Novel agonist and antagonist radioligands for the GLP-2 receptor. Useful tools for studies of basic GLP-2 receptor 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 DPP-4, forming GLP-2(3-33), having low intrinsic activity and competitive antagonism properties at GLP-2 receptors. We created radioligands 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) enabling 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. Receptor expression was determined by target-tissue autoradiography and immunohistochemistry.

Key results: Both M10Y-substituted peptides induced cAMP production via the GLP-2 receptor comparable to the wildtype peptides. GLP-2(3-33,M10Y) maintained the antagonistic properties of GLP-2(3-33). However, hGLP-2(1-33,M10Y) had lower arrestin recruitment than hGLP-2(1-33). High affinities for the hGLP-2 receptor were observed using [¹²⁵ I]-hGLP-2(1-33,M10Y) and [¹²⁵ I]-hGLP-2(3-33,M10Y) with K_D values of 59.3 and 40.6 nM. The latter (with antagonistic properties) had higher B_max and faster on and off rates compared to the former (full agonist). Both bound the hGLP-1 receptor with low affinity (K_i of 130 and 330 nM, respectively). Autoradiography in wildtype mice revealed strong labelling of subepithelial myofibroblasts, confirmed by immunohistochemistry using a GLP-2 receptor specific antibody that in turn was confirmed in GLP-2 receptor knock-out mice.

Conclusion and implications: Two new radioligands with different binding kinetics, one a full agonist and the other a weak partial agonist with antagonistic properties were developed and subepithelial myofibroblasts identified as a major site for GLP-2 receptor expression.