N-terminal alterations turn the gut hormone GLP-2 into an antagonist with gradual loss of GLP-2 receptor selectivity towards more GLP-1 receptor interaction.

BACKGROUND AND PURPOSE: To fully elucidate the regulatory role of the GLP-2 system in the gut and the bones, potent and selective GLP-2 receptor (GLP-2R) antagonists are needed. Searching for antagonist activity, we performed systematic N-terminal truncations of human GLP-2(1-33). EXPERIMENTAL APPROACH: COS-7 cells were transfected with the human GLP-2R and assessed for cAMP accumulation or competition binding using 125 I-GLP-2(1-33)[M10Y]. To examine selectivity, COS-7 cells expressing human GLP-1 or GIP receptors were assessed for cAMP accumulation. KEY RESULTS: Affinity of the N-terminally truncated GLP-2 peptides for the GLP-2 receptor decreased with reduced N-terminal peptide length (Ki 6.5-871 nM), while increasing antagonism appeared with inhibitory potencies (IC50 ) values from 79 to 204 nM for truncation up to GLP-2(4-33) and then declined. In contrast, truncation-dependent increases in intrinsic activity were observed from an Emax of only 20% for GLP-(2-33) up to 46% for GLP-2(6-33) at 1 µM, followed by a decline. GLP-2(9-33) had the highest intrinsic efficacy (Emax 65%) and no antagonistic properties. Moreover, with truncations up to GLP-2(8-33), a gradual loss in selectivity for the GLP-2 receptor appeared with increasing GLP-1 receptor (GLP-1R) inhibition (up to 73% at 1 µM). Lipidation of the peptides improved antagonism (IC50 down to 7.9 nM) for both the GLP-2 and the GLP-1R. CONCLUSION AND IMPLICATIONS: The N-terminus of GLP-2 is crucial for GLP-2R activity and selectivity. Our observations form the basis for the development of tool compounds for further characterization of the GLP-2 system.