b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1
Conformational state of human cardiac 5-HT4(g) receptorsinfluences the functional effects of polyclonal anti-5-HT4
receptor antibodies
Emmanuella Di Scala a, Stephanie Rose a, Olivier Herault b, Jorge Argibay a,Pierre Cosnay a, Veronique Bozon a,*aUMR CNRS 6542, Physiologie des Cellules Cardiaques et Vasculaires, Faculte des Sciences et Techniques, Universite Francois-Rabelais,
Tours 37200, Franceb INSERM ESPRI/UPRES-EA3249, Laboratoire d’Hematopoıese, Service d’Hematologie, Faculte de Medecine, Hopital Bretonneau,
CHU, Tours, France
a r t i c l e i n f o
Article history:
Received 25 July 2006
Accepted 7 December 2006
Keywords:
Antibody
Conformational state
Inverse-agonist
5-HT4 receptor
Serotonin
CHO cells
a b s t r a c t
The functional effects of the anti-G21V antibody directed against the second extracellular
loop of human heart 5-HT4 receptors can differ when the receptors are expressed in
different cell lines. Here, we extend these studies to show variation in the responses of
5-HT4(g) receptors to the antibody within the same expression system. In a previous report
no effect of the anti-G21V antibodies had been shown upon 5-HT4(g) receptors expressed in
CHO cells. Here the same antibodies alone or when added before 5-HT had a functional
‘‘inverse-agonist like’’ effect upon 5-HT4(g) receptors expressed in a separate line of CHO
cells. Although these CHO cells showed a lower efficacy of cAMP production evoked by 5-HT
than the previous report they express a similar h5-HT4(g) receptor density. Inhibition of
either phosphodiesterases or Gi proteins had no effect upon the action of the antibody.
Conformational states of the 5-HT4 receptor and/or equilibrium between different states of
receptors may then determine the functional effect of antibodies against this receptor.
# 2006 Elsevier Inc. All rights reserved.
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1. Introduction
Serotonin (5-HT) exerts positive inotropic and chronotropic
effects on human myocardium via 5-HT4 receptors (h5-HT4).
These receptors belong to the seven transmembrane-span-
ning G protein-coupled receptors superfamily (GPCR) [1]. In the
heart, functional 5-HT4 receptors are found in atria where they
are positively coupled to adenylate cyclase via Gs proteins [2–
5], activate cAMP-dependent protein kinase (PKA) and phos-
phorylate key proteins involved in cardiac excitation–con-
traction coupling including the L-type calcium channel [4,6].
Six 5-HT4 receptor isoforms, named 5-HT4(a), 5-HT4(b), 5-HT4(c),
5-HT4(g) originally called 5-HT4(e), 5-HT4(i) and 5-HT4(n), have
* Corresponding author. Tel.: +33 2 47 36 71 14; fax: +33 2 47 36 71 12.E-mail addresses: [email protected], veronique.bozon@univ-tou
0006-2952/$ – see front matter # 2006 Elsevier Inc. All rights reserveddoi:10.1016/j.bcp.2006.12.009
been identified in human atria [7–15]. Finally, activation of the
5-HT4 receptor has been suggested to contribute to proar-
rythmic activity such as atrial fibrillation [16,17].
Autoantibodies directed against cardiac G-protein-coupled
beta 1- and beta 2-adrenergic as well as M2 muscarinic
receptors have been implicated in some cardiopathologies
[18–24]. The human cardiac 5-HT4 receptor [25] may be a target
for autoantibodies in patients with atrial arrythmia. We have
previously shown that the polyclonal anti-G21V antibody
raised against the second extracellular loop of the cardiac h5-
HT4 receptor has diverse effects upon the h5-HT4 receptor.
Indeed, in human atrial myocytes the anti-G21V antibody
inhibited the 5-HT4 receptor mediated effect of serotonin [26],
rs.fr (V. Bozon).
.
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1 965
in COS-7 cells the transiently expressed h5-HT4(e/g) (originally
called 5-HT4(e)) receptor isoform was activated by this antibody
[27] while in CHO cells the same antibody showed no effect
upon the same stably expressed h5-HT4(g) receptor [6]. The
different behaviour of the recombinant h5-HT4(g) receptor
toward the anti-G21V antibody seems to be dependent on the
expression system, cellular and membrane environment.
To further explore this idea we took advantage of a separate
line of CHO cells which stably express the 5-HT4 receptor but
which shows a lower efficacy of cAMP production evoked by
5HT. Here we show that the anti-G21V antibody has functional
effects upon h5-HT4(g) receptors expressed in this line.
Therefore, the cell type does not seem to be the only parameter
implicated in the variability of the response of h5-HT4
receptors to the anti-G21V antibody. We examine the
possibilities that the density of expressed receptors, the signal
transduction pathway activated and/or the equilibrium
between the spontaneously active and the inactive forms of
the receptor, R/R*, could be factors contributing to the diverse
responses of h5-HT4 receptors.
2. Materials and methods
2.1. Materials
Purified anti-G21V polyclonal antibodies and Fab fragments of
anti-G21V antibodies were gracefully given by Dr. J. Hoebeke,
CNRS UPR 9021 (Institut de Biologie Moleculaire et Cellulaire,
Strasbourg, France). The CHO cell line stably expressing the h5-
HT4(g) receptor and the ligands GR127935 (N-[4-methoxy-3-(4-
methyl-1-piperazininyl)phenyl]-20-methyl-40-(5-methyl-1,2,4-
oxadiazol-3-yl)[1,1-bibhenyl]-4-carboxamide) and ML10375 (2-
[cis-3,5-dimethylpiperidino]ethyl 4-amino-5chloro-2methoxy-
benzoate) were nicely supplied by Dr. R. Fischmeister (INSERM
U-446, Laboratoire de Cardiologie Cellulaire et Moleculaire,
Faculte de Pharmacie, Chatenay-Malabry, France). FITC (fluor-
escein isothiocyanate), forskolin (7beta-acetoxy-1alpha,6be-
ta,9alpha-trihydroxy-8,13-epoxy-labd-14-en-11-one) and 5-HT
(5-hydroxytryptamine) were purchased from Sigma (Saint
Quentin Fallavier, France). GR113808 {[1-2-(methyl-sulfonyla-
mino)ethyl]-4 piperidinyl]methyl1-methyl-1H-indole-3 carbox-
ylate} was from TOCRIS (Illkirch, France). [3H]GR113808 was
purchased from Amersham Pharmacia Biotech Europe (Orsay,
France). Other compounds were of reagent grade obtained from
either Sigma or Invitrogen.
2.2. Cell culture
The CHO cell line that stably expressed h5-HT4(g) receptors
originally called h5-HT4(e) [11,12,28] was the gift of Dr. R.
Fischmeister (Chatenay-Malabry, France). CHO cells were
grown at 37 8C and 5% CO2 in Ham F12 medium (Life
Technologies, Cergy Pontoise, France) supplemented with
10% fetal calf serum (FCS), 10 mM HEPES, 1 mg/ml neomy-
cin, 2 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml
streptomycin. For transduction assays the cells were
harvested and seeded into 24-well plates with 1 � 105 cells
per well. Cells were then maintained for 24 h in medium
with or without the addition of FCS, this had no effect upon
on the results. In one series of experiments, 2 mg/ml of
Pertussis toxin (PTX) was added to the culture medium
for 24 h.
2.3. Adenylate cyclase activity
CHO cells expressing h5-HT4(g) receptors were preincubated
for 15 min in solution A, which consisted of Ham F12 medium
and 1 mM GR127935, the antagonist of endogenous 5-HT1(b)
receptors [28]. In some experiments 5 mM of the phospho-
diesterase inhibitor theophyllin was added to solution A. All
experiments were conducted at 37 8C in a 5% CO2 atmosphere.
One micro molar of 5-HT, 1 mM of the inverse agonist ML10375
[28,29], 10 mg/ml of polyclonal anti-G21V antibody, 30 mg/ml
of monovalent Fab fragments of anti-G21V antibody and
10 mM of forskolin (activator of adenylate cyclase) were added
to the culture medium as required. Control experiments
involved either CHO cells that expressed the h5-HT4(g)
receptor preincubated in solution A and then incubated in
culture medium alone for the duration of the respective
experiment or replicating experiments upon untransfected
CHO cells. cAMP accumulation in the culture supernatant was
measured with a radioimmunoassay kit (Beckmann Coulter,
Marseille, France).
2.4. Radiolabelled ligand binding and Kd determination
Binding of the specific 5-HT4 receptor antagonist,
[3H]GR113808 [12,30], to CHO cells that stably expressed
h5-HT4(g) receptors was determined. After aspiration of the
culture medium and washing, 4 � 105 cells were incubated
for 90 min at 37 8C with 0.1 nM [3H]GR113808 (specific
activity 84 Ci/mmol) per well, either alone or with an excess
of unlabelled ML10375 (10 mM) in a volume of 0.15 ml
PBS buffer (8.1 mM Na2HPO4, 1.9 mM NaH2PO4, 3.8 mM NaCl,
3 mM KCl, 0.9 mM CaCl2, 0.5 mM MgCl2) with 0.05%
bovine serum albumin. The cells were collected with 1N
NaOH and placed for 20 min in 4 ml Optiphase Hifase 2
scintillation cocktail (EG&G Wallac, France). Radioactivity
was measured using a liquid scintillation counter (LKB
Wallac, Rack beta). Untransfected CHO cells were used as
control. For Scatchard analysis, incubation conditions were
identical to those for ligand binding, except that 10
concentrations of [3H]GR113808 ranging from 0.02 to 3 nM
were used.
2.5. Flow cytometric analysis
CHO cells were collected by scraping and pelleted by
centrifugation at 1000 rpm for 10 min at room temperature.
4 � 105 cells were incubated with 100 mg/ml anti-G21V anti-
body or 1 mM 5-HT for 30 or 60 min at 4 8C without agitation. In
some experiments, cells were pretreated with 100 mg/ml anti-
G21V antibody for 30 min at 4 8C and were subsequently
incubated for another 30 min at 4 8C with 1 mM 5-HT and vice
versa. Cells were then washed in PBS and incubated in 1 ml of
a 1/160 dilution of affinity-purified goat anti-rabbit IgG
conjugated to fluorescein isothiocyanate (FITC) for 30 min at
4 8C. The cells were washed twice with PBS and immediately
analyzed on a FACScan1 flow cytometer using CellQuest
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1966
software (Becton Dickinson, Santa Clara, CA, USA). The
membrane expression of h5-HT4(g) receptor was analysed in
FL1(H) on the gate of live cells. Control experiments were done
with 100 mg/ml of rabbit purified IgG (control).
2.6. Statistical analysis
Data values are expressed as mean � S.E.M. of results obtained
from n experiments. Data were analysed with Student’s t-test.
Statistical significance was set at p < 0.05 (* in figures indicates
p < 0.05 compared with basal values).
Fig. 1 – Functional behaviour of 5-HT4(g) receptors
expressed in CHO cells. (A) The effects of forskolin (Fk), an
agonist (5-HT) and an antagonist (ML10375) upon cAMP
production. The amount of cAMP produced by each ligand
is expressed relative to that recorded in their absence
(basal). Cells were stimulated for 15 min with either 10 mM
forskolin, 1 mM 5-HT, 1 mM ML10375, or 1 mM ML10375
with 1 mM 5-HT (ML+5-HT). Columns and bars represent
mean W S.E.M. values of data obtained from duplicate
determinations of cAMP in five different experiments.
:* p < 0.05 compared with basal. (B) Dose-dependent cAMP
production by 5-HT. Data is normalized to the maximal
response (4 W 0.8 pmol cAMP/well). The symbols and bars
represent the mean W S.E.M. of data obtained in duplicate
measurements in five different experiments. The line
represents fitting a sigmoid equation to the data, the EC50
value was 32 W 10 nM.
Fig. 2 – Scatchard analysis of [3H]GR113808 binding to CHO
cells expressing the h5-HT4(g) receptor. The Scatchard plot
was derived from the mean of duplicate measurements of
a single representative experiment. The line represents
the fitting a linear regression equation to the data. High
affinity binding was represented by a Kd of 0.27 nM for a
calculated amount of 363 fmol receptors/mg protein.
3. Results
3.1. Characterisation of recombinant h5-HT4(g) receptorsexpressed in CHO cells
In this line of CHO cells expressing the h5-HT4(g) receptor, the
basal cAMP level in the absence of an agonist was about 1.4-fold
greater than in untransfected CHO cells. It therefore seems that
h5-HT4(g) receptorsshowedaconstitutiveactivity intheabsence
ofany5-HT4 ligandandthatafractionofthesereceptors isunder
a spontaneously active state (R*) in the membrane [31].
The ability of these cells to produce intracellular cAMP via
direct activation of the adenylate cyclase was determined with
10 mM forskolin. The cAMP level was increased by 945 � 10%
(Fig. 1A). This is not significantly different from our previous
results obtained in CHO cells (1148 � 290%) [6].
Functional coupling of the recombinant h5-HT4(g) receptor
isoform to adenylate cyclase was investigated by measuring
cAMP production in response to various experimental condi-
tions in presence of 5-HT1(b) antagonist, GR127935. Fig. 1B
shows that the h5-HT4(g) receptor responded to 5-HT in a dose-
dependent manner and induced cAMP synthesis with an
apparent EC50 value of 32 � 10 nM (n = 4) and a maximum of
cAMP production for 1 mM of 5-HT of about 136 � 13% (Fig. 1A).
These results are similar to that obtained in a previous CHO
cell line (EC50 value: 13 � 7 nM) [6]. Untransfected cells pre-
incubated with 1 mM of GR127935, 5-HT1(b) receptor antagonist,
did not respond to 5-HT.
The pharmacological characteristics of the h5-HT4(g) recep-
tor was determined with the specific h5-HT4 antagonist,
ML10375 [28,29]. This drug reduced basal cAMP to 28% and
inhibited 5-HT evoked cAMP production in CHO cells (Fig. 1A).
Neither 5-HT nor ML10375 had any effect upon cAMP levels in
untransfected CHO cells. ML10375 acts as an inverse-agonist of
functional activity of the h5-HT4(g) receptor [28,29].
Fig. 3 – Flow cytometric analysis of specific binding of anti-
G21V antibody to h5-HT4(g) receptors expressed in CHO
cells. Fluorescence intensity recorded from CHO cells
incubated with anti-G21V antibody (Aa and Bb) is
compared with that recorded in control experimental
conditions (control), and in the presence of either 5-HT
alone (Ab and Ba), anti-G21V antibody then 5-HT (Ac) or 5-
HT then anti-G21V antibody (Bc). Data are representative
of four separate experiments.
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b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1 967
To estimate the number of h5-HT4(g) receptors, transfected
CHO cells were incubated with increasing concentrations of
[3H]GR113808 in the presence of an excess of unlabelled
ML10375 (Fig. 2). A single high affinity binding component was
identified. Scatchard analysis of the results gave a Kd value of
0.27 nM for a calculated amount of 363 fmol receptor/mg of
total protein.
3.2. Recognition of the h5-HT4(g) receptor by the polyclonalanti-G21V antibody
h5-HT4(g) receptor expression at the surface of CHO cells was
analyzed by flow cytometry. Fig. 3 and Table 1 show that the
Fig. 4 – The effects of anti-G21V antibody (10 mg/ml) upon
cAMP production evoked by 1 mM 5-HT in CHO cells
expressing the 5-HT4(g) receptor. cAMP production is
expressed relative to that recorded in the absence of either
5-HT or anti-G21V antibody (basal). Schematic
representations of the experimental protocols are indicated
inset above each graph. Control protocols (a and b)
consisted of applying either anti-G21V antibody or 5-HT to
CHO cells for 20 min (b) or 40 min (a). (A) Anti-G21V antibody
was applied to CHO cells for 20 min prior to the addition of
5-HT for a further 20 min. (B) 5-HT was applied to CHO cells
for 20 min prior to the addition of anti-G21V antibody for a
further 20 min. The columns and bars represent the
mean W S.E.M. values of duplicate measurements in five
different experiments. *p < 0.05 compared with basal.
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1968
fluorescence intensity of CHO cells expressing the h5-HT4(g)
receptors incubated with anti-G21V antibodies (Fig. 3Aa and
Bb; Table 1 (Aa and Bb)) was higher than that of cells incubated
with purified IgG (Fig. 3A control and B control; Table 1).
Similar results were observed in cells incubated first with anti-
G21V and then 5-HT (Fig. 3Ac; Table 1 (Ac)), and in cells
incubated first in 5-HT and then anti-G21V (Fig. 3Bc; Table 1
(Bc)). Transfected cells incubated in 5-HT alone (Fig. 3Ab and
Ba; Table 1 (Ab and Ba)) yielded no significant specific
immunofluorescence. In this last condition, the fluorescence
intensity was less than that measured under control condi-
tions (Fig. 3A and B; Table 1) because the assay was realized
without purified IgG which had been present in the control.
This data showed that the polyclonal anti-G21V antibody
specifically recognized the recombinant receptor expressed in
CHO cells and this in the presence or in the absence of 5-HT.
Moreover, 5-HT did not modify the binding of the anti-G21V
antibody to the receptor.
3.3. Effects of polyclonal anti-G21V antibodies on h5-HT4(g) receptor activity
To test the effects of the polyclonal anti-G21V antibody on h5-
HT4(g) receptor activity, cAMP levels were measured in CHO
cells incubated with the antibody in the absence or in the
presence of 1 mM 5-HT. Two experimental protocols were
used: either cells were stimulated first with anti-G21V anti-
body and subsequently with 5-HT (Fig. 4A) or vice versa
(Fig. 4B). Anti-G21V antibody alone induced an ‘‘inverse-
agonist like’’ effect. The basal cAMP level of CHO cells was
decreased significantly when anti-G21V antibody was applied
for either 20 min (48 � 8%, Fig. 4Bb) or 40 min (60 � 15%,
Fig. 4Aa). Moreover, the application of the antibody before 5-
HT induced a decrease of cAMP production evoked by the
agonist (Fig. 4Ac, 86 � 7% versus 136 � 13% for 5-HT alone
Fig. 4Ab). However, there was no effect upon the increase of
cAMP induced by 5-HT when the anti-G21V antibody was
added in the continuous presence of the agonist (Fig. 4Bc,
210 � 16% versus 244 � 22% for 5-HT alone Fig. 4Ba), though
the antibody did bind to the 5-HT4(g) receptor (Fig. 3). These
antibodies had no effect (2.31 � 0.45 pmol/well versus
2.1 � 0.42 pmol/well in the absence of antibodies) on cAMP
concentration in untransfected CHO cells. We conclude that
the anti-G21V antibody had a functional and ‘‘inverse-agonist
like’’ effect upon h5-HT4(g) receptors expressed in transfected
CHO cells, both alone or when applied before 5-HT.
3.4. Are functional actions of anti-G21V antibodiesassociated with h5-HT4(g) receptor dimerization?
A number of studies have suggested that in order to transduce
extracellular signals GPCR receptors form dimers in the
membrane [32,33]. Moreover, antibodies directed against the
second extracellular loop of GPCRs could favor dimerization.
In order to test whether the anti-G21V antibody required
receptor dimers to be effective we investigated the effects of
monovalent Fab fragments of anti-G21V antibodies. Fig. 5
shows that the Fab fragments significantly decreased basal
cAMP (29 � 8%) in CHO cells. Figs. 4 and 5 therefore show that
monomeric and dimeric antibodies had similar functional
effects on recombinant h5-HT4(g) receptors and that the
action of anti-G21V antibodies on h5-HT4(g) receptors would
be independent of receptor dimerization.
3.5. Implication of phosphodiesterases or Gi-proteins inthe ‘‘inverse-agonist like’’ effects of the antibodies on h5-HT4(g) receptors
The effects of anti-G21V antibodies on h5-HT4(g) receptors are
similar to those of an inverse agonist. Two mechanisms could
Fig. 5 – The effects of monovalent Fab fragments of anti-
G21V antibody (30 mg/ml) upon cAMP production on CHO
cells. cAMP production is expressed relative to that
recorded in the absence of Fab antibody (basal). The
columns and bars represent the mean W S.E.M. values of
duplicate measurements in three different experiments.*p < 0.05 compared with basal.
Fig. 6 – The effects of anti-G21V antibodies upon h5-HT4(g)
receptors in the presence of either phosphodiesterase or
Gi-protein inhibitors. The cells were incubated either in
solution A supplemented with 5 mM theophyllin
(phosphodiesterase inhibitor) for 15 min (A) or in culture
medium with 2 mg/ml of Pertussis toxin (PTX, Gi protein
inhibitor) for 24 h (B). The incubations were then
continued for further 15 min with the addition of either
10 mg/ml anti-G21V, 1 mM 5-HT or 10 mg/ml anti-G21V
then 1 mM 5-HT [(anti-G21V)+5-HT]. cAMP production is
expressed relative to that recorded in the absence of either
5-HT or anti-G21V antibody (basal), and in absence or
presence of inhibitors. Columns and bars represent
mean W S.E.M. values of data obtained from duplicate
determination of cAMP in three different experiments.*p < 0.05 compared with basal.
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1 969
modulate intracellular cAMP level, the activation of phospho-
diesterases [34] and/or Gi proteins [35].
The broad spectrum phosphodiesterase inhibitor theo-
phyllin increased basal cAMP in CHO cells approximately 10-
fold to 60 � 2 pmol/well against 6.3 � 0.2 pmol/well in control
non treated cells. Fig. 6A shows that the anti-G21V antibody
decreased cAMP by equivalent percentages in the absence
(55 � 13%) or in the presence (33 � 8%) of theophyllin.
Theophyllin also had no effect upon results obtained with
anti-G21V then 5-HT, cAMP was 98 � 10% in the present of
theophyllin versus 86 � 7% in its absence.
Incubation of CHO cells in Pertussis toxin (PTX), the Gi-
protein inhibitor, had no significant effect upon basal cAMP.
Fig. 6B shows that there was no difference in the effect of anti-
G21V antibody upon h5-HT4(g) receptors in CHO cells which
had been incubated in PTX (69 � 7%) compared with control
(62 � 6%). Moreover, there was also no difference in the
amount of cAMP evoked by anti-G21V then 5-HT (74 � 2% in
treated cells versus 87 � 6% in untreated cells).
Therefore, the ‘‘inverse-agonist like’’ effect of the anti-
G21V antibody upon h5-HT4(g) receptors was not related to an
action which passed via either phosphodiesterases or Gi
proteins.
4. Discussion
This study extends the type of direct functional effects of
antibodies directed against the second extracellular loop of
cardiac GPCR receptors [23,36–38] to include inverse agonism.
The polyclonal anti-G21V antibodies react with their epitopes
in the presence or the absence of serotonin. However, their
functional effects were observed only when the cells had been
stimulated first with anti-G21V. These results suggest that in
the presence of 5-HT the anti-G21V antibodies were unable to
induce the conformational changes in the receptors which are
implicated in signal transduction. The action of these
antibodies was possible only in absence of 5-HT bound to
the 5-HT4 receptor. The specificity of the antibody against 5-
HT4 receptors has been shown by the affinity purification
method against the second extracellular loop peptide [26] of
the human 5-HT4 receptor and confirmed by Western blot
b i o c h e m i c a l p h a r m a c o l o g y 7 3 ( 2 0 0 7 ) 9 6 4 – 9 7 1970
studies in CHO cells [12,26] and our results with flow
cytometric analysis. An autoimmune origin for atrial fibrilla-
tion has been proposed. The cardiac h5-HT4 receptors could be
the target of autoantibodies and since, unlike 5-HT, antibodies
are continuously present in the circulation they would
therefore be able to induce their effects upon 5-HT4 receptor
activity.
The functional effects of antibodies directed against the
5HT4 receptor are different in human cardiomyocytes [26],
CHO cells [6] and COS-7 cells [27]. This could be attributed to
differences in the cellular system, the number or density of
receptors in the membrane or the conformational state of the
receptor. In different cell types the receptor could be expressed
in a different membrane environment and/or linked to a
different transduction pathway. The interest of the present
work resides in the use of the same expression system (CHO
cells) as a previous report [6], which allows the comparison of
results. Since the same antibodies had different effects in the
same cell type, the differences cannot be entirely attributed to
the cellular system. Our results indicate that the number of
receptors is identical here (363 pmol/mg), in COS-7 cells [27]
and the previous report using CHO cells [6]. Therefore, the
different behaviour of the receptor towards the anti-G21V
antibodies cannot be related to the receptor density. The
effects of anti-G21V antibody could be dependent on the
equilibrium between inactive (R) and active (R*) forms of the
h5-HT4(g) receptor in the membrane [31]. The allosteric
equilibrium constant J = [R]/[R*] can be estimated by
(Rtotal � R*)/R* [39]. Rtotal corresponds to Bmax obtained with
[3H]GR113808 and R* to Bmax obtained with [3H]5-HT. Using this
procedure a value of J = 3 was obtained in a previous study
using CHO cells [6]. In the present work, which used a different
line of transfected CHO cells the Bmax value for [3H]5-HT could
not be recorded, which suggests that the concentration of the
active receptor, R*, was too low to be detectable. But since the
value of Rtotal was the same in the two studies J must be larger
than 3 here. This suggests that the equilibrium between
conformational states of the receptor has been shifted
towards the inactive R form. This is in agreement with the
lower basal cAMP level, the lesser production of cAMP by 5-HT
and the more important effects of the inverse agonist ML10375
obtained in this study compared with [6] (28 � 11% in this
versus 69 � 3% in previous CHO cell line). The ‘‘inverse
agonist-like’’ effect obtained with the anti-G21V antibody is
also in agreement with a larger proportion of inactive
receptors (R) [40–42].
Alternative explanations of the ‘‘inverse agonist like’’ effect
of the anti-G21V antibody could be the activation of a Gi-
protein pathway or to enhanced cAMP degradation by
phosphodiesterases. The results obtained here with theo-
phyllin and PTX could indicate that this is not the case.
However, the presence of cell microdomains limited by PDE
activity has been postulated [43] and a global measurement of
cAMP will not be able to detect local concentration changes.
For other GPCR receptors the application of monocatenar
anti-receptor Fab fragments have no action compared with the
‘‘agonist-like’’ effect of bicatenar antibodies [44]. The present
work shows for the first time a similar effect of the Fab and
bicatenar forms of anti-G21V antibodies on the 5HT4 receptor.
Since a dimerized form of the receptor is favoured by the
bicatenar antibodies, this result indicates that dimerization is
not necessary for 5HT4 receptor activation. However, non
functional dimerized forms of the 5HT4 receptor have been
described using bioluminescence resonance energy transfer
(BRET) [45]. Further studies are necessary to elucidate the
interactions of anti-G21V antibodies with mono and/or
dimerized forms of the receptor.
Acknowledgments
We warmly acknowledge the skilful technical assistance of D.
Gennetay and M. Pingaud. We also thank Dr. I. Findlay for
helpful comments on the text. This research was supported in
part by Fondation pour la Recherche Medicale, la Fondation de
France, le Conseil General et Regional de la Region Centre
(France). E. Di Scala is a scholar of Ministere de l0Enseignement
Superieur et de la Recherche.
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