FIGURE 5.

Confocal microscopy evidence for an involvement of dynamin, clathrin, and β-arrestins in CCK-stimulated CCK2R internalization. A, HEK cells Flp-In^TM CCK2R-293 were incubated with Alexa F 647-CCK (0.1 μm) in the presence of the dynamin inhibitor (dynasore, 80 μm) or the inhibitor of clathrin-coated pit formation (chlorpromazine, 50 μm). B, Flp-In^TM CCK2R-293 cells transfected with β-arrestin1-GFP or β-arrestin2-GFP were stimulated with CCK (0.1 μm) for the times indicated. Images show rapid translocation of β-arrestin2-GFP from cytosol to plasma membrane later followed by the appearance of fluorescence-labeled vesicles. C, measurement of the decrease of cytosolic fluorescence showing that β-arrestin1- or β-arrestin2-GFP recruitment to the plasma membrane was rapid and identical (50% of decrease at 200 s). D, Flp-In^TM CCK2R-293 cells were transfected with dominant negative β-arrestin1-GFP and/or β-arrestin2-GFP. Confocal microscopy images show that overexpression of dominant negative β-arrestin1 or -2 significantly altered CCK2R internalization, and expression of both β-arrestin1 and -2 blocked internalization in HEK cells. Images are representative of at least three separate experiments.