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.