Journal of Neuroche,nist~’
Lippincott—Raven Publishers, Philadelphia
© 1997 International Society for Neurochemistry
Cloning and Expression of a Human Serotonin
5-HT4 Receptor cDNA
use Van den Wyngaert, l~WalterGommeren, *peter Verhasselt, lMirek Jurzak,
tJosée Leysen, Walter Luyten, and Eckhard Bender
Departments of Experimental Molecular Biology, *Applied Molecular Biology, and
tBiochemical Pharmacology, Janssen Research Foundation, Beerse, Belgium
Abstract: Using a combination of library screening and
nested PCR based on a partial human serotonin 5-HT4
receptor sequence, we have cloned the complete coding
region for a human 5-HT4 receptor. The sequence shows
extensive similarity to the published porcine 5-HT4A and
rat 5-HT4L receptor cDNA; however, in comparison with
the latter, we find an open reading frame corresponding
to only 388 amino acids instead of 406 amino acids. This
difference is due to a frame shift caused by an additional
cytosine found in the human sequence after position
1,154. Moreover, we also found the same additional cytosine in the rat 5-HT4 sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in
rat genomic DNA. Based on a part of the genomic 5HT4 receptor sequence that was identified in the cloning
process, there seem to be at least two possible splice
sites in the coding region of the gene. The human 5-HT4
receptor, transiently expressed in COS-7 cells, showed
radioligand binding properties3H]GR113808
similar to 5-HT4
revealed
receptors
KD
values
of 0.15
±0.01 tissue.
nMforthe
human receptor and 0.3
in guinea
pig striatal
[
±0.1 nM in the guinea pig tissue. Binding constants were
determined for four investigated 5-HT
4 antagonists and
three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT4-specific agonists caused
an increase in cyclic AMP levels. Key Words: Serotonin—5-HT4 receptor—Cloning-—Human—Rat.
J. Neurochem. 69, 181 0—1819 (1997).
Serotonin 5-HT4 receptors were first defined in primary cultures of mouse embryonic colliculi, based on
observed 5-HT-induced cyclic AMP formation (Dumuis et al., 1988a,b). A series of benzamides known
for their gastrokinetic properties mimicked the 5-HTinduced cyclic AMP formation in the colliculi neurons,
whereby the potencies correlated with effects on intestinal tissue (Dumuis et al., 1989). Hence, it was suggested that the therapeutically interesting gastrokinetic
properties of drugs like cisapride (Schuurkes et al.,
1985) could be mediated by agonistic action on gut 51810
HT4 receptors (Briejer et al., 1995). The advent of
selective 5-HT4 antagonists (Gaster and Sanger, 1994;
Sanger and Gaster,
and(Ford
of a radiolabeled
3H]GR 1994)
113808
and Clarke, 5-HT4
1993)
antagonist the
facilitated
[ further exploration of the 5-HT
4 receptors in the brain and in peripheral tissues (Ford and
Clarke, 1993; Eglen et al., 1995). The occurrence of
5-HT4 receptors, demonstrated in mammalian brain
(including human) using radioligand binding in regions of the extrapyramidal motor system of the basal
ganglia and the mesolimbic system, has suggested a
possible role of 5-HT4 receptors in affectiveand cognitive processes (Waeber et al., 1993; Eglen et al.,
1995). By using functional studies in isolated peripheral tissues, 5-HT4 receptor-mediated responses were
detected in the gastrointestinal tract (Ford and Clarke,
1993), the atrium (Kaumann et al., 1996), the bladder
(Candura et al., 1996), and the pulmonary vein (Cocks
and Arnold, 1992). Ullmer et al. (1995) and Schmuck
et al. (1996) showed expression of the rat, porcine,
and human 5-HT4 receptor in blood vessels without
including functional studies. The best characterized
property of 5-HT4 ligands probably concerns gastrokinetic effects. However, studies in various species suggest that there may be species differences with respect
to agonistic and antagonistic properties of ligands. To
allow appropriate investigation of the properties of ligands for therapeutic applications, cloning and expression of human 5-HT4 receptors are of prime importance.
Presently, six complete or partial cDNA sequences
Received April 24, 1997; revised manuscript received June 30,
1997; accepted June 30, 1997.
Address correspondence and reprint requests to Dr. E. Bender at
Department of Experimental Molecular Biology, Janssen Research
Foundation, Tumhoutseweg 30, B-2340 Beerse, Belgium.
Abbreviations used: AC, adenylyl cyclase; DMEM, Duibecco’s
modified Eagle medium; DMSO, dimethyl sulfoxide; 5-HT, 5-hydroxytryptamine, serotonin; K,, inhibition constant; 5-MeOT, 5methoxytryptamine; ON1 —14, oligonucleotides 1—14; ORF, open
reading frame; plC50, negative logarithm of the concentration that
inhibits 50% of specific binding by the radioligand.
CLONING OF A HUMAN 5-HT
4 RECEPTOR cDNA
1811
tories (Sulzfeld, Germany). The Bradford protein assay was
encoding 5-HT4 receptors are known: two splice variants from the rat (Gerald et al., 1995), one murine
sequence (Claeysen et al., 1996), two partial porcine
splice variants (Ullmer et al., 1995), and a partial
human sequence (Ullmer et al., 1995). The predicted
protein structures reveal seven transmembrane domains for the complete open reading frames (ORFs).
According to their structure and 5-HT4 receptor-coupled signal transduction events (increase in cyclic
AMP formation, opening of K + channels), 5-HT4 receptors have been classified as G protein-coupled receptors. To obtain a 5-HT4 receptor eDNA from a
human brain eDNA library, we used a combination of
library screening and nested PCR. The derived human
sequence was highly similar to the published rat 5HT4L sequence (Gerald et al., 1995); however, due to
a frame-shift in the 3’ region, a different stop codon
is used to terminate translation. Therefore, the human
and rat sequences were confirmed by sequencing corresponding PCR samples from genomic DNA from
blood of normal human volunteers and rats. In both
species, the found frame shift was confirmed. The human cDNA was subcloned into the expression vector
pcDNA3 and transfected transiently into COS-7 cells
for the investigation of the pharmacological profile and
second messenger coupling of the receptor. Radioligand binding properties of the cloned human receptor
were compared with those of 5-HT4 receptors in guinea
pig striatal tissue. The pharmacological profile of the
receptor was evaluated further in light of the functional
properties of ligands.
MATERIALS AND METHODS
Materials
dNTPs, MgC12, and PCR buffer II were obtained from
Perkin—Elmer Cetus (Foster City, CA, U.S.A.). Pfu polymerase and the Robocycler PCR block were products of
Stratagene (La Jolla, CA, U.S.A.). TaKaRa Ex Taq polymerase was obtained from TaKaRa (Shigu, Japan). TaqStart
antibody and human brain 5 ‘-STRETCH PLUS eDNA library (XgtlO) were from Clontech (Palo Alto, CA, U.S.A.).
T4 DNA ligase and restriction endonucleases were products
of Boehringer (Mannheim, Germany). The Multiprime
3H]GR
DNA
113808labeling
with a specific
system,activity
Hybond-N
of 3.07filters,
Tbq/mmol
and were
[
ob-
tained from Amersham (Little Chalfont, U.K.). [32PIdCTP
was purchased from NEN Du Pont (Wilmington, DE,
U.S.A.). The Qiagen Lambda Maxi kit, plasmid preparation
kits, and the Qiaquick PCR purification kit were from Qiagen
(Hilden, Germany). The PRISM Ready Reaction Dye Terminator Cycle Sequencing Kit and the ABI 377 or 373A
sequencing machines were from Applied Biosystems (Foster
City, CA, U.S.A.). The GeneAmp PCR System 9600 was
from Perkin—Elmer (Norwalk, CT, U.S.A.). The MicroSpin
G-50 columns were obtained from Pharmacia Biotech (Uppsala, Sweden), and the mammalian expression vector
pcDNA3 was obtained from Invitrogen (Carlsbad, CA,
U.S.A.). Dulbecco’s modified Eagle medium (DMEM) and
fetal calf serum were from Life Technologies (Gaithersburg,
MD, U.S.A.). Wistar rats were from Charles River Labora-
performed with the reagent supplied from Bio-Rad (Nazareth Eke, Belgium). The NEN flash plate assay was supplied
by Du Pont de Nemours (Brussels, Belgium). The liquid
scintillation spectrometer and the scintillation fluid Ultima
Gold MV were from Packard (Meriden, CT, U.S.A.). Cisapride was synthesized by Janssen Pharmaceutica NV
(Beerse, Belgium). Tropisetron was a gift from Sandoz (Basel, Switzerland), and GR 125487 was a gift from Glaxo
(Ware, U.K.). SB 204070 and GR 113808 were synthesized
by Janssen Pharmaceutica for its own purposes. All compounds were dissolved and diluted in dimethyl sulfoxide
(DMSO; except the indoleamines, which were dissolved in
water and protected from light throughout the experiment).
The final DMSO concentration in the tests did not exceed
0.5% (vol/vol). The GraphPad Prism program was from
GraphPad Software, Inc. (San Diego, CA, U.S.A.).
General molecular biological methods
Unless otherwise indicated, all PCR reactions were performed in a total volume of 50 ~.tl,containing 1 p~lof eDNA
and 2.5 U of Pfu polymerase in 1 x PCR buffer II, 200 ,uM
dNTPs, 200 nM primers, and 2.0 mM MgCl
2. PCR conditions were 5 mm of denaturation at 95°C, 1 mm at 54°C,
and 2 mm at 75°C, followed by a 10-mm incubation at
75°C.DNA manipulations were done according to standard
protocols (Maniatis et al., 1982).
DNA sequencing was carried out with reagents from the
PRISM Ready Reaction Dye Terminator Kit and run on a
GeneAmp PCR System 9600 according to the specifications
of the supplier.
Preparation of human 5-HT4 receptor probe and
eDNA library screening
Two primers [oligonucleotide 1 (ON1): 5’-CTG CTG
CCA GCC TTT GGT CTA T-3’; 0N2: 5 ‘-TCT CTG TCC
TCA TGC GAT GAG TG-3 ‘] were designed based on the
published partial human 5-HT4 receptor eDNA sequence
(Ullmer et al., 1995). PCR reactions were performed using
the human brain XgtlO 5 ‘-STRETCH PLUS eDNA library.
After the 398-bp PCR product was subcloned into pUCl8,
a 370-bp human 5-HT4 eDNA insert was removed from the
using
pUC18 construct by restriction digest using 32P]dCTP
StuI and KpnI.
TheMultiprime
the
eDNA was DNA
then randomly
labeling system.
labeled with
A human
[
brain kgtl0
5 ‘-STRETCH PLUS eDNA library was screened as described in the Lambda Library Protocol Handbook (Clontech). One positive plaque was observed, and after two
rounds of plaque purification a single positive plaque (clone
17) was cored out. Sequencing by primer walking of the X
clone 17 insert revealed that it contained part of the human
5-HT
4 receptor eDNA, from nucleotide position 507 to position 1,077 of the ORF.
Nested PCR amplification and cloning of the 5’
and 3’ ends of the human 5-HT4 ORE
Based on the sequence of the X clone 17 insert, four oligonucleotides were designed (0N5: 5 ‘-AAG GCC ACC ACA
GAG CAG-3’; 0N6: 5 ‘-TGC CCG TTG TAA CAT CTG
AA-3’; 0N7: 5’-TGT TCA ACC ACA ACC ATT AAT
GG-3’; 0N8: 5’-ATA GAC CAA AGG CTG GCA GC3’). PCR reactions combining these oligonucleotides with
one universal )~gtl0primer (0N3: 5 ‘-AGC AAG TTC AGC
CTG GTT AAG T-3’; 0N4: 5 ‘-TTA TGA GTA TTT CTT
CCA GGG-3’) were performed on the ~gtl0 human brain
J. Neurochem., Vol. 69, No. 5, 1997
1812
I. VAN DEN WYNGAERT ET AL.
5 ‘-STRETCH PLUS eDNA library to amplify the missing
5’ and 3’ parts of the human 5-HT
4 ORF by nested PCR.
To obtain the 5’ region, primers 0N3 and 0N6 were used
in a first round of PCR, followed by a secondary PCR using
1 p~lof the resulting product in combination with primers
0N3 and ONS. In an analogous fashion, the missing 3’
region was amplified by combining first 0N4 and 0N8 and
using the resulting product as substrate for the primer combi-
nation 0N4 and ON7. The PCR was performed under standard conditions, except that TaKaRa Ex Taq polymerase,
(1.25 U) was used. The reactions were cycled 35 times for
30 s at 95°C,30 s at 54, 55, 56, 57, 58, or 59°C,and 2 mm
at 72°Cin the Robocycler PCR block, followed by a 10-mm
incubation at 72°C.PCR products were ligated into pUC18.
Assembly of the full-length human 5-HT4
receptor coding region
The full-length eDNA was assembled by a two-step ligation. In the first step, the EcoRI/PfIMI restriction enzyme
fragment of nested PCR product 0N3/0N5 was ligated with
the XbaI/PflMI restriction enzyme fragment ONl /0N2 into
the EcoRIIXbaI large fragment of the mammalian expression vector pcDNA3, yielding pcDNA3/ON1ION2/0N31
0N5. In parallel, the EcoRIIAf1111 restriction enzyme fragment of the X clone 17 insert was ligated together with the
BstUIIAf1111 restriction enzyme fragment of the nested PCR
product 0N4/0N7 into the EcoRIIEcoRV large fragment
ofpcDNA3, yielding pcDNA3I l7/0N4/0N7. In the second
step, the EcoRIISphI small fragment of pcDNA3 /ON 1 /
0N2/0N3 IONS was ligated together with the SphI/NotI
small fragment of pcDNA3 /1 7/0N4/0N7 into the EcoRII
NotI large fragment of pcDNA3.
Sequence verification of the human and rat 5-HT4
ORF 3’ end
To verify the nucleotide sequence at the 3’ end of the
human and rat 5-HT4 coding region, control PCRs were set
up on genomic DNA to amplify the 3’ end as follows: two
oligonucleotides (0N9: 5’-AGA GTC AGT GTC ACC
CGC CAG-3’; ON1O: 5 ‘-AAG CAG CAG CTT AGG ACC
TG-3’) were designed downstream of the putative splice site
(assigned on the basis of the reported rat 5-HT4 receptor
sequence). PCR was carried out on genomic DNA samples
prepared from the blood of 10 human volunteers in a volume
of 100 ftl using standard conditions.
Similarly, PCR was carried out on genomic DNA prepared
from rat blood to amplify the short and long rat 5-HT4 splice
variants. This was performed using oligonucleotides ONll
(5’-TTT GCA TAG TGG TCA ACA CCA GG-3’), 0N12
(5’-CGT CTT CAA TCA AAA GCA TGA TTC C-3’),
0N13 (5’-AGA GTC GGT GTC ACC TCA CAG-3’), and
0N14 (5’-CAG CTT AGG ACT GGC TTC TTT TC-3’).
Expression of the human 5-HT4 receptor in
mammalian cells
COS-7 cells were grown in DMEM supplemented with
10% fetal calf serum. A large-scale plasmid preparation of
5-HT4/pcDNA3 was made using the Qiagen large-scale
plasmid prep kit. Plasmid DNA was
transfected
COSday beforeinto
transfec2 the
7tion)
cellsusing
(seeded
the DEAE-dextran
at 30,000 cells/cm
method (Huylebroeck et al.,
1988) in serum-free DMEM for 30 mm. The transfection
medium was removed and replaced by COS-7 medium supplemented with 100 ~iM chloroquine. After 4 h, the chloroquine medium was replaced by COS-7 medium containing
J. Neurochem., Vol. 69, No. 5, 1997
4 mM sodium butyrate. Sixteen hours later, this medium
was replaced by COS-medium containing only 2% fetal calf
serum. Incubation was continued for another 48 h before
the cells were harvested for binding and signal transduction
assays.
Membrane preparation
The transfected COS-7 cells were cultured on 150-mm
Petri dishes and washed twice with ice-cold phosphate-buffered saline. The cells were then scraped from the plates
with a cell scraper, suspended in 50 mM Tris-HC1 buffer,
pH 7.4, and harvested by centrifugation for 10 mm at 16,000
g. The pellet was resuspended in 5 mM Tris-HC1, pH 7.4,
and homogenized with an UltraTurrax homogenizer; the resulting membranes were collected by centrifugation for 20
mm at 25,000 g. Membranes were stored at —70°C in 50
mM Tris-HC1 buffer, pH 7.4, at a protein concentration of
1 mg/ml. For the preparation of membranes from guinea pig
striatum, the tissue was pooled in cold 50 mM Tris-HC1
buffer, pH 7.4, homogenized, centrifuged, and collected as
described for the cell membranes. The Bradford protein
assay was used for protein determination with bovine serum
albumin as a standard.
Radioligand binding
Assay mixtures (0.5 ml) contained [3H]GR 113808, an
aliquot of membrane preparation (25—75 ,ug of protein for
transfected COS-7 cells, 200—300 ,ug of protein for guinea
pig striatal tissue), and solvent for total binding or 10 pM
5-HT to determine nonspecific binding. The assay buffer
consisted of 50 mM HEPES/NaOH, pH 7.5, 1 ,aM pargyline
(monoamine oxidase inhibitor), and paroxetine (5-HT transporter inhibitor), and the mixture was incubated at 37°Cfor
30 mm. Competition binding experiments were performed
with a 0.2 nM concentration of the radioligand. Ligand concentration binding isotherms were obtained using 10 concentrations of [3H]GR 113808 in a concentration range of 16
pM to 1.6 nM. The incubation was terminated by rapid
filtration over Whatman GF/B filters presoaked in 0.1%
polyethylenimine, and three washing steps with 3 ml of icecold Tris-HC1 buffer, pH 7.4. Filter-bound radioactivity was
counted in a liquid scintillation spectrometer using 2 ml of
scintillation fluid.
Ligand concentration binding isotherms (rectangular hyperbola) and sigmoidal inhibition curves were calculated by
nonlinear regression analysis according to algorithms described by Oestreicher and Pinto (1987). The maximal number of binding sites (Bmax) and equilibrium dissociation constant (KD) of the radioligand and the plC
50 (negative logarithm of the concentration that inhibits 50% of specific
binding by the radioligand) values of competitors were derived from the curve fitting. Apparent inhibition constant
(K~)values were calculated according to the equation of
Cheng and Prusoff (1973). Graphs were prepared using the
GraphPad Prism program.
Measurement of cyclic AMP formation
For the stimulation of adenylyl cyclase (AC), COS-7 cells
were transfected and cultured in 24-multiwell plates (seeding
density of 30,000/well). The DMEM contained 1% dialyzed
calf serum (<0.1 nM 5-HT). On the third day following
transfection, the cells were washed with buffer consisting of
2S mM Tris-HC1, pH 7.4, 120 mM NaCl, 5 mM KCI, 0.8
mM MgC12, 1.8 mM CaC12, 15 mM glucose, and 0.04 mM
phenol red. Isobutylmethylxanthine at 1 mM, I 1iM pargy-
CLONING OF A HUMAN 5-HT
4 RECEPTOR cDNA
line, and 1 ~.sMparoxetine were then added to the buffer. To
estimate the maximal AC stimulation, 100 jiM forskolin was
used. The incubation with compounds was performed for 20
mm at 37°C.The final concentration of DMSO (whenever
needed to dissolve the compounds) did not exceed 0.5%
(vol/vol) and was also included in the corresponding control
samples. All points were from triplicate assays. The incubation was stopped by acidification with 0.1 ml of ice-cold 1
M HC1O4, followed by neutralization with K2HPO4. After
the formation of a KC1O4 precipitate, the plates were centrifuged for S mm at 2,000 g and the supernatant was assayed
for cyclic AMP content using the NEN flash plate assay
according to the protocol of the supplier.
RESULTS
Based on the partial eDNA sequence of the human
5-HT4 receptor (Ullmer et al., 1995), two PCR primers
were designed to amplify this 397-bp-long fragment
of human 5-HT4 eDNA, stretching from position 366
to position 762 of the ORF [following the alignment
of the published rat eDNA sequence (Gerald et al.,
1995)1. This PCR product was cloned, radiolabeled,
and then used to screen a commercial human brain
eDNA library, resulting in one positive clone (clone
17). Sequence analysis of the 1,650-bp-long insert
showed extensive similarity to the published rat and
pig sequences from position 507 to position 1,077 of
the rat 5-HT4 ORF (Fig. 1). However, downstream
of position 1,077 and upstream of position 507, no
significant similarity was detectable (Fig. 1). As the
known rat (Gerald et al., 1995) and porcine (Ullmer
et al., 1995) splice variants diverge at these points and
the sequences at these positions are in good agreement
with the consensus 5’ and 3’ splice sites (Fig. 1; Shapiro and Senapathy, 1987), the insert of clone 17 is
most likely a genomie DNA fragment, contaminating
the eDNA library.
To obtain the remaining parts of the human 5-HT4
eDNA sequence, primers were designed based on the
5’ and 3’ ends of the sequence of clone 17 that still
showed sequence similarity to the rat 5-HT4 eDNA.
These primers were used in combination with primers
complementary to the XgtlO vector sequence 5’ and
3’to the cloning site in a nested PCR reaction.
The sequence of the 0N3/0N5 PCR product cone-
1813
sponds to the 5’ region of the human 5-HT4 receptor
eDNA from position —289 to position +386, with the
A of the start eodon being position + 1. The 0N4/0N7
PCR product corresponds to the 3’ region of the human
5-HT4 receptor eDNA from position + 1,057 up to the
stop eodon and continuing for 301 nueleotides into the
3’ untranslated region. Therefore, the resulting prod-
ucts were shown to contain the missing parts of the
human 5-HT4 coding region, as well as part of the 5’
and 3’ untranslated regions. The full-length nueleotide
sequence of the coding region and derived amino acid
sequence are shown in Fig. 2.
The human sequence showed overall similarity to
the published rat 5-HT4 receptor long splice variant
sequence (Fig. 3), but it differed by one additional
cytosine after position 1,154 of the rat coding sequence
(Fig. 2). This reading frame shift resulted in a completely different amino acid sequence downstream and
usage of a different stop eodon, terminating the protein
after amino acid position 388 instead of amino acid
406. To verify the human sequence, the 3’ region of the
ORF was amplified from genomic DNA of 10 different
human volunteers. All samples showed the additional
cytosine after position 1,154.
To determine whether this was a species difference,
the long splice variant 3’ part of the rat 5-HT4 receptor
was amplified from two different rat genomie DNA
samples. In contrast to the published sequence (Gerald
et al., 1995), we also found a cytosine triplet instead
of a doublet present in the rat sequence. When the
additional eytosine is inserted into the rat nueleotide
sequence, the derived rat long splice variant is only
one amino acid longer than the short splice variant.
This shortened rat 5-HT4L splice variant displays an
ORF that corresponds closely to the cloned human 5HT4 receptor (see Fig. 3).
The pharmacological characteristics of the human 5HT4 receptor protein were investigated by radioligand
binding assays on membranes from COS-7 cells transiently transfeeted with the human 5-HT4/peDNA3 expression plasmid. Preliminary functional data were obtained by measuring 5-HT-induced cyclic AMP formation in the same cell population.
3H]Ligand concentration binding isotherms with [
FIG. 1. Nucleotide sequence comparison ofthe insert
from clone 17 with the porcine 5-HT4A (UlImer et al.,
1995) and rat 5-HT4L (Gerald et al., 1995) sequences.
The positions where the sequences diverge are indicated (A). Sequences that correspond to the 3’ or
5’ splice consensus site are boxed. Numbering of the
nucleotides is according to their position in the ORF;
for the porcine partial sequence, it is based on the
human 5-HT4 ORE.
J. Neurochem., Vol. 69, No. 5, 1997
1814
I. VAN DEN WYNGAERT ET AL.
GR 113808 on membranes from three independent
transfeetions in COS-7 cells revealed Bmax values ranging from 1,114 to 3,225 fmol/mg of protein and a
mean K value of 0.15 ±0.1 nM. The corresponding
0
values on preparations from the guinea pig striatum
were 182 ±30 fmol/mg of protein and 0.3 ±0.1 nM
for the Bmax and K0 values, respectively (n = 3). A
straight line in a Seatehard analysis suggested the presence of a single high-affinity binding site in both tissues. Representative curves are shown in Fig. 4. Membranes from COS-7 cells transfected with the pcDNA3
3HIGR
vector
show by
any5-HT).
specific [
113808 control
binding did
(i.e.,not
inhibited
Several selective ligands for the 5-HT
4 receptor
were used in competition binding experiments as reference compounds to define the pharmacological profile
of the cloned human 5-HT4 receptor (Briejer et al.,
1995). High-affinity antagonists with high selectivity
for the 5-HT4 receptors were used: SB 204070 (Wardle
et al., 1994), GR 125487 (Gale et al., 1994), and GR
113808 (Grossman et al., 1993), as well as the firstdescribed, but weaker and nonseleetive 5-HT4 antagonist tropisetron [ICS 205-930 (Dumuis et al., l988b)].
In addition, we investigated 5-HT, its natural derivative
5-methoxytryptamine (5-MeOT), and the benzamide
gastroprokinetie agent cisapride, known to be a partial
agonist of the receptor. Inhibition curves of radioligand
binding in membranes from transfeeted COS-7 cells
and from guinea pig striatum are depicted in Fig. 5,
and the mean plC50 and K values are presented in
Table 1.
The functional coupling of the cloned human receptor in COS-7 cells to the cyclic AMP pathway was
investigated in preliminary experiments. These experiments have clearly shown a stimulation of the AC
system by receptor-specific agonists (Fig. 6). Basal
cyclic AMP levels were 3—4 nM in the presence of
<0.1 nM 5-HT. Incubation with 5-HT, 5-MeOT, or
cisapride increased the levels of cyclic AMP up to
sevenfold. In veetor-transfected cells, cyclic AMP levels were not elevated following incubation with the 5HT4 agonists (data not shown). The forskolin-induced
cyclic AMP level in 5-HT4 receptor transfeeted cells
remained unchanged after 5-HT addition. Hence, we
found no indication for an inhibitory action of 5-HT
on the cyclic AMP system.
DISCUSSION
On the basis of the published partial human 5-HT4
eDNA sequence, we attempted to clone a full-length
FIG. 2. Nucleotide and deduced amino acid sequence of the
cloned human 5-HT4 receptor. Potential N-linked glycosylation
(•), protein kinase C phosphorylation (V), and palmitoylation
(~)sites are indicated. Positions of amino acids and nucleotides
are given at the end ofeach line. The additional C that was found
compared with the published rat 5-HT4L cDNA (Gerald et al.,
1995) is underlined.
J. Neurochem., Vol. 69, No. 5, 1997
CLONING OF A HUMAN 5-HT
4 RECEPTOR cDNA
1815
FIG. 3. Alignment of the six known partial [pig
5-HI45 (UlImer et al., 1995), and rat
study)) and complete [rat 5-HT45 (Gerald et al., 1995), mouse 5-HT4L (Claeysen et al.,
1996), rat 5-HT4L (Gerald et al., 1995), and a human
5-HT4 (this study)] 5-HT4 receptor amino acid sequences. The putative transmembrane domains
(TM) are boxed.
5-HT4A, pig
5-HT4L (this
eDNA by screening a commercially available human
brain eDNA library. However, the only clone that was
found contained a fragment of genomie DNA as an
insert. To some extent, this parallels the difficulties
described in finding a full-length rat eDNA clone in
four different rat eDNA libraries (Gerald et al., 1995),
most likely reflecting the low abundance of the 5-HT4
receptor mRNA.
The central sequence of the insert from our genomie
clone shows a high degree of sequence similarity to
the published rat 5-HT4 eDNA; however, at the 5’ and
3’ ends of this central segment, the sequence similarity
.1. Neurochem., Vol. 69, No. 5, 1997
1816
I. VAN DEN WYNGAERT ET AL.
amino acid level and confirming also the eytosmne doublet after position 1,152.
To confirm that our sequence is correct at the 3’
end, we examined this part of the sequence in genomic
DNA from 10 different human volunteers. In all eases,
we obtained our original sequence, suggesting that this
is the correct sequence. To investigate whether this
finding pointed toward a species difference, we ampli-
fied the same region from rat genomie DNA. In two
independent experiments, we found a sequence with
an additional eytosine after nueleotide position 1,154,
corresponding to our findings on the human receptor.
The amino acid sequence of the long splice variant of
the rat receptor derived from the nucleotide sequence
including the cytosine triplet is highly similar to our
human sequence (Fig. 2). However, this indicates that
our long rat splice variant is only 388 amino acids long
instead of 406. Further studies are needed to determine
whether the observed difference is based on a variant
DNA sequence or due to a sequencing error. Two partial 5-HT4 receptor eDNA splice variants are known
in the pig, where the alternative splicing occurs at a
different position from that in the rat sequence. Regard3HJGR113808 concentration binding isotherm for
FIG.
4. [ preparations from COS-7 cells transfected with the
membrane
human 5-HT
4 receptor (A) and from guinea pig striatum (B).
Total binding (•), nonspecific binding (•), and specific binding
(D) are presented as means of duplicate determinations. The
data represent a typical experiment out of three independent
experiments. Nonspecific binding was determined in the presence of 10 jiM 5-HT. KD and ~ values were derived from curve
fitting using nonlinear regression analysis. The mean values for
the 5-HT4 transfected COS-7 cells were a KD of 0.15 ±0.01 nM
and a ~ of 2,221 fmol/mg of membrane protein; for the guinea
pig striatum, a K0 of 0.3 ±0.1 nM and a Brna~of 182 ± 30 fmol/
mg of membrane protein were determined.
ing this variation, our clone is similar to the 5-HT4A
sequence (Fig. 3).
Taken together, the few known 5-HT4 receptor
eDNA sequences from different species show a re-
stops (Fig. 1). The 5’ site of sequence divergence
corresponds to the position in the porcine 5-HT4B
where an insertion is found, suggesting that an intron/
exon boundary is present at this point in the human
gene as well. At the 3’ end, the similarity stops where
the rat long and short splice variants diverge in sequence; therefore, this point also corresponds to an
exon/intron boundary. This interpretation is strengthened by the occurrence of sequences closely resembling splice consensus motifs at the site of sequence
divergence (Fig. 1; Shapiro and Senapathy, 1987).
The missing parts of the ORF at the 5’ and 3’ ends
were cloned by nested PCR performed on a human
brain eDNA library. The nucleic acid and derived
amino acid sequences showed a high degree of similar-
ity to the published rat 5-HT4 long splice variant; however, we also observed an additional cytosine after
position 1,154. The recently cloned murine 5-HT4 receptor eDNA (Claeysen et al., 1996) is to a large
extent similar to the published long splice variant of
the rat (Fig. 3), showing only 16 differences at the
J. Neurochem., Vol. 69, No. 5, 1997
3H]GR
113808
nM) binding
membrane preparations
FIG. 5. (0.2
inhibition
by 5-HT4to antagonists
and agonistsfrom
of [ COS7 cells transfected with the human 5-HT
4 receptor (A) and from
guinea pig striatum (B). Depicted points are means of two to
values
five independent experiments, and the calculated plC~~
are given in Table 1.
CLONING OF A HUMAN 5-HT
4 RECEPTOR cDNA
1817
3H]GR 113808 binding in membranes from COS-7 cells
TABLE I. Potency
transiently
of compounds
transfected
to compete
with the with
human
0.2 5-HT
nM [
4 receptor and from guinea pig ,ctriatum
Human 5-HT4 receptor in
Guinea pig 5-HT4 receptor in
Rat 5-HT4L
COS-7 cells
the striatum
receptor in
COS-7 cells”
plC
50
Compound
Antagonists
SB 204070
GR 125487
GR 113808
Tropisetron
Agonists
Cisapride
5-HT
5-MeOT
±SD
(n)
9.7
±0.2 (4)
9.5 ±0.1 (3)
9.4 ±0.03 (3)
6.4 ±0.1 (3)
6.9 ± 0.2 (4)
6.6 ±0.3 (5)
5.7 ± 0.2 (3)
pK1
plC50
10
10 ±0.3
9.6 ±0.2
9.3 ±0.3
6.6 ±0.3
9.8
9.6
6.7
7.2
6.9
6
al., 1993.
Literature data obtained for 0from
the rat Waeber
5-HT4L et
receptor
or on
From Gerald et al., 1995;
±SD
(n)
0
Human brain
pK,
membranes
pK
pK,
(4)
(3)
(2)
(2)
10.1
9.7
9.4
6.7
ND
ND
ND
6.7
ND
ND
9.8
6.7
7.1 ± 0.1 (3)
7.2 ±0.2 (3)
6.5 ±0.2 (2)
7.2
6.9
6.8
7.4
6.8
6.5
6.6
6.4
6.3
homogenates from human brain are included for comparison. ND, not determined.
markable sequence variation and various possibilities
to generate splice variants. Detailed analysis of the
species and tissue-dependent presence of these differ-
ent variants will be important for the elucidation of
the 5-HT
4 receptor physiology and pharmacology. For
the rat long and short 5-HT4 receptor splice variant,
different poteneies for AC stimulation have been described previously (Gerald et al., 1995). As we found
the long splice variant to be actually only 388 amino
acids long, however, this cannot be attributed to a
fourth, additional phosphorylation site. Further support
for the existence of 5-HT4 receptor variant sequences
can be drawn from reports on tissue-dependent desensitization rates of 5-HT4 receptor preparations (Ansanay et a]., 1992), which are attributed to differences
of the intercellular C-termini of the involved 5-HT4
receptors (Gerald et al., 1995). The coupling effi-
In addition to the discussed interspecies sequence
similarity, our pharmacological data strongly support
the classification of the cloned human receptor into
the
3H]GR
5-HT4
high affinity
of [ et al.,
113808 receptor
is a firstfamily.
line ofThe
evidence
(Grossman
1993). This radioligand has already proven its usefulness in the mapping of 5-HT
4 receptors in the rat,
guinea pig, and human brain (Grossman et al., 1993;
Waeber et al., 1993). The K0 values measured in this
study on the human receptor expressed in COS-7 cells
and on the guinea pig striatal receptor (0.15 and 0.3
nM, respectively) are in excellent accordance with a
reported K0 of 0.2 nM for the guinea pig striatum
and human brain membranes (Grossman et al., 1993;
Waeber et al., 1993). GR 113808 has also been reported to be a potent 5-HT4 receptor antagonist in the
guinea pig ascending colon and rat esophagus with
ciency of 5-HT4 receptors was also reported to be tissue-specific (Eglen et al., 1995), which is consistent
pA2 values of 9.3 and 9.5, respectively, and to have a
with a different GTP sensitivity of radioligand binding
in even closely related membrane preparations (Grossman et al., 1993).
man et a]., 1993; Sanger and Gaster, 1994). The pK,
>3,000-fold selectivity over other receptors (Grossvalue of cold GR 113808 in our binding tests on both
preparations (9.4 and 9.6) is in good agreement with
these functional data.
The rank order of potency
of the
compounds
113808
bindingused
on
3H]GR
for the cell
competition
of transfeeted
[
COS-7
membranes
with the 5-HT
4 receptor clone (and on the guinea pig tissue as well)
matches the expected pharmacological profile. The ligand found to have the highest pK, values on the human
and guinea pig receptor was SB 204070 (10 and 10.1,
respectively). This benzodioxane derivative is the
most potent and selective 5-HT4 antagonist described
so far (Gaster and Sanger, 1994) with an apparent pA2
of 10.8 on the guinea pig distal colon. It has a 5,000FIG. 6. Stimulation of AC in COS-7 cells transfected with the
human 5-HT4 receptor by specific agonists. Basal levels and
levels after stimulation with a 1 jiM concentration of the indicated
compounds from a triplicate determination are presented. cAMP,
fold selectivity over all other receptor preparations
tested (Wardle et al., 1994).
GR 125487 is a metabolically stable derivative of
GR 113808, with a prolonged in vivo biological halflife and slightly higher affinity (pK, = 10.4) on guinea
cyclic AMP.
pig striatum (Gale et a]., 1994).
J. Neurochen,., Vol. 69. No. 5, 1997
1818
I. VAN DEN WYNGAERT ET AL
Another competitor tested, the potent 5-HT3 receptor antagonist tropisetron (ICS 205-930) was the first
described 5-HT4 antagonist (Dumuis eta]., 1988b). Its
moderate potency in binding tests on both membrane
preparations (pK1 of 6.7 for the human and guinea pig
reeepior) is in good agreement with the first reports
about the inhibition of 5-HT-stimulated AC on mouse
eollieular neurons [pK, = 6.15 (Ansanay et al., 1992)]
and binding data (pK1 of 6.7) on human brain membranes (Waeber et a]., 1993). The tested agonists, including the full agonists 5-HT and 5-MeOT and the
partial agonist eisapride, exhibited lower affinities on
both membrane preparations in competition with a radiolabeled antagonist than reported in functional tests
on the guinea pig colon, where the pEC50 values were
8.0, 7.8, and 7.5, respectively (Schuurkes et al., 1985;
Leung et al., 1996). Agonists bind with high affinity
to the G protein-coupled state of the receptor and have
lower affinity for the uncoupled receptor. As radiolabeled antagonists detect both states of the receptor,
agonist competition often shows an overall lower plC5o
value, reflecting the affinity for the uncoupled receptor
(Adham eta]., 1996). Consistent with this assumption
and our data, pK~values of 7.5 and 6.8 for 5-HT and
5-MeOT, respectively, were observed in the guinea pig
striatum (Leung et al., 1996).
In summary, the pharmacological profile of the
cloned human receptor matches the profiles .found in
the guinea pig striatum and human brain membranes
(see Table 1; Waeber et a]., 1993), as well as that of
the cloned rat 5-HT4L receptor expressed in COS-7
cells (see Table 1; Gerald et al., 1995). The stimulatory effect of 5-HT on cyclic AMP formation observed
in 5-HT4/peDNA3 transfeeted COS-7 cells is an important criterion of the definition of the 5-HT4 receptor
family and is in agreement with many reports (Clarke
and Boekaert, 1993). The ability of the indole structure
5-MeOT and the benzamide eisapride to stimulate cyclic AMP production is a particular feature of the 5HT4 receptor and has also been shown for the cloned
rat 5-HT4L receptor (Gerald et al., 1995).
The characterization of a particular receptor is based
upon its structure, its second messenger coupling, and
its pharmacological profile. The human receptor described in this report meets all the criteria to classify
it as a 5-HT4 receptor.
Acknowledgment: The authors thank Dr. Michel Briejer
for critical reading of the manuscript.
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