Figure 9.

Inhibition of endosomal acidification prevents receptor recycling and resensitization. (A) DLD-1 cells transiently transfected with FLAG-hGLP-2R were pretreated with vehicle or 25 μM monensin for 30 min before and during assessment of hGLP-2R internalization and cell surface reappearance as described in Figure 4. Data (means ± SD of triplicate values) are expressed as percentage of the cell surface receptor level at time 0. ^*p < 0.05, ^***p < 0.001, vehicle vs. monensin treated. Inset, total cellular receptor protein following the hGLP-2 washout period (200 min) in vehicle- (open bar) and monensin (filled bar)-treated cells. (B) hGLP-2R resensitization, as described in Figure 8, was performed in the absence (vehicle) and presence of monensin. Data (means ± SD of triplicate values) are representative of three independent experiments and are expressed relative to their corresponding control. Statistical comparisons between recovery time and corresponding control values are indicated with asterisks (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). Statistical significance between recovery time points in the presence or absence of inhibitors is represented by ^### p < 0.001. (C) After a 30-min treatment with vehicle or monensin at 37°C, transfected BHK cells were incubated with anti-FLAG antibody on ice for 1 h media containing vehicle or monensin, as described in MATERIALS AND METHODS. Cells were treated with either media alone (control) or 10 nM hGLP-2 at 37°C for 20 min, washed, and allowed to recover for 0 or 2 h at 37°C in the absence (vehicle, top) or presence of monensin (bottom). After fixation and permeabilization, the receptor/anti-FLAG antibody complexes were visualized by confocal microscopy using a Cy3-conjugated secondary antibody.