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\begin{document}

\title{Development of high-affinity agonist- and antagonist radioligands for
the GLP-2 receptor - powerful tools for the study of GLP-2 pharmacology}



\author[1]{Sarina Gadgaard}%
\author[2]{Wijnand van der Velden}%
\author[2]{Sine Schiellerup }%
\author[2]{Jenna  Hunt }%
\author[2]{Maria  Gabe }%
\author[2]{Johanne Windeløv}%
\author[2]{Geke Boer}%
\author[2]{Hannelouise Kissow}%
\author[2]{Cathrine Ørskov}%
\author[2]{Jens Holst}%
\author[2]{Bolette Hartmann}%
\author[3]{Mette Rosenkilde}%
\affil[1]{University of Copenhagen Faculty of Health and Medical Sciences}%
\affil[2]{University of Copenhagen}%
\affil[3]{University of Copenhagen }%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Glucagon-like peptide-2 (GLP-2) is a 33 amino acid
pro-glucagon-derived hormone produced in the intestinal enteroendocrine
L-cells with trophic actions on both the gut and bones. GLP-2(1-33) is
cleaved by the ubiquitous protease dipeptidyl peptidase-4 (DPP-4),
resulting in GLP-2(3-33) with competitive antagonistic properties on the
GLP-2 receptor (GLP-2R). Here we present two new hGLP-2 radioligands
with different pharmacodynamic profiles. Experimental Approach: The
methionine in position 10 of GLP-2(1-33) was substituted with tyrosine
to enable oxidative iodination with incorporation of the iodine isotope
{[}125I{]}. Similar substitution was done in GLP-2(3-33), thereby
creating two new radioligands; an agonist {[}125{]}-hGLP-2(1-33,M10Y)
and an antagonist {[}125{]}-hGLP-2(3-33,M10Y). Both were characterized
regarding competition binding, binding kinetics and target tissue
autoradiography. Key results: High and similar binding affinities for
the human GLP-2R were observed for {[}125I{]}-hGLP-2(1-33,M10Y) and
{[}125I{]}-hGLP-2(3-33,M10Y) with KD values of 59.3 nM and 40.6 nM,
respectively. The M10Y substitution did not change the functional
properties of GLP-2(1-33) or GLP-2(3-33). The antagonist
{[}125I{]}-hGLP-2(3-33,M10Y) had higher Bmax and faster on-rate for the
hGLP-2R compared to the agonist {[}125I{]}-hGLP-2(1-33,M10Y). Using
autoradiography in mice strong labeling was observed in subepithelial
myofibroblasts (SEMF) and pancreas islet cells. Both radioligands were
selective for the GLP-2R, except for a low affinity binding to the
GLP-1R (IC50 of 130 and 330 nM, respectively) Conclusion and
implications: We successfully developed two new high affinity
radioligands for GLP-2R studies and identified SEMF and pancreatic
islets as target for GLP-2. It is uncertain whether binding in the
pancreas islets results from GLP-2R or GLP-1R binding.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Glucagon-like peptide-2 (GLP-2) is a gut hormone consisting of 33 amino
acids (GLP-2(1-33)), which is a product of the pro-glucagon gene
secreted from the enteroendocrine L-cells of the small intestine upon
nutrient ingestion. GLP-2(1-33) is cleaved by the ubiquitous protease
dipeptidyl peptidase-4 (DPP-4), resulting in the degradation product
GLP-2(3-33) (Hartmann et al. 2000; Holst et al. 2000). In mice,
administration of GLP-2(1-33) promotes growth of the small and large
intestine (Drucker et al. 1996; Thulesen et al. 2002), stimulates
proliferation of the crypt cell, nutrient absorption as well as
promoting healing and maintenance of epithelial integrity (Drucker,
2013; Dub\selectlanguage{ngerman}é, 2006). These intestinotrophic actions of GLP-2 have been
exploited therapeutically with the use of the DPP-4 resistant
GLP-2(1-33) analogue Teduglutide, that since 2012 has been used in the
treatment of short bowel syndrome (SBS) in adults (Jeppesen et al. 2001;
Kim et al. 2017). In addition, a four month clinical study showed that
GLP-2(1-33) has an anti-catabolic effect on the bone tissue by
inhibiting bone resorption (as measured by the bone marker CTX
(C-terminal telopeptide)), as shown by our group and others
(Askov-Hansen et al. 2013; Gottschalck et al. 2008; Gottschalck et al.
2008; Henriksen et al. 2009; Henriksen et al. 2003; Henriksen et al.
2007).

The metabolite GLP-2(3-33) acts as a partial agonist of the GLP-2
receptor (GLP-2R) (EC\textsubscript{50} of \textasciitilde{}6 nM and
Emax of \textasciitilde{}15\% of GLP-2(1-33)) with competitive
antagonistic properties on the human (hGLP-2R) \emph{in vitro}
and\emph{in vivo} (Thulesen et al. 2002). Structurally, GLP-2 is closely
related to the peptide hormones glucagon-like peptide-1 (GLP-1) and
glucose-dependent insulinotropic polypeptide (GIP). GIP (secreted from
enteroendocrine K-cells) and GLP-1 (co-secreted with GLP-2 from L-cells)
are important insulinotropic hormones, whereas GLP-2 is inactive in this
respect (Schiellerup et al. 2019). GLP-1 analogs are widely used as
treatment for type 2 diabetes mellitus (T2DM) and obesity, and more
recently, a dual-agonist of GLP-1 and GIP showed promising effects
within this field (Coskun et al. 2018; Willard et al. 2020)

The GLP-2R is a G protein-coupled receptor (GPCR), belonging to the
subclass B1 of the GPCR family, which comprises of 15 receptors
including the GLP-1 receptor (GLP-1R), the GIP receptor (GIPR), the
glucagon receptor (GCGR), the Secretin receptor (SecretinR), and the
vasoactive intestinal peptide 1 and 2 receptors (VPAC1R and -2R)
(Culhane et al. 2015; Gabe et al. 2020; Kenakin et al. 2010). High
resolution structures of class B1 GPCRs (but not yet GLP-2R) combined
with mutation studies, have enabled the analysis of the active,
intermediate and inactive conformations of the receptor, thereby
revealing residues that are essential for ligand binding and/or
activation (Gabe et al. 2020; Parthier et al. 2009; Song et al. 2017;
Zhang et al. 2018; Zhang et al. 2017). Currently, the leading paradigm
regarding activation of class B1 GPCRs is the ``two-step'' mechanism,
suggesting that the peptide ligand switches between an overall
disordered and more ordered alpha-helical secondary structure, where the
N-terminal part of the extracellular domain (ECD) of the receptor
recognizes the C-terminal or middle part of the ligand for interaction,
where after the N-terminal part of the ligand orientates towards and
docks into the transmembrane domains (TMDs) of the receptor (Gabe et al.
2020; Parthier et al. 2007). The two-step mechanism is a simplified
model of a complex network of conformational changes that takes place
upon activation (Liang et al. 2018; Liang et al. 2018; Sasaki et al.
1975; Schwartz et. al. 2017; Venneti et al. 2011; Zhang et al. 2017;
Zhao et al. 2019). Signaling through class B1 receptors, including the
GLP-2R, mainly occurs through G\selectlanguage{greek}α\selectlanguage{english}s-coupling, thereby evoking multiple
signaling cascades, including increased levels of the downstream second
messenger cyclic adenosine monophosphate (cAMP) (Correll et al. 2014).
By immunofluorescence microscopy, Estall et al. showed that the
C-terminus of the GLP-2R recruits \selectlanguage{greek}β-\selectlanguage{english}arrestin-2 following agonist
stimulation, but that the recruitment is not required for G\selectlanguage{greek}α\selectlanguage{english}s-coupling,
desensitization, and receptor endocytosis of GLP-2R (Estall et al.
2005). Functional consequences of \selectlanguage{greek}β-\selectlanguage{english}arrestin recruitment by the GLP-2R
have not yet been described, although important effects of recruitments
have been demonstrated for other class B1 GPCRs, including the GIPR
(Gabe et al. 2018) and the GLP-1R (Roussel et al. 2016; Whalen et al.
2011).

Although the GLP-2R was cloned for the first time in 1999, the precise
tissue and cellular localization of GLP-2R expression remains
controversial, undoubtedly due to the lack of specific antibodies.
Messenger RNA (mRNA) transcripts of the GLP-2R are found within
gastro-intestinal tissues (stomach, duodenum, jejunum, ileum, colon and
intestinal ganglion cells) of various species, including human and
rodents (Bjerknes et al. 2002; El-Jamal et al. 2014; Guan et al. 2006;
Munroe et al. 1999; \selectlanguage{ngerman}Ørskov et al. 2005; Pedersen et al. 2015; Yusta et
al. 2000). El-Jamal N et. al. have demonstrated GLP-2R mRNA transcripts
within the intestinal subepithelial myofibroblasts (SEMF) cell line,
CCD-19Co (El-Jamal et al. 2014), supporting earlier studies (Ørskov et
al. 2005), describing the expression of the GLP-2R protein throughout
the small and large intestine, and particularly within the SEMFs of the
GI tract by immunohistochemistry. Also, the mRNA transcript of the
GLP-2R has been reported in extraintestinal tissues (fat, lymph nodes,
bladder, spleen liver and hepatocytes) (El-Jamal et al. 2014; Yusta et
al. 2000; Yusta et al. 2019). Interestingly, De Heer et. al.
demonstrated the GLP-2R mRNA transcript in human and rat pancreas (De
Heer et al. 2007), a tissue that is known for high expression levels of
the GLP-1R (Richards et al. 2014). \selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}Thus, there seems to be
expression of the GLP-2R in several tissues.

Here, we present two new, high-affinity human GLP-2 (hGLP-2)
radioligands with tyrosine (Tyr)-substitution at position 10 in the two
naturally occurring GLP-2 peptides, the natural agonist GLP-2(1-33) and
the antagonist and partial agonist GLP-2(3-33). With these, we
determined active and in-active receptor conformations, the interchange
between the two, and differential binding kinetics for the two
radioligands. Furthermore, we performed autoradiography studies in mice
to determined GLP-2R protein expression \emph{in vivo} .

\par\null

\textbf{MATERIALS AND METHODS}

\textbf{Plasmids and peptides}

cDNA of the receptors were inserted into the pcDNA3.1(+) vector. All
ligand peptides were purchased from CASLO ApS (Technical University of
Denmark, DTU-Science Park) with a minimum purity of 95\%.

\textbf{Cell culturing, transfection and generation of stable cell
lines}

COS-7 cells were cultured at 10\% CO\textsubscript{2}, 95\% air humidity
and 37degC in Dulbecco's Modified Eagle Medium (DMEM) 1885 supplemented
with 10\% fetal bovine serum (FBS), 1\% penicillin (180
U/mL)/streptomycin (45 \selectlanguage{greek}µ\selectlanguage{english}g/mL). The cells were transfected using the
calcium phosphate precipitation method as previously described (Jensen
et al. 2008). Briefly, the cells were seeded in T175/T75/T25 flasks one
day before transfection with 40/20/10 \selectlanguage{greek}µ\selectlanguage{english}g receptor DNA. Transiently
transfected COS-7 cells were used in cAMP accumulation and for whole
cell homologous and heterologous binding\emph{.}

HEK-293 cells stably expressing hGLP-2R or pcDNA3.1(+) were generated by
transfection as described above. The cells were cultured at 10\%
CO\textsubscript{2}, 95\% air humidity and 37\selectlanguage{ngerman}°C in Dulbecco's Modified
Eagle Medium (DMEM), containing 1\% GlutaMAX, and supplemented with 10\%
fetal bovine serum (FBS) 1\% penicillin (180 U/mL)/streptomycin (45
\selectlanguage{greek}µ\selectlanguage{english}g/mL) and 0.4 mg/mL G418 for selection. Stably transfected HEK-293
cells were utilized for membrane preparation used in kinetic binding
experiments.

\textbf{Membrane preparation}

hGLP-2R and pcDNA3.1(+) membranes were prepared through several
centrifugation steps. The cells were scraped with PBS supplemented with
a cOmplete EDTA free protease inhibitor (Roche, Basel, Switzerland) and
then homogenized using a Douncer homogenizer. The homogenate was
centrifuged for 3 min at 500 rpm (54 g; 4\selectlanguage{ngerman}°C) and subsequently the
supernatant was centrifuged for 45 min at 14 500 rpm at (24 446g; 4°C).
The resulting pellet was resuspended in storage buffer (20 mM HEPES
buffer (pH 7.2), 0.4 mM CaCl\textsubscript{2}, 2 mM
MgCl\textsubscript{2} and cOmplete EDTA free protease inhibitor) and
stored at -80°C. Protein determination was performed according to a
standard Pierce BCA protein assay protocol (Thermo Scientific, Rockford,
IL).

\textbf{cAMP measurements}

For the cAMP measurements, COS-7 cells were transfected with receptor
plasmid or pcDNA3.1(+) and seeded with 25 000 cells per well in a
CulturPlate-96 (PerkinElmer; Waltham, MA) one day after transfection.
The following day, the cells were washed once with HEPES-buffered saline
and incubated for 30 min at 37°C with HEPES-buffered saline supplemented
with 1mmol/l 3-isobutyl-1-methylxanthine (IBMX) buffer. To test for
intrinsic activity, endogenous hGLP-2(1-33) or hGLP-2(3-33) or the
M10Y-substituted variants (hGLP-2(1-33,M10Y) or hGLP-2(3-33,M10Y)) were
added in increasing concentrations, and the plates were incubated for
additional 30 min at 37°C. To test hGLP-2(3-33) and hGLP-2(3-33,M10Y) as
antagonists the cells were preincubated for 10 min with a fixed
concentration of antagonist followed by the addition of increasing
concentrations of agonist. Afterwards, the HitHunter cAMP XS-assay
(DiscoverX, Birmingham, UK) was carried out according to the
manufacturer's instructions. All experiments were made in duplicates,
and luminescence was measured by a Perkin Elmer EnVision 2104 Multilabel
reader (PerkinElmer; Waltham, MA).

\textbf{Oxidative iodination}

The radioligands were created by oxidative iodination with the oxidizing
agent ChloramineT. Here the iodine isotope {[}\textsuperscript{125}I{]}
becomes incorporated in the Tyr residue at position 10 of
hGLP-2(1-33,M10Y) or hGLP-2(3-33,M10Y). 2nmol peptide was dissolved in
10\selectlanguage{greek}µ\selectlanguage{english}L iodination buffer (100 mM phosphate buffer) and 0.4 nCi
Na\textsuperscript{125}I was added. A stepwise, so-called stoichiometric
oxidation reaction was performed by sequential addition of 6 aliquots of
5 \selectlanguage{greek}µ\selectlanguage{english}L ChloraminT (30 \selectlanguage{greek}µ\selectlanguage{english}g/mL) with 1 min intervals during constant
stirring. Under these conditions {[}\textsuperscript{125}I{]} is
incorporated at the hydroxyl group in the ortho position of the Tyr
residue. The reaction was terminated by the addition of 400 \selectlanguage{greek}µ\selectlanguage{english}L phase A
(0,1\% trifluoracetic acid (TFA)). The reaction was carried out in at pH
7.4 to avoid labeling of histidine residues at basic conditions (pH
\textgreater{} 8.5). The product was fractionated on a high-performance
liquid chromatography (HPLC) (\selectlanguage{ngerman}Åkta, GE Healthcare, Boston US) with a C18
column for reverse-phase (RP) HPCL (RP-HPCL). The column was initially
flushed with 80\% phase A and 20\% phase B (Acetonitrile + 0,1\% TFA),
and terminally by applying increasing concentrations of phase B. The
pressure of the RP-HPCL was kept constant at 8 MPa with a flow of 1
mL/min. Before binding assays were performed the eluted fractions were
tested in homologous competition binding (see next section for method).

\textbf{Homologous and heterologous binding}

For the competition binding experiments, COS-7 cells were transfected
with receptor plasmid or pcDNA3.1(+) and seeded with 150 000 cells per
well in CulturPlate-24 (PerkinElmer; Waltham, MA) one day after
transfection. The number of cells seeded per well was selected on the
basis to obtaining 5-10\% specific binding of the radioligands. Day 2
after transfection, the cells were washed twice in prechilled binding
buffer (50 mM HEPES buffer, supplemented with 1 mM
CaCl\textsubscript{2}, 5 mM MgCl\textsubscript{2} and 0,5\% (w/v) BSA)
at pH 8 and incubated for 15 min at 4C°. Increasing concentration of
unlabeled ligand followed by a stable concentration of the radioligand
(20 000cpm/well) were added to the cells, which were then incubated for
additional 3 hours at 4C°. After incubation, the cells were washed twice
in prechilled binding buffer, lysed, and counted using a Wizard gamma
counter (PerkinElmer; Waltham, MA).

\textbf{Kinetic binding experiments}

The association assays were performed by preparing a mixture of 10 \selectlanguage{greek}µ\selectlanguage{english}g
hGLP-2R or pcDNA3.1(+) and 0.5 mg wheatgerm agglutinin coated (WGA) PVT
SPA beads (PerkinElmer; Waltham, MA). This mixture was pre-coupled on a
shaker in a total volume of 50 \selectlanguage{greek}µ\selectlanguage{english}L binding buffer (50 mM HEPES buffer (pH
7.2)), supplemented with 1 mM CaCl\textsubscript{2}, 5 mM
MgCl\textsubscript{2} and 0,5\% (w/v) BSA) for 30 min at 30\selectlanguage{ngerman}°C. The
pre-coupling was followed by the distribution of membrane suspension in
a CulturPlate-96 (PerkinElmer; Waltham, MA) in a total volume of 90 \selectlanguage{greek}µ\selectlanguage{english}L
binding buffer and spun down afterwards (1 500 RPM, 485 g, 5 min, room
temperature). The reaction was initiated by the addition of 0.19 \selectlanguage{ngerman}± 0.001
nM {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) or 0.21 ± 0.004 nM
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y), and the amount of
radioligand bound to receptor was measured every minute up to 100 min
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) or 120 min
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) at 30°C, using a TopCount
NXT Microplate Scintillation \& Luminescence Counter (PerkinElmer;
Waltham, MA).

For the dissociation assays, the membrane suspension was distributed to
the wells in a total volume of 85 \selectlanguage{greek}µ\selectlanguage{english}L binding buffer. The mixture was
then pre-incubated for 60 min at 30\selectlanguage{ngerman}°C after addition of 0.19 ± 0.001 nM
{[}\textsuperscript{125}I{]}GLP-2
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) or 0.21 ± 0.004 nM
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y). The dissociation was
initiated by the addition of 5 \selectlanguage{greek}µ\selectlanguage{english}L of 1 \selectlanguage{greek}µ\selectlanguage{english}M unlabeled hGLP-2(1-33,M10Y) or
hGLP-2(3-33,M10Y). The amount of receptor-bound radioligand was measured
every minute up to 500 min.

\textbf{Immunohistology and autoradiography}

All procedures in the mice were approved by the Danish National Animal
Experiments Inspectorate (license no. 2018-15-0201-01397). All mice were
kept in the animal facility and received tap water and standard chow ad
libitum.

\textbf{Surgical procedure.} Female C57BL/6JRj mice (n=9) weighing 18-26
g were purchased from Janvier (Saint Berthevin Cedex, France) and left
to acclimatize for at least one week before experimental procedures were
performed. The mice were anaesthetized with an intraperitoneal injection
of ketamine-xylazine (100:10 mg/kg, Pharma service SUND, UCPH,
Copenhagen, Denmark). The abdomen was opened by a midline incision, the
inferior caval vein exposed, and
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (2 pmol (3 mill cpm))
dissolved in 100 \selectlanguage{greek}µ\selectlanguage{english}L of 0.04 M phosphate buffer containing in addition
1\% HSA (pH 7.5) was injected slowly. Three of the animals also received
a 1000-fold excess of unlabeled hGLP-2(1-33,M10Y) (2 nmol) in
combination with the labelled peptide in the same injection to test for
specific binding. The actual amount of
{[}\textsuperscript{125}I{]}-labelled peptide administered was
calculated from the specific radioactivity of the radioligand. Before
injection, 10 \selectlanguage{greek}µ\selectlanguage{english}L of the {[}\textsuperscript{125}I{]}-labelled peptide
stock solution was counted in a gamma-counter to determine the amount of
radioactivity injected into the animals. Ten min after peptide
injection, the thorax was opened, after which the vascular system was
perfused at a constant flow with 0.9\% saline with an outlet through the
right ventricle. Next, the mice were fixated by flushing the system with
ice-cold 4\% paraformaldehyde. After fixation, the pancreas, small
intestine and kidneys (as positive control) were removed and stored in
45\% paraformaldehyde until further processing.

\textbf{Autoradiography.} Small intestinal, pancreatic and kidney tissue
samples were embedded in paraffin, and histological 4 \selectlanguage{greek}µ\selectlanguage{english}m sections were
cut with a microtome and placed on glass slides. The sections were
dewaxed and coated in a dark room with 43-45\textsuperscript{o}C Kodak
NTB emulsion (VWR, Herlev, Denmark) diluted 1:1 with
43-45\textsuperscript{o}C water, and subsequently dried and stored in
light-proof boxes at 5\textsuperscript{o}C for 6 weeks. After 6 weeks,
the tissue sections were developed in a dark room in Kodak D-19
developer (VWR, Herlev, Denmark) for 5 min, dipped 10 times in 0.5\%
acetic acid, and fixated in 30\% sodium thiosulphate for 10 min. The
sections were then washed, first in water for 10 min and then in 70\%
ethanol. Finally, the sections were lightly counterstained with
haematoxylin and examined with a light microscope (Orthoplan, leitz).
Images were taken with an AxioCam ICc5 camera (Zeiss) connected to the
light microscope.

\textbf{Data and statistical analysis}

IC\textsubscript{50} and EC\textsubscript{50} values were determined by
non-linear regression using GraphPad Prism 8, which was also used for
statistical calculation. Sigmoid curves were fitted logistically with a
Hill slope of 1 or -1 for the activation and binding curve,
respectively.

To calculate the total density of receptors expressed
(B\textsubscript{max}) we used equation (1) (Deblasi, Reilly et al.
1989):\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\mathbf{B}_{\mathbf{\max}}\mathbf{=}\frac{\mathbf{B}_{\mathbf{0}}\mathbf{\bullet}\mathbf{\text{IC}}_{\mathbf{50}}}{\mathbf{[L]}}\) & \textbf{(1)}\tabularnewline
\bottomrule
\end{longtable}

where B\textsubscript{0} is the total specific binding in CPM and
{[}L{]} is the concentration of radioligand in nM.

The association rate constant (k\textsubscript{on}) was calculated as
follows (2) (Velden et al. 2020):\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\(\mathbf{k}_{\mathbf{\text{on}}}\mathbf{=\ }\frac{\mathbf{k}_{\mathbf{\text{obs}}}\mathbf{-\ }\mathbf{k}_{\mathbf{\text{off}}}}{\left[\mathbf{L}\right]}\) & \textbf{(2)}\tabularnewline
\bottomrule
\end{longtable}

Where k\textsubscript{obs} is the observed association rate constant
(min\textsuperscript{-1}) and k\textsubscript{off} is the dissociation
rate constant (min\textsuperscript{-1}). Statistical significances
between dose-response curves were analyzed by two-way ANOVA. **** p
\textless{} 0.0001, *** p \textless{} 0.001, ** p \textless{} 0.01 and
*p \textless{} 0.05. ns indicates non-significant differences.

\textbf{Bioinformatics}

Sequence similarities (\%) were evaluated by protein blast at the
National Center for Biotechnology Information (NCBI)
https://www.ncbi.nlm.nih.gov/. The amino acid alignment were acquired my
Multiple Sequence Alignment using the Clustal Omega software available
at the EMBL-EBI website; ~http://www.ebi.ac.uk/Tools/msa/clustalo/.

\textbf{RESULTS}

\textbf{Introduction of Tyr in position 10 does not alter the
pharmacodynamic properties in terms of cAMP production}

The sequence of hGLP-2 (figure 1a) does not include a Tyr residues and
therefore unsuitable for oxidative iodination using iodine-125
{[}\textsuperscript{125}I{]}. hGLP-2 shows high sequence similarities to
the class B1 hormones hGIP, hGCG and hGLP-1 (figure 1a). Generally, a
high level of promiscuity among class B1 ligand-receptor pairs can be
found (Sandoval et al. 2015; Skov-Jeppesen et al. 2019; Svendsen et al.,
2018), which enable us to look for a suitable position for
{[}\textsuperscript{125}I{]}-labeling of hGLP-2 At position 10 in hGIP
and hGCG, a Tyr residue is found, which is the target for oxidative
{[}\textsuperscript{125}I{]}-labeling of these peptides (Sparre-Ulrich
et al. 2017, 2016). At the corresponding site in hGLP-2 a methionine
(Met) residue is found, which we replaced with a Tyr residue (referred
to as M10Y) (figure 1a). Because GLP-2(1-33) is rapidly cleaved into the
antagonist (and partial agonist) hGLP-2(3-33) by DPP-4, (Hartmann et al.
2000), we modified both peptides to create the two consecutive peptides
(hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y)) with the intension of creating
both an agonistic and an antagonistic radioligand.

First, the activity of the two altered peptides was measured in terms of
cAMP accumulation. COS-7 cells transiently expressing hGLP-2R were
stimulated with increasing concentrations of the two modified GLP-2
variants in comparison with the endogenous GLP-2 peptides. Endogenous
hGLP-2(1-33) and hGLP-2(3-33) accumulated cAMP as previously shown
(figure 1b,c and table 1) (Skov-Jeppesen et al. 2019). hGLP-2(1-33,M10Y)
displayed a strong and full activation of hGLP-2R with only a 2.5-fold
decreased potency compared to hGLP-2(1-33) (figure 1b and table 1).
Similar to the endogenous hGLP-2(3-33), hGLP-2(3-33,M10Y) was a partial
agonist with similar potency and efficacy as hGLP-2(3-33) (figure 1c and
table 1). These data show that the two M10Y-substituted variants
function in a similar manner as their corresponding endogenous peptides.
As GLP-2(3-33) has previously been described as a competitive antagonist
for the hGLP-2R (Skov-Jeppesen et al. 2019), we tested the antagonistic
properties of hGLP-2(3-33,M10Y) by determining the impact of increasing
concentrations (100 mM and 1 \selectlanguage{greek}µ\selectlanguage{english}M) of hGLP-2(3-33,M10Y), on the potency of
hGLP-2(1-33) on the hGLP-2R. Consistent with a competitive antagonistic
nature, hGLP-2(3-33,M10Y) resulted in a rightward shift of the
dose-response curve of hGLP-2(1-33) (figure 1d and table 1).

\textbf{High and similar binding properties of iodinated
hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y)}

Since both modified peptides had similar functional properties as the
endogenous peptides, we continued with hGLP-2(1-33,M10Y) and
hGLP-2(3-33,M10Y) for radioligand development using chloramineT for
stoichiometric oxidation of the Tyr residue. To verify the binding
properties of the two radioligands, we performed homologous whole cell
competition binding in cells transiently expressing the hGLP-2R. Both
radioligands showed high-affinity binding for hGLP-2R (figure 2 a,b and
table 2), thereby demonstrating successful development of two new
radioligands with high and similar binding affinities for the hGLP-2R. A
significant higher Bmax was found for the antagonist
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (96,6
fmol/10\textsuperscript{5}) compared to the agonist
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (58,0
fmol/10\textsuperscript{5}) (figure 2c). These data are in accordance
with a generally higher amount of binding sites for GPCR antagonists
compared to agonists (Baker et al. 2007).

Since ligand--receptor binding kinetics is a key determinant of ligand
efficacy (Velden et al. 2020), we determined the association
(k\textsubscript{on}) and dissociation (k\textsubscript{off}) rates of
both radioligands, using cell membranes stably expressing the hGLP-2R.
For both ligands, the kinetic profiles were best fitted with a one-phase
association and a one-phase dissociation. Saturation of
{[}\textsuperscript{125}I{]}-hGLP-2(1--33,M10Y) was reached at around 60
min, whereas for {[}\textsuperscript{125}I{]}-hGLP-2(3--33,M10Y)
saturation was reached already at 40 min (figure 2d). This was also
reflected in the observed on-rates with a \textasciitilde{}3 fold higher
k\textsubscript{obs} for {[}\textsuperscript{125}I{]}-hGLP-2(3--33,M10Y)
(0.076 \selectlanguage{ngerman}± 0.009 min\textsuperscript{-1}) compared to
{[}\textsuperscript{125}I{]}-hGLP-2(1--33,M10Y) (0.027 ± 0.003
min\textsuperscript{-1}). After reaching equilibrium, the binding was
reversed by the addition of 1 \selectlanguage{greek}µ\selectlanguage{english}M unlabeled hGLP-2(1--33,M10Y) and
hGLP-2(3--33,M10Y), respectively (figure 2e). The two ligands had
similar dissociation rates (k\textsubscript{off}) of 0.009 \selectlanguage{ngerman}± 0.004
min\textsuperscript{-1} and 0.010 ± 0.002 min\textsuperscript{-1},
respectively. Finally, we calculated the on-rate (k\textsubscript{on})
of both radioligands, and found a \textasciitilde{}3.5 fold higher
on-rate for the antagonist (0.329 ± 0.047
nM\textsuperscript{-1}*min\textsuperscript{-1}), compared to the agonist
(0.094 ± 0.014 nM* min\textsuperscript{-1}) (figure 2f). Thus, the
receptor binding of the antagonist is faster than that of the agonist,
presumably reflecting, that the receptor undergoes less conformational
changes upon antagonist binding compared to agonist binding.

\textbf{Native hGLP-2(1-33) binds to the hGLP2-R with highest affinity
among the tested ligands}

In order to determine whether agonists and antagonists competed
similarly, we measured heterologous binding (figure 3) by displacing the
two radioligands with the four unlabeled peptides; hGLP-2(1-33),
hGLP-2(3-33), hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y). Overall, all four
ligands were able to compete with both radioligands, with no significant
differences in their binding affinities whether using the agonist or the
antagonist radioligand (figure 3a and table 2). However, native
hGLP-2(1-33) had a 4- to 5-fold higher affinity compared to the other
three hGLP-2 variants (figure 3 and table 2). The decreased affinity of
hGLP-2(1-33,M10Y) compared to hGLP-2(1-33) is consistent with the
slightly decreased potency for hGLP-2(1-33,M10Y) compared to
hGLP-2(1-33) (figure 1b).

\textbf{Minimal binding to other class B1 GPCRs for
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y)}

Given the high sequence similarity between class B1 receptors and their
peptide ligands, we next determined whether the two radioligands
cross-react with the closely related class B1 GPCRs; hGIPR, hGLP-1R,
hGCGR and hSecretinR, VPAC-1R, and VPAC-2R (figure 4a). While we
observed no specific binding for five of the six receptors, a low, but
significant binding was observed for both radioligands to the hGLP-1R
(Emax of 37.8\% and 26.7\% of the hGLP-2R binding) (figure 4b and
supplementary figure 1).

Furthermore, we tested the binding properties of both radioligands on
the mouse and rat GLP. Also here, we observed high-affinity binding
(figure 5a,b and table 2). The similar IC\textsubscript{50} values of
hGLP-2(1-33) on the GLP-2R from the three species suggests overall
strong structural similarities between the receptors (table 2).

\textbf{Autoradiography in mice reveals receptor expression in
sub-epithelial myofibroblasts.}

Having confirmed the binding of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) to rodent GLP-2Rs, we did
autoradiography studies in mice with this radioligand. In all mice
examined (n=6), we observed strong labeling in the SEMFs of the
gastrointestinal (GI) tract (figure 5c,e and supplementary figure 2) and
in the islet cells of the endocrine pancreas (figure 5d,f and
supplementary figure 2). These data are consistent with previous data
(El-Jamal et al. 2014; De Heer et al. 2007; Ørskov et al. 2005), and
thereby confirming at the protein level what was shown at the level of
GLP-2R mRNA transcript. Injection of unlabeled GLP-2(1-33) prior to the
radioligand abrogated labeling in both tissues (figure 5e,f), supporting
the specific binding of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y).\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}

Since the pancreas is known for high expression levels of the GLP-1R and
given the observed binding of both hGLP-2-based radioligands to the
hGLP-1R (figure 4), we tested the binding of the radioligands to the
mouse and rat GLP-1R. Here, we were surprised by a very high specific
binding of both radioligands to the mouse GLP-1R (mGLP-1R), while no
binding was observed for the rat GLP-1R (rGLP-1R) (supplementary figure
3). The binding of both radioligands to mGLP-1R reached the same Emax as
for the mouse GLP-2R (mGLP-2R), but have a 32-fold lower affinity (table
2).

\textbf{Specific binding of GLP-2 to the GLP-1R}

The binding to the mGLP-1R combined with the low, but specific binding
to the hGLP-1R, inspired us to further characterize the action of hGLP-2
in the hGLP-1R system using the same experimental setup as for the
hGLP-2R (figure 1). The two full-length variants hGLP-2(1-33) and
hGLP-2(1-33,M10Y) both showed weak agonistic properties on the hGLP-1R
with \textgreater{}1000-fold lower potency compared to endogenous GLP-1
(figure 6a and table 1). These data corresponds to previous data
(Gasbjerg et al. 2018). In contrast, the two N-terminally truncated
variants hGLP-2(3-33) and hGLP-2(3-33,M10Y) did not activate the hGLP-1R
(figure 6b and table 1). To confirm that the hGLP-2 mediated cAMP
response was mediated through the GLP-1R, we reversed the signal by
employing the high affinity GLP-1R antagonist exendin(9-39) (Schirra et
al. 1998). A rightward shift was observed for the dose-response curve of
hGLP-2(1-33) in the presence of exendin(9-39) (figure 6c and table 1),
demonstrating that the cAMP accumulation induced by hGLP-2(1-33) is
mediated through the hGLP-1R, in a similar manner, as that of GLP-1. In
contrast, increasing concentrations (up to 1 \selectlanguage{greek}µ\selectlanguage{english}M) of hGLP-2(3-33,M10Y)
did not affect the activity of GLP-1 on the hGLP-1R (figure 6d and table
1).

\textbf{DISCUSSION}

Despite the emerging evidence for the biological importance of GLP-2 as
a trophic hormone for the gut and bones, very little structural
information is available of the GLP-2R. An increasing number of
high-resolution structures of class B1 GPCRs, have been published (Liang
et al. 2018; Qiao et al. 2020; Wu et al. 2020; Zhang et al. 2018; Zhang
et al. 2017; Zhao et al. 2020), yet the structure of the GLP-2R remains
to be determined. However, a handful of studies focusing on the GLP-2
structure and its interaction with the GLP-2R have elucidated structural
requirements for GLP-2's interaction with its receptor. In 2000,
DaCambra et. al. performed an Alanine(Ala)-scan within the DPP-4
resistant h{[}Gly\textsuperscript{2}{]}GLP-2(1-33) and showed reduced
receptor activation (cAMP accumulation), of the rGLP-2R by alterations
in the N-terminus part of the peptide (Dacambra et al. 2000). Here, Ala
replacement of the Histidine1 and the Asparticacid3 of hGLP-2, severely
reduced receptor activation with only modest changes in binding
affinity. These data demonstrate the importance of the GLP-2 N-terminus
for receptor activation, as also illustrated by the partial agonism (and
competitive antagonism) of GLP-2(3-33) (Thulesen et al. 2002) (figure
1). Ala-scan within the C-terminus part of
h{[}Gly\textsuperscript{2}{]}GLP-2 severely reduced the binding affinity
demonstrating a central role of the C-terminus part for receptor
binding. In 2011, Venneti et. al. presented the first three-dimensional
solution structure of GLP-2 by nuclear magnetic resonance (NMR) (Venneti
et al. 2011). This structure supported the distinct roles of the N- and
C-terminus part of GLP-2 and revealed a stable alpha-helical
conformation at the central region (between Phe6 and Ile27) and a less
well-defined helical conformation in the C-terminus region. The binding
interface with the extracellular domain (ECD) of the receptor was
predicted to be between Leucine17 and Lysine30, while the N-terminus
part of GLP-2 from Histiine1 to Aspargine16 lacked contact with the
extracellular domains of the GLP-2R. The central roles of the N- and
C-terminus part of GLP-2 in respectively, receptor activation and
receptor binding were supported by Yamazaki et. al. in 2013 (Yamazaki et
al. 2013), showing a decreased intrinsic placental alkaline phosphatase
(PALP) activity (driven by cAMP) for GLP-2(3-33), (6-33) and (11- to
13-33). Most recently, Wisniewski et. al. replaced each residue in the
DPP-4 resistant {[}Gly2,Nle10{]}hGLP-2(1-30) analog with its
d-enantiomer in a systematic approach to gain insight into the GLP-2R
recognition revealing a loss of potency at position 5, 8, 9, 12, and 14
in the N-terminus, and similar loss for position 17-20, 25, and 29 in
the C-terminus (Wisniewski et al. 2016). Consistent with this, the
C-terminal of GLP-2 orientates towards a hydrophilic cavity in the NMR
structure (Venneti et al. 2011). Thus, the N-terminal part of GLP-2
plays a central role in receptor activation, while the C-terminus adopts
an alpha-helical conformation that plays a central role of receptor
binding of GLP-2 consistent with the suggested ``two-step'' activation
model of class B1 GPCRs, a model that is now much more refined (Liang et
al. 2018; Qiao et al. 2020; Wu et al. 2020; Zhang et al. 2018; Zhang et
al. 2017; Zhao et al. 2020).

The M10Y-modification barely changed the functional properties of the
two endogenous hGLP-2 variants, demonstrating, in agreement with the
model discussed above, that the Met10 of GLP-2 neither plays an
important role in ligand binding nor receptor activation. According to
the NMR structure, Met10 is positioned at the beginning of the
alpha-helix and is not part of the binding interface of the GLP-2R
(Venneti et al. 2011). Consistent with this, Wisniewski et. al. replaced
the oxidation and alkylation-prone Met residue at position 10 of hGLP-2
by the isosteric Nor-leucine (Nle) (Wisniewski et al. 2016). Met is
characterized by a sulfur atom in the sidechain, which is highly
sensitive to reactive oxygen species (ROS) that often changes structural
and functional properties of proteins (Black et al. 1991; Kim et al.
2014). ROS-mediated oxidation occurs by the addition of a single oxygen
molecule to the sulfur atom, forming methionine sulfoxide (MetSO) (Kim
et al. 2014), which creates a chiral center around the sulfur atom and
overall results in a stiffer and more polar side chain compared to the
unoxidized Met residue (Black et al. 1991). These changes can have
profound structural and functional consequences (Chao et al. 1997; Hoshi
et al. 2001; Gu et al. 2015; Sugamura et al. 2011). To protect for
oxidative damage of the Met in GLP-2 during the oxidative iodination,
and since Met is dispensable for GLP-2 function (Drucker et al. 2013;
Venneti et al. 2011; Wisniewski et al. 2016; Yamazaki et al. 2013), we
replaced Met10 with a Tyr residue. Thereby we created a target site for
oxidative iodination using {[}\textsuperscript{125}I{]} in the full
agonist (GLP-2(1-33)) and in the antagonist and partial agonist
(GLP-2(3-33)). These modifications created the two peptides;
hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y). These two M10Y-substituted
peptides acted as their wildtype counterparts, and with these, we were
in a unique position allowing us to investigate both agonist
{[}I\textsuperscript{125}{]}-hGLP-2(1-33,M10Y) and antagonist
{[}I\textsuperscript{125}{]}-hGLP-2(3-33,M10Y) binding.

The similar affinities (K\textsubscript{D}) support the main role of the
N-terminus in receptor activation and not in receptor binding (Couvineau
et al. 2011). Moreover, the higher Bmax for the antagonist follows the
general trend for more antagonist binding conformations versus agonist
conformations of GPCRs ( Rosenkilde et al. 1994). Interestingly, for the
first time among class B1 GPCRs, we describe the binding kinetics of a
peptide agonist in comparison with a peptide antagonist and show, that
the on-rate for the antagonist is significantly faster than for the
agonist. Binding kinetics parameters, including k\textsubscript{on} and
k\textsubscript{off}, have been highlighted to be more important in
describing a ligand's \emph{in vivo} efficacy and the onset of action,
than the classical parameters such as K\textsubscript{D} and
K\textsubscript{I} (Velden et al. 2020). The slower on-rate for the
agonist could reflect a more complex binding compared to the antagonist
in line with expected induction of active receptor states (Zhang et al.
2018). When comparing the apparent affinities for the agonists and the
antagonists obtained in competition with two radioligands, we observed
similar affinities irrespective of choice of radioligand. This suggests
that all four ligands (initially) interact similarly with the ECDs of
the hGLP-2R, and that the receptor easily interchanges between
(sequential) conformations induced by the agonist and the antagonist.

The location of the GLP-2R remains controversial in both rodent and
humans. It has been reported that the GLP-2R mRNA transcript and protein
is expressed in SEMFs (El-Jamal et al. 2014; \selectlanguage{ngerman}Ørskov et al. 2005). Here
we confirm receptor expression at the protein level in SEMFs in the
intestine and in the pancreatic islet cells of mice by using the
agonistic radioligand {[}I\textsuperscript{125}{]}-hGLP-2(1-33,M10Y).
The prevention of {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y)
labeling by co-injection with excess amounts of unlabeled
hGLP-2(1-33,M10Y) demonstrates the specificity of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) binding. The strong
staining of the pancreatic islet cells by
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) could result from GLP-2R
expression in the pancreatic islet cells, in agreement with what was
previously shown at the mRNA level (De Heer et al. 2007). Alternatively,
it could result from cross-interaction of GLP-2 with the GLP-1R, or a
combination of the two. The strong binding properties of both
radioligands to the mGLP-1R, and the low potency activation of the
hGLP-1R by GLP-2(1-33) and GLP-2(1-33, M10Y), demonstrate that the
pancreatic staining could be a result of GLP-1R binding. Also,
promiscuity is known within GPCRs, demonstrated by the activation of the
GIPR by GLP-2 (Skov-Jeppesen et al. 2019), the binding and activation of
both the GLP-1R and the GCGR by oxyntomodulin (Holst et al. 2018;
Jorgensen et al. 2007), and the activation of the mGLP-1R by glucagon
(Svendsen et al. 2018). Thus, cross-activation is a common phenomenon
within class B1 GPCRs, which is reflected in the high sequence
similarities observed among the receptors and across species. For rodent
GLP-2R's, 81\% and 79\% sequence identities are found for the mGLP-2R
and rGLP-2R, respectively, explaining the high-affinity binding observed
for both radioligands to the rodent GLP-2R's. \selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}\selectlanguage{english}

As we observed no binding of either hGLP-2 radioligand to the rGLP-2R,
future autoradiography studies in rats would eliminate the binding of
GLP-2 to the GLP-1R. Another possibility would be to use GLP-1R
knock-out (KO)-mice, or eliminate hGLP-1R binding by modifications of
GLP-2 at the C-terminus, as suggested recently in study where
replacement of position 11 and/or 16 of hGLP-2(1-30) eliminated hGLP-1R
actively, while retaining high hGLP-2R activity (Wisniewski et al.
2016). Thus, it is possible to decrease GLP-2 binding to the GLP-1R
without compromising the GLP-2R binding.

\textbf{CONCLUSION}

We have developed two new radioligands (an agonist and an antagonist)
for the GLP-2R; both with high affinity to the human, rat and mouse
GLP-2R. With these, we show differential binding kinetics of agonist and
antagonist to the GLP-2R, and confirm GLP-2R expression at the protein
level in the GI tract's SEMFs and in the pancreatic islet cells.
Moreover, we demonstrate cross-activity with -binding and -activity of
GLP-2 within the GLP-1R system. These observations are of importance for
tissue localization and structural characterization for not only the
GLP-2R, but also for other class B1 GPCRs.

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\textbf{TABLE 1}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Table 1: cAMP accumulation\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Table 1: cAMP accumulation\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Table 1: cAMP accumulation\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Table 1: cAMP accumulation\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Table 1: cAMP accumulation\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Table 1: cAMP accumulation\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Ligand}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{pEC\textsubscript{50}} \selectlanguage{ngerman}\textbf{(M) ± SEM}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{EC50} \textbf{{[}nM{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\selectlanguage{ngerman}\textbf{Efficacy ± SEM}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
n\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2R\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9.7 \selectlanguage{ngerman}± 0.06\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.22\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
100 \selectlanguage{ngerman}± 2.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{10}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(1-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9.3 \selectlanguage{ngerman}± 0.06\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
96 \selectlanguage{ngerman}± 2.9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{4}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(3-33)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7.6 \selectlanguage{ngerman}± 0.28\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
27.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
11 \selectlanguage{ngerman}± 1.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7.5 \selectlanguage{ngerman}± 0.32\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
31.6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.1 \selectlanguage{ngerman}± 1.2\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{7}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2 + 100 nM hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.9 \selectlanguage{ngerman}± 0.15\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.4\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
93 \selectlanguage{ngerman}± 12*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2 + 1 \selectlanguage{greek}µ\selectlanguage{english}M hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.4 \selectlanguage{ngerman}± 0.20\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
4.3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
78 \selectlanguage{ngerman}± 7.7*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-1R\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-1\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
11 \selectlanguage{ngerman}± 0.04\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.03\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
100 \selectlanguage{ngerman}± 1.3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{9}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7.0 \selectlanguage{ngerman}± 0.12\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
98\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
88 \selectlanguage{ngerman}± 6.0\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{7}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(1-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7.0 \selectlanguage{ngerman}± 0.13\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
112\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
100 \selectlanguage{ngerman}± 8.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(3-33)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{N.A.}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{N.A.}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{N.A.}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{6}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{N.A.}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{N.A.}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{N.A.}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{6}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-2 +100nM exedin(9-39)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6.1 \selectlanguage{ngerman}± 0.83\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
881\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
31 \selectlanguage{ngerman}± 8,9\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-1 +1\selectlanguage{greek}µ\selectlanguage{english}M hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10 \selectlanguage{ngerman}± 0.14\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.07\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
100 \selectlanguage{ngerman}± 5.5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
hGLP-1 +100mM hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10 \selectlanguage{ngerman}± 0.22\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.05\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
100 \selectlanguage{ngerman}± 8.8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 1. cAMP accumulation table values.} Signaling values for
hGLP-2R and hGLP-1R in response GLP-2 and GLP-2 variants in cAMP
accumulation assays. All data were fitted with a three-parameter
logistic curve to obtain pEC\textsubscript{50}. Data represents the mean
\selectlanguage{ngerman}± SEM of at least three independent experiments performed in duplicates.
N.A. refers to no activation detected. *as saturation were not obtained
Emax ± SEM at 10nM hGLP-2(1-33) is given.

\textbf{TABLE 2}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Table 2: Competition binding\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{{[}\textsuperscript{125}I{]}-hGLP-2(1-33.M10Y)}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{{[}\textsuperscript{125}I{]}-hGLP-2(1-33.M10Y)}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{{[}\textsuperscript{125}I{]}-hGLP-2(1-33.M10Y)}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y)}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y)}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y)}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{Ligand}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{pEC\textsubscript{50}} \selectlanguage{ngerman}\textbf{(M) ± SEM}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{EC50} \textbf{{[}nM{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
n\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{pEC\textsubscript{50}} \selectlanguage{ngerman}\textbf{(M) ± SEM}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{EC50} \textbf{{[}nM{]}}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
n\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.9 \selectlanguage{ngerman}± 0.12\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
14\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.8 \selectlanguage{ngerman}± 0.09\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
15\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.2 \selectlanguage{ngerman}± 0.07\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
59\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.1 \selectlanguage{ngerman}± 0.13\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
78\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{6}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(3-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.2 \selectlanguage{ngerman}± 0.08\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
57\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.1 \selectlanguage{ngerman}± 0.13\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
81\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{6}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(3-33,M10Y)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.3 \selectlanguage{ngerman}± 0.10\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
49\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.4 \selectlanguage{ngerman}± 0.13\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
41\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{5}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
mGLP-2R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8.2 \selectlanguage{ngerman}± 0.13\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.6\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7.7 \selectlanguage{ngerman}± 0.16\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
18\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
rGLP-2R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8.3 \selectlanguage{ngerman}± 0.24\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
5.4\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8.2 \selectlanguage{ngerman}± 0.18\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-1R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.9 \selectlanguage{ngerman}± 0.50\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
130\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.5 \selectlanguage{ngerman}± 0.5\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{330}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
mGLP-1R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.7 \selectlanguage{ngerman}± 0.22\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
208\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6.7 \selectlanguage{ngerman}± 0.14\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{183}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
rGLP-1R\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
hGLP-2(1-33)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{N.B.}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{N.B.}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{N.B.}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{N.B.}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\emph{3}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 2. Competitive binding values.} Binding values
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) and
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) displaced by increasing
concentrations of GLP-2 and GLP-2 variants. All data were fitted with a
three-parameter logistic curve to obtain pIC\textsubscript{50}. Data
represents the mean \selectlanguage{ngerman}± SEM of at least three independent experiments
performed in duplicates. N.B. refers to no binding detected.

\textbf{FIGURE LEGENDS}

\textbf{Figure 1. Sequence alignment of GLP-2 and related peptides and
activity of hGLP-2 and variants at the hGLP-2R.} (a) Alignment of the
class B1 GPCR peptides; hGLP-2(1-33), hGIP(1-42), hGCG(1-29),
hGLP-1(7-36) (top panel) and the GLP-2 variants; hGLP-2(3-33),
hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y) (bottom panel). In the top
panel, dark grey refers to positions, which are fully conserved
(identical), medium grey refers to positions with strongly similar
residues, while light grey refers to positions with weakly similar
residues. The red box marks position 10 (counted from residue 1 of
hGLP-2(1-33)). (b-d) cAMP accumulation dose-response curve for hGLP-2R
stimulated with increasing concentration of (b) hGLP-2(1-33) \emph{(n =
10)} and hGLP-2(1-33,M10Y) \emph{(n =4)} , (c) hGLP-2(3-33) \emph{(n =
3)} and hGLP-2(3-33,M10Y) \emph{(n = 7)} , and (d) hGLP-2(1-33) in the
presence of 100 mM and 1 \selectlanguage{greek}µ\selectlanguage{english}M hGLP-2(3-33,M10Y) \emph{(n = 3)} . To
compensate for inter-assay variations, data have been normalized to
hGLP-2(1-33) within each experiment. The experiments were carried out in
duplicates and presented as mean \selectlanguage{ngerman}± SEM.

\textbf{Figure 2. Homologous competition binding and binding kinetic
experiments.} (a, b) Homologous binding curve using (a)
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) \emph{(n = 5)}and
(b) {[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red) \emph{(n = 5)}
. To compensate for inter-assay variations, the data have been
normalized to the specific binding to hGLP-2R within each assay. (c)
B\textsubscript{max} for {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y)
(black) and {[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red),
normalized to Bmax of {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y).
(d) Association \emph{(n = 4)} and (e) dissociation \emph{(n = 4)} of
{[}\textsuperscript{125}I{]}-hGLP-2(1--33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3--33,M10Y) (red) on/from hGLP-2R.
The dissociation was initiated by the addition of 1 \selectlanguage{greek}μ\selectlanguage{english}M unlabeled
hGLP-2(1--33,M10Y) or hGLP-2(3--33,M10Y). (f) Comparison of binding
kinetic parameters between
{[}\textsuperscript{125}I{]}-hGLP-2(1--33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3--33,M10Y) (red) obtained from
association and dissociation assays. To compensate for inter-assay
variations data have been normalized for each radioligand within each
assay. Differences were analyzed by paired t-test and significance
indicated by asterisks, **** p \textless{} 0.0001, *** p \textless{}
0.001, ** p \textless{} 0.01 and *p \textless{} 0.05. ns indicates
non-significant differences. The experiments were carried out in
duplicated and presented as mean \selectlanguage{ngerman}± SEM.

\textbf{Figure 3. Heterologous competition binding using radiolabeled
hGLP-2(1-33,M10Y) and hGLP-2(3-33,M10Y).} (a) Bar chart of the
pIC\textsubscript{50} values for binding of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red). (b-e) Competition
binding of {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red) displaced by
increasing concentrations of (b) hGLP-2(1-33) \emph{(n = 5)} , (c)
hGLP-2(1-33,M10Y) \emph{(n = 5 for
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) and n = 6 for
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y))} , (d)
hGLP-2(3-33)\emph{(n = 5 for
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) and n = 6 for
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y))} , and (e)
hGLP-2(3-33,M10Y) \emph{(n = 5)} . To compensate for inter-assay
variations the data were normalized to the specific binding of hGLP-2R
for each radioligand within each assay. Differences were analyzed by
paired t-test and significance indicated by asterisks, **** p
\textless{} 0.0001, *** p \textless{} 0.001, ** p \textless{} 0.01 and
*p \textless{} 0.05. ns indicates non-significant differences. The
experiments were carried out in duplicated and presented as mean ± SEM.

\textbf{Figure 4. Test for selectivity among class B1 GPCRs.} (a)
Phylogenetic tree of the class B1 subfamily GPCRs consisting of the
GLP-2R and 14 sequence related GPCRs (modified from (Gasbjerg et al..
2018)). (b) Heterologous binding of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red) to the
hGLP-1R\emph{(n = 3)} , hGIPR \emph{(n = 2)} , hGCGR \emph{(n = 2)} ,
hSecretinR\emph{(n = 2)} , VPAC-1R \emph{(n = 2)} and VPAC-2R \emph{(n =
2)}displaced by increased concentrations of endogenous hGLP-2(1-33). The
experiments were carried out in duplicated and presented as mean ± SEM.

\emph{See supplementary figure 1 for binding curves.}

\textbf{Figure 5. Binding of the two radioligands to rodent GLP-2Rs and
autoradiography} . (a, b) Competition binding of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red) to (a) the
mGLP-2R\emph{(n = 3)} and (b) the rGLP-2R \emph{(n = 3)} displaced by
increasing concentrations of endogenous hGLP-2(1-33). To compensate for
inter-assay variations the data were normalized to the specific binding
of respectively mGLP-2R and rGLP-2R for each individual radioligand
within each assay. The experiments were carried out in duplicated and
presented as mean ± SEM. Histological sections of the small intestine
(c, e) and endocrine pancreas (d, f) after autoradiography in mice
injected with (c, d) {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y), and
(e, f) {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) plus excess amount
of unlabeled hGLP-2(1-33,M10Y). The histological sections were
counterstained with hematoxylin. Scale bar 50\selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Figure 6. cAMP accumulation mediated by hGLP-2 variants at the
hGLP-1R.} cAMP accumulation for the hGLP-1R stimulated with increased
concentration of hGLP-1 \emph{(n = 9)} and, (a) hGLP-2(1-33) \emph{(n =
7)} and hGLP-2(1-33,M10Y) \emph{(n = 3)} , (b) hGLP-2(3-33) \emph{(n =
6)} and hGLP-2(3-33,M10Y) \emph{(n = 6)} , (c) hGLP-2(1-33) in the
presence of 100nM exendin(9-39) \emph{(n = 5)} , and (d) hGLP-1 in the
presence of 1\selectlanguage{greek}µ\selectlanguage{english}M hGLP-2(3-33,M10Y) and 100nM hGLP-2(3-33,M10Y) \emph{(n =
3)} for the hGLP-1R. To compensate for inter-assay variations data have
been normalized to hGLP-1 within each separate experiment. The
experiments were carried out in duplicated and presented as mean \selectlanguage{ngerman}± SEM.

\textbf{Supplementary figure 1. Test for selectivity among class B1
GPCRs.} (a-f) Competition binding curves of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red) to (a)
hGLP-1R\emph{(n = 3)} , (b) hGIPR \emph{(n = 2)} , (c) hGCGR \emph{(n =
2)} , (d) hSecretinR \emph{(n = 2)} , (e) VPAC-1R \emph{(n = 2)} , and
(f) VPAC-2R\emph{(n = 2)} displaced by increasing concentrations of
endogenous hGLP-2(1-33). To compensate for inter-assay variations data
have been normalized for each individual radioligand within each assay.
The experiments were carried out in duplicated and presented as mean ±
SEM.

\textbf{Supplementary figure 2. Autoradiography in mice using
{[}125I{]}-hGLP-2(1-33,M10Y).} Histological sections of the small
intestine (a-f) and pancreatic islet cells (g-l) after autoradiography
in mice, injected with {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y)
(a-c and g-i) or {[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) plus
excess amount of unlabeled hGLP-2(1-33,M10Y) (d-f and j-l). The
histological sections were counterstained with hematoxylin. Scale bar
50\selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Supplementary figure 3. Binding of the two hGLP-2 radioligands
to rodent GLP-1Rs.} (a, b) Competition binding of
{[}\textsuperscript{125}I{]}-hGLP-2(1-33,M10Y) (black) and
{[}\textsuperscript{125}I{]}-hGLP-2(3-33,M10Y) (red) to (a) the
mGLP-1R\emph{(n = 3)} and (b) the rGLP-1R \emph{(n = 3)} displaced by
increasing concentrations of endogenous hGLP-2(1-33). To compensate for
inter-assay variations the data were normalized to the specific binding
of, respectively, mGLP-1R and rGLP-1R for each individual radioligand.
The experiments were carried out in duplicated and presented as mean \selectlanguage{ngerman}±
SEM.

\textbf{Hosted file}

\verb`manuscript figures.docx`  available at \url{https://authorea.com/users/352361/articles/476666-development-of-high-affinity-agonist-and-antagonist-radioligands-for-the-glp-2-receptor-powerful-tools-for-the-study-of-glp-2-pharmacology}

\selectlanguage{english}
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