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).