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.