Novel agonist- and antagonist-based radioligands for the GLP-2 receptor - useful tools for studies of basic GLP-2R pharmacology

BACKGROUND: Glucagon-like peptide-2(GLP-2) is a pro-glucagon-derived hormone secreted from intestinal enteroendocrine L-cells with actions on gut and bones. GLP-2(1-33) is cleaved by the enzyme DPP-4, forming GLP-2(3-33) which has low intrinsic activity and competitive antagonistic properties on the GLP-2 receptor (GLP-2R). We created radioligands with different pharmacodynamic profiles based on these two molecules.

EXPERIMENTAL APPROACH: The methionine in position 10 of GLP-2(1-33) and GLP-2(3-33) was substituted with tyrosine(M10Y) to enable oxidative iodination, creating [125 I]-hGLP-2(1-33,M10Y) and [125 I]-hGLP-2(3-33,M10Y). Both were characterized by competition binding, on-and-off-rate determination and receptor activation (using the unlabeled peptides). Receptor expression was determined by target-tissue autoradiography and immunohistochemistry.

KEY RESULTS: Both M10Y-substituted peptides induced cAMP production via the GLP-2R comparable to the wildtype peptides, and GLP-2(3-33,M10Y) maintained the antagonistic properties of GLP-2(3-33). However, lower arrestin recruitment was observed for hGLP-2(1-33,M10Y) compared to hGLP-2(1-33). High affinities for the hGLP-2R were observed using [125 I]-hGLP-2(1-33,M10Y) and [125 I]-hGLP-2(3-33,M10Y) with KD values of unlabeled homologous peptides of 59.3nM and 40.6nM, however with higher Bmax , and faster on- and-off-rate for the latter (with antagonistic properties) compared to the former (full agonist). Moreover, both bound the hGLP-1R with low affiinity (Ki of 130nM and 330nM, respectively). Autoradiography in wildtype mice revealed strong labeling of subepithelial myofibroblasts (SEMF), confirmed by immunohistochemistry using a GLP-2R specific antibody. Specificity was confirmed in GLP-2R knock-out mice.

CONCLUSION AND IMPLICATIONS: We successfully developed two new radioligands and identified SEMF as a major site for GLP-2R expression. Moreover, we showed different binding kinetics of full agonist versus weak partial agonist with antagonistic properties.