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).