Agonism, antagonism and inverse agonism bias at the Ghrelin receptor
, Jean-Philippe Leyris
, Lauriane Onfroy
, Céline Galés
, Aude Saulière
, Marjorie Damian
, Sophie Mary
, Mathieu Maingot
, Séverine Denoyelle
, Pascal Verdié
, Jean Martinez
, Jean-Louis Banères
and Jacky Marie
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Université
Montpellier-ENSCM, Faculté de Pharmacie, 15 avenue Charles Flahault, BP 14491, 34093
Montpellier cedex 05, France.
Institut des Neurosciences de Montpellier, Hôpital Saint-Eloi, 80 avenue Augustin Fliche, BP 74103,
34091, Montpellier cedex 05, France.
Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la
Recherche Médicale, U1048, Université Toulouse III Paul Sabatier, Centre Hospitalier Universitaire
de Toulouse, 31432, Toulouse, France
Running title: Biased signaling at the ghrelin receptor
To whom correspondence should be addressed: Jacky Marie, Institut des Biomolécules Max
Mousseron (IBMM), UMR 5247 CNRS-Université Montpellier-ENSCM, Faculté de Pharmacie, 15
avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 05, France. Tel: 33 4 11759753, Fax: 33
4 1175 9760, Email: firstname.lastname@example.org
Keywords: G protein-coupled receptor (GPCR), ghrelin, signaling bias, G protein subtypes,
bioluminescence resonance energy transfer (BRET).
Background: GHS-R1a activates multiple
signaling pathways mediating feeding and
Results: Some GHS-R1a ligands activate Gq but
not Gi/o and fail to recruit β-arrestin2, others act
as selective inverse agonists at Gq compared to
Conclusion: Synthetic ligands can selectively
activate or reverse Gq-dependent signaling at
Significance: Ligand-biased signaling can be
exploited for the development of selective drugs
to treat GHS-R1a-mediated disorders.
The G protein-coupled receptor GHS-R1a
mediates ghrelin-induced growth hormone
secretion, food intake and reward-seeking
behaviours. GHS-R1a signals through Gq,
Gi/o, G13 and arrestin. Biasing GHS-R1a
signaling with specific ligands may lead to the
development of more selective drugs to treat
obesity or addiction with minimal side effects.
To delineate ligand selectivity at GHS-R1a
signaling, we analysed in detail the efficacy of
a panel of synthetic ligands at activating the
different pathways associated to GHS-R1a in
HEK293T cells. Besides β−arrestin2
recruitment and ERK1/2 phosphorylation, we
monitored activation of a large panel of G
protein subtypes using a BRET
with G protein-activation biosensors. We first
found that unlike full agonists, Gq partial
agonists were unable to trigger β-arrestin2
recruitment and ERK1/2 phosphorylation.
Using G protein-activation biosensors, we
then demonstrated that ghrelin promoted
activation of Gq, Gi1, Gi2, Gi3, Goa, Gob and
G13 but not Gs and G12. Besides, we
identified some GHS-R1a ligands that
preferentially activated Gq and antagonized
ghrelin-mediated Gi /Go activation. Finally,
we unambiguously demonstrated that in
addition to Gq, GHS-R1a also promoted
constitutive activation of G13. Importantly,
we identified some ligands that were selective
inverse agonists toward Gq but not of G13.
This demonstrates that bias at GHS-R1a
signaling can occur not only with regard to
agonism but also to inverse agonism. Our
data, combined to further in vivo studies, may
facilitate the design of drugs selectively
targeting individual signaling pathways to
treat only the therapeutically relevant
Ghrelin, a peptide hormone mainly produced by
the stomach (1), has emerged as an important
gut-brain signal to control growth hormone
secretion, food intake and reward seeking
behaviours (2,3). Ghrelin mediates these actions
through the growth hormone secretagogue type
1a receptor (GHS-R1a) receptor, a family A G-
protein coupled receptor (GPCR) (4,5). Because
of its possible implication in several
physiological disorders such as obesity, drug and
alcohol addiction, GHS-R1a represents a major
target for the development of therapeutic
molecules (6). So far, several academic
laboratories and pharmaceutical companies have
developed synthetic molecules that display
agonist, antagonist and inverse agonist properties
toward GHS-R1a intracellular signalling
pathways. Some of these molecules display
interesting properties with regard to food intake
stimulation or inhibition (7-9), addiction to drugs
including alcohol and cocaine (10,11) or growth
hormone secretion (12). Given the pleiotropic
actions of ghrelin, GHS-R1a synthetic ligands
can be useful to block or activate the targeted
physiological effect but can also lead to
undesirable side effects. For instance, synthetic
GHS-R1a antagonists that decrease food intake
and fat storage may be good candidate to treat
obesity but could have side effects due to their
inhibitory action on growth hormone secretion.
In the other way, agonists developed with the
aim to stimulate hormone secretion for treating
postmenopausal osteoporosis may have adverse
effect by increasing body weight (13). Therefore,
development of biased ligands that will
selectively inhibit or activate only one or a
subset of the GHS-R1a dependent physiological
responses could have significant therapeutic
advantages. This aim is certainly now attainable
because during the last decade, many
independent studies on GPCRs have described
biased agonists that are selective of a given
down-stream signaling pathways (14,15). Unlike
the endogenous ligand that usually activates all
signaling pathways the receptor is associated
with (G protein and β-arrestin dependent
pathways), synthetic ligands could thus
selectively activate only some of them (for
instance activation of!β -arrestin with no effect on
G protein or conversely) (16). Although
molecular mechanisms responsible for ligand-
directed functional selectivity are not fully
understood, there are increasing evidences that
biased activity results from selective stabilization
of different receptor conformations that differ
into their ability to couple to different
downstream effectors (17-21). Importantly, in
vivo studies demonstrated that side effects of
classical drugs might be diminished by the use of
biased molecules, suggesting this kind of
molecules could have potential clinical
application (16,22). As for many other GPCR,
rational design of biased ligands of the ghrelin
receptor first requires identification of lead
pathway-selective compounds. To identify such
lead compounds, careful dissection of the
different intracellular down-stream signaling
pathways of GHS-R1a is required. GHS-R1a is
coupled to Gq signaling pathway to trigger
inositol phosphate production and intracellular
calcium release (23).In the context of Gq-
signaling, an interesting particularity of GHS-
R1a is its exceptionally high constitutive
activity. Indeed, high basal levels of inositol
phosphate production were detected in GHS-
R1a-transfected cell lines (24,25). Significant
ligand independent Gq activation and AP2
recruitment were also clearly demonstrated to
occur with the purified GHS-R1a inserted in
lipid disc (26). As many other GPCRs, GHS-R1a
activates other G protein dependent and
independent pathways besides the Gq-associated
one. Indeed, following ghrelin stimulation, GHS-
R1a activates ERK1/2 through β-arrestin
dependent (27,28) and β-arrestin independent
(29) pathways. It also activates PI3 kinase,
PKCε, Src through a Gi/o protein dependent
In this context, we investigated here the
selectivity of a panel of GHS-R1a synthetic
ligands toward arrestin and G protein dependent
pathways. We paid particular attention to the
selectivity of ligands toward activation of several
G protein subtypes and isoforms thanks to the
use of recently developed G protein BRET-based
biosensors that were recently developed (31,32).
Our data suggest that some synthetic GHS-R1a
ligands are selective Gq-agonists. We also
identified ligands that displayed potential inverse
agonist selectivity toward Gq compared to G13.
The occurrence of such pathway-selective
ligands questions about the connection that could
exist between the biased behaviour of some
ligands toward intracellular pathways and their
selectivity toward food intake and GH secretion
already demonstrated in vivo.
Ghrelin 1-28 was purchased from polypeptide
group, MK-0677 (33) from Axon MedChem and
[D-Arg1-D-Phe5,D-Trp7,9,Leu11] substance P
(SPA) from Bachem. JMV compounds were
synthesized in our laboratory (IBMM, France).
The pseudopeptide JMV 1843 was previously
described by Guerlavais et al. (34), JMV 2959 by
Moulin et al. (35), JMV 3002, JMV 3018 and
JMV 3011 by Moulin et al. (36) In compound
JMV 4484, a second chiral center was introduced
in position 3 of the 1,2,4-triazole scaffold. This
chiral center contains an amino function, which
was elongated by the Leu-Leu dipeptide and a
lysine residue was then introduced in the N-
terminal part to mimic the peptide core of the
substance P analog. Peptide KwFwLL-NH
were synthetized at
IBMM as described in (37) and (38)
The thromboxane A2 receptor agonist U46619
was purchased from Cayman chemical. Arginine
vasopressin (AVP) was provided by Dr. B.
Mouillac (IGF, Montpellier, France).
Lipofectamine 2000, foetal bovine serum,
antibiotics (penicillin, streptomycin) and DMEM
medium were purchased from Invitrogen.
Coelenterazine 400a (DeepblueC) was
purchased from Interchim. IPone HTRF kit and
-cryptate were provided by
CisBio. BODIPY FL GTPγS was from
For experiments in lipid disc, Gαq and Gβ1γ2
subunits were produced in sf9 cells as described
(18). Gα13 was from Kerafast.!
RLuc-β-arrestin2 cloned in PRK6 vector was a
generous gift of Dr. M.A Ayoub (IGF,
Montpellier, France). Human vasopressin V2
receptor cloned in PRK5 vector was provided by
Dr. T. Durroux (IGF, Montpellier, France).
Human GHS-R1a cloned in pcDNA3.1+
(pcDNA-GHS-R1a) was purchased from the
cDNA resource Center (University of Missouri).
HA-GHS-R1a was generated by PCR using: 1) a
sense oligonucleotide primer containing HindIII
and EcoRI sites followed by a Kozac sequence,
an ATG codon, a HA sequence and nucleotides
1-25 of GHS-R1a; 2) an antisense
oligonucleotide primer containing a BamHI site
followed by a stop codon and nucleotides 1098-
1072 of GHS-R1a. The PCR product was
digested with HindIII and BamHI and cloned in
HindIII-BamHI sites of pcDNA3+ vector
(pcDNA-HA-GHS-R1a). To generate GHS-R1a-
YFP, GHS-R1a sequence was amplified by PCR
with a sense oligonucleotide containing an
EcoRI site and an antisense oligonucleotide
containing a BamHI site. The PCR fragment was
digested with EcoRI and BamHI and inserted
into EcoRI and BamHI sites of PRK6-YFP
vector provided by Dr. M.A Ayoub (IGF,
Montpellier, France).! A204E mutation was
introduced in GHS-R1a (GHS-R1a-A204E) by
PCR using pcDNA-GHS-R1a as a template and
30 mer forward and reverse oligonucleotide
primers in Accuprime Pfx SuperMix solution
(Invitrogen). SNAP-GHS-R1a was already
HEK293T cells were maintained in DMEM
Glutamax (Invitrogen) supplemented with
antibiotics (penicillin 50 µg/ml, streptomycin 50
µg/ml), HEPES 2 mM, 1% non-essential amino
acids and 10% heat inactivated Foetal Calf
For IP and binding assays, transfections were
performed in 96 well plates using cell density of
50,000 cells per well. Prior to cell plating, wells
were pre-coated with Poly-L-Ornithine (50 µl of
10 mg/ml) for 30 minutes at 37°C. Transfection
mixes were prepared using cDNA encoding
GHS-R1a, GHS-R1a A204E or SNAP-GHS-R1a
(200 ng-300 ng), lipofectamine 2000
(Invitrogen) with a ratio 0.4 for cDNA (µg) /
lipofectamine (µl) in a total volume of 50 µl
optiMEM culture medium per well. Prior to its
addition in plates, the transfection mixture was
pre-incubated for 20 min at room temperature.
Then 100 µl of HEK293T cells at a density of
500,000 cells/ml were plated in each well and
were incubated at 37°C under 5% CO
for 48 h.
Transfection condition for HTRF ligand binding
was performed as previously described (39).!
Ligand binding assay.
Ki values were determined from binding
competition experiments performed on intact
HEK293T cells expressing the GHS-R1a using a
Homogenous Time Resolved Fluorescence
(HTRF) assay previously described (39). HTRF
signal was collected in a PHERAstar microplate
reader (BMG LABTECH). Ki values were
obtained from binding curves using GraphPad
Prism software (GraphPad Software, Inc., San
The expression level (B
) of GHS-R1a
expressed in HEK293T cells was determined by
radioactive assay using [
previously described (39). !
Inositol Phosphate assay.
Inositol phosphate accumulation assay was
carried out 48 h after transfection on adherent
cells in 96-well plate at a density of 50,000
cells/well. IP1 production was measured using
the IP-One HTRF kit (Cisbio Bioassays Ref.
621PAPEC) as previously described (39).
Briefly, cells were stimulated for 30 min at 37°C
with the ligand to be tested in 70 µl of IP1
stimulation buffer. An anti-IP1 antibody labelled
with Lumi4-Tb (15 µl) and an IP1-d2 derivative
(15 µl) were added on cells. The medium was
incubated for 1 h at room temperature. Signals at
665 nm and 620 nm were detected using a
PHERAstar (BMG LABTECH) fluorescence
reader. Values are expressed as ∆F. ∆F
corresponded to: (Ratio 665 nm/620 nm of the
assay - Ratio 665 nm/620 nm of the negative
control)/ Ratio 665nm/620nm of the negative
The negative control corresponded to the Lumi4-
Tb blank and was used as an internal assay
control. Inositol phosphate accumulation was
expressed as the percentage of the maximal
ghrelin response using the formula: (∆F mock
cells-∆F receptor transfected cells)/(∆F mock
cells-∆F maximal! ghrelin stimulation for
receptor transfected cells).!!
ERK1/2 assay was carried out 48 h after
transfection on adherent cells in 96-well white
plates (Greiner Bio One) at a density of 50,000
cells/well. ERK1/2 phosphorylation was
measured after 10 min stimulation with ligands
using an HTRF-based Phospho-ERK
(Thr202/Tyr204) cellular assay kit according to
manufacturer instructions (Cisbio Bioassays).
Briefly signal is detected between anti-phospho
ERK antibody labelled with Eu3
and anti ERK1/2 antibody labelled with d2
acceptor. Signals at 665 nm and 620 nm were
measured using a PHERAstar (BMG
LABTECH) fluorescence reader. Values were
expressed as ratio of 665 nm/ 620 nm x 1000.
Arrestin recruitment assay.
The interaction between GHS-R1a-YFP and
Rluc-β-arrestin2 was measured in HEK 293T by
in 96 well white plates (Greiner Bio
One). Briefly cells were transfected by
lipofectamine with 100 ng of GHS-R1a–YFP
and 5 ng Rluc-β-arrestin2. 48 h after transfection
cells were washed with PBS and then incubated
for 45 min at 37°C with 50 µ l of ligand in
DMEM, 0.1% BSA. After stimulation, cells were
washed with 100 µl PBS. 50 µl of a 0.5 mM
coelenterazine H (Interchim) solution in PBS
was then added to the cells and the signal
measured with a Mithras LB 940 plate reader
(Berthold Biotechnologies) that allows
sequential integration of luminescence signal (5
cycles of 0.05 sec) with two filter settings (Rluc
filter, 485 ± 20 nm and YFP filter, 530 ± 25 nm).
The BRET ratio was defined as the difference of
the ratio 530 nm/485 nm of the co-transfected
Rluc and YFP proteins and the ratio of the Rluc
protein alone. Results are expressed in mBRET
corresponding to the ratio (530 nm/485 nm)
G protein activation BRET assay.
G protein activation was measured with the
BRET assay previously described (31,32).
Briefly HEK293T cells grown in 10 cm culture
dishes were co-transfecetd by lipofectamine
2000 with GHS-R1a and G protein subunits
(Rluc8-α, β1 and γ2-GF10). 48 h after
transfection, cells were washed with PBS,
detached with PBS containing 5 mM EDTA, and
resuspended in PBS supplemented with 5 mM
EDTA and 0.1% (w/v) glucose (buffer A) at
room temperature. Cells were then distributed in
a 96-well white plate (300,000 cells per well).
For kinetic analyses, 5 µM of deep blue C
(coelenterazine 400a, Interchim) were added and
the plate was immediately loaded in a Mithras
LB 940 multimode microplate reader (Berthold)
or a PHERAstar microplate reader (BMG
LABTECH). Then, 10 µl of ligand solution (1
µM) was injected after 30 sec of reading and
signal was recorded for 90 sec. The BRET signal
was obtained by calculating the ratio of GFP10
emission (515 ± 10 nm) over Rluc8 light
emission (400 ± 10 nm) at 1.6 sec intervals. For
end point measurements, cells (300,000) in 80
µl of buffer A were incubated in a 96 well plate
with 10 ml of ligand solution in buffer A at room
temperature for 3 min to 15 min. Then, 10 µl of
deep blue C solution (50 mM) was added and the
signal was recorded in a PHERAstar
microplate reader (BMG labtech). The BRET
signal was calculated as the ratio of emission
GFP10 (510-530 nm) to RLuc8 (410-480 nm)
recorded 5 times at 0.5 sec intervals.
Quantification of cell surface receptors by
24 h post-transfection with pcDNA3.1 (+)
(control) or vectors encoding N-terminally HA-
tagged GHSR1a either in presence of Gαq-Rluc8
or Gα13-Rluc8, untagged GFP10-Gγ2 and Gβ1
untagged, cells were split into 24-well plates.
Cells were fixed in 4 % paraformaldehyde,
saturated with PBS containing 1 % bovine serum
albumin and incubated with the primary anti-HA
antibody (Clone 16B12, Covance) and then with
HRP-labeled secondary antibody (Sigma). After
washing, cells were incubated with HRP
substrate: TMB (3,39,5,59-
tetramethylbenzidine). The reaction was stopped
with 1N HCl, and the plates were read at 450 nm
in a microplate reader (Varioscan Flash, thermo
electron). The 570-nm optic density
(background) was subtracted.
G-protein activation assay in lipid discs.
The human GHS-R1a receptor was expressed in
E. coli and assembled as a monomer into lipid
discs as described (26). GTPγS binding assays
were carried out by monitoring changes in the
fluorescence emission of bodipyGTPγS (26). In
these assays, ligand concentrations of 1µM and
1:10 receptor:G protein molar ratios with
receptor concentrations in the 20 nM range were
Quantification of ligand bias.
values were estimated from dose-
response curves using the nonlinear curve fitting
equation (three parameters) in GraphPad Prism
Ligand bias was quantify by fitting ligand
concentration-response curve using the method
developed by Kenakin et al. (40), which is based
on the operational model of agonism (41). The
transduction coefficient log (τ/Κ
) was derived
using the operational model equation in
GraphPad Prism. This transduction coefficient
represents the ability of an agonist to stimulate a
given signaling pathway. τ represents an index
of coupling efficiency of the agonist, K
functional equilibrium dissociation constant of
the agonist. To eliminate the impact of the
different sensitivities of the assays used, the [log
)] value determined for one ligand at a
given pathway was normalized to that
determined for a reference ligand at the same
pathway. In our study, the reference ligand was
ghrelin. By subtracting log (τ/Κ
) of ghrelin
from log (τ/Κ
) of each compound, a ∆log (t/K
value was obtained that gave a within-pathway
comparison of transduction efficiency of ligands:
) = log (τ/Κ
- log (τ/Κ
Finally, the bias of each ligand between different
signaling pathways was obtained in the form
) which was:
= ∆log (τ/K
where P1 is pathway 1 and P2 is pathway 2.
No ligand bias at two different pathways
compared to ghrelin will result in a value of
not significantly different from
0. Statistical analysis was performed using a
two-way unpaired Student’s t test. Difference
were considered significant when p was < 0.05.
Bias factor (BF) (or fold change in bias) was
calculated as follows: BF= 10
When a ligand promoted a detectable stimulation
of one pathway (P1) but did not promoted any
detectable stimulation at the other pathway (i.e
P2), a bias factor of this ligand between these
two pathways could not be calculated.
Binding properties of GHS-R1a ligands.
The binding affinities (Ki values) of the GHS-
R1a ligands used all along this study are reported
in Table 1.
Efficacy of GHS-R1a ligands toward Inositol
First, we tested a panel of GHS-R1a ligands for
their efficacy to stimulate Gq/G11 signaling by
measuring inositol phosphate production in
HEK-293T cells transiently expressing GHS-
As already published, GHS-R1a expressing cells
displayed a high basal level of inositol phosphate
production compared to un-transfected cells and
this basal activity was reversed in the presence of
the inverse agonist SPA (Fig. 1A). Thus, in the
HEK293T cells used all along this study, the
GHS-R1a displayed high constitutive activity
that amounts 50 to 70 % of the maximal
response promoted by ghrelin (Fig. 1A). The
pseudo-peptide JMV 1843 stimulated inositol
phosphate production to the same extent than
ghrelin, whereas the efficacy of the non-peptide
MK-0677 was slightly higher than that of ghrelin
(150 ± 25 % compared to 100% ghrelin
stimulation over basal) (Fig. 1A, Table 2).
Compared to ghrelin, JMV 1843 and MK-0677
compounds thus behaved as full and super
agonist respectively compared to ghrelin on
inositol phosphate pathway. When tested at a 10
M maximal dose, JMV 3011 had no effect
whereas JMV 3002, JMV 3018 and JMV 2959
induced an increase of inositol phosphate
production over basal with a partial agonist
effect compared to ghrelin (Fig. 1A, Table 2).
Thus, JMV 3002, JMV 3018 and JMV 2959 can
thus be considered as partial agonists on the IP
pathway (Fig. 1A, Table 2). However, the partial
agonist efficacy of these ligands was sometime
undetectable when the basal IP1 production
reached 60 % of the maximal ghrelin response, a
level often found in these experiments. We thus
postulated that the high level of basal IP1
production might mask this partial agonist effect
of ligands. Therefore, we checked whether we
could decrease the constitutive activity of GHS-
R1a by decreasing the expression level of GHS-
The basal level of IP1 production of HEK293T
cells expressing the GHS-R1a represented 55 ± 3
% and 62 ± 4 % of the maximal ghrelin for a
receptor expression level of 2.5 fmol/10
and 74 fmol/10
cells respectively. Thus,
decreasing the receptor expression level by a
factor 29.6 did not change significantly the level
of constitutive activity expressed as the % of
maximal ghrelin (Fig. 1B). This is due to the fact
that decreasing the receptor expression level
induced a concomitant decrease of the basal
level of IP1 production with a concomitant
decrease of the maximal level promoted by
ghrelin. Decreasing the receptor expression level
did not allow to better visualize the partial
agonist character of the ligands (Fig. 1C). We
therefore decided to evaluate the efficacy of
these ligands to promote IP1 production on a
GHS-R1a mutant (GHS-R1a A204E) with a
decreased basal activity. The A204E mutation
decreased the basal activity of the receptor
without changing its ability to respond to ghrelin
compared to the WT receptor (27,42). As shown
in Fig. 1D, the basal level of IP1 production of
HEK293T cells expressing the GHS-R1a A204E
was highly decreased compared to the WT
receptor, whereas both the efficacy and potency
of full agonists to induce IP1 production were
unaffected. Interestingly, as shown in Fig 1D,
whereas JMV 3011 remained neutral, the partial
agonist activity of JMV 3002, JMV 3018 and
JMV 2959 unambiguously detected with the
GHS-R1a A204E mutant. Although binding
characteristic (Ki) of ligands are quite similar
between the WT and the A204E mutant
receptors (Table 1), we cannot totally exclude,
however, that the A204E mutation induces
discreet changes in binding and signaling
properties of ligands. Nevertheless, our
observation suggested that an extremely high
constitutive activity might hide the partial
agonist character of some ligands. The behaviour
of the different ligands with regard to GHS-R1a
A204 was confirmed by testing them in
competition with ghrelin. In this cas, JMV 3011
fully antagonized the effect of ghrelin at
promoting IP1 production whereas ligands JMV
3002, JMV 3018 and JMV 2959 antagonized
only partially the ghrelin effect (Fig. 3A).
Efficacy of GHS-R1a ligands toward β-
arrestin2 recruitment and ERK1/2 activation.
As previously reported, ghrelin stimulation
promotes β-arrestin2 recruitment to GHS-R1a
(26,27). The level of constitutive β-arrestin2
recruitment was lower than that of IP1
production. SPA reduced almost to zero the basal
level of β-arrestin2 recruitment, suggesting that
this ligand displayed inverse agonist efficacy
toward β-arrestin2 recruitment also (Fig. 2A and
C). As expected, ghrelin, JMV 1843 and MK-
0677 promoted a large increase in β-arrestin2
recruitment to GHS-R1a (Fig. 2A and C). As
observed for IP1 production, MK-0677 behaved
as a super-agonist toward β-arrestin-2
recruitment compared to ghrelin and JMV 1843.
In contrast, no β-arrestin2 recruitment was
observed upon stimulation with JMV 3011, JMV
3002, JMV 3018 and JMV 2959 (Fig. 2A and C).
Thus, JMV 3011 was neutral with regard to both
IP1 production and β-arrestin2 recruitment
whereas JMV 3002, JMV 3018 and JMV 2959
were partial agonists on IP1 production but
neutral toward β-arrestin2 recruitment (Fig. 1,
2A and B). Because it was reported that GHS-
R1a activated ERK1/2 through Gq/11, Gi and
arrestin dependent pathways (28,43,44), we
tested the efficacy of our ligands toward ERK1/2
activation. All compounds displayed comparable
efficacy to promote β-arrestin2 recruitment
(Table 2, Fig. 2A and C) and ERK1/2
phosphorylation (Fig. 2B and D). Whereas
ghrelin, MK-0677 and JMV 1843 promoted
ERK1/2 phosphorylation, ligands JMV 3002,
JMV 3018, JMV 2959 and JMV 3011 were
neutral toward ERK1/2 phosphorylation (Fig. 2B
and D). When tested in competition with ghrelin,
JMV 3002, JMV 3018, JMV 2959 and JMV
3011 totally antagonized both β-arrestin2
recruitment (Fig. 3B) and ERK1/2
phosphorylation promoted by ghrelin (Fig. 3C).
Taken together, these data indicate that GHS-
R1a partial agonists of IP1 production are
neutral/antagonists of β-arrestin2 recruitment
and ERK1/2 phosphorylation. Although these
results suggest that these ligands are de facto
biased toward IP1 production over β-arrestin-2
recruitment and ERK1/2 phosphorylation, a bias
factor could not be calculated because no dose
response-curve could be obtained for β -arrestin2
and ERK1/2 (Table 3). Quantification of bias
could be done for JMV 1843 and MK-0677 only
and indicated that these ligands were not
significantly biased toward Gq activation and
IP1 production relative to β-arrestin2 recruitment
and ERK1/2 phosphorylation (Table 3).
Activation of G protein subtypes and isoforms
by GHS-R1a: a study with G protein
activation BRET biosensors.
To test GHS-R1a ligands on the activation of
different G protein subtypes besides Gq, we then
used a BRET
based assay that monitors
conformational changes of G proteins upon
activation (31,32). This BRET assay consisted in
measuring a BRET signal between Rluc8 fused
to the α subunit and GFP10 fused to the β2
subunit, in a Rluc8-α, β1, γ2-GFP10 complex
(see experimental procedures). When the three G
protein subunits were transfected in HEK293T
cells, a high BRET basal signal was detected due
to the close proximity of RLuc8 and GFP10 in
the inactive Gαβγ trimer. G-protein activation
then resulted in a large decrease of the BRET
signal due to conformational changes within the
G protein trimer with α subunit moving away
from the βγ complex (31).!!
Using this approach we revisited the GHS-R1a-
G protein coupling. As expected, GHS-R1a
stimulation with either ghrelin or MK-0677
agonists induced a large decrease of the BRET
signal with the Gq biosensor confirming that
GHS-R1a was indeed a typical Gq coupled
GPCR (Fig. 4). A decrease of the BRET signal
was also detected for Gi1, Gi2, Gi3, Goa, Gob
and G13 isoforms, confirming more directly than
in previous studies, that GHS-R1a can couple to
Gi and Go proteins. Importantly, a modest albeit
significant decrease of the BRET signal was
observed for G13, confirming that GHS-R1a
activates G13 also (Fig. 4A and B). In contrast,
no variation of the BRET signal was observed
for G12 and Gs. Because it was reported in the
literature that GHS-R1a activates Gs dependent
pathways in some cell systems, we questioned
whether the absence of Gs activation could result
from a default in the biosensor. To address this
point, we applied our BRET approach to the
detection of Gs coupling to a typical Gs-coupled
receptor control, the vasopressin V2 receptor. As
shown in Fig. 4C, a large decrease of the BRET
signal was observed on HEK293T cells co-
transfected with V2R and the Gs biosensor
following vasopressin stimulation. This
confirmed that the Gs biosensor was an
appropriate tool to detect Gs coupling and
therefore suggested that GHS-R1a was unable to
directly activate Gs, at least in HEK293T cells.
We also confirmed that the absence of G12
stimulation was not due to a non-functioning
G12 biosensor. Indeed, using the same
experimental conditions with HEK293T cells co-
expressing the thromboxane A2 α subtype (TPα)
receptor and the G12 sensor, stimulation with the
agonist U46619 triggered a decrease of BRET
signal was easily detected as already described
(32). This indicates that the G12 biosensor is
also functional, suggesting that GHS-R1a is not
coupled to G12 (Fig. 4D).
Selectivity of GHS-R1a ligands toward Gq
and IP1 production.
Although IP production was routinely used to
measure Gq/11 mediated signaling pathways, it
was interesting to compare the behaviour of our
ligands in the Gq activation and IP1 production
assays to assess wether the efficiency of ligands
to activate Gq is transmitted all along the PLCβ-
inositol phosphate pathway. Comparison of
transduction efficiency (∆log(τ/K
)) at Gq
activation and IP1 production was only possible
for ghrelin, MK-0677 and JMV 1843. Indeed, no
robust IP1 production dose-response curves
could be obtained for JMV 2959, JMV 3002 and
JMV 3018. Ghrelin, MK-0677 and JMV 1843
displayed similar potency and efficacy for both
Gq activation and IP1 with MK-0677 displaying
a higher efficacy and being more potent than
JMV 1843 and ghrelin at both pathways (Fig. 5
and Table 2 for EC
Quantification of bias demonstrated no
selectivity of MK-0677 and JMV 1843 toward
Gq activation and IP1 production compared to
the reference ligand ghrelin (Table 3). As
indicated above, quantification of bias was not
possible for JMV 2959, JMV 3002, JMV 3011
and JMV 3018 (Table 3).
Selectivity of GHS-R1a ligands toward Gq, Gi
and Go activation.
We performed dose-response curves for Gi and
Go isoforms (Fig. 6). EC
values reported in
Table 2, showed that ghrelin was equally potent
to stimulate Gq, Gi and Go, whereas MK 0677!
and JMV 1843 were more potent to stimulate Gq
than Gi and Go. As shown in Fig. 6 and Table 2,
MK 0677 displayed a higher potency toward Gq
than toward Gi2, Gi3, Goa and Gob.
Quantification of bias confirms that, MK-0677 is
indeed slightly but significantly biased toward
Gq relative to Gi2, Gi3, Goa and Gob with a bias
factor (BF) of 6.46, 10.11, 12.59 and 18.84
respectively (Fig. 6, Table 3). We then
investigated the selectivity of GHS-R1a ligands
to promote activation of Gq, Gi2 and Gob, three
G protein subtypes for which the highest BRET
variation was observed following ghrelin
As already discussed above, ghrelin, MK-0677
and JMV 1843 behaved as full agonists toward
Gq, Gi2 and Gob activation (Fig 6A, B and C).
JMV 3011, which was classified as neutral and
antagonist based on the IP1 production assay
(Fig. 1 and 3), had indeed no action by its own
on Gq, Gi2 and Gob activation. However, as
expected, it antagonized ghrelin-induced Gq, Gi2
and Gob activation (Fig. 6 and 7). JMV 3011 is
thus neutral and antagonist at Gq, Gi2 and Gob.
In contrast, JMV 2959, JMV 3002, JMV 3018,
which behaved as partial agonists of Gq, were
neutral at Gi2 and Gob-activation (Fig. 6 and 7).
When we tested them at a 10
M maximal dose
in competition with 10
M ghrelin, these
compounds partially inhibited ghrelin-promoted
Gq activation and completely suppressed
ghrelin-evoked Gi2 and Gob activation (Fig. 7).
Taken together, these results suggest that JMV
2959, JMV 3011 and JMV 3018 are de facto
biased agonists toward Gq relative to Gi2 and
Gob. However, one can consider this as an
observational bias. Indeed, no quantification of
bias was possible because these ligands did not
induce any significant signals at Gi2a and Gob.
Selectivity of inverse agonists toward Gq and
Interestingly, we found that the G-protein BRET
assay was also suitable for detection of inverse
agonists. Indeed, in contrast to agonists, SPA
induced an increase of the BRET signal on cells
co-expressing GHS-R1a and the Gq biosensor
(Fig. 8), confirming that SPA acts as an inverse
agonist toward GHS-R1a-promoted Gq
constitutive activity. To determine if using the
BRET assay could be a general way to identify
inverse agonists, we applied it to a panel of
compounds that had been classified as inverse
agonists based on IP1 production (Fig. 8A, Table
2). All these ligands promoted an increase of the
BRET signal in cells co-expressing GHS-R1a
and the Gq biosensor (Fig. 8B). This confirms
that SPA, KwFwLL-NH
and JMV 4484 are inverse agonists toward
GHS-R1a-mediated Gq constitutive activity.
Interestingly, for different classes of GHS-R1a
ligands, a close correlation was obtained
between their efficacy to modulate Gq activity
and their efficacy to modulate IP1 production
Beside constitutive activity at the Gq-Inostitol
phosphate signaling pathway, it had been
previously suggested, based on SRE reporter,
that GHS-R1a also constitutively activated
G12/G13-dependent pathways (25). Therefore,
we checked whether the G13 biosensor could be
used to bring a more direct proof of GHS-R1a-
mediated G13 constitutive activation. As was the
case for Gq, the basal level of G13 activity was
strictly dependent on the amount of GHS-R1a
receptors expressed at the surface of the cells.
Indeed, the BRET ratio decreased gradually
upon increasing the amount of GHS-R1a at the
cell surface (Fig. 9 A-F). These results
confirmed that GHS-R1a constitutively activated
G13 and, as expected, the level of this
constitutive activity correlated to the receptor
expression level. Based on this result, we
investigated the behaviour of inverse agonists of
Gq on G13 activation (Fig. 9G and H).
Interestingly, compound K-(D-1Nal)-FwLL-NH
promoted an increase of the BRET signal at both
Gq and G13 whereas SPA, KwFwLL-NH
JMV 4484 behaved only inverse agonists on Gq
only (Fig. 9G and H). Although this suggest that
and JMV 4484 are biased
toward Gq over G13, it is difficult to firmly
conclude at this stage if the analysis. Indeed, the
BRET signal was 10 fold lower for G13 than for
Gq. The observational bias could thus result
from a difference in sensitivity between Gq and
G13 sensors. In order to confirm the results
obtained with the BRET sensors, we then used a
totally different assay. This assay consisted in
measuring GTPγS binding to purified Gq and
G13 proteins, in the presence of purified GHS-
R1a embedded in lipid discs. As expected, the
agonist MK-0677 promoted an increase of
GTPγS binding at both Gq and G13. However,
decrease of GTPγS binding for both Gq and G13
compared to the unliganded receptor, SPA,
and JMV 4484 promoted a
decrease of GTPγS binding to Gq only (Fig. 10A
and B). These results were confirmed by kinetic
analyses of GTPγS binding (Fig. 10C and D).
Indeed, stimulation of GHS-R1a with K-(D-
promoted a decrease of the
Kact value for both Gq and G13 compared to
unliganded receptor,, whereas stimulation with
and JMV 4484 induced
decrease of the Kact value for Gq only (Fig. 10C
and D). Thus, these data confirmed that SPA,
and JMV 4484 compounds acted
as biased inverse agonists toward Gq relative to
G13. Although the raw fluorescence intensity
increase values upon GTP binding to Gq are two
fold higher than those obtained with G13, both
signal are nevertheless well above the signal to
noise ratio. This suggest that a difference in
sensitivity cannot explain the absence of change
in receptor-catalyzed GTPγS binding to G13
observed for SPA, KwFwLL-NH2 and JMV
4484. Unfortunately, no quantification of bias for
the inverse agonists could be done due to any
detectable effect of these ligands on G13.
In the present work we revisited the
pharmacological behaviour of ligands targeting
GHS-R1a by deeper exploring their efficacy
toward G protein dependent and independent
signaling pathways. We took a particular
attention to the selectivity of ligands toward a
panel of G-proteins subtypes thanks to G-protein
activation biosensors that were recently
developed (31,32). As already reported, GHS-
R1a displays one of the highest constitutive
activity (24,26) in the GPCR family (45,46). In
HEK293T cells, the model we used in this study,
the basal level of IP1 production represented 50-
70% of the maximal level promoted by ghrelin,
the endogenous ligand of GHS-R1a. In this
particular situation, detection of partial agonists
was problematic because the high basal level of
IP1 production might hide the partial agonist
character of some ligands. This was the case for
JMV 2959, firs reported as an antagonist (35,47)
but which displayed no or partial agonist activity
depending whether the basal activity was higher
or lower than 60 % of the maximal ghrelin
response. Indeed, we unambiguously
highlighted here the partial agonist character of
JMV 2959 and of other compounds (JMV 3002
and JMV 3018) by testing their efficacy on the
GHS-R1a-A204E mutant, which exhibits a low
constitutive activity (42). These results
highlighted the fact that ligands targeting GHS-
R1a might be classified as neutral or partial
agonists depending of the level of constitutive
activity of the receptor. This point is of
importance because constitutive activity of the
GHS-R1a was demonstrated in vivo in rat brain
(48) and in human somatotroph adenomas (49).
However, the exact level of constitutive activity
of the receptor is not ascertained due to the
difficulty to its in vivo quantification.
Furthermore, one can imagine that the level of
constitutive activity of the GHS-R1a varies with
its tissue or cellular localization. Indeed, it had
been repeatedly demonstrated that the level of
constitutive activity of a GPCR depends on the
cell content in various protein partners such as
other GPCRs or intracellular proteins (G
proteins, scaffolding proteins..) (45,46).
Therefore, the lack of knowledge on the ligand
independent activity of GHS-R1a in vivo should
make us cautious about classifying GHS-R1a
antagonists until a deep investigation has been
performed. Interestingly we found that JMV
compounds that behaved as partial agonists on IP
signaling were unable to promote both β-
arrestin2 recruitment and ERK1/2 activation, in
contrast to the full agonists ghrelin, MK-0677
and JMV1843. However, these partial agonists
totally inhibited ghrelin-promoted ERK1/2
phosphorylation and β-arrestin2 recruitment.
Thus, this suggests that JMV 2959, JMV 3018,
JMV 3002 are biased agonists toward IP
pathway compared to arrestin recruitment and
ERK1/2 activation and behave as biased
antagonists of arrestin recruitment and ERK1/2
activation compared to the IP pathway.
However, one can also consider this bias as an
observational bias. Indeed, no quantification of
bias was possible because these ligands did not
induce any detectable IP1 production, β-arrestin2
recruitment and ERK1/2 phosphorylation. We
took advantage here of G protein activation
biosensors (31,32) that directly report on
the conformational change of G protein upon
activation, to assess whether the partial agonist
behaviour of these ligands toward IP1 production
resulted from their Gq partial agonism. Although
it was reported that GHS-R1a activated G
protein dependent signalling pathways through
Gq, Gi, Go (23,30,50) and G13 (51), these
conclusions were! drawn from studies that
indirectly measured G protein activation. We
monitored here the selective coupling of GHS-
R1a to G protein family using activation
biosensors for a panel of G protein subtypes and
isoforms (Gq, Gi1, Gi2, Gi3, Goa, Gob, Gs, G12,
G13). As expected, GHS-R1a interaction with
either ghrelin, MK 0677 and JMV1843 resulted
in an efficient Gq activation. We also confirmed
that stimulation of GHS-R1a either with ghrelin
or MK-0677 promoted activation of Gi proteins
with a better efficacy for Gi2 and Gi3 than for
Gi1. We also found that ghrelin had the ability to
promote Goa, Gob and G13 activation, but was
unable to promote G12 and Gs activation. Thus,
our data demonstrate that ghrelin-stimulated
GHS-R1a activates Gq, Gi, Go and G13 proteins,
confirming that GHS-R1a signals through
intracellular pathways governed by these G
protein types. Although it was previously
reported that GHS-R1a activated G13 dependent
signaling pathways, these conclusions were
drawn from indirect measurement of SRE
(25,52) and Rho-kinase activation or by the use
of a dominant negative mutant of Gα13 (51).
Our data confirm more directly that ghrelin
stimulates GHS-R1a-mediated G13 activation. In
contrast, no Gs activation was detected,
confirming our previous data that concluded to
the inability of the purified GHS-R1a to couple
to Gs in lipid nanodiscs (26). Taken together our
data suggest that GHS-R1a does not interact with
Gs in contrast to published data indicating that!
ghrelin activated cAMP production in pancreatic
HIT-T25 beta cells (53,54). Although we found
that MK-0677 and JMV 1843 displayed similar
efficacy toward several G protein subtypes and
isoforms, quantification of bias demonstrate that
MK-0677 is slightly biased compared to ghrelin
toward Gq over Gi2, Gi3, Goa and Gob.
Interestingly, the neutral/antagonist character of
JMV 3011 demonstrated on IP1 production,
arrestin and ERK1/2 pathways was also
confirmed on Gq, Gi2 and Gob, making this
ligand a good lead for the design of GHS-R1a
signaling neutral/antagonist. More importantly,
the partial agonist behaviour of JMV 2959, JMV
3002 and JMV 3018 first assessed with the IP1
production assay was further confirmed with the
Gq biosensor. However, and interestingly,
ligands displaying partial agonist efficacy toward
Gq, were silent toward Gi2 and Gob. Moreover,
we clearly demonstrate that these ligands fully
inhibit Gi2 and Gob activation promoted by
ghrelin and MK-0677, whereas they only
partially inhibited Gq activation. Since, JMV
2959, JMV 3002 and JMV 3018 did not promote
any detectable activation of Gi2 and Gob, no
quantification of bias between Gq and Gi/o could
be obtained for these compounds. Nevertheless,
on can consider that up to 10
M, JMV 2959,
JMV 3002 and JMV 3018 behaved as: (i) biased
agonists toward Gq activation and IP1
production relative to Gi2, Gob activation,
arrestin recruitment and ERK1/2 activation; (ii)
biased antagonists toward Gi2, Gob activation,
arrestin recruitment and ERK1/2 activation
relative to Gq activation and IP production. It is
obvious that these results need to be confirmed
in a more physiologically relevant natural system
than HEK293 cells. It also remains to assess
whether the ligand-directed functional selectivity
toward down-stream signaling pathways leads to
a functional selectivity toward physiological
functions controlled by the ghrelin/GHS-
R1aaxis. One can nevertheless mention that in
vivo studies in rat demonstrated that JMV 2959
inhibited food intake, addiction but not GH
secretion promoted by ghrelin (35,47). This
observation is of importance because it suggests
that a potential link may exist between the
selective action of JMV 2959 toward the
signaling pathways activated by GHS-R1a and
its selective antagonist action toward
physiological responses promoted by ghrelin
(Fig. 11). Very interestingly, the synthetic ligand
GSK1614343 described as antagonist on calcium
release and IP1 production behaved in vivo as an
antagonist against GH secretion although it
stimulated food intake (55,56). In the same line,
another ghrelin analog, BIM-28163 considered
as a ghrelin receptor antagonist based on in vitro
calcium release assays, behaved in vivo as a
ghrelin-induced GH secretion antagonist, but as
an agonist on stimulation of food intake (57).
Therefore, GSK and BIM compounds behaved in
vivo in an opposite way of JMV 2959. Whether
the in vivo selective action of GSK and BIM
compounds results from a signaling bias opposite
to that of JMV 2959 remains to be determined.
Altogether, these observations suggest that it will
certainly be possible in a near future to design
new selective therapeutic drugs for pathologies
associated to ghrelin/GHS-R1a interactions. In
this context, it could be useful to selectively
block some of the signaling pathways linked to
constitutive activation of the GHS-R1a. Indeed,
GHS-R1a is one of the most constitutively active
GPCR and the potential role of its constitutive
activity in the “snacking” behaviour between
meals is questioned (58). So far, to our
knowledge, inverse agonists that display a
functional selectivity toward signaling pathways
have been only described for the ghrelin
receptor. Indeed it was reported that compound
, a GHS-R1a inverse agonist on
IP production was neutral in a SRE luciferase
assay, suggesting that this ligand is a biased
inverse agonist favouring inhibition of the
constitutive GHS-R1a-mediated Gq-dependent
pathway compared to that of the G13-dependent
pathway (52). Thanks to G-protein BRET
biosensors, we directly demonstrated that GHS-
R1a constitutively activates both Gq and G13.
We found that K-(D-1Nal)-FwLL-NH
had been previously characterized as a GHS-R1a
inverse agonist on IP pathway (38), is indeed an
efficient and potent inverse agonist toward Gq
activity but behaves also as an inverse agonist
toward G13. We also found that SPA as well as
and JMV 4484 behave as inverse
agonists toward IP production and Gq activation
but were silent toward G13 activation. However,
it was difficult to interpret these data as resulting
from a real functional selectivity because the
intensity of G13 signal was much lower than that
of Gq. Nevertheless, these results were
confirmed by directly measuring receptor
catalysed GTPγS binding to Gq and G13 with
GHS-R1a reconstituted in lipid discs, an assay
for which Gq and G13 signals were comparable.
Thus, using two different approaches that
directly report on G protein activity, some
inverse agonists appear selective toward Gq over
G13. However, we could not confirm with these
assays that SPA behaves as a modest inverse
agonist at G13 signaling as previously suggested
from data that measured indirectly G13 activity
by recording SRE activity (25). A possibility
would be that this discrepancy results from the
different methods used in the two studies.
Indeed, the method we have employed in the
present study monitors the activity of the G
protein itself whereas the SRE reporter assay
monitors an activity resulting from the activation
of various G proteins, including Gq, G13, Gi and
Gβγ of Gi. Therefore, it may be possible that the
modest inverse agonist activity of SPA observed
in the SRE reporter assay did not result from the
G13 activation but rather from the activation of
other G protein-dependent pathways. In
summary, we have identified in this study, a
series of synthetic ligands that behave as partial
agonists at Gq but are silent toward β-arrestin2
recruitment and Gi/Go activation. One of them,
JMV 2959, appears particularly attractive
because this ligand selectively blocks ghrelin-
evoked food intake and addictions without
altering GH secretion. It should be now of
interest to further explore the in vivo behaviour
of the other molecules identified in this study
that display a bias behaviour similar to that of
JMV 2959. Finally, our data suggest that GHS-
R1a-dependent constitutive activation of Gq and
G13 can be selectively modulated by synthetic
ligands. It would be also important to test in
future studies whether the signaling bias
promoted by some of our ligands can result from
their action on allosteric binding sites and not
through their direct action on the orthosteric site,
an issue that has not been explored in this study.
Indeed, for a given GPCR, allosteric agonists can
promote different signaling profiles compared to
the orthosteric agonist, and there are several
examples of allosteric ligands that change the
coupling preference of the endogenous agonist
(59). Finally, although our study brings new
information on the selectivity of ligands at the
GHS-R1a signaling, the data reported in this
work were obtained in a single cell system
model, HEK293. This cell system is certainly far
from representing the physiological context of
GHS-R1a-dependent signaling. Furthermore, the
selectivity of action of ligands on various
signaling pathways varies depending on the
cellular context (60,61). Therefore, before
drawing any definitive conclusions on the
physiological reality of the signaling selectivity
of our ligands that was only observed so far in
HEK293, further studies should be carried out in
other heterologous cell systems, or even better in
primary cells that endogenously express the
GHS-R1a. It is obvious that the development of
functional selective drugs that could be
therapeutically useful will require further studies
to better understand the contribution of
individual signaling pathways to the diverse
physiological responses controlled by GHS-R1a.
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale
(INSERM), Centre National de la Recherche Scientifique (CNRS), Université de Montpellier, Agence
Nationale de la Recherche (PCV08-323163).
We thank Eric Trinquet (Cisbio) for providing us materials for HTRF-based assays.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
C.M’K., J.P.L. and J. Marie designed research; C.M’K, J.P.L, L.O., C.G., A.S., D.G., M.D. and S.M.
performed research; M.M., S.D., P.V., J.A.F, J.M. synthetized ligands; C.M’K., J.L.B. and J. Marie
analyzed the data and J. Marie wrote the paper. All the authors reviewed the results and approved the
final version of the manuscript.
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The abbreviations used are: GHS-R1a, growth hormone secretagogue receptor type 1a; GH, growth
hormone, GPCR, G protein-coupled receptor; PBS, phosphate-buffered saline; IP1, inositol-1-
phosphate; HEK, human embryonic kidney; SRE, serum-responsive element; ERK, extracellular signal-
regulated kinase, BRET, bioluminescence resonance energy transfer; HTRF, homogenous time resolved
fluorescence; GTPγS, guanosine 5’-O-(3-thiotriphosphate); PKC, protein kinase C; PI3K,
phosphatidylinositol 3 kinase; YFP, yellow fluorescent protein; Luc, luciferase.!
Figure 1. Efficacy of GHS-R1a ligands to promote inositol phosphate production. Inositol
phosphate (IP1) production promoted by ligands at maximal dose (10
M) in cells expressing the GHS-
R1a (A) or the A204E GHS-R1a mutant (D). Basal level of IP1 production measured in cells expressing
different amounts of GHS-R1a (B). IP1 production promoted by ligands at maximal dose (10
cells expressing low and high amount of GHS-R1a (C). IP1 production was measured with an HTRF
assay in HEK293T cells treated with ligands for 30 min at 37°C. Data are expressed as the % of ghrelin
maximal response. Basal represents IP1 production measured in non-stimulated HEK293T cells
expressing GHS-R1a receptors. 0% is defined as the basal of IP1 production of mock-transfected
HEK293T cells (cells transfected with an empty pcDNA3.1 (+) vector). Values are mean ± S.E.M of
three independent experiments performed in triplicate. Statistical significance between stimulated and
non-stimulated cells was assessed using a one-way ANOVA followed by Dunnett’s post-hoc test
(***P<0.001, **P<0.01, *P<0.1).
Figure 2. Efficacy of GHS-R1a ligands to promote β-arrestin2 recruitment and ERK1/2
β-arrestin2 recruitment dose-response curves (A) and maximal responses with 10
M of ligand (C). β-
arrestin2 recruitment to GHS-R1a was measured with a BRET
assay upon ligand stimulation for 45
min at 37°C in HEK293T cells expressing the GHS-R1a. ERK1/2 phosphorylation dose-response
curves (B) and maximal responses with 10
M of ligand (D). ERK1/2 phosphorylation was measured
with an HTRF assay upon ligand stimulation for 10 min at 37°C in HEK293T cells expressing the
GHS-R1a. All data are expressed as the % of maximal ghrelin-induced stimulation. Dose response
curves (A, B) are representative of three experiments and graphs of maximal responses (C, D). Values
are mean ± SEM of three independent experiments performed in triplicate. 0% represents the basal of
mock-transfected HEK293T cells. Statistical significance between stimulated and non-stimulated cells
was assessed using a one-way ANOVA followed by Dunnett’s post-hoc test (***P<0.001, **P<0.01,
Figure 3. Antagonist efficacy of JMV compounds toward ghrelin-promoted IP1 production, β -
arrestin2 recruitment and ERK1/2 phosphorylation. IP1 production (A), β-arrestin2 recruitment (B)
and ERK1/2 phosphorylation (C) measured as described in Figure 1 and 2 and in the experimental
procedures section. HEK293T cells expressing the GHS-R1a were stimulated with ghrelin at 10
the absence or in the presence of 10
M JMV compounds. Data are expressed as the % of maximal
ghrelin-induced stimulation. Bars and error bars represent the mean ± S.E.M of three independent
experiments, each performed in triplicate. Statistical significance between stimulated and non-
stimulated cells was assessed using a one way ANOVA followed by Dunnett’s post-hoc test
(***P<0.001,**P< 0.01, *P<0.1).
Figure 4. Activation of G proteins subtypes and isoforms by GHS-R1a. A, G protein activation
kinetics was measured by BRET
using G protein activation biosensors as described in experimental
procedures section. HEK293T cells co-expressing both the GHS-R1a and the G protein biosensor were
stimulated by the GHS-R1a agonist MK-0677 (10
M). Data are representative of three to eight
independent experiments. B, BRET maximal signal promoted by 10
M MK-0677 on HEK293T cells
co-expressing GHS-R1a and G protein biosensors. Results are expressed as the difference in BRET
ratio measured in the presence