Kinetic binding experiments
The association assays were performed by preparing a mixture of 10 µg hGLP-2R or pcDNA3.1(+) and 0.5 mg wheatgerm agglutinin coated (WGA) PVT SPA beads (PerkinElmer; Waltham, MA). This mixture was pre-coupled on a shaker in a total volume of 50 µL binding buffer (50 mM HEPES buffer (pH 7.2)), supplemented with 1 mM CaCl2, 5 mM MgCl2 and 0,5% (w/v) BSA) for 30 min at 30°C. The pre-coupling was followed by the distribution of membrane suspension in a CulturPlate-96 (PerkinElmer; Waltham, MA) in a total volume of 90 µL binding buffer and spun down afterwards (1 500 RPM, 485 g, 5 min, room temperature). The reaction was initiated by the addition of 0.19 ± 0.001 nM [125I]-hGLP-2(1-33,M10Y) or 0.21 ± 0.004 nM [125I]-hGLP-2(3-33,M10Y), and the amount of radioligand bound to receptor was measured every minute up to 100 min [125I]-hGLP-2(3-33,M10Y) or 120 min [125I]-hGLP-2(1-33,M10Y) at 30°C, using a TopCount NXT Microplate Scintillation & Luminescence Counter (PerkinElmer; Waltham, MA).
For the dissociation assays, the membrane suspension was distributed to the wells in a total volume of 85 µL binding buffer. The mixture was then pre-incubated for 60 min at 30°C after addition of 0.19 ± 0.001 nM [125I]GLP-2 [125I]-hGLP-2(1-33,M10Y) or 0.21 ± 0.004 nM [125I]-hGLP-2(3-33,M10Y). The dissociation was initiated by the addition of 5 µL of 1 µM unlabeled hGLP-2(1-33,M10Y) or hGLP-2(3-33,M10Y). The amount of receptor-bound radioligand was measured every minute up to 500 min.