Figure 6.

hGLP-2R endocytosis is inhibited by disruption of lipid raft integrity. DLD-1(A) or BHK (B) cells transiently transfected with the FLAG-hGLP-2R were pretreated with either 4 μg/ml filipin, 10 mM MβCD, or vehicle for 30 min before and during a 20-min incubation with media alone (control) or 10 nM hGLP-2. Cell surface receptor levels were quantified by enzyme-linked immunoassay as described in MATERIALS AND METHODS. Data (means ± SEM of 3 independent experiments each performed in triplicate or quadruplicate) are expressed relative to control and compared with vehicle-treated cells (^***p < 0.001). The normalized control value represents cell surface receptor levels in cells exposed to each of the indicated inhibitors or vehicle, but not to agonist. (B) After a 30-min inhibitor treatment at 37°C, transfected BHK cells were incubated with anti-FLAG antibody on ice for 1 h in the presence of vehicle, filipin, or MβCD, as described in MATERIALS AND METHODS. After a wash with PBS, cells were treated with media alone (control) or 10 nM hGLP-2 at 37°C for 20 min in the absence (vehicle) or presence of inhibitors. After fixation and permeabilization, the receptor/anti-FLAG antibody complexes were visualized by confocal microscopy using a Cy3-conjugated secondary antibody.