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
[125I]. 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
[125I]-labeling of hGLP-2 At position 10 in hGIP
and hGCG, a Tyr residue is found, which is the target for oxidative
[125I]-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 µM) 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).