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.

avai lable at www.sc iencedi rec t .com

journal homepage: www.e lsev ier .com/ locate /b iochempharm

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: bozon@univ-tours.fr, 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.

r e f e r e n c e s

[1] Bockaert J, Pin JP. Use of a G protein-coupled receptor tocommunicate. An evolutionary success. C R Acad Sci III1998;321:529–51.

[2] Kaumann AJ, Sanders L, Brown AM, Murray KJ, Brown MJ. A5-hydroxytryptamine receptor in human atrium. Br JPharmacol 1990;100:879–85.

[3] Jahnel U, Rupp J, Ertl R, Nawrath H. Positive inotropicresponse to 5-HT in human atrial but not in ventricularheart muscle. Naunyn-Schmiedebergs Arch Pharmacol1992;346:482–5.

[4] Ouadid H, Seguin J, Dumuis A, Bockaert J, Nargeot J.Serotonin increases calcium current in human atrialmyocytes via the newly described 5-hydroxytryptamine4receptors. Mol Pharmacol 1992;41:346–51.

[5] Sanders L, Kaumann AJ. A 5-HT4-like receptor in humanleft atrium. Naunyn-Schmiedebergs Arch Pharmacol1992;345:382–6.

[6] Di Scala E, Rose S, Herault O, Argibay J, Cosnay P, Bozon V.Polyclonal antibody effects on the human cardiac 5-HT4(e)receptors depend upon the expression system. ReceptorsChannels 2004;10(3–4):125–9.

[7] Blondel O, Vandecasteele G, Gastineau M, Leclerc S,Dahmoune Y, Langlois M, et al. Molecular and functionalcharacterisation of a 5-HT4 receptor cloned from humanatrium. FEBS Lett 1997;412:465–74.

[8] Claeysen S, Faye P, Sebben M, Lemaire S, Bockaert J,Dumuis A. Cloning and expression of human 5-HT4sreceptors. Effect of receptor density on their coupling toadenylyl cyclase. Neuroreport 1997;8:3189–96.

[9] Van den Wyngaert I, Gommeren W, Verhasselt P, Jurzak M,Leysen J, Walter Luyten W, et al. Cloning and expression ofa human serotonin 5-HT4 receptor cDNA. J Neurochem1997;69(5):1810–9.

[10] Blondel O, Gastineau M, Dahmoune Y, Langlois M,Fischmeister R. Cloning, expression, and pharmacology offour human 5-hydroxytryptamine4 receptor isoformsproduced by alternative splicing in the carboxyl terminus. JNeurochem 1998;70(6):2252–61.

[11] Bender E, Pindon A, Van Oers I, Zhang YB, Gommeren W,Verhasselt P, et al. Structure of the human serotonin 5-HT4receptor gene and cloning of a novel 5-HT4 splice variant. JNeurochem 2000;74(2):478–89.

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 1 971

[12] Mialet J, Berque-Bestel I, Eftekhari P, Gastineau M, Giner M,Dahmoune Y, et al. Isolation of the serotoninergic 5-HT4(e)receptor from human heart and comparative analysis of itspharmacolgical profile in C6-glial and CHO cell lines. Br JPharmacol 2000;129:771–81.

[13] Bach T, Syversveen T, Kvingedal AM, Krobert KA, BrattelidT, Kaumann AJ, et al. 5-HT4(a) and 5-HT4(b) receptors havenearly identical pharmacology and are both expressed inhuman atrium and ventricle. Naunyn-Schmiedebergs ArchPharmacol 2001;363:146–60.

[14] Vilaro MT, Domenech T, Palacios JM, Mengod G. Cloningand characterization of a novel human 5-HT4 receptorvariant that lacks the alternatively spliced carboxyterminal exon. RT-PCR distribution in human brain andperiphery of multiple 5-HT4 receptor variants.Neuropharmacology 2002;42(1):60–73.

[15] Brattelid T, Kvingedal AM, Krobert KA, Andressen KW, BachT, Hystad ME, et al. Cloning, pharmacologicalcharacterisation and tissue distribution of a novel 5-HT4receptor splice variant, 5-HT4(i). Naunyn SchmiedebergsArch Pharmacol 2004;369(6):616–28.

[16] Kaumann AJ. Do human atrial 5-HT4 receptors mediatearrhytmias? Trends Pharmacol SCI 1994;5:451–5.

[17] Grammer JB, Zeng X, Bosch RF, Kuhlkamp V. Atrial L-typeCa2+-channel, b-adrenoreceptor, and 5-hydroxytryptaminetype 4 receptor mRNAs in human atrial fibrillation. BasicRes Cardiol 2001;96(1):82–90.

[18] Borda E, Pascual J, Cossio P, De la Vega M, Arana R, Sterin-Borda L. A circulating IgG in Chagas’ disease which binds tobeta-adrenoceptors of myocardium and modulates theiractivity. Clin Exp Immunol 1984;57(3):679–86.

[19] Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD.Molecular biology of the cell, 3rd ed. NY; 1994.

[20] Goin JC, Borda E, Leiros CP, Storino R, Sterin-Borda L.Identification of antibodies with muscarinic cholinergicactivity in human Chagas’ disease: pathologicalimplications. J Auton Nerv Syst 1994;47(1–2):45–52.

[21] Ferrari I, Levin MJ, Wallukat G, Elies R, Lebesgue D, Chiale P,et al. Molecular mimicry between the immunodominantribosomal protein P0 of Trypanosoma cruzi and afunctional epitope on the human beta 1-adrenergicreceptor. J Exp Med 1995;182(1):59–65.

[22] Elies R, Ferrari I, Wallukat G, Lebesgue D, Chiale P, Elizari M,et al. Structural and functional analysis of the B cellepitopes recognized by anti-receptor autoantibodies inpatients with Chagas’ disease. J Immunol 1996;157(9):4203–11.

[23] Mijares A, Lebesgue D, Argibay J, Hoebeke J. Anti-peptideantibodies sensitive to the ‘active’ state of the beta2-adrenergic receptor. FEBS Lett 1996;399(1–2):188–91.

[24] Lebesgue D, Wallukat G, Mijares A, Granier C, Argibay J,Hoebeke J. An agonist-like monoclonal antibody against thehuman beta2-adrenoceptor. Eur J Pharmacol1998;348(1):123–33.

[25] Bockaert J, Claeysen S, Compan V, Dumuis A. 5-HT4receptors. Curr Drug Targets CNS Neurol Disord2004;3(1):39–51.

[26] Salle L, Eftekhari P, Aupart M, Cosnay P, Hoebeke J, ArgibayJ. Inhibitory activity of antibodies against the human atrial5-HT(4) receptor. J Mol Cell Cardiol 2001;33(3):405–17.

[27] Bozon V, Di Scala E, Eftekhari P, Hoebeke J, Lezoualc’h F,Fischmeister R, et al. Agonist-like activity of antibodiesdirected against the second extracellular loop of the humancardiac serotonin 5-HT4(e) receptor in transfected COS-7cells. Receptors Channels 2002;8(2):113–21.

[28] Mialet J, Berque-Bestel I, Eftekhari P, Gastineau M, Giner M,Dahmoune Y, et al. Isolation of the serotoninergic 5-HT4(e)receptor from human heart and comparative analysis of its

pharmacological profile in C6-glial and CHO cell lines. Br JPharmacol 2000;129(4):771–81.

[29] Blondel O, Gastineau M, Langlois M, Fischmeister R. The 5-HT4 receptor antagonist ML10375 inhibits the constitutiveactivity of human 5-HT4(c) receptor. Br J Pharmacol1998;125(4):595–7.

[30] Ansanay H, Sebben M, Bockaert J, Dumuis A.Pharmacological comparison between [3H]GR 113808binding sites and functional 5-HT4 receptors in neurons.Eur J Pharmacol 1996;298(2):165–74.

[31] Fong TM. Mechanistic hypotheses for the activation of Gprotein-coupled receptors. Cell Signal 1996;8:217–24.

[32] Lee SP, O’Dowd BF, George SR. Homo- and hetero-oligomerization of G protein-coupled receptors. Life Sci2003;74(2–3):173–80.

[33] Bai M. Dimerization of G-protein-coupled receptors: roles insignal transduction. Cell Signal 2004;16(2):175–86.

[34] Nascimento JH, Salle L, Hoebeke J, Argibay J, Peineau N.cGMP-mediated inhibition of cardiac L-type Ca(2+) currentby a monoclonal antibody against the M(2) ACh receptor.Am J Physiol Cell Physiol 2001;281(4):C1251–8.

[35] Kilts JD, Gerhardt MA, Richardson MD, Sreeram G,Mackensen B, Grocott HP, et al. Beta(2)-adrenergic andseveral other G protein-coupled receptors in human atrialmembranes activate both G(s) and G(i). Circ Res2000;87(8):705–9.

[36] Mijares A, Verdot L, Peineau N, Vray B, Hoebeke J, Argibay J.Antibodies from Trypanosoma cruzi infected micerecognize the second extracellular loop of the beta 1-adrenergic and M2-muscarinic receptors and regulatecalcium channels in isolated cardiomyocytes. Mol CellBiochem 1996;163–164:107–12.

[37] Baba A, Yoshikawa T, Fukuda Y, Sugiyama T, Shimada M,Akaishi M, et al. Autoantibodies against M2-muscarinicacetylcholine receptors: new upstream targets in atrialfibrillation in patients with dilated cardiomyopathy. EurHeart J 2004;25(13):1108–15.

[38] Dobrev D, Christ T, Ravens U. Muscarinic subtype-2receptor autoantibodies: actors or bystanders in humanatrial fibrillation? Eur Heart J 2004;25(13):1091–2.

[39] Pindon A, Van Hecke G, Van Gompel P, Lesage AS, LeysenJE, Jurzak M. Differences in signal transduction of two 5-HT4 receptor splice variants: compound specificity anddual coupling with Galphas- and Galphai/o-proteins. MolPharmacol 2002;61:85–96.

[40] Daeffler L, Landry Y. Inverse agonism at heptahelicalreceptors: concept, experimental approach and therapeuticpotential. Fundam Clin Pharmacol 2000;14:3–87.

[41] Kenakin T. Inverse, protean, and ligand-selective agonism:matters of receptor conformation. FASEB J 2001;15(3):598–611.

[42] Joubert L, Claeysen S, Sebben M, Bessis AS, Clark RD, MartinRS, et al. A 5-HT4 receptor transmembrane networkimplicated in the activity of inverse agonists but notagonists. J Biol Chem 2002;277(28):25502–11.

[43] Georget M, Mateo P, Vandecasteele G, Lipskaia L, Defer N,Hanoune J, et al. Cyclic AMP compartmentation due toincreased cAMP-phosphodiesterase activity in transgenicmice with a cardiac-directed expression of the humanadenylyl cyclase type 8 (AC8). FASEB J 2003;17(11):1380–91.

[44] Mijares A, Lebesgue D, Wallukat G, Hoebeke J. From agonistto antagonist: Fab fragments of an agonist-like monoclonalanti-beta(2)-adrenoceptor antibody behave as antagonists.Mol Pharmacol 2000;58(2):373–9.

[45] Berthouze M, Ayoub M, Russo O, Rivail L, Sicsic S,Fischmeister R, et al. Constitutive dimerization of humanserotonin 5-HT4 receptors in living cells. FEBS Lett2005;579(14):2973–80.