cAMP measurements
For the cAMP measurements, COS-7 cells were transfected with receptor plasmid or pcDNA3.1(+) and seeded with 25 000 cells per well in a CulturPlate-96 (PerkinElmer; Waltham, MA) one day after transfection. The following day, the cells were washed once with HEPES-buffered saline and incubated for 30 min at 37°C with HEPES-buffered saline supplemented with 1mmol/l 3-isobutyl-1-methylxanthine (IBMX) buffer. To test for intrinsic activity, endogenous hGLP-2(1-33) or hGLP-2(3-33) or the M10Y-substituted variants (hGLP-2(1-33,M10Y) or hGLP-2(3-33,M10Y)) were added in increasing concentrations, and the plates were incubated for additional 30 min at 37°C. To test hGLP-2(3-33) and hGLP-2(3-33,M10Y) as antagonists the cells were preincubated for 10 min with a fixed concentration of antagonist followed by the addition of increasing concentrations of agonist. Afterwards, the HitHunter cAMP XS-assay (DiscoverX, Birmingham, UK) was carried out according to the manufacturer’s instructions. All experiments were made in duplicates, and luminescence was measured by a Perkin Elmer EnVision 2104 Multilabel reader (PerkinElmer; Waltham, MA).