Structural basis for constitutive activity and agonist-induced activation of the enteroendocrine fat sensor GPR119

Article abstract of DOI:10.1111/bph.12877

Background and Purpose GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts in the field, very little is known about the basic molecular pharmacology of GPR119.

Full text of DOI:10.1111/bph.12877

DOI:10.1111/bph.12877
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
RESEARCH PAPER
Thue Schwartz, University of
Copenhagen, Metabolic
Receptology and
Structural basis for
Enteroendocrinology,
Blegdamsvej 3, Copenhagen
DK-2200, Denmark. E-mail:
tws@sund.ku.dk
constitutive activity and
agonist-induced activation
of the enteroendocrine fat
sensor GPR119
----------------------------------------------------------------
*Present address: Weizmann
Institute of Science, 234 Herzl
Street, Rehovot 7610001, Israel.
----------------------------------------------------------------
†Present address: Lundbeck
Pharma A/S, Ottiliavej 9, 2500
Valby, Denmark.
----------------------------------------------------------------
Received
31 March 2014
Revised
4 August 2014
Accepted
6 August 2014
M S Engelstoft1,2, C Norn1,3*, M Hauge1,2, N D Holliday4, L Elster5†,
J Lehmann6, R M Jones6, T M Frimurer1,3 and T W Schwartz1,2
1Novo Nordisk Foundation Center for Basic Metabolic Research, Section for Metabolic Receptology
and Enteroendocrinology, 2Laboratory for Molecular Pharmacology, Department of Neuroscience
and Pharmacology, 3Novo Nordisk Foundation Center for Protein Research, Faculty of Health
and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 4School of Life Sciences,
University of Nottingham, Nottingham, UK, 57TM Pharma A/S, Hørsholm, Denmark, and
6Medicinal Chemistry, Arena Pharmaceuticals, San Diego, CA, USA
BACKGROUND AND PURPOSE
GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary
triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite
massive drug discovery efforts in the field, very little is known about the basic molecular pharmacology of GPR119.
EXPERIMENTAL APPROACH
GPR119 receptor signalling was studied in transfected cells. Mutational mapping (30 mutations in 23 positions) was
performed on residues required for ligand-independent and agonist-induced GPR119 activation (AR231453 and OEA). Novel
Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray
structures and fully flexible ligand docking.
KEY RESULTS
The increased signalling induced by increasing the cell surface expression of GPR119 in the absence of agonist and the
inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity
through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the
mutational map for residues affecting the constitutive signalling – albeit with key differences. Surprisingly, almost all residues
in extracellular loop-2b were important for the constitutive activity. The molecular modelling and docking demonstrated that
AR231453 binds in a ‘vertical’ pocket in between mutational hits reaching from the centre of the receptor out to extracellular
loop-2b.
CONCLUSIONS AND IMPLICATIONS
The high constitutive activity of GPR119 should be taken into account in future drug discovery efforts, which can now be
guided by the detailed knowledge of the physiochemical properties of the extended ligand-binding pocket.
Abbreviations
7TM, seven transmembrane; ECL, extracellular loop; OEA, oleoylethanolamide; REU, Rosetta energy units; RMSD, root
mean square deviation.
© 2014 The British Pharmacological Society
5774 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
Tables of Links
TARGETS
LIGANDS
β2-adrenoceptor
κ-opioid receptor
A2A receptor
D3 receptor
Ghrelin receptor
GPR119
Adenine
AR231453
ATP
GLP-1
Insulin
OEA
Chemokine receptors
CXCR4
H1 receptor
M2 receptor
cAMP
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://
www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are
permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).
(Hansen et al., 2012). Importantly, a number of synthetic
GPR119 agonists have been developed, which are highly
Introduction
By use of bioinformatics, GPR119 was originally identified as
an orphan, rhodopsin-like seven transmembrane (7TM)
segment, GPCR, which is not closely related to any other
7TM receptor (Fredriksson et al., 2003). However, GPR119
had in fact been identified previously in the patent literature
as an orphan receptor, that is highly expressed in pancreatic
islets, under various names such as: SNORF25 (Bonini et al.,
2001; Adham et al., 2003), RUP3, 19AJ, GPCR2, PFI-007
(Reilly, 2001; Ohishi et al., 2002), PS1, AXOR20, OSGPR116
(Griffin, 2006) and the glucose-dependent insulinotropic
receptor (Jones, 2006). The ability of GPR119 to act as a
pancreatic insulinotropic receptor was subsequently rediscov-
ered after the receptor had obtained its official name, GPR119
(Soga et al., 2005; Sakamoto et al., 2006; Chu et al., 2008).
Although the expression and function of GPR119 in beta cells
has been repeatedly shown, it is still unclear to what extent
GPR119 is also expressed in the other endocrine islet cells
(Jones et al., 2009). However, GPR119 has been shown to be
highly expressed in enteroendocrine cell lines such as
GLUTag and STC-1 cells (Chu et al., 2008), as well as in
natural enteroendocrine cells (Reimann et al., 2008; Parker
et al., 2009).
GPR119 is activated by a number of different lipid
metabolites, some of which are derived from dietary fat and
some of which may be generated locally in the tissue (Hansen
et al., 2012). Among the locally generated lipid metabolites,
which presumably act in a paracrine or autocrine fashion,
oleoylethanolamide (OEA) is particularly potent and could
potentially be generated under physiological conditions in
sufficient amounts to be a valid GPR119 agonist (Overton
et al., 2006; Hansen et al., 2012). The same is true for
2-monoacylglycerols, which are generated in large amounts
in the lumen of the gastrointestinal tract from dietary triglyc-
erides by the action of pancreatic lipase and which conse-
quently could act as luminal stimulators of enteroendocrine
cells (Hansen et al., 2012). In contrast, N-oleoyldopamine and
5-hydroxy-eicosapentaenoic acid, which also have been
reported to be GPR119 agonists (Chu et al., 2010; Kogure
et al., 2011), are probably not generated in sufficient amounts
under physiological conditions to be valid GPR119 agonists
selective and consequently have been crucial for the charac-
terization of GPR119 function in vitro and in vivo (Jones et al.,
2009).
GPR119 couples to Gαs (Chu et al., 2007) and is an effi-
cient stimulator of cAMP production both in transfected
cells (Soga et al., 2005; Overton et al., 2006; Chu et al., 2007)
and in endocrine cell lines expressing GPR119 where the
rise in cAMP levels is associated with hormone secretion.
Thus, GPR119 activation results in the secretion of insulin
from insulinoma cell lines (Soga et al., 2005; Chu et al.,
2007; Ning et al., 2008) and GLP-1 from enteroendocrine
cell lines (Chu et al., 2008; Lauffer et al., 2009) and primary
intestinal cell cultures (Lan et al., 2012). The prototype
GPR119 agonist, AR231453, has been shown to ameliorate
glucose excursions in both normal and diabetic mice (Chu
et al., 2007). Importantly, this effect was diminished when
glucose was administered i.p. instead of p.o. GPR119 ago-
nists induce both insulin and GLP-1 secretion in vivo and
importantly, neither of these effects are observed in
GPR119-deficient mice (Chu et al., 2007; 2008). Thus, both a
direct effect on the beta cells and an indirect incretin-based
effect appear to contribute to the GPR119-mediated
improvement in oral glucose tolerance. It has been sug-
gested that GPR119 also plays a role in the control of food
intake (Overton et al., 2006), but this is less certain than its
well-established role in glucose control (Jones et al., 2009;
Lan et al., 2009).
Early on, GPR119 became a major target for the develop-
ment of novel anti-diabetic agents and several selective
potent and efficacious GPR119 agonists have been in clinical
trials (Overton et al., 2008; Shah, 2009; Dhayal and Morgan,
2010; Ohishi and Yoshida, 2012). But despite their apparent
beneficial effects in various ex vivo and in vivo models, they
have apparently not lived up to the high expectations, which
originally surrounded GPR119. Several programmes have
been terminated or have been given back to the biotech
companies, from which they originated (Hansen et al., 2012).
However, it is still unclear whether this apparent lack of
clinical efficacy was caused by the broad pharmacology of
GPR119 agonists stimulating not only incretin and insulin
British Journal of Pharmacology (2014) 171 5774–5789 5775

M S Engelstoft et al.
BJP
L
G
F
S
L
I
M
P
G
L
D
S
F
L
V
I
Y
L
W
S
G
V
T
A
IV.
L
GPR119
A
A
S
A
T
S
L
II.
L
I
A
F
T
T
L
P
L
P
L
III.
Q
V
W
L
G
I
F
V
D
L
T
A
L
I
M
S
S
T
F
V
G
A
N
L
I
A
H
R
S
C
A
V
S
V
P
V
G
R
V.
W
L
I.
H
C
S
E
G
N
V
3
L
A
L
G
L
M
G
VII.
S
F
V
L
P
F
I
T
F
L
A
L
F
Y
Q
L
P
L
R
T
I
L
VI.
N
L
S
P
S
A
Y
Q
K
T
K
T
T
NH
T
2
G
Q
S
E
Q
C
G
I
P
M
E
S
V
Q
F
C
H
F F
Q
L
S
A
S
L
Q
A
V
Q
C
Y
S
F
V
L
C
R
1
1
F
M
S
L
H1
P
P
V
Extracellular
F
1 L
S
S
I
D
T
L
S
L
F
I
V
P
G
E
L
H
L
I
V
M
G
L
R
Y
L
G
A
L
F
V
G
F
T
S
L
L
V
S
I
A
L
W
G
I
T
F
T
L
V
F
A
L
V
F
G
I
L
L
T
A
V
L
S
P
S
T
A
G
C
F
M
F
A
T
S
I
V
A
L
Y
W
G
N
L
G
A
S
F
I
T
S
S
S
L
I
V
D
V
A
L
L
A
A
P
L
W
V
A
N
T
N
L
A
T
V
G
T
G
P
Y
L
F
M
L
A
L
L
I
S
R
K
N
C
L
L
L
V
I
L
V
L
I
V
I
A
C
A
T
V
V
A
V
Y
F
G
W
F
D
V
A
Q
Y
M
A
1
A
R
1
L
Intracellular
C
K
1
Y
L
D
T
D
E
L
L
F
P
F
G
S
M
I
L
V
R
L
L
A
S
K
I
H
S
R
S
V
M
L Y
I
I
P
Q
S
G
H
M
H
S
R
K
Q
P
D
Q
K
N
Q
K V G L A
Y
K
K
V
I
R
G
G
A
L
F
R Y
L
F
T S
S
L
K
M
L
F
M
A
HOOC- G
E
G
H A
D
F
L
E
S
S
S
A
S
R
I
N
I H C S S E R P R E P G C
T V
Figure 1
Helical wheel and serpentine model of the human GPR119. The sequence of GPR119 was depicted schematically from the membrane bilayer and
from the extracellular side. The residues selected for the mutational analysis are marked with blue and white letters. Residues conserved in family
A 7TM receptors and present in the GPR119 receptor are marked with grey. The Schwartz/Baldwin generic numbering system for 7TM receptors
(Schwartz et al., 2006) is used to denote the residues in the transmembrane helices throughout the paper. The residues designated as the first of
each helix are marked with 1. The mutations in ECL-2b are numbered according to their relative position from the conserved cysteine.
secretion but also counter-regulatory gut and pancreatic hor-
mones such as somatostatin and glucagon, or whether the
Methods
apparent lack of clinical efficacy of the present GPR119 ago-
Molecular biology
nists was related to difficulties in developing agonists for 7TM
GPR119 receptor cDNA was cloned into pcDNA3.1(+) and
receptors with appropriate signalling properties.
FLAG tagged. A modified signal peptide from viral haemag-
Very little has been published about the basic molecular
glutinin was inserted in front of the FLAG tag to enhance
and cellular pharmacology of GPR119 despite the massive
surface expression (Guan et al., 1992). Mutations were intro-
efforts invested in drug discovery processes targeting this
duced by site-directed mutagenesis using PCR. The resulting
receptor (Jones et al., 2009). In the present study, we focused
construct was transformed into competent Escherichia coli
on the apparent constitutive signalling of GPR119, and
XL1 blue, cultured and purified with the maxi prep kit from
through extensive mutational mapping (Figure 1) combined
Qiagen (Copenhagen, Denmark).
with molecular modelling and computational chemistry, we
characterized the molecular basis for constitutive versus
agonist-induced signalling of GPR119. The broad conclusion
is that both the prototype synthetic agonist AR231453 and the
most potent endogenous agonist OEA bind in a similar vertical
fashion to the main ligand-binding pocket of GPR119, and
that overlapping – but not identical – residues in this pocket
including key residues in extracellular loop-2b (ECL-2b) are
also important for the constitutive signalling of the receptor.
Cell culture and transfection
COS7 cells were grown in DMEM (low glucose) supplemented
with 10% fetal calf serum, 2 mM glutamine and 0.01 mg·mL−1
pen-strep at 37°C, 10% CO2. Cells were transfected with 20 μg
DNA/75 cm2 using calcium phosphate precipitation (Graham
and van der Eb, 1973) with chloroquine addition. For the
‘gene dosing’ experiment, cells were transfected with 0, 5, 10,
5776 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
20 or 40 μg DNA/75 cm2 GPR119 plasmid or 40 μg DNA/
sine A2A receptors (PDBID: 3QAK) to model the transmem-
brane helices TM-I to TM-VIII, including ICL1-3, ECL-1 and
ECL-2b; (ii) the chemokine receptor CXCR4 (PDBID: 3ODU)
to model ECL-2a; and (iii) dopamine D3 receptor (PDBID:
3PBL) to model ECL-3. The second model was based on: (i)
A2A receptor template to model TM-I-III and TM-V-VIII,
including ICL1-3, ECL-1 and ECL-2b; (ii) CXCR4 template to
model TM-IV including ECL-2a; and (iii) dopamine D3 recep-
tor to model ECL-3. The resulting models were optimized in
1000 steps of full-atom structure relaxation using Rosetta
version 3.2.1 and the membrane force field (Barth et al., 2007;
Leaver-Fay et al., 2011). Through the relaxation protocol, a
disulphide bridge between CysIII:01 and Cys155 (ECL-2) was
specified as a structural constraint. The top 300 (30%) best
scored models (Rosetta energy) were clustered based on the
root mean square deviation (RMSD) between main chain
atoms of residue positions involved in direct ligand interac-
tions in the various template structures using Rosetta cluster
routine and a 1.0 Å cluster threshold. A total of 43 non-
redundant energetically feasible GPR119 models (correspond-
ing to the lowest energy structures from each cluster) were
used as a receptor ensemble for docking.
75 cm2 empty plasmid.
Cell surface expression measurements (ELISA)
One day after transfection, cells were seeded in 96-well plates.
The next day, cells were washed with Tris-buffered saline
(TBS), fixed in 3.7% formaldehyde for 30 min, and washed
and incubated with blocking buffer (2% BSA, 0.5 mM CaCl2
in TBS) for 1 h. Next, cells were incubated with a FLAG-M1
antibody (2 μg·mL−1) from mice for 2 h, washed, and incu-
bated with a goat-anti-mouse antibody conjugated to HRP at
4°C overnight.
Then cells were washed and incubated with 150 μL tetram-
ethylbenzidine for 5 min before the reaction was stopped with
0.2 M H2SO4. Absorbance at 450 nm was quantified using a
multilabel counter (Wallac Victor2; Perkin Elmer, Broendby,
Denmark). Determinations were made in quadruplicate.
cAMP accumulation assay
One day after transfection, cells were seeded in 12-well plates,
2 × 105 cells per well in 0.5 mL medium supplemented with
2 μL·mL−1 adenine-[2,8-3H] (PerkinElmer). The next day, cells
were washed with HEPES-buffered saline and incubated with
1 mM IBMX in HEPES-buffered saline for 30 min at 37°C.
Ligands were added, and cells were incubated for another
30 min at 37°C. Medium was removed, and cells were lysed
with 5% trichloroacetic acid containing 0.1 mM ATP and
0.1 mM cAMP. The lysate was transferred to Dowex columns
(50WX4; Sigma-Aldrich, Broendby, Denmark), washed with
2 mL H2O and eluted into Alumina columns (A9003; Sigma)
with 10 mL H2O, from where cAMP was eluted with 6 mL
0.1 M imidazole. Then 15 mL of scintillation liquid (Gold
Star; Meridian Biotechnology, Chessington, UK) was added to
the eluate and radioactivity in the sample was measured on a
scintillation counter. Determinations were made in duplicate.
Full flexible receptor-ligand docking of
AR231453 into the GPR119-binding pocket
An ensemble of AR231453 containing nine conformations
within 3 kcal·mol−1 of the global minimum was generated
using the Merck Molecular Force Field as implemented in the
ICM software package version 3.7-2 with a generalized born
implicit solvation model. The conformational ensemble of
AR231453 was docked 1000 times to each of the 43 possible
binding pocket models using RosettaLigand with full receptor
and ligand flexibility (Meiler and Baker, 2006; Davis and
Baker, 2009; Fleishman et al., 2011; Lemmon and Meiler,
2012), producing a total of 43.000 receptor-ligand complexes.
The top 10% of the best scored complexes with respect to
ligand interaction energy were selected for further analysis,
which previously have been demonstrated to enrich native-
like ligand-binding conformations in a recent modelling and
docking assessment study on a large set of distinct 7TM
receptor X-ray structures (Nguyen et al., 2013). The RMSD
between distinct ligand-binding modes was calculated for all
complexes using an in-house script, and clustered with a
hierarchical agglomerative clustering algorithm as imple-
mented in BCL::Cluster (Alexander et al., 2011) using a clus-
tering threshold of 2.5 Å. Low energy complexes from the 10
largest clusters were visually inspected and analysed in the
context of the mutation analysis.
Data analysis
Data were analysed using the Prism 5.0 software (GraphPad
Software, San Diego, CA, USA). Surface expression of the
mutant receptors was normalized to wild-type receptor
surface expression (100%) and the signal from an empty
vector (0%) tested in parallel. cAMP counts were normalized
to the maximum AR231453-induced activation of the wild-
type receptor tested in parallel (100%) and the mean value of
cAMP accumulation from cells transfected with an empty
vector (0%). EC50 values were determined by nonlinear regres-
sion. The statistical significance of the effects of the muta-
tions on surface expression, basal activity, AR231453/OEA-
induced activity and potency (logEC50) was assessed using
ordinary one-way ANOVA with Dunnett’s post hoc test with
correction for multiple comparisons.
Materials
AR231453 was synthesized as previously described (Semple
et al., 2008). The synthesis of AR436352 is described in Sup-
porting Information Appendix S1. TM43718 was identified
by virtual screening of worldwide vendor libraries of small
molecule compounds based on ligand-based pharmacophore
multiplet searches around the two lead structures AR246881
(Arena Pharmaceuticals, San Diego, CA, USA) and PSN632408
(Prosidion, Oxford, UK) using the UNITY modelling packages
(Tripos, St. Louis, MO, USA). TM43718 was purchased as
E897-0145 from ChemDiv (San Diego, CA, USA) and OEA
from Sigma-Aldrich.
GPR119 receptor modelling
A multiple alignment between the sequence of the human
GPR119 receptor (accession code: Q8TDV5) and the sequence
of 14 distinct template structures was generated using ICM
version 3.7-2 (Molsoft LLC, San Diego, CA, USA). Two com-
parative GPR119 receptor models were generated based on a
hybrid multiple template approach (Mobarec et al., 2009;
Worth et al., 2011). The first model was based on: (i) adeno-
British Journal of Pharmacology (2014) 171 5774–5789 5777

M S Engelstoft et al.
BJP
substitutions as well as steric hindrance mutagenesis were
performed. The residues selected for the mutational analysis
were located in the main ligand-binding pocket distributed
on TM-II, TM-III, TM-V, TM-VI and TM-VII (Figure 1). In
addition, residues in ECL-2b were mutated, as this loop has
been shown to be a generally important structural element
involved in ligand binding at the entrance to the main
ligand-binding pocket (Nygaard et al., 2009). The effect of the
mutations on the constitutive and ligand-induced receptor
activation was examined by cAMP accumulation assays in
transfected COS7 cells. In the following, we first describe the
effects of the mutations on the ligand-independent, consti-
tutive activity of GPR119 and then the effects upon agonist-
induced receptor activation.
Results
GPR119 is constitutively active
Transfection experiments with increasing doses of expression
plasmid demonstrated that GPR119 signals with a high
degree of constitutive activity through the Gαs pathway. That
is, increasing cell surface receptor expression as determined
by ELISA in the absence of agonist was associated with increas-
ing stimulation of cAMP accumulation in transfected COS7
cells (Figure 2A and B). The degree of constitutive activity for
GPR119 was 37 0.8% of the Emax obtained with the proto-
type small molecule synthetic agonist AR231453 (Figure 2B
and Table 1). The relative constitutive activity of GPR119
determined as percentage of Emax was quite independent of
receptor expression (Figure 2B).
During a ligand-based drug discovery process for GPR119
agonists, it was discovered that one of the compounds –
TM43718 (Figure 3A) – surprisingly instead functioned as a
full-inverse agonist for GPR119, as it inhibited the spontane-
ECL-2b is broadly important for the
constitutive activity of GPR1119
Surprisingly, Ala substitutions of each of the residues in
ECL-2b strongly reduced the constitutive activity of
GPR119, that is, from nearly 40% to 5–7.5% of Emax, except
for the AlaC+4 (AlaC+4 was mutated to Gly) and the
HisV:01 mutant at the end of ECL-2b in which the consti-
tutive activity was reduced only down to 18 and 20% of
Emax respectively (Figure 4 and Table 1). The surface expres-
sion of the ECL-2b-mutated receptors was between 56 and
87% of wild type, indicating that the decrease in constitu-
tive activity was not resulting (solely) from a decrease in
surface expression.
ous ligand-independent signalling in
a dose-dependent
manner (EC50 = 1.6 μM), that is, down to the level observed in
cells transfected with empty vector (Figure 3B and C).
TM43718 also functioned as an antagonist of the AR231453-
induced Gαs signalling (Figure 3C).
In the original screening hit in the drug discovery process
for AR231453, it was described as being an inverse agonist,
although it was not further characterized as such at that time
(Semple et al., 2008). However, as shown in Figure 3D and E,
a distantly related second-generation tri-substituted pyrimi-
dine, AR436352, functioned as a 20 nM potent partial inverse
agonist. Like TM43718, AR436352 also acted as an antagonist
against AR231453 (Figure 3E). AR436352 also functioned as
an antagonist against OEA, but in contrast to TM43718,
The strong decrease in constitutive activity resulting from
Ala substitution at multiple positions suggests that ECL-2b
functions as a ‘tethered agonist’ in GPR119, and is important
for stabilization of an active conformation of the receptor.
AR436352 seemed to be
(Figure 3G).
Thus, both ‘gene-dosing’ experiments and experiments
with two different inverse agonists indicate that GPR119
signals with high constitutive activity through Gαs.
a non-competitive antagonist
Key residues in the main ligand binding
pocket are also important for constitutive
activity of GPR119
The mutational analysis revealed three positions in the main
ligand binding pocket where Ala substitution completely
abolished the constitutive activity of GPR119. These included
not only the highly conserved TrpVI:13 located in the middle
of the receptor, which previously has been implicated as a
‘micro-switch’ in the general 7TM activation mechanism
Mutational analysis of structural basis for
the constitutive activity of GPR119
To examine the structural basis for the constitutive activity
and to map ligand-receptor interactions in GPR119, alanine
0.15
A ELISA
B cAMP
4000
3000
2000
1000
0
AR231453 10-7
M
0.10
0.05
0.00
Basal activity
5
10
20
40
0
10
20
30
40
DNA (µg)
DNA (µg)
Figure 2
Constitutive activity of GPR119. (A) Surface expression of FLAG-tagged GPR119 resulting from transfections with increasing concentrations of a
GPR119 construct and measured with ELISA. (B) Basal level and AR231453-induced cAMP accumulation from cells transfected with increasing
concentrations of the GPR119 construct – 20 μg of DNA was chosen as the optimal amount for further studies.
5778 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
Table 1
Surface expression and basal activity of wild-type GPR119 and receptor mutants along with efficacy and potency of AR231453 and the potency
fold change between wild type and mutants (Fmut
)
Surface expression
Basal activity
Emax
AR231453 EC50
Mutant
(%)
SEM
(%)
SEM
(%)
SEM
Log
SEM
nM
Fmut
n
Wild type
100
2.1
37.0
0.7
96
0.9
−8.73
0.03
1.9
26
Binding pocket
GlnII:24Ala (65)
ArgIII:04Ala (81)
MetIII:05Ala (82)
ValIII:08Ala (85)
ThrIII:09Ala (86)
ThrIII:09Val (86)
AlaIII:12Val (89)
HisV:01Ala (162)
ValV:05Ala (166)
SerV:09Ala (170)
SerV:09Val (170)
TrpVI:13Ala (238)
PheVI:16Ala (241)
GlyVI:20Ala (245)
GlyVI:20Ser (245)
GlyVI:20Val (245)
GlnVI:23Ala (248)
GluVII:02Ala (261)
ArgVII:03Ala (262)
TrpVII:06Ala (265)
GlyVII:09Ala (268)
GlyVII:09Val (268)
GlyVII:09Phe (268)
ECL-2b
122
9.4
2.4
31.0
1.9*
5.3
1.5
1.4
6.0
2.1
7.2
6.0
1.9
6.6
7.5
3.5
–
99
41*
24*
88
7.1
2.5
1.9
7.2
4.5
6.2
32.0
2.4
8.5
7.4
5.0
–
−8.9
0.24
0.15
0.19
0.28
0.36
0.63
0.81
0.08
0.61
0.38
0.16
–
1.2
11.6
4.3
1.6
15.8
1.3
103.0
1.8
9.3
2.3
9.7
–
0.6
6.1
2.3
0.8
8.3
0.7
54.0
0.9
4.9
1.2
5.1
–
4
4
4
4
4
4
4
3
3
4
4
4
3
5
4
5
3
4
4
5
4
4
3
51*
−7.94*
−8.37
−8.80
−7.80*
−8.90
−6.99*
−8.75
−8.03*
−8.64
−8.02*
–
78
5.8
8.3*
115
9.2
29.0
13.0*
32.0
27.0
20.0*
35.0
57.0*
12.0*
–
55*
12.0
14.0
43.0
4.3
39*
61*
90
71
231*
58*
73
53*
9.1
70*
108
77
157*
164*
40*
17.0
30.0
2.6
–
72
4.7
11.0*
48.0*
57.0*
30.0
31.0
13.0*
−0.1*
34.0
19.0*
20.0*
20.0*
1.8
3.1
3.4
1.9
7.9
1.3
0.7
2.7
2.5
2.8
1.6
27*
106
111
61*
87
4.4
4.1
4.1
2.4
11.0
3.3
1.2
–
−7.47*
−8.78
−8.75
−8.82
−8.07
−7.75*
−7.94*
–
0.50
0.16
0.15
0.16
0.41
0.16
0.13
–
34.0
1.7
1.8
1.5
8.6
17.7
11.6
–
18.0
0.9
0.9
0.8
4.5
9.3
6.1
–
136*
116
71
3.7
5.6
4.5
111
54*
18.0
5.1
54*
18*
–
50*
4.1
40*
2.8
148*
206*
155*
6.1
78
3.8
–
−8.46
–
0.13
–
3.4
–
1.8
–
7.0
–
16.0
57*
8.4
−7.10*
0.38
80.0
42.0
SerC+1Ala (156)
PheC+2Ala (157)
PheC+3Ala (158)
AlaC+4Gly (159)
ValC+5Ala (160)
PheC+6Ala (161)
71
69
73
87
66
56*
3.5
4.4
3.6
3.8
3.4
2.3
6.9*
6.4*
1.3
0.5
0.6
3.3
0.9
1.0
48*
23*
24*
75
2.2
1.7
1.2
4.6
1.4
1.2
−8.44
−6.83*
−7.35*
−8.60
−8.13*
−8.65
0.12
0.18
0.13
0.17
0.11
0.15
3.7
147.0
45.0
2.5
1.9
77.0
24.0
1.3
3
5
5
3
3
3
3.9*
18.0*
5.7*
34*
21*
7.4
3.9
5.6*
2.2
1.2
Statistically significant changes from wild type are marked with an asterisk (*). Residue positions are given in parentheses after the
Schwartz/Baldwin name.
(Nygaard et al., 2009; Holst et al., 2010), but also rather sur-
prisingly two Arg residues located at the extracellular ends of
TM-III and TM-VII – ArgIII:04 and ArgVII:03 respectively
(Figure 5 and Table 1). Ala substitution of the third charged
residue of the main ligand-binding pocket, GluVII:02, located
at the extracellular end of TM-VII in close spatial proximity to
the two Arg residues, also decreased the constitutive activity
albeit only down to 13% (Table 1). Similarly, Ala substitution
of the neighbouring residue to ArgIII:04, MetIII:05, reduced
the constitutive activity to 8.3%. Ala substitution of PheVI:16
and ThrIII:09, located deeper in the binding pocket, reduced
the constitutive activity of the receptor to 11 and 13% of Emax
respectively. In the case of SerV:09, Ala substitution in fact
increased the constitutive signalling of the receptor whereas
substitution with Val decreased the activity to 12% (Figure 5
and Table 1).
In conclusion, a cluster of residues located at the upper
part of the main ligand-binding pocket in between TM-III,
TM-VI and TM-VII are highly important for maintaining the
constitutive activity of GPR119.
British Journal of Pharmacology (2014) 171 5774–5789 5779

M S Engelstoft et al.
BJP
A
B
C
TM43718
AR231453 (n = 21)
TM43718 (n = 6)
No antagonist
TM43718 10–5.5
100
80
60
40
20
0
100
M
TM43718 10–5
M
EC50: 1.9 nM
80
60
40
20
0
EC50: 1.6 mM
–11 –10 –9
–8
–7
–6
–5
–11
–10
–9
–8
–7
–6
0
0
log[ligand] (M)
log[AR231453] (M)
D
CF3
N
E
F
AR436352
No antagonist
AR231453 (n = 21)
AR436352 (n = 3)
100
80
60
40
20
0
100
80
60
40
20
0
AR52 10–8
AR52 10–7
M
M
M
N
N
AR52 10–6 .5
O
AR52 10–6
M
Me
N
N
O
F
EC50: 20 nM
S
O
O
H3C
–11 –10 –9
–8
–7
–6
–5
–10
–9
–8
–7
–6
–5
0
0
log[ligand] (M)
log[AR231453] (M)
G
No antagonist
120
100
80
60
40
20
0
–7
AR52 10
AR52 10
M
M
–6
–9
–8
–7
–6
–5
–4
0
log[OEA] (M)
Figure 3
Constitutive activity of GPR119 – effect of combined small molecule antagonists and inverse agonists. (A, D) Structure of TM43718 and AR436352.
(B, E) Normalized concentration–response curves for TM43718 and AR436352 measured by cAMP accumulation in GPR119-transfected cells and
for AR231453. (C, F, G) Normalized concentration–response curves for AR231453 or OEA measured by cAMP accumulation in GPR119-transfected
cells treated with TM43718 or AR436352 before the AR231453 or OEA treatment.
Mutational mapping of the ligand
vation differed at key positions from the mutational map of
residues being important for the constitutive activity of
GPR119 (Figure 6, Table 1). Three mutations in the main
ligand-binding pocket completely abolished AR231453-
induced receptor activation: TrpVI:13Ala, TrpVII:06Ala and
interactions in the main ligand-binding
pocket of GPR119
Although there was a large degree of overlap, the mutational
map for residues involved in agonist-induced receptor acti-
5780 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
EC50: 3.7 nM
ELISA: 71%
EC50: 147 nM
ELISA: 69%
(n = 5)
EC50: 65 nM
ELISA: 73%
SerC+1Ala
PheC+2Ala
A
PheC+3Ala
5–10%
10–15%
15– %
Y
K
T
Q
G
100 (n = 3)
100
80
60
40
20
0
100 (n = 5)
Q
F F
Q
80
60
40
20
0
80
60
40
20
0
S
A
C
3
F 4
2
1
V
F
M
5
HV:01
P
6
I
P
H
G
L
TMIV
TMV
0
–11 –10 –9
EC50: 7.4 nM
–8
–7
–6
–5
0
–11 –10 –9
–8
–7
–6
–5
0
–11 –10 –9
–8
–7
–6
–5
EC50: 2.2 nM
ELISA: 56%
(n = 3)
EC50: 2.5 nM
ELISA: 87%
(n = 3)
ValC+5Ala
PheC+6Ala
EC50: 1.8 nM
ELISA: 58%
(n = 3)
AlaC+4Gly
HisV:01Ala
ELISA: 66%
(n = 3)
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
0
–11 –10 –9
–8
–7
–6
–5
0
–11 –10 –9
–8
–7
–6
–5
0
–11 –10 –9
–8
–7
–6
–5
0
–11 –10 –9
–8
–7
–6
–5
log[AR231453] (M)
Figure 4
Structural basis for the constitutive activity – ECL-2b. The graphs show normalized concentration–response curves for AR231453 on GPR119
receptors mutated in ECL-2b (black). On each graph, the wild-type curve is shown in stippled grey for comparison. The EC50 values are indicated
in grey stippled lines and can also be found in the upper left corner of each graph together with the surface expression level of the mutant and
number of repetitions. The figure on the upper left shows the ECL-2 of GPR119, colour coded for the level of constitutive activity obtained with
the receptor mutants. The constitutive activity was calculated by setting the Emax for AR231453-induced activation of the wild-type receptor to 100
and the mean value from cells transfected with an empty vector to 0.
GlyVII:09Val. As described above, TrpVI:13 is involved in the
general 7TM activation mechanism, but could also be inter-
acting directly with the ligand from its position in the middle
Two phenylalanines in ECL-2b of GPR119
are important for ligand potency
In contrast to the constitutive signalling of GPR119, where
of the receptor. The indole side chain of TrpVII:06 is located
nearly all substitutions in ECL-2b had major effects, only Ala
in TM-VII two helical turns above TrpVI:13 and faces into the
substitutions of the two neighbouring phenylalanines,
main ligand-binding pocket, thus TrpVII:06 likely interacts
PheC+2 and PheC+3, seriously affected the agonist-induced
directly with the AR231453 agonist. In the case of GlyVII:09,
signalling, that is, the potency of AR231453 was impaired 77-
introduction of Ala or Phe resulted in an 18- and 50-fold
and 34-fold, respectively, and OEA had no effect in the
decrease in AR231453 potency, respectively, while the
PheC+2 to Ala mutant and was impaired 22-fold by the
PheC+3 mutant. Ala substitutions of SerC+1, AlaC+4, PheC+6
and HisV:01 had no effect on agonist potency, whereas Ala
substitution of ValC+5 decreased the AR231453 potency 3.9-
β-branched side chain of Val completely abolished
AR231453-induced receptor activation.
In addition to TrpVII:06, Ala substitution of three
charged residues, ArgIII:04, GluVII:02 and ArgVII:03,
fold (Figure 6, Table 1).
located at the interface between TM-VII and TM-III and the
It should be noted, however, that nearly all of the substi-
interface between TM-VII and TM-VI each decreased the
tutions in ECL-2b, which decreased the constitutive activity,
potency of AR231453 6- to 10-fold (Figure 6, Table 1).
also decreased the efficacy of AR231453, that is, by around
Similarly, mutations of a group of residues located fairly
40% (Figure 4), indicating that ECL-2b is generally important
deep in the main ligand-binding pocket at the interface
for the stabilization of the active conformation of GPR119,
between TM-III and TM-V also decreased AR231453 potency
although many of the mutations did not affect agonist
4- to 10-fold, that is, ThrIII:09, ValV:05 and SerV:09,
potency.
and in the case of AlaIII:12 to Val even 54-fold (Figure 6,
Table 1).
Molecular models of predicted binding modes
With regard to OEA, the mutational map closely resem-
bled that of AR231453 although subtle differences were
observed (Figure 6, Table 2). The most noticeable was that the
AlaIII:12 to Val and the GlyVII:09 to Val mutations had a
greater effect on AR231453-induced activation than on OEA-
induced activation, whereas the PheVI:16 to Ala mutation,
which decreased the potency of AR231453 18-fold, resulted
in a complete lack of activation by OEA.
of AR231453 in GPR119
Initially, we compared the molecular models built over single
GPCR X-ray structures with two models, Combi1 and
Combi2, based on a composed multiple template approach in
which structural fragments from three different GPCR X-ray
structures were joined: A2A, CXC4 and dopamine D3 recep-
tors. For example, the structure of ECL-3 from the D3 receptor
British Journal of Pharmacology (2014) 171 5774–5789 5781

M S Engelstoft et al.
BJP
EC50: 11.6 nM
ELISA: 51%
(n = 4)
EC50: 103 nM
ELISA: 231%
(n = 4)
AlaIII:12Val
ArgIII:04Ala
EC50: 4.3 nM
100 ELISA: 78%
MetIII:05Ala
ThrIII:09Ala/Val
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
(n = 4)
80
60
40
20
0
TIII:09V
TIII:09A
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
EC50: 9.8 nM
ELISA: 50%
(n = 4)
ArgVII:03Ala
120
100
80
60
40
20
0
SerV:09Ala/Val
100
80
60
40
20
0
L
G
F
L
I
M
G
L
S
F
L
D
Y
V
SV:09A
A
SV:09V
L
T
IV.
L
A
A
S
V
I
W
A
T
L
II.
L
I
S
G
S
A
P
T
L
P
L
F
S
P
L
III.
Q
V
M
G
I
F
V
D
L
T
A
T
I.
L
S
I
S
T
V
G
0
–11 –10
–9
–8
–7
–6
–11 –10
–9
–8
–7
–6
A
0
H
R
W
C
S
A
F
V
A
P
G
V
R
V.
F
V
S
L
N
H
V
S
EC50: -
ELISA: 40%
(n = 4)
EC50: -
ELISA: 40%
(n = 5)
TrpVII:06Ala
TrpVI:13Ala
L
C
L
E
A
100
80
60
40
20
0
100
80
60
40
20
0
L
L
M
I
G
W
VII.
S
F
V
G
L
P
F
I
L T
G
L
F
N
Y
A
I
T
Q
L
0–5%
L
L
L
VI.
N
S
5–10%
T
G
S
10–15%
15– %
I
P
L
V
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
EC50: 34 nM
ELISA: 72%
(n = 3)
EC50: 18 nM
ELISA: 54%
(n = 4)
120
100
80
60
40
20
0
PheVI:16Ala
GluVII:02Ala
GlyVII:09Ala/Phe/Val
GlyVI:20Ala/Val
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
GVII:09A
GVII:09F
GVI:20A
GVI:20V
GVII:09V
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
0
–11 –10
–9
–8
–7
–6
log[AR231453] (M)
Figure 5
Structural basis for the constitutive activity – main ligand-binding pocket. The graphs show normalized concentration–response curves for
AR231453 on selected receptors with mutations in the main ligand-binding pocket (black). On each graph, the wild-type curve is shown in
stippled grey for comparison. The EC50 values are indicated in grey stippled lines and can also be found in the upper left corner of each graph
together with the surface expression level of the mutant and number of repetitions, except for graphs showing more than one mutant receptor
– see Table 1. The helical wheel in the middle shows the location of the mutated residues, and a colour code denotes the level of constitutive
activity obtained with the GPR119 receptor mutants. The colours are based on alanine substitutions. The constitutive activity was calculated by
setting the Emax for AR231453-induced activation of the wild-type receptor to 100 and the mean value from cells transfected with an empty vector
to 0.
was selected because it has an internal disulphide bridge
similar to the one in GPR119 (Figure 1). The models were
optimized using full-atom structure relaxation with Rosetta,
and the 30% best scoring models were subjected to cluster
analysis and 43 non-redundant energetically feasible models
corresponding to the lowest energy structures from each
cluster were used as a receptor ensemble for ligand docking
(see Methods for details). The energy of the models based on
multiple templates was approximately 10 Rosetta energy
units (REU) lower compared with the best models developed
from single-template structures (Figure 7A). The Combi2
model was used for further analysis.
A
conformational ensemble of low-energy structures
of AR231453 in Combi2 was generated and docked 1000
times to each of the 43 selected binding pocket ensembles
using RosettaLigand with full receptor and ligand flexibility.
To elucidate possible binding modes of AR231453 in the
GPR119 receptor, we analysed the top 10% best scoring
complexes (4300) with respect to ligand interaction
energy.
5782 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
A
AR231453
O
L
G
F
CH
L
3
M
O
S
G
L
Val
S
2-fluoro-4-
methanesulfonyl-
S
F
D
Y
V
A
L
L
T
IV.
L
A
A
phenyl
V
W
A
T
L
II.
F
L
S
I
G
S
A
F
I
I
P
T
L
P
L
HN
S
P
L
Q
III.
5-nitro-pyrimidine
V
G
N
I
F
T
V
D
L
A T
I
L
M
S
O
F
S
V
T
V
L
G
A
H
R
N
N
C
S
A
V
S
V
F
A
A
P
V
G
R
O
C
V.
W
F
L
N
I.
H
N
S
V
F
S
L
C
L
A
E
L
G
M
W G
I
S
VII.
F
V
G
L
F
P
I
F
Isopropyl-oxadiazole
T
L
L
F
N
Y
A
I
T
Q
S
L
O
L
Potency shiꢀ
N
L
VI.
N
L
Val
L
N
0-4
T
4-10
10-20
>20
G
S
Val,Phe
CH
P
3
I
L
V
H
C
3
L
G
F
B
OEA
M
G
Val
S
L
S
F
D
Y
V
A
A
L
L
W
T
IV.
L
HO
A
V
A
T
L
II.
L
I
I
S
G
S
A
F
I
T
V
P
NH
L
L
P
S
P
L
III.
Q
G
G
I
F
T
V
D
L
O
A
T
L
M
S
I
F
S
V
T
V
L
A
H
R
C
S
A
V
F
A
A
P
V
G
R
C
V.
W
F
V
L
N
I.
H
S
V
F
S
L
S
C
L
E
A
L
M
I
S
G
VII.
W
F
V
G
L
F
P
I
F
G
T
L
L
F
N
Y
A
I
Q
L
L
Potency shiꢀ
L
VI.
N
L
S
Val
0-4
T
T
4-10
10-20
>20
G
S
Val
I
P
L V
Figure 6
Structural basis for agonist-induced activation. The structures of AR231453 or OEA are shown to the left. In the helical wheel shown together with
ECL-2b, the mutated residues are coloured corresponding to their effect on the potency of (A) AR231453 or (B) OEA in the activation of GPR119.
The colours are based on alanine substitutions, except for AlaIII:12, SerV:09 and GlyVII:09, for which the valine substitution determines the colour.
Two overall ligand-binding possibilities were identified in
which AR231453 binds in the main ligand-binding pocket in
an extended conformation perpendicular to the plane of the
membrane between TM-III, -V, -VI and -VII, where the sulfo-
nyl moiety of the ligand is either pointing to the ECL region,
which we call SO2-up, or towards the bottom of the main
ligand-binding pocket, SO2-down. The SO2-up binding pose
was observed in approximately 80% of the top 10% best
scored models energy-wise from the docking simulation
(Figure 7B). The analysis of the 10 most populated binding
modes identified by clustering based on RMSD between dis-
tinct ligand-binding modes showed that six clusters repre-
sented the SO2-up pose and four represented the SO2-down
pose. Model scores and ligand interaction energies associated
with models in the most populated clusters were surprisingly
similar, although a slightly better score (−36.6 REU) was
observed for the best SO2-up docking pose (Figure 7D.2) com-
pared with the best (−35.3 REU) SO2-down (Figure 7D.3). The
largest, most populated cluster was with AR231453 in a
SO2-up docking pose of which the best scored model is shown
in Figure 7D.1.
The major AR231453 binding
mode – ‘SO2-up’
Although the detailed receptor-ligand interaction varied
among the best scoring clusters and models, the overall
SO2-up binding mode represented by the largest clusters
was similar, as illustrated in the ‘lig-plot’ (Figure 7C) and
shown in the two models in Figure 7D.1–2. In this binding
pose, the isopropyl-oxadiazole moiety is located deep in the
main binding pocket involved in hydrophobic/aromatic
interactions with AlaIII:12 and the highly conserved
TrpVI:13
– and is making hydrogen bond interactions
with ThrIII:09 and SerV:09 in most models. In contrast,
analysis of the top scored models suggests that the effect of
the GlyVII:09 mutations is most likely related to indirect
effects.
British Journal of Pharmacology (2014) 171 5774–5789 5783

M S Engelstoft et al.
BJP
Table 2
Efficacy and potency of OEA on wild-type GPR119 and receptor mutants as well as the potency fold change between wild type and mutants (Fmut
)
Emax
OEA EC50
SEM
Mutant
(%)
SEM
Log
nM
290
Fmut
n
Wild type
97
2.0
−6.54
0.13
7
Binding pocket
GlnII:24Ala (65)
ArgIII:04Ala (81)
MetIII:05Ala (82)
ValIII:08Ala (85)
ThrIII:09Ala (86)
ThrIII:09Val (86)
AlaIII:12Val (89)
ValV:05Ala (166)
SerV:09Ala (170)
SerV:09Val (170)
TrpVI:13Ala (238)
PheVI:16Ala (241)
GlyVI:20Val (245)
GlnVI:23Ala (248)
GluVII:02Ala (261)
ArgVII:03Ala (262)
TrpVII:06Ala (265)
GlyVII:09Val (268)
ECL-2b
101
34*
26*
97
5.6
2.4
1.5
3.4
2.1
8.7
19.0
13.0
16.0
17.0
–
−6.23
−5.83
−6.01
−6.18
−6.11
−6.59
−5.80
−6.07
−6.73
−5.76
–
0.26
0.12
0.08
0.12
0.20
0.77
0.42
0.88
0.65
0.70
–
596
1470
975
663
784
257
1600
861
185
1760
–
3.5
8.6
5.7
3.9
2.0
0.67
4.1
2.2
0.5
4.6
3
3
3
3
4
4
4
3
4
4
3
3
3
3
3
3
4
3
37*
63*
116
69
120
47*
–
–
–
–
–
–
–
–
80
2.6
8.4
9.2
2.7
–
−6.58
−5.90
−5.69
−5.54
–
0.28
0.39
0.28
0.17
–
262
1270
2040
2860
–
1.5
3.3
73
71
12.0
17.0
–
20*
–
43*
2.5
−5.79
0.30
1630
9.6
PheC+2Ala (157)
PheC+3Ala (158)
–
–
–
–
–
–
3
4
19*
2.9
–5.12
0.51
7816
21.0
Statistically significant changes from wild type are marked with an asterisk (*). Residue positions are given in parentheses after the
Schwartz/Baldwin name.
The 5-nitro-pyrimidine scaffold of AR231453 is located
centrally in the ligand-binding pocket and predicted to be
involved in substantial aromatic/hydrophobic interactions
with the key residues PheVI:16, TrpVII:06 and PheC+2 (ECL-
2b) across the clusters (Figure 7D.1–3), supporting the impor-
tance of these residues in the mutational analysis. In the
models associated with the cluster including the best scored
SO2-up binding pose (Figure 7D.2), the nitro group is
involved in polar interactions with GlnVI:23, while either of
the pyridyl nitrogens (in the pyrimidine scaffold) make
hydrogen bond interactions with the amine group of the
indole side chain of TrpVII:06. Accordingly, GluVII:02
(located opposite to GlnVI:23) is involved in hydrogen bond
interactions with the adjacent aniline linker. In models asso-
ciated with the most populated binding conformation
(Figure 7D.1), AR231453 is basically rotated 180° around its
‘vertical’ isopropyl-sulphonyl axis. In these models, GlnVI:23
is involved in the hydrogen bond interaction with GluVII:02
and/or either of the pyridyl nitrogens. Both of these SO2-up
possibilities are supported by the effect of mutations of key
residues, PheVI:16, TrpVII:06 and PheC+2, and by the
moderate effect of the GlnVI:23 and GluVII:02 mutations.
Importantly, no obvious polar contact seems to be formed
between these residues and AR231453 in the models associ-
ated with the best scored reversed SO2-down binding mode
(Figure 7D.3). In these SO2-down models, GlnVI:23 and
GluVII:02 were often found to interact with each other
instead of with AR231453.
With regard to ECL-2, the terminal 2-fluoro-4-
methanesulfonyl-phenyl moiety of AR231453 is in the
SO2-up pose surrounded by PheC+2 and PheC+3 as well as
hydrophobic residues at the extracellular end of TM-VII and
ECL-3, indicating that the pronounced effects of Ala substi-
tutions of PheC+2 and PheC+3 in ECL-2b are due to the
disruption of direct interactions with the agonist. In contrast,
the modest effects of mutations of ValC+5 ArgIII:04 and
ArgVII:03 on the potency of AR231453 are likely to be indi-
rect as these residues mainly make interactions with other
residues in the receptor.
The structural basis for the mutations which did not affect
the potency of AR231453 (e.g. GlnII:24, MetIII:05, ValIII:08,
HisV:01 and GlyVI:20) could in most cases be explained by
their limited interaction or remote positions to AR231453
(Figure 7C and D.1–3).
5784 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
Figure 7
Molecular model of proposed binding mode for AR231453 in GPR119. (A) Rosetta conformational energies of relaxed GPR119 homology models
based on the single-template structures of adenosine A2A receptors (PDBID:3EML), CXCR4 (PDBID:3ODU), dopamine D3 receptors (PDBID:3PBL),
histamine H1 receptors (PDBID:3RZE) and muscarinic M2 receptors (PDBID:3UON), and two GPR119 models (Combi1 and Combi2) based on
hybrid template structures assembled by fragments from the adenosine A2A, CXCR4 and the dopamine D3 receptors. (B) Density of computed
distances between the isopropyl moiety of the ligand and a point in the extracellular loop region and computed distances between the sulfonyl
moiety and a point in the bottom of the main binding site for the top 10% complexes with the best scored docking pose. (C) Ligplot illustrating
predicted receptor-ligand interactions in good agreement with the mutational data. (D) Extracellular and membrane view for (i) the most
populated AR231453 binding conformation based on clustering; (ii) the best scored SO2-up; and (iii) the best scored SO2-down binding mode
of AR231453 among the 10 most populated clusters. AR231453 is shown as sphere with green carbons. Mutated residues are shown in sticks and
colour coded as described in previous figures.
In conclusion, although both SO2-down and SO2-up poses
are energetically allowed and found in surprisingly compara-
Discussion and conclusions
ble numbers of clusters, the best scoring models and the most
populated clusters of models for AR231453 in its ‘vertical’
binding pocket in GPR119 correspond to SO2-up poses, where
it, for example, makes strong polar interactions with key
residues in the middle of the pocket.
In the present study, the structural basis for both the ligand-
induced and constitutive signalling of the enteroendocrine
lipid sensor GPR119 was characterized by mutational analysis
and molecular modelling. Although there are key differences,
a surprisingly large degree of overlap was found between
British Journal of Pharmacology (2014) 171 5774–5789 5785

M S Engelstoft et al.
BJP
residues located at the extracellular ends of TM-III, TM-V,
TM-VI and TM-VII, and in ECL-2b being important for both
ligand-independent and ligand-induced signalling. For the
prototype synthetic GPR119 ligand AR231453, a picture is
emerging, where it activates the receptor by binding in a tight
vertical pocket, most likely with its isopropyl-oxadiazole
moiety penetrating deep into the receptor interacting with
key micro-switch residues, and with the 2-fluoro-4-
methanesulfonyl-phenyl moiety at the opposite end of the
molecule pointing towards the extracellular space interacting
with residues in ECL-2b.
Structural basis for agonist activation
of GPR119
The mutational mapping identified residues located all the
way from the bottom of the main ligand-binding pocket,
[i.e. TrpVI:13 (6.50)] to the ECL structures (i.e. PheC+2
and PheC+3 in ECL-2b) to be important for agonist-induced
activation of GPR119. Between these sites, in particular
AlaIII:12, PheVI:16, TrpVII:06 and GlyVII:09 were important
for AR231453-induced activation of GPR119. These are all
residues facing the main ligand-binding pocket, which
recently have been confirmed to be involved in ligand
binding in a large series of X-ray structures of receptor-
ligand complexes (Nygaard et al., 2009; Katritch et al., 2013).
In fact, the elongated overall shape of AR231453 and its
proposed vertical binding mode in the main ligand-binding
pocket is somewhat similar to the antagonist ZM241385 and
its binding in the A2A receptors (Jaakola et al., 2008; Nygaard
et al., 2009). Interestingly, the mutational map for the
endogenous high-potency agonist OEA and for the proto-
type synthetic agonist AR231453 was similar – including the
two Phes in ECL-2b. This is rather surprising because the
structures of the two ligands are very different and because
OEA is a highly flexible ligand (Figure 6). In relation to
agonist binding, it should be noted that activating metal ion
sites have been built between positions VI:16 (6.45) and
III:08 (3.32) in the β2-adrenoceptor and the κ-opioid receptor
(Thirstrup et al., 1996; Elling et al., 2006), that is, right in the
middle of the proposed binding site for AR231453 in
GPR119. An interesting key residue involved in agonist
binding in GPR119 is VII:06. This position is well known,
particularly in chemokine receptors, which nearly all have a
glutamic acid residue at this position serving as an anchor
point for a centrally located positively charged nitrogen of
the different types of ligands (Rosenkilde and Schwartz,
2006; Jensen et al., 2007; Thiele et al., 2012). GPR119 is
unique in having a Trp residue at position VII:06, which is
not found in any other 7TM receptors (Mirzadegan et al.,
2003).
The many high-resolution X-ray structures of different
GPCRs have revealed that ECL-2b is a very important struc-
tural element, as it not only borders the entrance to the
main ligand-binding pocket, but importantly, residues in
this loop are involved in direct ligand interactions in most
receptors that have, at present, been characterized (Nygaard
et al., 2009; Peeters et al., 2011). For GPR119, it is interesting
that key residues in ECL-2b, that is, PheC+2, PheC+3 and
ValC+5, are conserved between not only the human and
mouse homologues but even in zebrafish GPR119, despite
the fact that human and zebrafish GPR119 only share 35%
identity. Normally, residues in the extracellular domains of
7TM receptors vary the most. The surprising conservation
of these residues in ECL-2b supports the notion that this
loop has a major functional role in GPR119 either as a ‘teth-
Structural basis for GPR119
constitutive activity
Although it was initially reported that one of the earliest
compounds selective for GPR119 was an inverse agonist
(Semple et al., 2008), very little focus has since been placed
on the fact that GPR119 is highly constitutively active, at
least when expressed in transfected cells. As GPR119 is acti-
vated by a range of different fatty acid derivatives (Hansen
et al., 2012), the observed apparent constitutive activity
might in fact instead be mediated by some unknown lipid
ligand produced by the cells. However, we did observe key
differences between residues being important for constitutive
activity and residues important for OEA-induced activation.
This is most clear for the TrpVII:06 to Ala substitution, which
did not affect the high constitutive activity of the receptor,
but eliminated the activity of OEA. This suggests that GPR119
is truly constitutively active. But it is still possible that some
other cellular-derived, unidentified ligand binding slightly
different from OEA is constitutively activating the receptor.
To elucidate this, an inverse agonist without antagonistic
properties would be useful, so that inhibition of constitutive
activity could be distinguished from that induced by blocking
endogenously produced GPR119 ligands, as demonstrated in
the ghrelin system by Holst and co-workers (Holst et al., 2003;
Petersen et al., 2009).
The structural basis for the constitutive activity of
GPR119 is particularly dependent upon two arginine residues
in the TM region – ArgIII:04 and ArgVII:03 – and surprisingly
almost the entire ECL-2b. Because ECL-2b displays great aro-
maticity, it could be speculated that cationic π-interactions
between the arginine residues in the upper part of the main
ligand-binding pocket and the phenylalanines in ECL-2b
could be stabilizing the ligand-independent active receptor
conformation. However, our current models of GPR119 in
complex with agonist ligands do not support that notion.
In the ghrelin receptor, it is a particular aromatic cluster of
residues – PheVI:16, PheVII:06 and PheVII:09 – located rela-
tively deep in the main ligand-binding pocket at the interface
between TM-VI and TM-VII that are important for the con-
stitutive activity (Holst et al., 2003). Two of these residues are
also found in GPR119; however, even though the PheVI:16 to
Ala mutation in GPR119 also decreases the constitutive activ-
ity, the receptor still displays a constitutive activity of 11%
and the TrpVII:06 to Ala mutation does not affect the ligand-
independent signalling at all. Position VII:09 is a glycine in
GPR119, and introduction of a phenylalanine here results in
a decrease in constitutive activity. Thus, although being struc-
turally similar, the interface of TM-VI and VII does not serve
the same function in GPR119 as in the ghrelin receptor.
ered agonist’
– leading to the apparent high ligand-
independent constitutive activity receptor – or is involved in
binding some unknown autocrine or paracrine lipid agonist.
In the ghrelin receptor, a naturally occurring mutation in
ECL-2b has been shown to completely eliminate the consti-
tutive activity (Holst and Schwartz, 2006; Mokrosinski et al.,
2012).
5786 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL,
Spedding M et al. (2013). The Concise Guide to PHARMACOLOGY
2013/14: G protein-coupled receptors. Br J Pharmacol 170:
1459–1581.
Molecular modelling of GPR119
agonist complex
In general, molecular models are built over the X-ray struc-
ture of the receptor which is most closely related to the target
of interest. However, here we found that molecular models
with apparent lower energy can be generated when different
segments are sampled from different receptors based on local
and not overall homology, that is, using a composed template
as proposed by Worth et al. (2011). Docking of ligands in
molecular models is far from trivial, particularly when no
experimental data are available (Kufareva et al., 2011).
However, as demonstrated in a recent benchmark study
where we ‘re-docked’ ligands into known X-ray structures as
if they were molecular models using full flexible receptor-
ligand simulations, the results are much improved if experi-
mental data are available concerning ligand-receptor
interactions (Nguyen et al., 2013). Consequently we believe
that the preferred docking pose for AR4231453 presented in
the present study (Figure 7C and D.1) is probably quite close
to the real binding mode for this prototype GPR119 agonist.
In conclusion, the present study demonstrates that
GPR119 signals with high constitutive activity through Gαs
which should be taken into account in the drug discovery
process. Moreover, the mutational mapping and novel
insight into the receptor binding mode of agonists will
improve structure-based drug discovery efforts for GPR119 to
control gut hormone and insulin secretion.
Alexander N, Woetzel N, Meiler J (2011). BCL::Cluster: a method
for clustering biological molecules coupled with visualization in the
Pymol Molecular Graphics System. Computational Advances in Bio
and Medical Sciences (ICCABS), IEEE 1st International Conference.
13–18.
Barth P, Schonbrun J, Baker D (2007). Toward high-resolution
prediction and design of transmembrane helical protein structures.
Proc Natl Acad Sci U S A 104: 15682–15687.
Bonini JA, Borowsky BE, Montclair NA, Ridgewood NB, Cliffside P,
Thompson TO (2001). DNA encoding SNORF25 receptor. United
States Patent US 6,221,660 B1.
Chu ZL, Jones RM, He H, Carroll C, Gutierrez V, Lucman A et al.
(2007). A role for beta-cell-expressed G protein-coupled receptor
119 in glycemic control by enhancing glucose-dependent insulin
release. Endocrinology 148: 2601–2609.
Chu ZL, Carroll C, Alfonso J, Gutierrez V, He H, Lucman A et al.
(2008). A role for intestinal endocrine cell-expressed g
protein-coupled receptor 119 in glycemic control by enhancing
glucagon-like Peptide-1 and glucose-dependent insulinotropic
peptide release. Endocrinology 149: 2038–2047.
Chu ZL, Carroll C, Chen R, Alfonso J, Gutierrez V, He H et al.
(2010). N-oleoyldopamine enhances glucose homeostasis through
the activation of GPR119. Mol Endocrinol 24: 161–170.
Davis IW, Baker D (2009). RosettaLigand docking with full ligand
and receptor flexibility. J Mol Biol 385: 381–392.
Acknowledgements
Dhayal S, Morgan NG (2010). The significance of GPR119 agonists
as a future treatment for type 2 diabetes. Drug News Perspect 23:
418–424.
The Novo Nordisk Foundation Center for Basic Metabolic
Research (http://www.metabol.ku.dk) is supported by an
unconditional grant from the Novo Nordisk Foundation to
University of Copenhagen. M. S. E. and M. H. P. were sup-
ported by a PhD scholarship from the Faculty of Health and
Medical Sciences, University of Copenhagen.
Elling CE, Frimurer TM, Gerlach LO, Jorgensen R, Holst B, Schwartz
TW (2006). Metal ion site engineering indicates a global toggle
switch model for seven-transmembrane receptor activation. J Biol
Chem 281: 17337–17346.
Fleishman SJ, Leaver-Fay A, Corn JE, Strauch EM, Khare SD, Koga N
et al. (2011). RosettaScripts: a scripting language interface to the
Rosetta macromolecular modeling suite. PLoS ONE 6: e20161.
Author contributions
Fredriksson R, Hoglund PJ, Gloriam DE, Lagerstrom MC, Schioth
HB (2003). Seven evolutionarily conserved human rhodopsin G
protein-coupled receptors lacking close relatives. FEBS Lett 554:
381–388.
M. S. E., C. N., M. H. P., N. D. H. and T. M. F. performed the
research. M. S. E., C. N., N. D. H., T. M. F. and T. W. S. designed
the research study. L. E., J. L., R. M. J., C. N. and T. M. F.
contributed essential reagents or tools. M. S. E., C. N., N. D.
H., T. M. F. and T. W. S. analysed the data. M. S. E., C. N., T.
M. F. and T. W. S. wrote the paper.
Graham FL, van der Eb AJ (1973). A new technique for the assay of
infectivity of human adenovirus 5 DNA. Virology 52: 456–467.
Griffin G 2006. Methods for identification of modulators of
OSGPR116 activity. United States Patent US 7083933 B1.
Guan XM, Kobilka TS, Kobilka BK (1992). Enhancement of
membrane insertion and function in a type IIIb membrane protein
following introduction of a cleavable signal peptide. J Biol Chem
267: 21995–21998.
Conflict of interest
None.
Hansen HS, Rosenkilde MM, Holst JJ, Schwartz TW (2012). GPR119
as a fat sensor. Trends Pharmacol Sci 33: 374–381.
Holst B, Schwartz TW (2006). Ghrelin receptor mutations – too
little height and too much hunger. J Clin Invest 116: 637–641.
References
Holst B, Cygankiewicz A, Jensen TH, Ankersen M, Schwartz TW
(2003). High constitutive signaling of the ghrelin receptor –
identification of a potent inverse agonist. Mol Endocrinol 17:
2201–2210.
Adham N, Bonini J, Borowsky B, Boyle N, Thompson T (2003).
DNA encoding SNORF25 receptor. U.S. Pat. Appl. Publ.
US20030125539 A1.
British Journal of Pharmacology (2014) 171 5774–5789 5787

M S Engelstoft et al.
BJP
Holst B, Nygaard R, Valentin-Hansen L, Bach A, Engelstoft MS,
Petersen PS et al. (2010). A conserved aromatic lock for the
tryptophan rotameric switch in TM-VI of seven-transmembrane
receptors. J Biol Chem 285: 3973–3985.
Nguyen ED, Norn C, Frimurer TM, Meiler J (2013). Assessment and
challenges of ligand docking into comparative models of G-protein
coupled receptors. PLoS ONE 8: e67302.
Ning Y, O’Neill K, Lan H, Pang L, Shan LX, Hawes BE et al. (2008).
Endogenous and synthetic agonists of GPR119 differ in signalling
pathways and their effects on insulin secretion in MIN6c4
insulinoma cells. Br J Pharmacol 155: 1056–1065.
Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EY, Lane
JR et al. (2008). The 2.6 angstrom crystal structure of a human A2A
adenosine receptor bound to an antagonist. Science 322:
1211–1217.
Nygaard R, Frimurer TM, Holst B, Rosenkilde MM, Schwartz TW
(2009). Ligand binding and micro-switches in 7TM receptor
structures. Trends Pharmacol Sci 30: 249–259.
Jensen PC, Nygaard R, Thiele S, Elder A, Zhu G, Kolbeck R et al.
(2007). Molecular interaction of a potent nonpeptide agonist with
the chemokine receptor CCR8. Mol Pharmacol 72: 327–340.
Ohishi T, Yoshida S (2012). The therapeutic potential of GPR119
agonists for type 2 diabetes. Expert Opin Investig Drugs 21:
321–328.
Jones RM 2006. Discovery of agonists of the glucose dependent
insulinotropic receptor, GPR119, a pancreatic beta-cell oGPCR, for
the treatment of NIDDM. 232nd ACS National Meeting, San
Francisco, CA, United States, MEDI-275.
Ohishi T, Takasaki J, Matsumoto M, Saito T, Kamohara M, Soga T
et al. (2002). Method of screening remedy for diabetes. PCT
International Application. WO 2002044362 A1.
Jones RM, Leonard JN, Buzard DJ, Lehmann J (2009). GPR119
agonists for the treatment of type 2 diabetes. Expert Opin Ther Pat
19: 1339–1359.
Overton HA, Babbs AJ, Doel SM, Fyfe MC, Gardner LS, Griffin G
et al. (2006). Deorphanization of a G protein-coupled receptor for
oleoylethanolamide and its use in the discovery of small-molecule
hypophagic agents. Cell Metab 3: 167–175.
Katritch V, Cherezov V, Stevens RC (2013). Structure-function of
the G protein-coupled receptor superfamily. Annu Rev Pharmacol
Toxicol 53: 531–556.
Overton HA, Fyfe MC, Reynet C (2008). GPR119, a novel G
protein-coupled receptor target for the treatment of type 2 diabetes
and obesity. Br J Pharmacol 153 (Suppl. 1): S76–S81.
Kogure R, Toyama K, Hiyamuta S, Kojima I, Takeda S (2011).
5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist
and enhances glucose-dependent insulin secretion. Biochem
Biophys Res Commun 416: 58–63.
Parker HE, Habib AM, Rogers GJ, Gribble FM, Reimann F (2009).
Nutrient-dependent secretion of glucose-dependent insulinotropic
polypeptide from primary murine K cells. Diabetologia 52:
289–298.
Kufareva I, Rueda M, Katritch V, Stevens RC, Abagyan R,
Participants GD (2011). Status of GPCR modeling and docking as
reflected by community-wide GPCR Dock 2010 assessment.
Structure 19: 1108–1126.
Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP,
Buneman OP et al.; NC-IUPHAR (2014). The IUPHAR/BPS Guide to
PHARMACOLOGY: an expert-driven knowledgebase of drug targets
and their ligands. Nucl. Acids Res. 42 (Database Issue): D1098–106.
Lan H, Vassileva G, Corona A, Liu L, Baker H, Golovko A et al.
(2009). GPR119 is required for physiological regulation of
glucagon-like peptide-1 secretion but not for metabolic
homeostasis. J Endocrinol 201: 219–230.
Peeters MC, van Westen GJ, Li Q, Ap IJ (2011). Importance of the
extracellular loops in G protein-coupled receptors for ligand
recognition and receptor activation. Trends Pharmacol Sci 32:
35–42.
Lan H, Lin HV, Wang CF, Wright MJ, Xu S, Kang L et al. (2012).
Agonists at GPR119 mediate secretion of GLP-1 from mouse
enteroendocrine cells through glucose-independent pathways. Br J
Pharmacol 165: 2799–2807.
Petersen PS, Woldbye DP, Madsen AN, Egerod KL, Jin C, Lang M
et al. (2009). In vivo characterization of high Basal signaling from
the ghrelin receptor. Endocrinology 150: 4920–4930.
Lauffer LM, Iakoubov R, Brubaker PL (2009). GPR119 is essential for
oleoylethanolamide-induced glucagon-like peptide-1 secretion from
the intestinal enteroendocrine L-cell. Diabetes 58: 1058–1066.
Reilly MA (2001). G-protein receptor. Eur. Pat. Appl. EPO Patent
1092727-A2.
Leaver-Fay A, Tyka M, Lewis SM, Lange OF, Thompson J, Jacak R
et al. (2011). ROSETTA3: an object-oriented software suite for the
simulation and design of macromolecules. Methods Enzymol 487:
545–574.
Reimann F, Habib AM, Tolhurst G, Parker HE, Rogers GJ, Gribble
FM (2008). Glucose sensing in L cells: a primary cell study. Cell
Metab 8: 532–539.
Rosenkilde MM, Schwartz TW (2006). GluVII:06 – a highly
conserved and selective anchor point for non-peptide ligands in
chemokine receptors. Curr Top Med Chem 6: 1319–1333.
Lemmon G, Meiler J (2012). Rosetta Ligand docking with flexible
XML protocols. Methods Mol Biol 819: 143–155.
Meiler J, Baker D (2006). ROSETTALIGAND: protein-small molecule
docking with full side-chain flexibility. Proteins 65: 538–548.
Sakamoto Y, Inoue H, Kawakami S, Miyawaki K, Miyamoto T,
Mizuta K et al. (2006). Expression and distribution of Gpr119 in the
pancreatic islets of mice and rats: predominant localization in
pancreatic polypeptide-secreting PP-cells. Biochem Biophys Res
Commun 351: 474–480.
Mirzadegan T, Benko G, Filipek S, Palczewski K (2003). Sequence
analyses of G-protein-coupled receptors: similarities to rhodopsin.
Biochemistry 42: 2759–2767.
Mobarec JC, Sanchez R, Filizola M (2009). Modern homology
modeling of G-protein coupled receptors: which structural template
to use? J Med Chem 52: 5207–5216.
Schwartz TW, Frimurer TM, Holst B, Rosenkilde MM, Elling CE
(2006). Molecular mechanism of 7TM receptor activation – a global
toggle switch model. Annu Rev Pharmacol Toxicol 46: 481–519.
Mokrosinski J, Frimurer TM, Sivertsen B, Schwartz TW, Holst B
(2012). Modulation of constitutive activity and signaling bias of the
ghrelin receptor by conformational constraint in the second
extracellular loop. J Biol Chem 287: 33488–33502.
Semple G, Fioravanti B, Pereira G, Calderon I, Uy J, Choi K et al.
(2008). Discovery of the first potent and orally efficacious agonist
of the orphan G-protein coupled receptor 119. J Med Chem 51:
5172–5175.
5788 British Journal of Pharmacology (2014) 171 5774–5789

GPR119 constitutive activity and ligand binding
BJP
Shah U (2009). GPR119 agonists: a promising new approach for the
treatment of type 2 diabetes and related metabolic disorders. Curr
Opin Drug Discov Devel 12: 519–532.
Worth CL, Kreuchwig A, Kleinau G, Krause G (2011). GPCR-SSFE: a
comprehensive database of G-protein-coupled receptor template
predictions and homology models. BMC Bioinformatics 12: 185.
Soga T, Ohishi T, Matsui T, Saito T, Matsumoto M, Takasaki J et al.
(2005). Lysophosphatidylcholine enhances glucose-dependent
insulin secretion via an orphan G-protein-coupled receptor.
Biochem Biophys Res Commun 326: 744–751.
Supporting information
Thiele S, Malmgaard-Clausen M, Engel-Andreasen J, Steen A,
Rummel PC, Nielsen MC et al. (2012). Modulation in selectivity and
allosteric properties of small-molecule ligands for CC-chemokine
receptors. J Med Chem 55: 8164–8177.
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
http://dx.doi.org/10.1111/bph.12877
Thirstrup K, Elling CE, Hjorth SA, Schwartz TW (1996).
Construction of a high affinity zinc switch in the kappa-opioid
receptor. J Biol Chem 271: 7875–7878.
Appendix S1 Supplementary methods.
British Journal of Pharmacology (2014) 171 5774–5789 5789