British Journal of Pharmacology (2010), 159, 1450–1462

© 2010 The Authors

Journal compilation © 2010 The British Pharmacological Society All rights reserved 0007-1188/10
www.brjpharmacol.org

RESEARCH PAPER
Inverse agonist and neutral antagonist actions of
synthetic compounds at an insect 5-HT1 receptor bph_638 1450..1462

B Troppmann1, S Balfanz2, A Baumann2 and W Blenau1

1
Institute of Biochemistry and Biology, Universität Potsdam, Potsdam, Germany, and 2Institute of Structural Biology and
Biophysics 1, Forschungszentrum Jülich, Jülich, Germany

Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and
behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an
insect model for neurobiology, physiology and pharmacology.
Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The
receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP
assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal
antiserum.
Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mam-
malian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was
expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse
agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies
showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands.
Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral
antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in
mediating central and peripheral effects of 5-HT in insects.
British Journal of Pharmacology (2010) 159, 1450–1462; doi:10.1111/j.1476-5381.2010.00638.x; published online 3
March 2010

Keywords: biogenic amine; constitutive activity; cellular signalling; G-protein-coupled receptor; insect; methiothepin; salivary
gland; 5-HT; serotonin; WAY 100635
Abbreviations: [cAMP]I, intracellular concentration of 3′-5′-cyclic adenosine monophosphate; 5-CT,
5-carboxamidotryptamine; 5-MeOT, 5-methoxytryptamine; 8-OH-DPAT, (+/-)-8-hydroxy-2-
(dipropylamino)tetralin; AD, antibody diluent; Dm5-HT1A/1B/2/7, Drosophila melanogaster 5-HT1A/1B/2/7 receptor;
GPCRs, G-protein-coupled receptors; HA, hemagglutinin epitope; IBMX, isobutylmethylxanthine; NCC 1,
corpora cardiaca nerve 1; Pea5-ht1, gene, mRNA or cDNA of the Periplaneta americana 5-HT1 receptor;
Pea5-HT1, P. americana Pea5-HT1 receptor; RACE, rapid amplification of cDNA ends; TBS-T, Tris-buffered saline
containing Tween 20; TM, transmembrane domain; ZT, zeitgeber time

Introduction depression, suicidal behaviour, infantile autism, eating disor-

ders and obsessive–compulsive disorder (Jones and Blackburn,
The biogenic amine 5-hydroxytryptamine (5-HT) acts as a 2002). In insects, 5-HT signalling has been ascribed as being
chemical messenger in most animal phyla in which it con- involved in the modulation of the heart rate (Zornik et al.,
trols and modulates a great variety of important physiological 1999), secretory processes (Just and Walz, 1996), development
and behavioural processes (Weiger, 1997). Disruption of the (Colas et al., 1999), circadian rhythms (Page, 1987; Yuan et al.,
5-hydroxytryptaminergic system has been linked to several 2005), aggression (Dierick and Greenspan, 2007), behavioural
human disease states, such as schizophrenia, migraine, gregarization in locusts (Anstey et al., 2009) and learning and
memory (Sitaraman et al., 2008). The multifaceted actions of
5-HT are mediated by binding to integral membrane recep-
Correspondence: Dr Wolfgang Blenau, Universität Potsdam, Institute of tors, most of which, except for the 5-HT3 receptor ion
Biochemistry and Biology, -Animal Physiology-Karl-Liebknecht-Str. 24-25,
channel, belong to the superfamily of G-protein-coupled
D-14476 Potsdam, Germany. E-mail: blenau@uni-potsdam.de
Received 29 June 2009; revised 10 November 2009; accepted 13 November receptors (GPCRs). In vertebrates, six main classes of
2009 G-protein-coupled 5-HT receptors have been classified on the
 Characterization of an insect 5-HT1 receptor
B Troppmann et al 1451

basis of their sequence similarities, gene organization, second- molecular and pharmacological details of an insect 5-HT1
messenger coupling pathways and pharmacological charac- receptor, and advances our knowledge concerning the
teristics (Hoyer et al., 2002; nomenclature follows Alexander complexity of the 5-hydroxytryptaminergic system in
et al., 2009). The 5-HT1 and 5-HT5 receptors couple preferen- insects.
tially to Gi/o proteins, and inhibit cAMP synthesis. The 5-HT2
receptors couple preferentially to Gq/11 proteins, which
mediate the hydrolysis of inositol phosphates and a sub- Methods
sequent increase in cytosolic Ca2+ levels. The 5-HT4, 5-HT6
and 5-HT7 receptors all couple preferentially to Gs proteins, Cloning of Pea5-ht1 cDNA
and promote cAMP formation. In invertebrates, the Degenerate primers (DF1: 5′-TGYTGGBTICCITTYTT-3′; DR1:
5-hydroxytryptaminergic system might be similarly complex 5′-TTDATISHRTADATIAYIGGRTT-3′) corresponding to highly
(Hauser et al., 2006). For example, the fruit fly Drosophila conserved amino acid sequences in TM 6 and TM 7 of bio-
melanogaster is known to express at least four 5-HT receptor genic amine receptors were designed to amplify receptor frag-
subtypes that are predicted to be orthologs of the mammalian ments (Walz et al., 2006). The PCR was performed on a
5-HT1A, 5-HT2 and 5-HT7 receptors. These are the Dm5-HT1A P. americana brain cDNA library (Blenau and Baumann, 2005).
and Dm5-HT1B (Saudou et al., 1992), Dm5-HT2 (Colas et al., Amplification was carried out for 2.5 min at 94°C (one cycle),
1995) and Dm5-HT7 (Witz et al., 1990) receptors, respectively followed by 35 cycles of 40 s at 94°C, 40 s at 55–65°C and 30 s
(see Nichols, 2006; Nichols and Nichols, 2008). The sequences at 72°C, and a final extension of 10 min at 72°C. The PCR
and the signal transduction mechanisms of 5-HT receptors are product was cloned into pGEM-T vector (Promega, Man-
generally highly conserved between vertebrates and inverte- nheim, Germany), and subsequently analysed by DNA
brates (Hen, 1992). In contrast, the pharmacological proper- sequencing (AGOWA, Berlin, Germany). Based on this
ties of vertebrate and invertebrate receptors vary significantly sequence information, specific primers for rapid amplification
in many cases (see Blenau and Baumann, 2001; Tierney, of cDNA ends (RACE) PCR experiments were designed. To
2001). The 5-HT1 receptors form the largest class of 5-HT amplify the missing 5′-region of the cDNA, two consecutive 5′
receptors (Gerhardt and van Heerikhuizen, 1997). In verte- RACE experiments were performed with specific reverse
brates, several members of this class show agonist- primers (S5-1: 5′-GAGTTGAAATAGCCGAGCC-3′, S5-2:
independent activation of associated G proteins, and thus are 5′-CACTAGGAGCGTTGTGTCC-3′). Amplification of the 3′
constitutively active (McLoughlin and Strange, 2000; Martel end was performed by 3′ RACE by using a specific forward
et al., 2007). Constitutive activity is now accepted as being a primer (S3: 5′-GGAGAGCTTCTTTCTGTGG-3′). Finally, a PCR
common property of many GPCRs (Seifert and Wenzel-Seifert, was performed on single-stranded P. americana brain cDNA to
2002), but has not, as yet, been shown for any insect 5-HT1 amplify the entire coding region of Pea5-ht1 by using two
receptor. gene-specific primers annealing in the 5′- and 3′-untranslated
In the present study, we have cloned and characterized a regions (SF1: 5′-GTGCGGTGCTGTCGACGCC-3′; SR1:
5-HT receptor of the cockroach Periplaneta americana with 5′-CTCCGTTAATATAGCGCAC-3′). The nucleotide sequence
significant homologies to members of the 5-HT1 receptor of Pea5-ht1 has been submitted to the EBI database (accession
class. Cockroaches have been widely used as a model organ- no. FN298392).
ism for basic research in physiology and neurobiology
(Downer, 1990; Watanabe and Mizunami, 2007). In particu-
lar, the salivary gland of P. americana is a well-established Multiple sequence alignment and phylogenetic analysis
model system for studying excitation–secretion coupling in Amino acid sequences used for phylogenetic analyses were
epithelia and aminergic signal transduction (see House and identified by protein–protein BLAST searches of the NCBI
Ginsborg, 1985; Walz et al., 2006). Information has thus been database with the deduced amino acid sequence of Pea5-ht1
accumulated on the pharmacology of amine receptors in the (Pea5-HT1) as ‘bait’. Multiple sequence alignments of the com-
salivary glands and other tissues of cockroaches (Downer, plete amino acid sequences were performed with ClustalW.
1990; Walz et al., 2006; Troppmann et al., 2007). Compara- Values for identity (ID) and similarity (S) were calculated by
tively little is known, however, concerning the exact reper- using the BLOSUM62 substitution matrix in BioEdit 7.0.5
toire and molecular properties of amine receptors in (Hall, 1999). MEGA 4 (Tamura et al., 2007) was used to calcu-
P. americana (Bischof and Enan, 2004; Rotte et al., 2009), and, late the genetic distances between the core sequences, and to
until this study, no molecular data on 5-HT receptors have construct neighbour-joining trees with 2000-fold bootstrap
been available. resampling. The D. melanogaster ninaE-encoded rhodopsin 1,
In this investigation, we show that the mRNA encoding a and the D. melanogaster FMRFamide receptor were used as
cockroach 5-HT1 receptor is expressed in the brain, salivary outgroups.
gland and midgut tissue. Immunohistochemical analysis has
revealed the presence of the receptor protein in a specific
subset of pars intercerebralis cells of the cockroach brain. RT-PCR amplification of Pea5-ht1 fragments
When stably expressed in HEK 293 cells, the receptor inhib- Total RNA was isolated from brain, salivary glands, midgut,
its the formation of cAMP with an EC50 of ~130 nM for Malpighian tubules and flight muscle of adult male cock-
serotonin. The receptor shows constitutive activity, which roaches by using TRIZOL LS (Invitrogen, Karlsruhe,
can be blocked by the 5-HT1A receptor antagonist WAY Germany). The samples were either digested with DNase I
100635. Our study has therefore elucidated unique (Ambion, Huntingdon, UK) to degrade contaminating

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
1452 B Troppmann et al

genomic DNA or with DNase I and an RNase Cocktail sections on a vibratome (Pelco 101, series 1000; Pelco, St
(Ambion) for negative controls. Pea5-ht1-specific fragments Louis, MO, USA). Mildly agitated, free-floating sections
were amplified from 100 ng total RNA by using the Super- were subsequently exposed to the following steps: (i)
Script One-Step RT-PCR System (Invitrogen). The sense primer de-gelatinization in warm buffer 1 for 2 ¥ 10 min; (ii) rinsing
was 5′-GACACTAGTGGTGCTTCTGG-3′, and the antisense in buffer 1 for 2 ¥ 15 min; (iii) incubation in 50 mM NH4Cl in
primer was 5′-GTCATGGGACCTACGCCATC-3′. Amplifica- buffer 1 for 10 min; (iv) washing in buffer 1 for 3 ¥ 10 min;
tion resulted in a fragment of 243 bp. RT-PCR was also per- (v) incubation for 90 min in a blocking solution (buffer 1
formed with primers for the P. americana actin gene (accession containing 3% normal goat serum and 0.5% Triton X-100),
no. AY116670) as an internal control (ActinF: 5′-CGAGT which was also used as antibody diluent (AD); (vi) overnight
AGCTCCTGAAGAGC-3′; ActinR: 5′-GGCCTCTGGACAACGG incubation at 4°C in primary antisera (rabbit anti-Pea5-HT1
AACC-3′). cDNA was synthesized for 30 min at 50°C, followed 1:1000 and rat anti-5-HT 1:100 (Chemicon, Temecula, CA,
by a single denaturation step at 94°C for 2 min. Amplification USA) in AD; (vii) washing in buffer 2 (50 mM Tris, pH 7.5;
of Pea5ht-1 or Peaactin fragments was performed for 30 cycles 145 mM NaCl) for 3 ¥ 15 min; (viii) incubation in Alexa Fluor
at 94°C for 40 s, 60°C for 40 s and 72°C for 40 s, followed by 488 goat anti-rabbit IgG 1:100 (Molecular Probes, Eugene,
a final extension at 72°C for 10 min. OR, USA) and Cy3-conjugated AffiniPure goat anti-rat IgG
1:400 (Jackson Immunoresearch, West Grove, PA, USA)
diluted in AD for 3 h at room temperature; (ix) washing in
Antibody production and purification buffer 2 for 3 ¥ 15 min; and (x) mounting on slides with
The anti-Pea5-HT1 receptor polyclonal rabbit antiserum was Mowiol 4.88 (Farbwerke Hoechst, Frankfurt, Germany)
produced commercially (Pineda-Antikörper-Service, Berlin, containing 2% n-propyl-gallate as an anti-fading reagent.
Germany). Antibodies were raised against a synthetic peptide Sections from 15 individual brains were treated with the
(NH2-CFITKRRFRRMKSNKKSS-CONH2) corresponding to a Pea5-HT1 receptor antiserum and sections from five brains
region within the 3rd cytoplasmic loop of the Pea5-HT1 recep- with pre-absorbed antiserum. Fluorescence images were
tor (Figure 1). A cysteine residue was added N-terminally to recorded with a Zeiss LSM 510 confocal microscope (Carl
the peptide for coupling to the protein carrier, viz., keyhole Zeiss, Jena, Germany).
limpet haemocyanin. The monospecific IgG fraction was puri-
fied via affinity chromatography.
Construction of pcPea5-ht1-HA expression vector
An expression-ready construct of Pea5-ht1 cDNA was gener-
Western blot analysis ated by PCR. To monitor transfection efficiency and receptor
Entire cockroach brains were homogenized in 150 mL Roti- protein expression, a hemagglutinin (HA) epitope tag was
load sample buffer (Roth, Karlsruhe, Germany), and incu- engineered onto the 3′ end of the cDNA. PCR was performed
bated for 5 min at 95°C, or membrane proteins were isolated with a sense primer 5′-TATGATGTGCGGCCGCCCACCATG
and incubated for 5 min at 60°C. Proteins were separated by GATCTCCTGAGC-3′ and the antisense primers, 5′-TGGGAC
sodium dodecyl sulphate–polyacrylamide gel electrophoresis GTCGTatGGGTaTCTAAGCTTTCCCGGCCTG-3′ (first-round
on 12% gels. Approximately 10 mg total protein, as deter- PCR) and 5′-TTTTCTAGATTAAGCGTAGTCTGGGACGTCG
mined by a modified Bradford assay, was run per lane. Pro- TATGGGTA-3′ (second-round PCR). The PCR product was
teins were transferred to polyvinylidene fluoride membranes digested with Not I and Xba I, and subcloned into
(Roth). Membranes were blocked with 5% dry milk in Tris- pcDNA3.1(+) vector (Invitrogen) yielding pcPea5-ht1-HA. The
buffered saline containing Tween 20 (TBS-T; 10 mM Tris–HCl, correct insertion was confirmed by DNA sequencing.
pH 7.5, 150 mM NaCl, 0.01% Tween 20) for 30 min at room
temperature. Membranes were probed with affinity-purified
anti-Pea5-HT1 antibodies (dilution 1:20 000 in TBS-T). For Functional expression of the Pea5-HT1-HA receptor
controls, antibodies were pre-absorbed to the synthetic Approximately 8 mg pcPea5-ht1-HA vector was introduced
peptide (15 mg·mL–1). Membranes were washed with TBS-T, into exponentially growing HEK 293 cells (~4 ¥ 105 cells per
followed by incubation with a secondary antibody conju- 5 cm Petri dish) by a modified calcium phosphate method
gated to horseradish peroxidase (1:20 000; American Qualex, (Chen and Okayama, 1987). Stably transfected cells were
La Mirada, CA, USA). Signals were visualized with an selected in the presence of the antibiotic G418 at 0.8 mg·mL-1.
enhanced chemiluminescence detection system (Super Signal Isolated foci were propagated and analysed for the expression
West Pico Chemiluminescent Substrate, Pierce, Rockford, IL, of Pea5-HT1-HA by immunocytochemistry and Western blot
USA). Each analysis was performed three times with indepen- with a commercial anti-HA antibody (Anti-HA High Affinity,
dently isolated protein samples. Roche, Penzberg, Germany).

Immunofluorescence staining of brain sections Functional characterization of Pea5-HT1 receptors

Cockroach brains were dissected, fixed overnight at 4°C in Assays to determine the ability of Pea5-HT1-HA receptors
4% paraformaldehyde in phosphate-buffered saline (pH 7.4) to attenuate adenylyl cyclase activity were performed
and washed 3 ¥ 5 min with buffer 1 (50 mM Tris–HCl, pH as described earlier (Grohmann et al., 2003). Pea5-HT1-
7.5). Brains were embedded in 18% gelatin (250 g Bloom, expressing cells were grown in minimal essential medium
Fluka, Buchs, Switzerland), and cut into 50 mm thick serial with GlutaMAXTMI, 10% (v/v) fetal bovine serum (FBS), 1%

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
B Troppmann et al 1453

Figure 1 Amino acid sequence alignment of Pea5-HT1 and orthologous receptors from Drosophila melanogaster (Dm5-HT1A; accession no.
CAA77570), Dm5-HT1B (no. CAA77571), Papilio xuthus (Pxu5-HT1, no. BAD72868) and Penaeus monodon (Pem5-HT1, no. AAV48573). Identical
residues (ⱖ80%) between the receptors are shown as white letters against black, whereas conservatively substituted residues are shaded.
Putative transmembrane domains (TM 1–TM 7) are indicated by grey bars. Potential N-glycosylation sites (+) and putative palmitoylation sites
(*) of Pea5-HT1 are labelled. Underlined letters represent the region within the third cytoplasmic loop from which the Pea5-HT1-specific peptide
antigen was derived. The amino acid position is indicated on the right.

(v/v) non-essential amino acids and antibiotics (all from Invit- In earlier experiments with a HEK 293 cell line expressing
rogen). Cells were incubated with ligands for 30 min at 37°C an insect 5-HT7 receptor (Am5-HT7, Schlenstedt et al., 2006),
in the presence of the phosphodiesterase inhibitor, isobutyl- we observed desensitization effects of Am5-HT7, most likely
methylxanthine (IBMX; final concentration 100 mM) and due to the presence of 5-HT in FBS. For that reason, we
lysed by adding 0.5 mL ice-cold ethanol. After 1 h at 4°C, the determined dose–response curves for 5-HT with Pea5-HT1
lysate was transferred to a reaction tube, and lyophilized. The receptor-expressing cells in medium supplemented with
amount of cAMP produced was determined with the TRK 432 either 10% FBS or 2% Ultroser G (Pall Bioserpa, Cergy-Saint-
cyclic AMP assay kit (GE Healthcare, Freiburg, Germany). Christophe, France). As EC50 values were not significantly
Mean values of cAMP·mg-1 protein were determined from different under these conditions (data not shown), all experi-
four independent measurements, performed in duplicate to ments were performed with cells grown in medium contain-
quadruplicate. ing 10% (v/v) FBS as indicated above.

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
1454 B Troppmann et al

Data analysis toylation (Papoucheva et al., 2004). Anchorage of palmitic

Data shown represent the mean values ⫾ SEM, and were acid in the membrane would create a fourth intracellular
analysed (sigmoidal dose–response curves, calculation of EC50 loop, which is believed to stabilize the structure of the recep-
values, statistics) and displayed by using PRISM 4.01 software tor. Together, these results demonstrate that the deduced
(GraphPad, San Diego, CA, USA). Statistical significance of amino acid sequence of the Pea5-HT1 receptor displays
differences between means was determined by using one-way the characteristic features of seven-transmembrane-domain
ANOVA followed by Dunnett’s multiple comparison test with P receptors with qualities appropriate to 5-HT receptors.
values ⱕ 0.05 being considered significant. BLAST searches with the Pea5-HT1 sequence indicated that
it shared pronounced homology with 5-HT1 receptors of crus-
taceans and insects. High amino acid identity (ID)/similarity
Materials (S) was found with the 5-HT1 receptors of the holometabolous
The pharmacological ligands 5-carboxamidotryptamine insects Papilio xuthus (Px5-HT1; ID 44%, S 53%) and D. mela-
maleate salt (5-CT), 5-HT hydrochloride, nogaster (Dm5-HT1A; ID 39%, S 50%; Dm5-HT1B; ID 39%, S
5-methoxytryptamine (5-MeOT) (+/-)-8-hydroxy-2- 52%) and for the crustaceans Penaeus monodon (Pem5-HT1;
(dipropylamino)tetralin hydrobromide (8-OH-DPAT), bus- ID 42%, S 52%), Procambarus clarkii (Pro5-HT1; ID 45%,
pirone hydrochloride, dopamine hydrochloride, histamine S 54%) and Panulirus interruptus (Pan5-HT1; ID 43%, S 51%)
dihydrochloride, lysergic acid diethylamide (LSD), methio- (Figure 1). A multiple amino acid sequence comparison
thepin mesylate salt, mianserin hydrochloride (+/-)- within the conserved transmembrane domains and short
octopamine hydrochloride, spiperone, sumatriptan succinate, linker regions of Pea5-HT1 with invertebrate and human 5-HT
tyramine hydrochloride and WAY 100635 maleate salt receptors was used to calculate a phylogenetic tree (Figure 2).
were all purchased from Sigma-Aldrich Chemie GmbH The Pea5-HT1 receptor was incorporated into the comprehen-
(Taufkirchen, Germany). sive branch of the 5-HT1 receptor class, and was robustly
placed in a clade of invertebrate 5-HT1 receptors.

Results
Tissue distribution of Pea5-ht1 mRNA
Cloning and sequence analysis of a 5-HT1 receptor from The expression pattern of Pea5-ht1 mRNA in various tissues of
P. americana P. americana was investigated by RT-PCR with specific primers
Initially, a 97 base pair long cDNA fragment of a putative 5-HT corresponding to sequences within the third cytoplasmic
receptor of P. americana was amplified by using degenerate loop. The transcript of the Pea5-HT1 gene was detected in
primers (see Methods). The full-length cDNA was obtained by samples of the brain, salivary glands and midgut (Figure 3).
5′ and 3′ RACE experiments. Sequence analysis revealed that Conversely, no receptor mRNA expression was detected in
the P. americana receptor had highest similarity to 5-HT recep- samples of Malpighian tubules and leg muscle. To ensure that
tors of the 5-HT1 subtype. The deduced amino acid sequence the fragments had not been amplified from genomic DNA,
is characterized by a long third cytoplasmic loop and a short samples were treated with DNase I. The negative control PCR
C-terminal region, consistent with the structure of 5-HT1 on samples treated additionally with an RNase cocktail did
receptors, which are coupled to Gi proteins (Gerhardt and van not result in the amplification of any PCR product (data not
Heerikhuizen, 1997). Accordingly, the P. americana receptor shown).
was named Pea5-HT1. The nucleotide sequence of Pea5-ht1
consists of 2564 nucleotides. The longest open reading frame
comprises 2049 nucleotides encoding a predicted protein of Generation of an anti-Pea5-HT1 antibody and
683 amino acids with a calculated molecular weight of 74 kDa immunohistochemical localization of Pea5-HT1 receptors
(Pea5-HT1). Upstream of the translation initiation codon We generated a polyclonal antiserum directed against a part
(ATG, position 193–195), stop codons were identified in all of the third cytoplasmic loop of Pea5-HT1 receptors, as
three reading frames. The flanking sequence of this ATG described in Methods. Western blots of P. americana brain
triplet agrees well with the consensus sequence for the eukary- proteins showed that the antibody recognized a single band
otic translation start site (CCACCATGG; Kozak, 1984). Analy- of ~80 kDa (Figure 4A). After pre-absorption with the peptide
sis of the deduced amino acid sequence with the topology (15 mg·mL-1) used for immunization, the signal was com-
predictor Phobius (Käll et al., 2004) led to the prediction that pletely lost. This result demonstrated that the antibody spe-
the polypeptide contains an extracellular N-terminus, a cyto- cifically recognized the Pea5-HT1 receptor protein. In addition
plasmic C-terminal region and seven hydrophobic helical to its presence in brain tissue, the 5-HT1 receptor was detected
domains. Within these transmembrane segments, the typical on Western blots with membrane proteins from salivary
sequence motifs of 5-HT1 receptors were well conserved gland tissue. Here, the receptor protein was less abundantly
(Figure 1). 5-HT receptors are known for their numerous post- expressed. Furthermore, we studied whether the receptor
translational modifications. The N-terminal region of the protein was glycosylated in these tissues. De-glycosylation of
Pea5-HT1 receptor contains seven consensus sites for N-linked membrane proteins from brain and salivary gland tissue by
glycosylation (Figure 1). The third cytoplasmic loop com- PNGase F digest resulted in a slight shift of the apparent
prises four consensus sites for phosphorylation by protein molecular weight from ~80 to ~75 kDa (Figure 4B). This
kinase C ([S/T]-x-[R/K]). The C-terminus of Pea5-HT1 receptors molecular weight was in good accordance with the predicted
harbours a cysteine residue (C666), a potential target for palmi- molecular weight of 74 kDa for the Pea5-HT1 receptor.

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
B Troppmann et al 1455

Figure 2 Phylogenetic analysis of Pea5-HT1 and various 5-HT receptors. Alignments were performed with BioEdit (version 7.0.5; Hall, 1999)
using the core amino acid sequences lacking the variable regions of the amino and carboxyl terminus, and the third cytoplasmic loop. The
genetic distance was calculated with MEGA4 (Tamura et al., 2007). The receptor sequences followed by their accession numbers are listed in
the order illustrated: Panulirus interruptus (Pan5-HT1, no. AY528822); Procambarus clarkii (Pro5-HT1, no. ABX10973), Penaeus monodon
(Pem5-HT1, no. AAV48573), Periplaneta americana (Pea5-HT1, no. FN298392), Drosophila melanogaster (Dm5-HT1A, no. CAA77570), Dm5-HT1B
(no. CAA77571), Papilio xuthus (Pxu5-HT1, no: BAD72868), Manduca sexta (Ms5-HT1A, no. DQ840515), Bombyx mori (Bm5-HT, no.
CAA64862), Ms5-HT1B (no. DQ840516), Lymnaea stagnalis (Lym5-HT1, no. L06803), human 5-HT1A (no. NP_000515), human 5-HT1B (no.
NP_000854), human 5-HT1D (no. NP_000855), human 5-HT1E (no. NP_000856), human 5-HT1F (no. NP_000857), human 5-HT7 isoform a
(no. NP_000863), human 5-HT7 isoform d (no. NP_062873), human 5-HT7 isoform b (no. NP_062874), Apis mellifera (Am5-HT7, no.
AM076717), Dm5-HT7 (no. A38271), Aedes aegypti (Aae5-HT7, no. AAG49292), Pan5-HT2 (no. AY550910), Pro5-HT2 (no. ABX10972),
Dm5-HT2 (no. CAA57429), Lym5-HT2 (no. U50080), human 5-HT2B (no. NP_000858), human 5-HT2A (no. NP_000612), human 5-HT2C (no.
NP_000859), D. melanogaster ninaE-encoded rhodopsin 1 (DmninaE, no. NM_079683) and D. melanogaster FMRFamide receptor (DmFR, no.
AAF47700). The numbers at the nodes of the branches represent the percentage bootstrap support for each branch. The scale bar allows
conversion of branch lengths in the dendrogram to genetic distance between clades.

To determine the cellular distribution of the Pea5-HT1 these neurons pass down the anterior surface of the brain and
receptor within the brain of P. americana, vibratome sections cross over in the chiasma region of the corpora cardiaca nerve
were immunostained with the anti-Pea5-HT1 antibody and 1 (NCC 1). Extensive arborization is observed in the protoce-
with an anti-5-HT antibody for orientation purposes. The rebral region ventral of the pars intercerebralis. The labelled
receptor antibody specifically labelled some large somata in axons then proceed posteriorly and continue to the corpora
the pars intercerebralis (Figure 5A and C). Labelled axons of cardiaca complex. Accordingly, in more posterior frontal

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
1456 B Troppmann et al

Figure 3 Tissue distribution of Pea5-ht1 mRNA. The 100 bp DNA

ladder (marker) is shown on the left. Detection of PCR products
amplified on total RNA isolated from brain, salivary glands, Mal-
pighian tubules, midgut and muscle. Amplification failed when
samples were digested with an RNase cocktail prior to RT-PCR (data
not shown). The lower panel shows RT-PCR products amplified by
using actin (accession no. AY116670)-specific primers as a control.

Figure 5 Immunohistochemical localization of Pea5-HT1 receptors

in brain sections. Vibratome sections of brains were double labelled
with anti-5-HT (red) and anti-Pea5-HT1 (green), and imaged by con-
focal microscopy. Each image shows the sum of multiple consecutive
optical sections representing a total thickness of ~20 mM. (A) Central
part of an anterior frontal section of the cockroach brain (for sche-
matic drawing, see C). (B) Central part of a more posterior frontal
section of the cockroach brain. (C) Schematic drawings of sections of
Figure 4 Western blot analysis with the anti-Pea5-HT1 receptor anti- the cockroach brain. Grey boxes highlight the details displayed in A
body. Molecular weight marker in kDa. (A) Specificity of anti-Pea5- + B. Abbreviations: V, vertical lobe; AL, antennal lobe; Ca, calyx; Ch,
HT1 receptor antibody tested on Periplaneta americana brain proteins. chiasm region of nervi coporis cardiaci I; M, medial lobe; NCC I, nervus
Lane 1: Western blot analysis with anti-Pea5-HT1 antibody corporis carciaci I; PI, pars intercerebralis.
(1:20 000). A single band of ~80 kDa was detected. Lane 2: Western
blot analysis with anti-Pea5-HT1 antibody (1:20 000) pre-absorbed
with 15 mg·mL-1 of the peptide used for immunization; no band was
detected. (B) Western blot of membrane proteins (10 mg per lane) confirmed by Western blotting and immunocytochemistry
from brain tissue (b) and salivary glands (sg) treated without (–) or
with (+) PNGase F. De-glycosylation resulted in a slight shift of the (data not shown). Basal levels of intracellular cAMP ([cAMP]i)
protein band from ~80 to ~75 kDa. were not significantly different in non-transfected (12.9 ⫾
0.5 pmol cAMP·mg-1 protein, n = 4) and Pea-5-HT1-expressing
cells (16.3 ⫾ 0.7 pmol cAMP·mg-1 protein, n = 4). However,
sections of the brain, the cell cluster in the pars intercerebralis Pea5-HT1-expressing cells treated with a non-saturating con-
and NCC 1 profiles were stained (Figure 5B and C). centration of NKH-477 (10 mM; Wachten et al., 2006) exhib-
ited significantly higher levels of cAMP (154.4 ⫾ 7.3 pmol
cAMP·mg-1 protein, n = 4) than NKH-477-treated controls
Functional analyses of Pea5-HT1 receptors in HEK 293 cells (76.7 ⫾ 2.3 pmol cAMP·mg-1 protein, n = 4). To analyse the
For functional and pharmacological characterization, we gen- ligand specificity of the cloned receptor, various biogenic
erated a HEK 293 cell line stably expressing the Pea5-HT1 amines (5-HT, dopamine, tyramine, octopamine and hista-
receptor (see Methods). Expression of Pea5-HT1 receptors was mine, each at a concentration of 10 mM) were tested for the

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 Characterization of an insect 5-HT1 receptor
B Troppmann et al 1457

Figure 6 Modulation of intracellular cAMP levels in HEK 293 cells stably expressing the Pea5-HT1 receptor and in non-transfected cells. The
amount of cAMP is given as the percentage of the value obtained after incubation with 10 mM NKH-477 (100%), a water-soluble forskolin
analogue. Error bars indicate SEM and are in some cases too small to be represented. The statistical analysis is based on a one-way ANOVA
followed by Dunnett’s multiple comparison test; ***P < 0.0001. (A) Effect of NKH-477 and 5-HT (10 mM) on cAMP levels in non-transfected
cells and in Pea5-HT1-expressing cells. To determine the basal [cAMP]i, cells were incubated with 100 mM IBMX only (basal). Data represent
the mean ⫾ SEM of 16 values from four experiments each performed in quadruplicate. Asterisks indicate statistically significant differences for
drug versus NKH-477 (100%) for a given cell line. (B) Dose-dependent effect of 5-HT (10-9–3 ¥ 10-5 M) on [cAMP]i. Data represent the mean
⫾ SEM of eight replicates from four experiments each performed in duplicate. (C) Effects of 5-HT receptor agonists (10 mM) on NKH-477-
stimulated cAMP production in Pea5-HT1-expressing cells. Data represent the mean ⫾ SEM of 14 values from four experiments each performed
in either triplicate or quadruplicate. Asterisks indicate statistically significant differences for drug versus NKH-477. (D) Effects of putative
antagonists (10 mM) on 5-HT-mediated (500 nM) inhibition of NKH-477-stimulated cAMP production in Pea5-HT1-expressing cells. Data
represent the mean ⫾ SEM of 16 values from four experiments each performed in quadruplicate. Asterisks indicate statistically significant
differences for drug versus 5-HT.

inhibition of NKH-477-stimulated cAMP production in non- (Figure 6B). Half-maximal reduction of cAMP production
transfected and Pea5-HT1-expressing cells. Only 5-HT signifi- (EC50) was observed with ~130 nM 5-HT (logEC50 = -6.89 ⫾
cantly decreased the NKH-477-induced production of cAMP 0.08, mean ⫾ SEM). Maximal attenuation of cAMP synthesis
in Pea5-HT1-expressing cells (Figure 6A). No effect of 5-HT was (by ~75%) was attained with 5-HT concentrations of ⱖ10 mM.
observed in non-transfected cells. The dose–response relation- To characterize the pharmacological profile of Pea5-HT1
ship of 5-HT on the cAMP level was examined for 5-HT receptors in detail, the effect of various 5-HT receptor agonists
concentrations ranging from 1 nM to 30 mM. In Pea5-HT1- and antagonists on NKH-477-induced cAMP production was
expressing cells, the 5-HT effect was concentration dependent investigated. The 5-HT derivative 5-MeOT, a non-selective
and saturable, resulting in a sigmoidal dose–response curve 5-HT receptor agonist, acted as partial agonist and decreased

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
1458 B Troppmann et al

Figure 7 Modulation of intracellular cAMP levels in HEK 293 cells

stably expressing the Pea5-HT1 receptor by WAY 100635 and methio-
thepin. (A) Dose-dependent effects of the neutral antagonist methio-
thepin and the inverse agonist WAY 100635 on NKH-477-stimulated
cAMP production in the presence of 500 nM 5-HT. Data represent the
mean ⫾ SEM of eight replicates from two experiments representative
of four similar experiments. (B) Dose-dependent effects of methio-
thepin and WAY 100635 on NKH-477-stimulated cAMP production in
the absence of 5-HT. Data represent the mean ⫾ SEM of eight
replicates from two experiments representative of four similar experi-
ments. (C) Inhibition of the effect of WAY 100635 (30 mM) on the
NKH-477-stimulated cAMP production by methiothepin (10 mM).
Data represent the mean ⫾ SEM of eight replicates from two experi-
ments representative of four similar experiments. Asterisks indicate
statistically significant differences for drug versus NKH-477 (one-way
ANOVA followed by Dunnett’s multiple comparison test; **P < 0.01).
䉴

the NKH-477-mediated cAMP production by ~40%

(Figure 6C). 5-CT and 8-OH-DPAT, selective agonists for mam-
malian 5-HT1 and 5-HT7 receptors, respectively, showed no
significant effect on [cAMP]i. Furthermore, the selective 5-HT1A
receptor agonist buspirone, the 5-HT1B/D/F agonist sumatriptan
and the ergoline LSD (a 5-HT2 receptor agonist) also showed
no detectable effect. In addition, the ability of four putative
antagonists to inhibit the 5-HT effect on NKH-477-stimulated
cAMP production was assayed. Mianserin and spiperone
showed no antagonistic activity at the tested concentration of
10 mM (Figure 6D). On the contrary, the inhibitory effect of
5-HT (500 nM) was antagonized by methiothepin, a non-
selective antagonist at mammalian 5-HT receptors (Hoyer
et al., 1994), and by WAY 100635, a potent and selective
5-HT1A receptor antagonist (Fletcher et al., 1996). The effect of
both substances was dose dependent (Figure 7A). Methio-
thepin precisely compensated for the 5-HT effect, which
resulted in a [cAMP]i of ~100%. In contrast, WAY 100635 not
only compensated for the 5-HT effect, but increased [cAMP]i
even further to more than 100%. These results indicated that
methiothepin acted as a neutral antagonist, whereas WAY
100635 acted as an inverse agonist on the Pea5-HT1 receptor,
which is constitutively active when expressed in HEK 293 cells
(see above). To confirm this hypothesis, the effect of both
substances on the NKH-477-induced cAMP production was
examined in the absence of 5-HT. As expected, the neutral
antagonist methiothepin did not affect [cAMP]i. WAY 100635,
in contrast, increased [cAMP]i in a dose-dependent manner,
thus antagonizing the constitutive activity of Pea5-HT1 recep-
tors by acting as an inverse agonist (Figure 7B). In non-
transfected control cells, WAY 100635 (30 mM) did not
significantly influence the NKH-477-stimulated cAMP level
(data not shown). Finally, the neutral antagonist methio-
thepin was able to block the effect of the inverse agonist WAY
100635 in Pea5-HT1-expressing cells (Figure 7C).
HEK 293 cells expressing the Pea5-HT1 receptor did not
show 5-HT-dependent Ca2+ signals (data not shown). Applica-
tion of the Ca2+ ionophore, ionomycin (2 mM), served as a
positive control to demonstrate that the Ca2+ detection system
worked properly.

Discussion and conclusions

In the present study, we have characterized a 5-HT1 receptor

of the American cockroach, P. americana. Orthologous

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 Characterization of an insect 5-HT1 receptor
B Troppmann et al 1459

receptors have been reported from several other invertebrate to most of the tested compounds. Only 5-MeOT, which is a
species, ranging from insects to molluscs (Saudou et al., 1992; non-selective agonist of various vertebrate and invertebrate
Olde and McCombie, 1997; Barbas et al., 2002; Spitzer et al., 5-HT receptors, inhibited cAMP production and acted as a
2008). These invertebrate receptors share pronounced partial agonist. Our search for substances that could counter-
sequence and functional similarity with mammalian 5-HT1 act the effect of 5-HT led to the discovery that both methio-
receptors. However, not all of these invertebrate receptors thepin and WAY 100635 display this property. Interestingly,
have been characterized in detail. the selective 5-HT1A receptor ligand WAY 100635 (Fletcher
et al., 1996; Newman-Tancredi et al., 1997) blocked not only
the 5-HT-induced inhibition of adenylyl cyclase activity via
Structural characteristics of Pea5-HT1 receptors
Pea5-HT1 receptors, but also agonist-independent activity of
The amino acid sequence of Pea5-HT1 receptors displays char-
this receptor, resulting in cAMP levels above 100%. This result
acteristic properties of amine-activated GPCRs in general
confirms our assumption that Pea5-HT1 receptors are consti-
(Strader et al., 1995) and 5-HT1 receptors in particular (Kroeze
tutively active, and demonstrates that WAY 100635 acts as an
et al., 2002; Nichols and Nichols, 2008). The presence of a
inverse agonist on this insect 5-HT1 receptor. The latter is
large third cytoplasmic loop and a short C-terminal region is
surprising, as WAY 100635 has been shown to act as a neutral
typical for the 5-HT1 receptors and for other biogenic amine
antagonist on mammalian 5-HT1A receptors in most studies
receptors that couple to Gi proteins (Kroeze et al., 2002;
(Fletcher et al., 1996; Newman-Tancredi et al., 1997; Martel
Nichols and Nichols, 2008). Furthermore, signature sequence
et al., 2007). However, under certain conditions, it can also
motifs for co- and post-translational modifications, ligand
display inverse agonist properties in mammals (Cosi and
binding and receptor activation are well conserved in Pea5-
Koek, 2000). Methiothepin has been described as an inverse
HT1 receptors
agonist at mammalian 5-HT1 receptors (McLoughlin and
Strange, 2000; Martel et al., 2007). With respect to the Pea5-
Functional and pharmacological properties of Pea5-HT1 receptors HT1 receptor, however, methiothepin is a neutral agonist that
We have established a HEK 293 cell line that stably expresses is able to compensate for the effects of the full agonist 5-HT
the cloned Pea5-HT1 receptor in order to examine the func- and the inverse agonist WAY 100635.
tional and pharmacological properties of the receptor. When
compared with non-transfected cells, Pea5-HT1-expressing
cells show an increased sensitivity to NKH-477, a direct acti- Expression pattern of Pea5-HT1 receptors
vator of adenylyl cyclase. This supersensitization of adenylyl Clues to the possible functions of a receptor might be
cyclase is a typical property of cells expressing constitutively obtained from its cellular localization. In D. melanogaster, the
active Gi/o-coupled receptors (Johnston and Watts, 2003; Dm5-HT1A receptor is predominantly expressed in the mush-
Beggs et al., 2005). Constitutive activity has been described for room bodies (Yuan et al., 2006), whereas Dm5-HT1B is
mammalian and crustacean 5-HT1 receptors (Newman- expressed not only in the mushroom bodies, but also strongly
Tancredi et al., 1997; Spitzer et al., 2008) and for various other in pars intercerebralis neurons and certain clock neurons (Yuan
GPCRs (see Seifert and Wenzel-Seifert, 2002). Interestingly, et al., 2005). Nothing is known regarding the expression of
mammalian 5-HT1 receptors display constitutive activity not 5-HT1 receptors in other insects. In P. americana, we have been
only in heterologous expression systems, but also in vivo able to show expression of Pea5-HT1 receptors in a subset of
(Martel et al., 2007). High levels of GPCR constitutive activity pars intercerebralis cells and a neural tract connecting these
are assumed to be associated with certain human pathological cells with the retro-cerebral complex and the stomatogastric
states, including metabolic diseases and some forms of cancer nervous system. The labelling of only a few cells within the
(Seifert and Wenzel-Seifert, 2002). In insects, constitutive brain of P. americana by the Pea5-HT1-specific antibody might
activity has been reported only for a few amine receptors so be considered surprising with respect to the widespread dis-
far, namely, for a D1-like dopamine receptor, a D2-like dopam- tribution of 5-HT. Two explanations for this apparent discrep-
ine receptor and a 5-HT7 receptor of the honeybee Apis mel- ancy come to mind. First, additional 5-HT receptors probably
lifera (Mustard et al., 2003; Beggs et al., 2005; Schlenstedt exist that might also be expressed in the brain in P. americana.
et al., 2006). Second, the anti-Pea5-HT1 receptor antibody might only label
Activation of 5-HT1 receptors, including Pea5-HT1, results in cells that express the receptor polypeptide at high density.
the inhibition of cAMP accumulation. Application of 5-HT to Our inability to detect the receptor immunocytochemically in
Pea5-HT1-expressing HEK 293 cells attenuated the NKH-477- the salivary glands, where its expression has been established
induced cAMP formation by 67%. Two D. melanogaster 5-HT1 by RT-PCR and Western blotting, argues in favour of this
receptors (Dm5-HT1A and Dm5-HT1B) inhibit adenylyl cyclase option.
in a comparable manner (Saudou et al., 1992). The EC50 of
5-HT for Pea5-HT1 receptors was 130 nM, thus demonstrating
a relatively low potency compared with other arthropod Possible physiological roles of Pea5-HT1 receptors
orthologues, for example, the 5-HT1 receptors of D. melano- As Pea5-HT1 receptors are expressed in the salivary glands of
gaster (30 nM for Dm5-HT1A, 18 nM for Dm5-HT1B; Saudou P. americana, these receptors probably participate in the
et al., 1992) and Boophilus microplus (83 nM; Chen et al., control of 5-HT-stimulated salivary secretion. In physiological
2004). experiments investigating the pharmacology of 5-HT-induced
Our attempts to identify full agonists that mimic the inhibi- salivary secretion, we have established a pharmacological
tory effect of 5-HT have revealed that the receptor is resistant profile similar to that of the heterologously expressed

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
1460 B Troppmann et al

Pea5-HT1 receptor: 5-MeOT acts as a partial agonist and pro- receptor now provide the basis for new studies regarding the
vokes saliva secretion, whereas 5-CT and 8-OH-DPAT show significance of this particular receptor for cockroach behav-
only minor effects (Troppmann et al., 2007). Furthermore, the iour and physiology. For example, the consequences of inter-
non-selective antagonist methiothepin blocks 5-HT-induced ference with Pea5-HT1 expression (application of the RNAi
saliva secretion. However, we have also shown that mianserin technique) or receptor activation (application of identified
inhibits salivary secretion, whereas it is not a potent antago- Pea5-HT1 receptor ligands) for rhythmic and aggressive behav-
nist at the Pea5-HT1 receptor expressed in HEK 293 cells. In ioural patterns can now be analysed.
the salivary gland, 5-HT induces the secretion of proteins
from a specific cell type, viz., the central cells (Just and Walz,
1996; Walz et al., 2006). However, because this effect is mim- Acknowledgement
icked by interventions that elevate intracellular cAMP levels
and are modulated by increased intracellular Ca2+ (Rietdorf This study was supported by a grant from the German
et al., 2005), the Pea5-HT1 receptor is unlikely to be the media- Research Foundation (BL 469/4).
tor of this effect. For these reasons, we expect that one or
more additional 5-HT receptors (5-HT7 and/or 5-HT2) are
expressed in P. americana salivary glands. We have meanwhile
Conflict of interest
cloned a putative 5-HT7 receptor fragment of the cockroach,
and have detected expression of the respective mRNA in sali- The authors state no conflict of interest.
vary gland tissue by RT-PCR (Troppmann and Blenau, unpub-
lished data). This receptor and 5-HT2 receptors of P. americana
remain to be characterized in the future in order to complete
our understanding of the complex effects of 5-HT in the
References
control and modulation of salivary secretion in this insect.
Alexander SPH, Mathie A, Peters JA (2009). Guide to Receptors and
From the expression of Pea5-HT1 in pars intercerebralis neurons
Channels (GRAC), 4th edition. Br J Pharmacol 158 (Suppl. 1): S1–
projecting to the retrocerebral complex and stomatogastric S254.
nervous system, we conclude that the Pea5-HT1 receptor is Anstey ML, Rogers SM, Ott SR, Burrows M, Simpson SJ (2009). 5-HT
probably involved in the 5-HT-mediated control or modula- mediates behavioral gregarization underlying swarm formation in
tion of neuroendocrine secretion processes and/or the motion desert locusts. Science 323: 627–630.
of the gut and foregut. 5-HT stimulates both the fore- and Barbas D, Zappulla JP, Angers S, Bouvier M, Castellucci VF, Des-
hindgut of cockroaches (Brown, 1965, 1975; Cook et al., Groseillers L (2002). Functional characterization of a novel 5-HT
receptor (5-HTap2) expressed in the CNS of Aplysia californica.
1969). As the effect has been observed in denervated prepa-
J Neurochem 80: 335–345.
rations, this action of 5-HT appears to be directly on the gut
Beggs KT, Hamilton IS, Kurshan PT, Mustard JA, Mercer AR (2005).
muscle (Cook et al., 1969; Brown, 1975). As the Pea5-HT1 Characterization of a D2-like dopamine receptor (AmDOP3) in
receptor is also expressed in the gut, it is a likely candidate in honey bee, Apis mellifera. Insect Biochem Mol Biol 35: 873–882.
mediating this effect of 5-HT on visceral muscle activity. Bell WJ, Sams GR (1973). Aggressiveness in the cockroach Periplaneta
Considerably, more is known about the functions of the americana (Orthoptera, Blattidae). Behav Biol 9: 581–593.
two 5-HT1 receptors in the genetic model organism D. mela- Bischof LJ, Enan EE (2004). Cloning, expression and functional analy-
nogaster. Dm5-HT1A mRNA has been found to oscillate with a sis of an octopamine receptor from Periplaneta americana. Insect
Biochem Mol Biol 34: 511–521.
phase of ZT18 (Claridge-Chang et al., 2001), whereas there is
Blenau W, Baumann A (2001). Molecular and pharmacological prop-
no circadian variation in mRNA or protein levels of Dm5-HT1B
erties of insect biogenic amine receptors: lessons from Drosophila
(Yuan et al., 2005). Furthermore, the Dm5-HT1B receptor is melanogaster and Apis mellifera. Arch Insect Biochem Physiol 48:
expressed in the clock network, and affects circadian light 13–38.
sensitivity by decreasing the activity of the protein kinase Blenau W, Baumann A (2005). Molecular characterization of the ebony
SHAGGY, which, in turn, produces increased stability of the gene from the American cockroach, Periplaneta americana. Arch
transcription factor TIMELESS (Yuan et al., 2005). In contrast, Insect Biochem Physiol 59: 184–195.
the Dm5-HT1A receptor seems to have a sleep-regulating role, Brown BE (1965). Pharmacologically active constituents of the cock-
roach corpus cardiacum: resolution and some characteristics. Gen
because Dm5-HT1A mutant flies have short and fragmented
Comp Endocrinol 5: 387–401.
sleep patterns (Yuan et al., 2006). Recently, Johnson et al.
Brown BE (1975). Proctolin: a peptide transmitter candidate in insects.
(2009) have postulated a role of 5-HT1 receptors in the modu- Life Sci 17: 1241–1252.
lation of aggressive behaviour in D. melanogaster. Interest- Chen C, Okayama H (1987). High-efficiency transformation of mam-
ingly, these authors postulate a ‘certain constitutive level of malian cells by plasmid DNA. Mol Cell Biol 7: 2745–2752.
activation’ of 5-HT1-like receptors (Dm5-HT1A and Dm5-HT1B) Chen A, Holmes SP, Pietrantonio PV (2004). Molecular cloning and
such that increased activation of these circuits (by 8-OH- functional expression of a serotonin receptor from the Southern
DPAT) increases certain forms of aggressive behaviour, cattle tick, Boophilus microplus (Acari: Ixodidae). Insect Mol Biol 13:
45–54.
whereas inactivation of these circuits (by WAY 100635)
Claridge-Chang A, Wijnen H, Naef F, Boothroyd C, Rajewsky N, Young
decreases this behaviour (Johnson et al., 2009).
MW (2001). Circadian regulation of gene expression systems in the
Cockroaches are also established model organisms for Drosophila head. Neuron 32: 657–671.
studying both circadian rhythms (Helfrich-Förster et al., 1998) Colas JF, Launay JM, Kellermann O, Rosay P, Maroteaux L (1995).
and aggressive behaviour (Bell and Sams, 1973). The pharma- Drosophila 5-HT2 5-HT receptor: coexpression with fushi-tarazu
cological characterization and localization of the Pea5-HT1 during segmentation. Proc Natl Acad Sci USA 92: 5441–5445.

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
B Troppmann et al 1461

Colas JF, Launay JM, Vonesch JL, Hickel P, Maroteaux L (1999). 5-HT receptors – structure and function at the molecular level. Curr Top
synchronises convergent extension of ectoderm with morphoge- Med Chem 2: 507–528.
netic gastrulation movements in Drosophila. Mech Dev 87: 77– Martel JC, Ormière AM, Leduc N, Assié MB, Cussac D, Newman-
91. Tancredi A (2007). Native rat hippocampal 5-HT1A receptors show
Cook BJ, Eraker J, Anderson GR (1969). The effect of various biogenic constitutive activity. Mol Pharmacol 71: 638–643.
amines on the activity of the foregut of the cockroach, Blaberus McLoughlin DJ, Strange PG (2000). Mechanisms of agonism and
giganteus. J Insect Physiol 15: 445–455. inverse agonism at 5-HT 5-HT1A receptors. J Neurochem 74: 347–357.
Cosi C, Koek W (2000). The putative ‘silent’ 5-HT1A receptor antago- Mustard JA, Blenau W, Hamilton IS, Ward VK, Ebert PR, Mercer AR
nist, WAY 100635, has inverse agonist properties at cloned human (2003). Analysis of two D1-like dopamine receptors from the honey
5-HT1A receptors. Eur J Pharmacol 401: 9–15. bee Apis mellifera reveals agonist-independent activity. Brain Res Mol
Dierick HA, Greenspan RJ (2007). 5-HT and neuropeptide F have Brain Res 113: 67–77.
opposite modulatory effects on fly aggression. Nat Genet 39: 678– Newman-Tancredi A, Conte C, Chaput C, Spedding M, Millan MJ
682. (1997). Inhibition of the constitutive activity of human 5-HT1A
Downer RGH (1990). Octopamine, dopamine, and receptors by the inverse agonist, spiperone but not the neutral
5-hydroxytryptamine in the cockroach nervous system. In: Huber I, antagonist, WAY 100,635. Br J Pharmacol 120: 737–739.
Masler EP, Rao BR (eds). Cockroaches as Models for Neurobiology: Nichols CD (2006). Drosophila melanogaster neurobiology, neurophar-
Application in Biomedical Research. CRC Press: Boca Raton, FL, macology, and how the fly can inform central nervous system drug
pp. 103–124. discovery. Pharmacol Ther 112: 677–700.
Fletcher A, Forster EA, Bill DJ, Brown G, Cliffe IA, Hartle JE et al. Nichols DE, Nichols CD (2008). 5-HT receptors. Chem Rev 108: 1614–
(1996). Electrophysiological, biochemical, neurohormonal and 1641.
behavioural studies with WAY-100635, a potent, selective and silent Olde B, McCombie WR (1997). Molecular cloning and functional
5-HT1A receptor antagonist. Behav Brain Res 73: 337–353. expression of a 5-HT receptor from Caenorhabditis elegans. J Mol
Gerhardt CC, van Heerikhuizen H (1997). Functional characteristics of Neurosci 8: 53–62.
heterologously expressed 5-HT receptors. Eur J Pharmacol 334: 1–23. Page TL (1987). 5-HT phase-shifts the circadian rhythm of locomotor
Grohmann L, Blenau W, Erber J, Ebert PR, Strünker T, Baumann A activity in the cockroach. J Biol Rhythms 2: 23–34.
(2003). Molecular and functional characterization of an octopam- Papoucheva E, Dumuis A, Sebben M, Richter DW, Ponimaskin EG
ine receptor from honeybee (Apis mellifera) brain. J Neurochem 86: (2004). The 5-hydroxytryptamine(1A) receptor is stably palmitoy-
725–735. lated, and acylation is critical for communication of receptor with
Hall TA (1999). BioEdit: a user-friendly biological sequence alignment Gi protein. J Biol Chem 279: 3280–3291.
editor and analysis program for Windows 95/98/NT. Nucleic Acids Rietdorf K, Blenau W, Walz B (2005). Protein secretion in cockroach
Symp Ser 41: 95–98. salivary glands requires an increase in intracellular cAMP and Ca2+
Hauser F, Cazzamali G, Williamson M, Blenau W, Grimmelikhuijzen concentrations. J Insect Physiol 51: 1083–1091.
CJP (2006). A review of neurohormone GPCRs present in the fruitfly Rotte C, Krach C, Balfanz S, Baumann A, Walz B, Blenau W (2009).
Drosophila melanogaster and the honey bee Apis mellifera. Prog Neu- Molecular characterization and localization of the first tyramine
robiol 80: 1–19. receptor of the American cockroach (Periplaneta americana). Neuro-
Helfrich-Förster C, Stengl M, Homberg U (1998). Organization of the science 162: 1120–1133.
circadian system in insects. Chronobiol Int 15: 567–594. Saudou F, Boschert U, Amlaiky N, Plassat JL, Hen R (1992). A family of
Hen R (1992). Of mice and flies: commonalities among 5-HT recep- Drosophila 5-HT receptors with distinct intracellular signalling prop-
tors. Trends Pharmacol Sci 13: 160–165. erties and expression patterns. EMBO J 11: 7–17.
House CR, Ginsborg BL (1985). Salivary gland. In: Kerkut GA, Gilbert Schlenstedt J, Balfanz S, Baumann A, Blenau W (2006). Am5-HT7:
LI (eds). Comprehensive Insect Physiology, Biochemistry and Pharmacol- molecular and pharmacological characterization of the first 5-HT
ogy, Vol. 11. Pergamon Press: Oxford, pp. 195–224. receptor of the honeybee (Apis mellifera). J Neurochem 98: 1985–
Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ 1998.
et al. (1994). International union of pharmacology classification of Seifert R, Wenzel-Seifert K (2002). Constitutive activity of G-protein-
receptors for 5-hydroxytryptamine (serotonin). Pharmacol Rev 46: coupled receptors: cause of disease and common property of wild-
157–203. type receptors. Naunyn Schmiedebergs Arch Pharmacol 366: 381–416.
Hoyer D, Hannon JP, Martin GR (2002). Molecular, pharmacological Sitaraman D, Zars M, Laferriere H, Chen YC, Sable-Smith A, Kitamoto
and functional diversity of 5-HT receptors. Pharmacol Biochem Behav T et al. (2008). 5-HT is necessary for place memory in Drosophila.
71: 533–554. Proc Natl Acad Sci USA 105: 5579–5584.
Johnson O, Becnel J, Nichols CD (2009). 5-HT 5-HT2 and 5-HT1A-like Spitzer N, Edwards DH, Baro DJ (2008). Conservation of structure,
receptors differentially modulate aggressive behaviors in Drosophila signaling and pharmacology between two 5-HT receptor subtypes
melanogaster. Neuroscience 158: 1292–1300. from decapod crustaceans, Panulirus interruptus and Procambarus
Johnston CA, Watts VJ (2003). Sensitization of adenylate cyclase: a clarkii. J Exp Biol 211: 92–105.
general mechanism of neuroadaptation to persistent activation of Strader CD, Fong TM, Graziano MP, Tota MR (1995). The family of
Gai/o-coupled receptors? Life Sci 73: 2913–2925. G-protein-coupled receptors. FASEB J 9: 745–754.
Jones BJ, Blackburn TP (2002). The medical benefit of 5-HT research. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: Molecular
Pharmacol Biochem Behav 71: 555–568. Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol
Just F, Walz B (1996). The effects of 5-HT and dopamine on salivary Biol Evol 24: 1596–1599.
secretion by isolated cockroach salivary glands. J Exp Biol 199: Tierney AJ (2001). Structure and function of invertebrate 5-HT recep-
407–413. tors: a review. Comp Biochem Physiol A Mol Integr Physiol 128: 791–
Käll L, Krogh A, Sonnhammer EL (2004). A combined transmembrane 804.
topology and signal peptide prediction method. J Mol Biol 338: Troppmann B, Walz B, Blenau W (2007). Pharmacology of serotonin-
1027–1036. induced salivary secretion in Periplaneta americana. J Insect Physiol
Kozak M (1984). Compilation and analysis of sequences upstream 53: 774–781.
from the translational start site in eukaryotic mRNAs. Nucleic Acids Wachten S, Schlenstedt J, Gauss R, Baumann A (2006). Molecular
Res 12: 857–872. identification and functional characterization of an adenylyl
Kroeze WK, Kristiansen K, Roth BL (2002). Molecular biology of 5-HT cyclase from the honeybee. J Neurochem 96: 1580–1590.

British Journal of Pharmacology (2010) 159 1450–1462

 Characterization of an insect 5-HT1 receptor
1462 B Troppmann et al

Walz B, Baumann O, Krach C, Baumann A, Blenau W (2006). The that activates adenylate cyclase. Proc Natl Acad Sci USA 87: 8940–
aminergic control of cockroach salivary glands. Arch Insect Biochem 8944.
Physiol 62: 141–152. Yuan Q, Joiner WJ, Sehgal A (2006). A sleep-promoting role for the
Watanabe H, Mizunami M (2007). Pavlov’s cockroach: classical con- Drosophila 5-HT receptor 1A. Curr Biol 16: 1051–1062.
ditioning of salivation in an insect. PLoS ONE 2: e529. Yuan Q, Lin F, Zheng X, Sehgal A (2005). 5-HT modulates circadian
Weiger WA (1997). Serotonergic modulation of behaviour: a phyloge- entrainment in Drosophila. Neuron 47: 115–127.
netic overview. Biol Rev Camb Philos Soc 72: 61–95. Zornik E, Paisley K, Nichols R (1999). Neural transmitters and a
Witz P, Amlaiky N, Plassat JL, Maroteaux L, Borrelli E, Hen R (1990). peptide modulate Drosophila heart rate. Peptides 20: 45–51.
Cloning and characterization of a Drosophila 5-HT receptor

British Journal of Pharmacology (2010) 159 1450–1462