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
[125I]-hGLP-2(3-33,M10Y) (96,6
fmol/105) compared to the agonist
[125I]-hGLP-2(1-33,M10Y) (58,0
fmol/105) (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
(kon) and dissociation (koff) 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
[125I]-hGLP-2(1–33,M10Y) was reached at around 60
min, whereas for [125I]-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 ~3 fold higher
kobs for [125I]-hGLP-2(3–33,M10Y)
(0.076 ± 0.009 min-1) compared to
[125I]-hGLP-2(1–33,M10Y) (0.027 ± 0.003
min-1). After reaching equilibrium, the binding was
reversed by the addition of 1 µM unlabeled hGLP-2(1–33,M10Y) and
hGLP-2(3–33,M10Y), respectively (figure 2e). The two ligands had
similar dissociation rates (koff) of 0.009 ± 0.004
min-1 and 0.010 ± 0.002 min-1,
respectively. Finally, we calculated the on-rate (kon)
of both radioligands, and found a ~3.5 fold higher
on-rate for the antagonist (0.329 ± 0.047
nM-1*min-1), compared to the agonist
(0.094 ± 0.014 nM* min-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.