila enzymes. Moderate stringency hybridization conditions of the filters were performed at 42°C in 50% formamide, 3 × SSPE, 50 mM Tris-HCL (pH 7.4), 1 × Denhardt’s, 1 mM EDTA, and 20 µg/ml salmon sperm DNA. The filters were then washed at 65°C in 2 × SSPE, 0.2% SDS. The same conditions were used to identify both DTH and DTPH using probes generated from the mammalian homologues (Neckameyer and Quinn, 1988; Neckameyer and White, 1992). 15 Kinase Experiments Twenty-five micrograms of protein extract was incubated for 5 min at 30°C in kinase buffer (5 mM Tris-HCl, pH 7.0, 2 mM MgCl2, 0.5 mM spermidine, 0.1 mM EDTA, 0.1 mM EGTA, 0.25 mM cAMP, 0.5 mM ATP) in the presence or absence of 18.2 pmol units of the catalytic subunit of PKA (Sigma) prior to addition of the reaction mixture. End Product Inhibition Assay of Enzymatic Activity Protein extracts (0.1 to 100 µg) were added to the assay reaction composed of 50 mM HEPES (pH 7.0), 50 µM substrate (L-tryptophan or L-phenylalanine), 5 mM dithiothreitol, 10 µM Fe(NH4)2(SO4)2, 50 µM BH4 (the biopterin cofactor), 0.1 mg/ml catalase, and 1 µCi 3H-tryptophan or phenylalanine (~30 Ci/mmol; Amersham, Arlington Heights, IL). The reactions (final volume of 50 µl) were incubated at room temperature for 10 min. A 10× volume of 7.5% charcoal in 1M HCl (DarcoG-60) was added to stop the reaction. The mixture was vortexed and the supernatant clarified by centrifugation at 14,000g for 2 min, and an aliquot of the clarified supernatant was carefully removed to a vial containing scintillation fluid for counting. To establish background levels, a 10× volume 1M HCl was added to tubes containing only the reaction mix without the crude protein extract. Each point was performed in duplicate; the data represents of the average value of each point. Each assay was repeated 4–6 times. Substrate Concentration Curves Substrate (phenylalanine or tryptophan) was added at a range of 1 µM to 200 µM using 25 µg protein extract and 250 µM BH4. Cofactor Concentration Curves Tetrahydrobiopterin (BH4) was added at a range of 1 to 200 µM using 25 µg protein extract and 50 µM substrate. May 2005 Twenty-five micrograms of protein extract was preincubated on ice with 10 µM Fe (NH4)2(SO4)2, 50 mM HEPES (pH 7.0), and 200 µM dopamineHCl, serotonin-HCl, or L-tyrosine, for 15 min prior to addition of the reaction mixture. Incubation of DTRH With a-Methyl Tryptophan and Parachlorophenylalanine Twenty-five micrograms of protein extract was incubated on ice with Fe(NH4)2(SO4)2, 50 mM HEPES (pH 7.0) and 200 µM α-methyl-DL- tryptophan or parachlorophenylalanine (Sigma) for 15 min prior to addition of the assay mixture. Preparation of Antibody Against DTRH Protein to be used as antigen was generated by inducing log-phase cells (pET11a-DTRH in BL21 DE3) with IPTG (0.6 mg/ml culture) and allowing them to grow at 37°C for an additional 2.5 h. The protein was pelleted, resuspended in 1× SDSreducing buffer, and loaded onto a 1.5-mm 10% SDS-polyacrylamide gel. After electrophoresis, the gel was submerged in ice-cold 250 mM KCl for at least 20 min. The bands representing bacteriallygenerated DTRH (which were recognized by molecular weight and the banding pattern) were excised, minced in phosphate buffered saline [PBS, 34.24 mM NaCl, 6.626 mM KCl, 3.6737 mM KH2PO4, 18.386 mM NAH2PO4 (pH 7.0)], mixed with an equal volume of Freund’s adjuvant, and injected subcutaneously into two New Zealand White rabbits. After serum collection and clot re- 16 Coleman and Neckameyer moval, the serum was clarified by centrifugation at 7,600g at 4°C for 10 min. The antibodies were stored at –20°C after quick freezing in a dry iceethanol bath. The recognition pattern of the DTRH antibodies by Western analyses of in vitro generated protein is identical, so results from only one antibody are reported in this study. Cross-Linking of Antibody DTRH antiserum (~100 µg) was bound to 300 µl of Protein A sepharose beads in PBS for 1 h at room temperature with gentle mixing. The beads were washed 2× with 0.2M NaBO4 (pH 9) and suspended in a final volume of 1 ml 0.2M NaBO4 (pH 9). Dimethylpimelmidate (6.4 mg) was added to the solution and incubated for 30 min at room temperature with gentle rotation. The beads were gently pelleted and resuspended in 0.2 M ethanolamine (pH 8), followed by a 2-h incubation at room temperature with gentle rotation. The beads were then washed 3 times with PBS and stored at 4°C (final volume, 1 mL). Before use, the beads were washed 6× with an equal volume of 300 mM glycine (pH 3). Two PBS washes followed to neutralize the pH. Immunoprecipitation One milliliter of IP buffer (1 × PBS, 0.1 µg/ml leupeptin, 10 mM PMSF, 1 mM sodium ortho-vanadate, 50 mM sodium fluoride, 6% sucrose) was added to 25 µl of cross-linked beads and 30 µl of DTRH protein (~200 µg), incubated at 4°C for a period of 4–18 h, then centrifuged briefly to bring down the beads. The beads were then washed 3 times in washing buffer (1 × PBS, 30 mM PMSF, 6% sucrose). SDS non-reducing buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.2% bromophenol blue) was added to the samples, after which the tubes were placed at 100°C for 5 min, spun briefly, and the supernatants were transferred to new tubes. For samples that were analyzed by 2D, the markers used were IEF mix pH 3.6–9.3 (Sigma), two-dimensional electrophoresis markers (pI range 7.6–3.8; MW range 17,000–89,000; Sigma), and glyceraldehyde-3-phosphate dehydrogenase (Sigma). Analysis of 32P Incorporation of Kinased Recombinant DTPH Phosphorylated by cAMP-Dependant Protein Kinase Ten micrograms of pET-DTRH extract was incubated in kinase buffer (5 mM Tris-HCl, pH 7.0, 2 mM MgCl2, 0.5 mM spermidine, 0.1 mM EDTA, 0.1 mM EGTA, 0.05 mM cAMP), 3.3 µM ATP mix (2 µCi γ-32P-ATP + 1 mM ATP ), and 18.2 pmol units of PKA (Sigma). The samples were placed at 30°C for 15 min and were terminated by the addition of 2 × SDS-reducing buffer. Inhibition of Phosphorylation by PKA Ten micrograms of pET-DTRH extract was incubated in kinase buffer in the presence or absence of 18.2 pmol units of PKA. Protein kinase inhibitor (18.2 pmol units, from rabbit, Sigma) was added to one set of reactions. The samples were loaded onto 10% SDS-PAGE gels and electrophoresed at 135 V for 10 h. The gels were dried for 1 h at 80°C and exposed to autoradiographic film (XOMAT-AR, Kodak) at –80°C. 2-Dimensional Polyacrylamide Gel Electrophoresis The first dimension was run with a 20 mM NaOH upper running buffer (cathode), and a 10 mM H3PO4 lower running buffer (anode), and was carried out in cylindrical rod isoelectric focusing gels cast in glass capillary tubes (30% acrylamide with 4% crosslinker in 8M urea and 2% nonidet P-40). The first dimension gels were run at 800V (~1.7 mA) for 16 h and a water-filled syringe was used to extract the gels from the capillary tubes. The second dimension was an overlay of the first dimension capillary gels on 10% SDS-PAGE vertical slab gels. To analyze 32P incorporation into the Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila separated proteins, the gels were dried for 1 h at 80°C and exposed to autoradiographic film (XOMAT-AR, Kodak) at –80°C. Western analysis was performed on unlabelled phosphorylated extracts using standard procedures. The DTRH antibody was used at a dilution of 1:2,000 and the secondary antibody at a dilution of 1:5,000 (αrabbit IgG, horseradish peroxidase linked whole antibody, from donkey, Amersham). The blots were then incubated for 1 min with ECL chemiluminescent substrate (Amersham) and exposed to film (X-OMAT AR, Kodak). RESULTS Sequence Alignment of DTRH With TRH, PAH, and DTPH DTPH, a dual-function enzyme capable of hydroxylating both tryptophan and phenylalanine, was originally hypothesized to be the only enzyme in Drosophila capable of these activities. Analysis of the Drosophila genome project identified an additional transcript with striking homology to mammalian tryptophan hydroxylase. This cDNA localized to 61F on the left arm of the third chromosome, while DTPH is located at position 66A. Although the cDNA sequence of DTPH shares greater DNA sequence homology with mammalian tryptophan hydroxylase than does DTRH, the protein sequence of DTRH is more highly conserved with mammalian tryptophan hydroxylase. This would explain why only DTPH was identified during the library screen using mammalian tryptophan hydroxylase as probe (Neckameyer and White, 1992). There is 56.8% identity between DTRH and Tph1 (Fig. 2a), 57.2% identity between DTRH and Tph2 (Fig, 2b), and 47.9% identity between DTRH and mammalian PAH. There is 52.8% identity between DTRH and DTPH (data not shown) and 43.9% identity between DTRH and DTH (data not shown). There is 55.9% identity between DTPH and Tph1, 54.8% identity between DTPH and Tph2 (data not shown), and 64.7% between DTPH and PAH (see Table 1). These comparisons suggest that DTRH is more closely related to mammalian tryptophan hydroxylase than is DTPH. May 2005 17 DTRH and DTPH Recognize Distinct and NonOverlapping Patterns by Southern Analysis Genomic DNA was digested with restriction enzymes, electrophoresed through an agarose gel, transferred to nitrocellulose, and probed with either 32P-DTRH or 32P-DTPH using the same conditions used to identify DTPH using mammalian tryptophan hydroxylase as probe (Neckameyer and White, 1992). The pattern of restriction fragments in Drosophila genomic DNA recognized by the fulllength rabbit tryptophan hydroxylase cDNA is the same as that recognized by DTPH (Neckameyer and White, 1992); this is also the same pattern observed using mammalian PAH as probe (Morales et al., 1990). A pattern distinct from that of DTPH is detected by DTRH (Fig. 3). The observed patterns are indicative of a single gene for each locus, and suggest that the gene at 61F is not simply a duplication of the gene at 66A. Recombinant DTRH Exhibits Substrate Specificity, Responds Differently to Increasing Concentrations of Substrate, and Exhibits Time Dependence. Increasing amounts of E. coli protein extracts containing the induced DTRH protein (1–100 µg) were assayed for the ability to hydroxylate tryptophan, phenylalanine, or tyrosine (Fig. 4a). When similarly induced E. coli containing the pET-11a plasmid was assayed using the same substrates, no detectable activity was observed (data not shown), consistent with the fact that these bacteria do not contain iron-dependent aromatic amino acid hydroxylases or monoamine biosynthetic machinery. Therefore, any hydroxylation activity detected can be solely attributed to the introduction of the pETDTRH plasmid. DTRH exhibited high levels of tryptophan hydroxylase activity (average specific activity = 1.62 pmol product/minute/mg protein), but was also capable of hydroxylating phenylalanine (average specific activity = 0.52 pmol product/min/ mg protein). DTRH has no detectable TH activity in vitro and thus is unlikely to use tyrosine as a substrate. This confirms that DTRH is a dual-function hydroxylase and not a promiscuous enzyme. 18 Coleman and Neckameyer Fig. 2. Alignment of DTRH with mammalian Tph1 and Tph2. a: Amino acid number is given preceding each row of sequence for Drosophila TRH (DTRH) and mammalian Tph1. b: Tph2. Best-fit analysis was performed to determine the regions of similarity between DTRH, DTPH, Homo sapiens Tph1, and Homo sapiens Tph2 [GenBank Ac- cession numbers NM_004179 (Tph1), NM_173357 (Tph2)]. Canonical PKA sites are underlined and the associated serine residue is denoted in boldface. c: PKA phosphorylation sites are denoted by asterisks (*) and to show regions of similarity between DTRH and Tph1 and Tph2. DTRH was then incubated with increasing concentrations of substrate (1 to 200 µM of tryptophan, phenylalanine, or tyrosine). No enzymatic activity was seen below 10 µM substrate, demonstrating that enzymatic activity does not occur in the absence of substrate (Fig. 4b). Substrate inhibition was observed in the tryptophan hydroxylase reaction; this inhibition is also observed with mammalian TRH (Johanssen et al., 1991). pET-DTRH tryptophan and phenylalanine hydroxylase activities also increase with increasing time (0–30 min) (Fig. 4c). The kinetic parameters of DTRH were determined for tryptophan and phenylalanine hydroxylation (data not shown). The Km for DTRH tryptophan hydroxylase activity is 4.72 µM compared with a Km for mammalian TRH of 12.5–32 µM for the substrate tryptophan (Hufton et al., 1995). The Km for DTRH phenylalanine hydroxylase activity is 9.46 µM compared with a Km for mammalian PAH of 200– 300 µM for the substrate phenylalanine (Hufton et al., 1995) (data not shown). We estimate the induced DTRH protein comprises about 50% of Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila Fig. 2. May 2005 continued 19 20 Coleman and Neckameyer TABLE 1. Identity Comparisons of DTRH and DTPH With Other Aromatic Amino Acid Hydroxylases % Identity* DTRH Tph1 Tph2 PAH DTPH DTH 56.8 57.2 47.9 52.8 43.9 DTPH Tph1 Tph2 PAH DTRH DTH 55.9 54.8 59.9 52.8 47.8 *The % identity of DTRH and DTPH is shown when compared to Tph1 and Tph2 and PAH, all from Homo sapiens. Also shown is the % identity between DTRH and DTPH as well as their identity with DTH, Drosophila tyrosine hydroxylase. the E. coli protein preparation (data not shown). That the Km for DTRH tryptophan hydroxylase activity is more consistent with the reported values for mammalian tryptophan hydroxylase suggests that DTRH tryptophan hydroxylase activity is more similar to that of mammalian tryptophan hydroxylase than DTRH phenylalanine hydroxylase activity is to that of PAH. For direct comparisons of the phenylalanine and tryptophan hydroxylase activities, all subsequent assays were performed under the same conditions (10 min; 50 µM substrate). Determining the Cofactor, Iron and Catalase Requirements for DTRH The aromatic amino acid hydroxylase superfamily members are characterized by their ability to bind reduced iron for optimal enzymatic activity; the tetrahydrobiopterin cofactor maintains iron in the reduced form. The enzymatic activity of DTRH was assessed in the absence of the tetrahydrobiopterin cofactor, reduced iron and catalase (Fig. 5). Very small amounts of activity can be detected in the absence of cofactor (specific activity = 0.38 pmol 5-OH tryptophan/min/mg protein; specific activity = 0.29 pmol tyrosine/min/mg protein), iron (specific activity = 0.22 pmol 5-OH tryptophan/minute/mg protein; specific activity = 0.30 pmol tyrosine/min/mg protein) and catalase (specific activity = 0.23 pmol 5-OH tryptophan/ minute/mg protein; specific activity = 0.29 pmol tyrosine/min/mg protein). Significant activity is only seen in the presence of all assay components (specific activity = 1.59 pmol 5-OH tryptophan/ min/mg protein; specific activity = 0.845 pmol tyrosine/min/mg protein). The kinetic parameters of DTRH for the cofactor were also determined for both phenylalanine and tryptophan hydroxylation. The Km for the tryptophan hydroxylase activity of DTRH is 3.07 µM Fig. 3. DTPH and DTRH represent the products of two distinct genetic loci in Drosophila. Genomic DNA was digested with restriction enzymes, electrophoresed through a 0.7% agarose gel, transferred to nitrocellulose, and probed with either (a) 32P-DTRH or (b) 32P-DTPH. Moderate stringency hybridizations conditions of the filters were performed at 42°C in 50% formamide,3 × SSPE, 50 mM TrisHCl (pH 7.4), 1 × Denhardt’s, 1 mM EDTA, and 20 µg/ml salmon sperm DNA. The filters were then washed at 65°C in 2 × SSPE, 0.2% SDS. Equivalent exposures are shown. B, Bam HI; E, Eco RI; H, Hind III; P, Pst I; Pv, Pvu II; Sc, SacI; Sm, Sma I. Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila 21 compared with a Km for mammalian tryptophan hydroxylase of 20–30 µM for the cofactor (21) (data not shown). The Km for phenylalanine hydroxylase activity of DTRH is 2.03 µM compared with a Km for the mammalian PAH of 15–25 µM for the cofactor (21) (data not shown). Given the estimation that the induced DTRH protein comprises 50% of the crude E. coli protein preparation, the Kms for the cofactor for DTRH tryptophan and phenylalanine hydroxylase activities are consistent with what has been observed for the mammalian enzymes. The kinetic data, in summary, suggest that DTRH is very likely to function as a real hydroxylase in vivo. Effects of Reaction End Products on Tryptophan and Phenylalanine Hydroxylase Activities of DTRH We examined DTRH enzymatic activity in the presence of serotonin, dopamine, and tyrosine (the Fig. 4. Recombinant DTRH responds differently to increasing concentrations of protein and substrate, and exhibits time dependence and substrate specificity. pET-DTRH protein extract was incubated using standard assay conditions with phenylalanine, tryptophan, or tyrosine as substrate. (a) Increasing concentrations of the recombinant protein (1–100 µg, with 50 µM substrate) and (b) increasing concentrations of substrate (1 to 200 µM phenylalanine, tryptophan, or tyrosine with 25 µg protein) were added to the reaction mixture and incubated at 25°C for 10 min. c: The enzymatic activity of pET-DTRH was also examined at increasing time intervals (0–30 min with 25 µg protein and 50 µM substrate incubated at 25°C) DTRH exhibits high levels of tryptophan hydroxylase activity (specific activity = 1.62 pmol product/minute/mg protein), while also showing a significant ability to hydroxylate phenylalanine (specific activity = 0.52 pmol); DTRH has no detectable TH activity in vitro. Substrate inhibition is observed for tryptophan hydroxylation; there is no apparent substrate inhibition for DTRH phenylalanine hydroxylation. d: 25 µg protein and 50 µM substrate were incubated under standard conditions. Five replicate assays were performed for each concentration point. Values represent the mean of replicate samples under the specified conditions. Standard error of the mean is denoted by the error bars. May 2005 22 Coleman and Neckameyer Fig. 5. Optimal recombinant DTRH activity requires cofactor and iron. pET-DTRH protein extract (25 µg) was incubated using standard assay conditions as described in Materials and Methods and assayed for (a) tryptophan hydroxylase activity or (b) phenylalanine hydroxylase activity. The assay was also performed in the absence of the tetrahydrobiopterin cofactor or reduced iron. Though some activity can be detected in the absence of these components, significant activity is only seen in the presence of BH4, catalase, and reduced Fe. Activity is expressed in pmol product produced per minute per mg protein. *P < 0.0001. end products of the tryptophan and phenylalanine hydroxylase reactions) with both tryptophan (Fig. 6) and phenylalanine (Fig. 7) as substrate. Both the tryptophan and the phenylalanine hydroxylase activities of DTRH are inhibited by dopamine and tyrosine (Fig. 6), the end products of the phenylalanine hydroxylase reaction, but not by serotonin, the end product of the tryptophan hydroxylase reaction (Fig. 7). Fig. 6. DTRH tryptophan hydroxylation is subject to end product inhibition by dopamine and tyrosine, but not serotonin. pET-DTRH (25 µg) was incubated for 10 min at 25°C following a 15-min preincubation in the presence or absence of (a) serotonin, (b) dopamine, or (c) tyrosine. End product inhibition is observed in the tryptophan hydroxylase reaction when DTRH is assayed in the presence of dopamine and tyrosine, while preincubation with serotonin has no effect on tryptophan hydroxylase activity. *P < 0.005. Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila 23 cAMP-Dependant Protein Kinase (PKA) Phosphorylation of DTRH Phosphorylation of DTRH by the catalytic subunit of PKA was carried out in the presence of cold or radioactive ATP and analyzed by Western analysis or autoradiography (data not shown). The BL21/ DE3 strain of E. coli lacks several kinases (including PKA), so any induced proteins are unphosphorylated. Our data show that DTRH is capable of being phosphorylated in vitro by PKA; this phosphorylation is not observed when DTRH is incubated in the presence of both PKA and a specific inhibitor of PKA (Fig. 8). DTRH incubated under phosphorylation conditions with PKA exhibits five distinct isoforms revealed by two-dimensional electrophoresis and Western blot analysis (Fig. 9); these isoforms are in the range of 61-kDa molecular mass and have a pI of 5–6, which is consistent with the predicted pI for this protein of 5.61. The same pattern is seen following 32P incorporation (data not shown). Fig. 7. DTRH phenylalanine hydroxylation is subject to end product inhibition by dopamine and tyrosine, but not serotonin. pET-DTPH (25 µg) was incubated for 10 min at 25°C with phenylalanine as substrate following a 15-min preincubation in the presence or absence of (a) serotonin, (b) dopamine, or (c) tyrosine.**P < 0.0001, *P < 0.001. May 2005 Fig. 8. In vitro phosphorylation of DTRH by PKA. pETDTRH extract (10 µg) was incubated with 32P-γ-ATP in the presence or absence of 18.2 pmol units of catalytic subunit of PKA. Protein kinase inhibitor was added to one set of reactions. Following the kinase assay, the samples were immunoprecipitated using antibodies raised against DTRH, and electrophoresed through a 10% SDS-PAGE gel, which was dried and exposed to autoradiographic film. 24 Coleman and Neckameyer Fig. 9. Two-dimensional electrophoretic analysis of PKA phosphorylation of DTRH. Recombinant DTRH protein extract (10 µg) was used in each experiment. The recombinant protein was subjected to phosphorylation by PKA for 15 min at 30°C as described in Experimental Proce- dures, followed by immunoprecipitation with the DTRH antibodies, then analyzed by Western immunoblot analysis. The pH scale is denoted above the figure and highlights that DTRH has a pI of 5.61. We also examined the enzymatic activity of DTRH following phosphorylation by PKA. DTRH tryptophan hydroxylation is unaffected by PKA phosphorylation (Fig. 10a), while phosphorylation by PKA causes a significant increase in phenylalanine hydroxylase activity (Fig. 10b; specific activity in the absence of PKA = 0.17 pmol 5-OH tryptophan/min/ mg protein; specific activity in the presence of PKA = 1.47 pmol 5-OH tryptophan/min/mg protein). Effect of Serotonin Synthesis Inhibitors on DTRH Tryptophan and Phenylalanine Hydroxylation We have examined the enzymatic activity of DTRH following incubation with the serotonin synthesis inhibitors α-methyl-DL- tryptophan (AMTP) and parachlorophenylalanine (pCPA) using tryptophan (Fig. 11a,b) or phenylalanine (data not shown) as substrate. The inhibitors significantly affected DTRH tryptophan hydroxylase activity, but the phenylalanine hydroxylase activity was significantly affected by AMTP (specific activity in the absence AMTP = 1.29 pmol 5-OH trp/min/mg protein; specific activity in the presence of AMTP = 0.0075 ;specific activity in the absence of pCPA = 1.013 pmol 5-OH trp/min/mg protein; specific activity in the presence of pCPA = 0.081 pmol 5-OH trp/min/protein) (specific activity in the absence of AMTP = 0.665 pmol tyr/min/mg protein; specific activity in the presence of AMTP = 0.154; specific activity in the absence of pCPA = 0.664 pmol tyr/min/mg protein; specific activity in the presence of pCPA = 0.691 pmol tyr/min/mg protein). Fig. 10. PKA phosphorylation has no effect on DTRH tryptophan hydroxylase activity and increases DTRH phenylalanine hydroxylase activity. pET-DTRH protein extract (25 µg) was incubated using (a) tryptophan or (b) phenylalanine as substrate for 10 min at 30°C following a 15-min preincubation in the presence or absence of PKA. *P < 0.0001. Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila 25 Fig. 11. The effect of serotonin synthesis inhibitors on DTRH tryptophan hydroxylation and phenylalanine hydroxylation. pET-DTRH (25 µg) was incubated for 10 min at 25°C following a 15-min preincubation in the presence or absence of (a) AMTP or (b) pCPA. AMTP and pCPA cause a decrease in DTRH tryptophan hydroxylase activ- ity. pET-DTPH (3 µg) was incubated for 10 min at 25°C following a 15-min preincubation in the presence or absence of (c) AMTP or (d) pCPA. AMTP has no effect on DTPH tryptophan hydroxylase activity and pCPA cause a increase in DTPH tryptophan hydroxylase activity. **P < 0.0001, *P < 0.05. The effect of these inhibitors on DTPH enzymatic activity was also examined using tryptophan (Fig. 11c,d) or phenylalanine (data not shown) as substrate. The tryptophan hydroxylation of DTPH was not significantly affected by AMTP (specific activity in the absence of AMTP = 0.326 nmol tyr/ min/mg protein; specific activity in the presence of AMTP = 0.331 nmol tyr/min/mg protein. It is interesting to note that the tryptophan hydroxylase activity of DTPH was significantly increased in the presence of pCPA (specific activity in the absence of pCPA = 0.217 nmol 5-OH tryptophan/ min/mg protein; specific activity in the presence of pCPA = 0.431 nmol 5-OH tryptophan/min/mg protein). The phenylalanine hydroxylase activity of DTPH was significantly affected by AMTP, but not affected by pCPA (specific activity in the absence of AMTP = 0.738 nmol tyr/min/mg protein; specific activity in the presence of AMTP = 0.212 nmol tyr/min/mg protein; specific activity in the May 2005 26 Coleman and Neckameyer presence of pCPA= 0.478 nmol tyr/min/mg protein; specific activity in the presence of pCPA = 0.433. DISCUSSION It was originally thought that there were only two amino acid hydroxylases in Drosophila: DTH, which hydroxylates tyrosine, and DTPH, a dualfunction enzyme responsible for the hydroxylation of both tryptophan and phenylalanine (Neckameyer and White, 1992). This was attributed to the hypothesis that tyrosine hydroxylase diverged from the ancestral gene before tryptophan hydroxylase and phenylalanine hydroxylase diverged from each other (Neckameyer and White, 1992). This hypothesis was based on several pieces of evidence: (1) DTPH exhibited phenylalanine as well as tryptophan hydroxylase activities; (2) DTPH was expressed in both dopaminergic and serotonergic neurons; (3) DTPH deletion strains phenocopied the 5-HT2Dro receptor mutants, demonstrating that it functioned in vivo as a tryptophan hydroxylase, and (4) mammalian phenylalanine hydroxylase and tryptophan hydroxylase probes only recognized DNA corresponding to the DTPH locus. Recent studies in mice have suggested the presence of two distinct tryptophan hydroxylase isoforms: Tph1 and Tph2. To study the physiological impact of the loss of 5-HT synthesis, Walther and colleagues (2003) generated mice genetically deficient for tryptophan hydroxylase (Tph –/–). These mice still expressed normal amounts of 5-HT in classical serotonergic brain regions, but lacked 5HT in the periphery except in the duodenum (which contained about 4% of normal 5-HT levels). These mice exhibited no differences in serotonergic-modulated behaviors. After database screening, a human genomic clone was obtained and this sequence was used to perform 5′ and 3′ RACE experiments with brain RNA from Tph –/– mice, and a full-length cDNA was obtained (referred to as Tph2), which was different from the known Tph (now referred to as Tph1), as well as mouse PAH and TH. Tph1 mRNA was detected in the duodenum, while Tph2 was detected exclusively in the brain. Brain stem total RNA samples from wild-type mice revealed about 150 times more Tph2 than Tph1 mRNA (Walther et al., 2003). Analysis of the Drosophila genome project identified an additional transcript, DTRH, distinct from DTPH, with striking homology to mammalian tryptophan hydroxylase. Data presented in this report provide evidence that DTRH has tryptophan hydroxylase activity in vitro. This work, and previous work from our lab, suggests that in Drosophila, two enzymes (DTRH and DTPH) must be regulated to synthesize 5-HT in the presence of the appropriate substrate. This is consistent with recent work from mammalian studies that have demonstrated the presence of at least two tryptophan hydroxylase enzymes. Alignment of the core of the deduced protein sequence of DTRH with mammalian tryptophan hydroxylase (Tph1 and Tph2; Fig. 1; Table 1) and PAH show that there is greater identity between DTRH and mammalian tryptophan hydroxylase (56.8% when compared with Tph1 and 57.2% when compared with Tph2) than between DTRH and PAH (47.9%), implying that DTRH may act primarily as a tryptophan hydroxylase in Drosophila although it has PAH activity in vitro. Given the relative sequence identity between DTRH, DTPH, and DTH, it is likely that there has been an ancient divergence from the ancestral hydroxylase gene to generate three enzymes with differential hydroxylase activities. Further evidence to support the hypothesis that DTRH may act in the same manner as Tph2 and that DTPH may act in the same manner as Tph1, was found when DTPH was compared to mammalian TRH and mammalian PAH, since there is greater identity between DTPH and mammalian PAH (59.9%) than DTPH and mammalian tryptophan hydroxylase (55.9% when compared to Tph1 and 54.8% when compared to Tph2). This evidence also supports the idea that DTPH is likely to function as the PAH in vivo. Interestingly, DTRH shares slightly more identity with Tph2, while DTPH shares slightly more identity with Tph1 (though greater identity is still seen when DTPH is compared with mammalian PAH). These slight similarities may lend credence to the Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila hypothesis that DTRH may act in a similar manner to Tph2. The observation from the Southern analysis that probes against DTRH and DTPH do not recognize the same restriction pattern not only confirms that DTRH and DTPH represent the products of distinct genetic loci, but suggests that these enzymes have differential function and activity in Drosophila, and that the divergence is indeed ancient. To examine this hypothesis, it was first important to show that DTRH and DTPH act similarly to the members of the aromatic amino acid hydroxylase family. Increasing tryptophan and phenylalanine hydroxylase activity is observed for DTRH in the presence of increasing concentrations of protein, substrate, and with increasing time. Acting similarly to mammalian TRH, substrate inhibition is seen when DTRH is assayed for its TRH activity. The observation that DTRH exhibits greater tryptophan hydroxylase activity while also displaying a significant ability to hydroxylate phenylalanine, but not tyrosine, lends further credence to the observation that tyrosine hydroxylase may be the more evolutionarily divergent hydroxylase since DTH does not hydroxylate phenylalanine or tryptophan in vitro (Neckameyer et al., 2005). Although DTRH has been shown in vitro to act as a dual-function hydroxylase, this may not be the case in vivo. The aromatic amino acid hydroxylases share structural similarities and thus common reaction mechanisms, and are characterized by their ability to bind reduced iron as well as by their requirement for a tetrahydrobiopterin cofactor. The enzymes are composed of multiple subunits, each with an iron binding site. It has been shown that iron binding is necessary for activity, and that the bound iron must be in the reduced (ferrous) state for catalysis to occur (Fitzpatrick, 1999). The cofactor for these enzymes, tetrahydrobiopterin (BH4), is necessary to keep iron in the reduced state and is, therefore, required along with molecular oxygen for the hydroxylase reactions (Fitzpatrick, 1999). Catalase is provided in the reaction as an antioxidant. Therefore, pET-DTRH was assayed in the presence or absence of reduced iron, BH4 or May 2005 27 catalase (Fig 5). Consistent with what is seen with all other analyzed aromatic amino acid hydroxylases, DTRH requires iron, cofactor, and catalase for optimal enzymatic activity to occur. We examined the inhibition profile of DTRH in the presence of serotonin, dopamine, or tyrosine (the end products of the tryptophan and phenylalanine hydroxylase reactions) with tryptophan (Fig. 6) or phenylalanine (Fig. 7) as substrate. It has been shown that all three mammalian aromatic amino acid hydroxylases are inhibited by catecholamines (Shiman, 1985; Martinez et al., 1991). It is not surprising that DTRH tryptophan hydroxylase activity is not inhibited by 5-HT since this is also observed with mammalian Tph2 prepared from rat homogenates (Johansen et al., 1991). The inhibition when assayed with dopamine points to another similarity between DTRH and mammalian tryptophan hydroxylase, and to another difference between the tryptophan hydroxylation of DTRH and that of DTPH, where no inhibition by 5-HT or dopamine is observed (Coleman and Neckameyer, 2004). TRH activity is modulated post-transcriptionally by phosphorylation (Ehret et al., 1991; Makita et al., 1990; Johansen et al., 1996), and differentially phosphorylated protein isoforms from the brainstem and pineal gland, differing in physiochemical properties, have been identified (Kim et al., 1991). PAH is regulated by phosphorylation of Serine-16 (Wretborn et al., 1980). The regulatory influence of PKA on tryptophan hydroxylase activity has long been under investigation. That tryptophan hydroxylase is phosphorylated but not activated by PKA in vitro points to an interesting parallel between mammalian tryptophan hydroxylase and the tryptophan hydroxylase activity of DTRH that is not seen with DTPH tryptophan hydroxylase activity. From the predicted amino acid sequence for DTRH, the consensus sites for PKA phosphorylation are at serines 80, 163, 183, 329, and 509. An additional canonical site for PKA phosphorylation can be found at Serine-52, but this lies outside of the region of homology with the other hydroxylase enzymes, therefore we do not expect this resi- 28 Coleman and Neckameyer due to be phosphorylated. We have observed the presence of five isoforms following two-dimensional analysis of PKA phosphorylation of DTRH (Fig. 9). The possibility exists, as has been found for PKA phosphorylation of mammalian tryptophan hydroxylase, that there is one major site of phosphorylation; in mammals, this corresponds to Serine-58, which is in the proposed N-terminal domain, and Serine-260 and -443, which are outside of the N-terminal domain, may confer conformational and, thus, regulatory controls. Upon examination of Tph2 from several species, the consensus sites for PKA are not conserved when compared with Tph1. When DTRH and Tph1 are compared, Serine-58, the major site of phosphorylation for mammalian Tph1, lies in a similar region to DTRH Serines-163 and -183 (Fig. 2a). The other PKA phosphorylation sites in Tph1 that have been identified, Serines-260 and -443, show the most identity with DTRH. Serine-260 of Tph1 perfectly aligns with Serine-329 of DTRH and Serine-443 of Tph1 closely aligns with Serine-509 of DTRH (Fig. 2a). The consensus sites for PKA phosphorylation of Tph2 lie at Serines-99, -103, 159, -228, -306, and -443. Serines-99 and -103 of Tph2 lie in a similar region to DTRH Serine-163, and Serine-159 of Tph2 shares exact consensus sequence identity and alignment with DTRH Serine183 (Fig. 2b). Serine-306 of Tph2 shares very similar identity and alignment with DTRH Serine329 and Serine-443 of Tph2 is closely associated with DTRH Serine-509 (Fig. 2b). It is also important to note that the sites for PKA phosphorylation for Tph1 are known but those for Tph2 and DTRH are only deduced. The greatest homology when comparing the PKA consensus sequences of these enzymes can be observed with Serine-260 (Serine-306 in Tph2) and Serine-443. In DTRH (as well as in DTPH, data not shown), there is greater homology at these residues than at any other consensus sites throughout the protein. These serine residues (Serines-260 and -443) have been hypothesized to confer regulatory controls, and the similarities found in the sequences in these regions again suggests that both DTPH and DTRH may share in the production of 5-HT. If the residues involved in the regulation of these enzymes lie in the region with the greatest homology, then it should follow that these enzymes must be under similar strict controls to yield the proper response in the presence of the substrate, tryptophan, and to regulate the production of 5-HT in target tissues. Phosphorylation of DTRH by PKA has no effect on tryptophan hydroxylase activity, but increases phenylalanine hydroxylase activity (Fig. 10). PKA has been shown to increase both mammalian tryptophan hydroxylase and phenylalanine hydroxylase activity (Yamauchi and Fujisawa, 1979; Abita et al, 1976), but given that previous studies were performed with what we now know is Tph1, it is difficult to draw comparisons with the mammalian enzymes. Administration of the serotonin synthesis inhibitor AMTP resulted in a decrease in serotonin content in the pineal gland and brain of rats (Sourkes et al, 1970; Montine et al, 1992.) It has been found that AMTP causes an activation of the escape activity in socially naive crickets (Dyakonova et al., 1999; Stevenson et al., 2000), but AMTPtreated crickets show a decreased ability to exhibit dominant behavior in response to tactile stimulation in a behavioral paradigm of social rank (Dyakonova et al, 1999), as well as a decrease in the intensity and duration of their fights (Stevenson et al, 2000). The host-seeking response of mosquitoes treated with AMTP was not altered, but bloodfeeding success was reduced significantly. The AMTP-treated mosquitoes responded positively when placed in close proximity to a host, but fewer treated than untreated mosquitoes fed to repletion, or fed at all, and those animals that fed to repletion took longer to do so (Novak and Rowley, 1994; Novak et al., 1995). Together, these studies show that AMTP administration results in a decrease in 5-HT synthesis, leading to aberrant responses in 5-HT regulated behaviors. pCPA is by far the most well-known and widely used inhibitor of tryptophan hydroxylase. The administration of this derivative to rats causes a rapid, simultaneous decrease in the brain levels of 5-HT and its major metabolite 5-hydroxyindoleacetic Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila acid (Koe and Weismann, 1966). TRH activity in the rat brain is irreversibly inhibited by the in vivo administration of pCPA, resulting in a long-lasting depletion of 5-HT (Koe and Weisenman, 1966). However, pCPA acts in vitro as a competitive inhibitor of TRH (Jequier et al., 1967). These differences can be explained as resulting from an in vivo interference of pCPA with the synthesis of TPH (Cortes et al., 1993). It has also been shown that pCPA administration provokes a large decrease in the concentration of TRH in the raphe nuclei (Richard et al., 1990). Systemic attempts to deplete 5-HT in the Drosophila CNS by feeding pCPA or AMTP did not result in diminished 5-HT immunoreactivity (Accardo and Neckameyer, unpublished data), although these drugs inhibit 5-HT synthesis when targeted to specific areas of the mammalian brain (Koe and Weismann, 1966; Sourkes et al., 1970; Montine et al., 1992). DTRH tryptophan hydroxylase activity was drastically inhibited by pCPA and AMTP (Fig. 11a,b), while DTPH was not (Fig. 11c,d). If DTRH does represent neuronal tryptophan hydroxylase in Drosophila, and DTPH represents peripheral tryptophan hydroxylase, then diminished neuronal 5-HT synthesis would not be observed in the presence of these inhibitors and 5-HT synthesized by peripheral DTPH could be taken up by the CNS since there is no blood-brain barrier in Drosophila. Mammalian tryptophan hydroxylase was never thought to be a strong target for drug discovery because of its wide-ranging function; however, an isoform expressed primarily in the periphery and pineal gland may be a candidate for novel antiemetic or gut motility agents. Conversely, the identification of Tph2 might foster drug development in which the neuronal form of tryptophan hydroxylase could be modulated with little or no peripheral effects. The differences in regulation, response to substrate, end product, synthesis inhibitors, and phosphorylation by PKA seen with DTRH and DTPH in vitro suggest how these enzymes may function in 5-HT synthesis in vivo, and provide a model for how the two isoforms may be regulated in mammals. The studies presented in this report allow for an initial understanding of the actions May 2005 29 of the enzymes in Drosophila and provide a framework from which to investigate the functional roles of these enzymes in vivo. ACKNOWLEDGMENTS The authors thank Dr. Alger S. Coleman, Jr., and Dr. Mattie J. Coleman for advice and encouragement. REFERENCES Abita JB, Milstein S, Chang N, Kaufman S.1976. In vitro activation of rat liver phenylalanine hydroxylase by phosphorylation. J Biol Chem 251:5310–5314. Azmitia EC, Segal M.1978. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J Comp Neurol 179:641–667. Azmitia EC, Whitaker-Azmitia PM. 1991. Awakening the sleeping giant: anatomy and plasticity of the brain serotonergic system. J Clin Psychiatry 52(Suppl):4–16. Ballion B, Branchereau P, Chapron J, Viala D. 2002. Ontogeny of descending serotonergic innervation and evidence for intraspinal 5-HT neurons in the mouse spinal cord. Brain Res Dev Brain Res 137:81–88. Bellipanni G Rink E, Bally-Cuif L. 2003. Cloning of two tryptophan hydroxylase genes expressed in the diencephalons of the developing zebrafish brain. Mech Dev 119S:S215– S220. Brown M, Nestler E. 1985. Cathecholamines and indolalkymines. In: Kerkut GA, Gilbert LJ, editors. Comprehensive insect physiology, biochemistry and pharmacology, vol. 11. Oxford: Pergamon Press. p 435–497. Coleman CM, Neckameyer WS. 2004. Substrate regulation of serotonin and dopamine synthesis in Drosophila. Invertebr Neurosci 5:85–96. Cortes R, Mengod G, Celada P, Artigas F. 1993. p-chlorophenylalanine increases tryptophan-5-hydroxylase mRNA levels in the rat dorsal raphe: a time course study using in situ hybridization. Neurochemistry 60:761–764. Côté F, Thévenot E, Fligny C, Frome Y, Darmon M, Ripoche M, Bayard E, Hanoun N, Saurini F, Lechat P, Dandolo L, 30 Coleman and Neckameyer Hamon M, Mallet J, Vodjdani G. 2003. Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc Natl Acad Sci 100:13525–13530. Dyakonova VE, Schurmann F, Sakharov DA. 1999. Effects of serotonergic and opioidergic drugs on escape behaviors and social status of male crickets. Naturwissenschaften 86:435–437. Ehret M, Pevet P, Maitre M. 1991. Tryptophan hydroxylase synthesis is induced by 3',5'-cyclic adenosine monophosphate during circadian rhythm in the rat pineal gland. J Neurochem 57:1516–1521. Fitzpatrick PF. 1999. Tetrahydrobiopterin-dependent amino acid hydroxylases. Annu Rev Biochem 68:355–381. Hufton SE, Jennings IG, Cotton RG. 1995. Structure and function of the aromatic amino acid hydroxylases. Biochem J 311:353–366. Jequier E, Lovenberg W, Sjoerdsma A. 1967.Tryptophan hydroxylase inhibition: the mechanism by which p-chlorophenylalanine depletes rat brain serotonin. Mol Pharmacol 3:274–278. Johanssen PA, Wolf WA, Kuhn DM. 1991. Inhibition of tryptophan hydroxylase by benserazide and other catechols. Biochem Pharmacol 41:625–628. Johansen PA, Jennings I, Cotton RGH, Kuhn DM. 1996. Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase. J Neurochem 66:817–823. Karlin S, Bergman A, Gentles AJ. 2001. Genomics. Annotation of the Drosophila genome. Nature 411:259–260. Kim KS, Wessel TC, Stone DM, Carver CH, Joh TH, Park DH. 1991. Molecular cloning and characterization of cDNA encoding tryptophan hydroxylase from rat central serotonergic neurons. Brain Res Mol Brain Res 9:277–283. Koe BK, Weissman A. 1966. p-Chlorophenylalanine: a specific depletor of brain serotonin. J Pharmacol Exp Ther 154:499–516. Makita Y, Okuno S, Fujisawa H. 1990. Involvement of activator protein in the activation of tryptophan hydroxylase by cAMP-dependent protein kinase. FEBS Lett 268:185–188. Martinez A, Andersson KK, Haavik J, Flatmark T. 1991. EPR and 1H-NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors. Eur J Biochem 198:675–682. Montine TJ, Missala K, Sourkes TL. 1992. Alpha-methyltryptophan metabolism in rat pineal gland and brain. J Pineal Res 12:43–48. Morales G, Requena JM, Jimenez-Ruiz A, Lopez MC, Ugarte M, Alonso C. 1990. Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA. Gene 93: 213–219. Neckameyer W, Quinn WG. 1989. Isolation and characterization of the gene for Drosophila tyrosine hydroxylase. Neuron 2:1167–1175. Neckameyer W, White K. 1992. A single locus encodes both phenylalanine hydroxylase and tryptophan hydroxylase activities in Drosophila. J Biol Chem 267:4199–4206. Neckameyer W, Holt B, Paradowski T. 2005. Biochemical conservation of recombinant Drosophila tryptophan hydroxylase with its mammalian cognates. Biochemical Genetics (in press). Novak MG, Rowley WA. 1994. Serotonin depletion affects blood-feeding but not host seeing ability in Aedes triseriatus. J Med Entomol 31:600–606. Novak MG, Ribeiro JM, Hildebrand JG. 1995. 5-hydroxytryptamine in the salivary glands of adult female Aedes aegypti and its role in regulation of salivation. J Exp Biol 198:167–174. Peroutka SJ. 1990. Receptor “families” for 5-hydroxytryptamine. J Cardiovasc Pharmacol 16(Suppl 3):S8–14. Richard F, Sanne JL, Bourde O, Weissman D, Ehret M, Cash C, Maitre M, Pujol JF. 1990. Variation of tryptophan-5hydroxylase concentration in the rat raphe dorsalis nucleus after p-chlorophenylalanine administration. I. A model to study the turnover of the enzymatic protein. Brain Res 536:41–45. Saudou F, Boschert U, Amlaiky N, Plassat JL, Hen R. 1992. A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns. EMBO J 1:7–17. Shiman R. 1985. Tetrahydrobiopteran dependent aromatic amino acid hydroxylases. In: Blakely RL, Benkovic SJ, editors. Folates and Pterins, 2. New York: John Wiley & Sons. p 179–249. Archives of Insect Biochemistry and Physiology Serotonin Synthesis in Drosophila Sourkes TL, Missala K, Oravec M. 1970. Decrease of cerebral serotonin and 5-hydroxyindolylacetic acid caused by (-)alpha-methyltryptophan. J Neurochem 17:111–115. Stevenson PA, Hofmann HA, Schoch K, Schildberger K. 2000. The fight and flight responses of crickets depleted of biogenic amines. J Neurobiol 43:107–120. 31 Wretborn M, Humble E, Ragnarsson U,d Engstrom L. 1980. Amino acid sequence at the phosphorylated site of rat liver phenylalanine hydroxylase and phosphorylation of a corresponding synthetic peptide. Biochem Biophys Res Commun 93:403–408. Valles AM, White K. 1988. Serotonin-containing neurons in Drosophila melanogaster: development and distribution. J Comp Neurol 268:414–428. Yamauchi T, Fujisawa H .1979. Regulation of bovine adrenal tyrosine 3-monooxygenase by phosphorylation-dephosphorylation reaction, catalyzed by adenosine 3’5’-monophosphate- dependent protein kinase and phosphoprotein phosphatase. Arch Biochem Biophys 198:219–226. Walther DJ, Peter J, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M. 2003. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76. Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG. 2004. Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217. May 2005
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