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Serotonin synthesis by two distinct enzymes in Drosophila melanogaster.

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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-
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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.
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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.
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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
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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.
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May 2005
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two, synthesis, distinct, enzymes, melanogaster, serotonin, drosophila
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