Figure 4

Representative photomicrographs and FACS analysis illustrating receptor and agonist dependent internalization. (A) The cells displayed colocalization at the membrane in non-permeabilized and intracellularly in permeabilized cells with distinct populations of hSSTR5 (green) and β₂AR (red). Following treatment with SST, β₂AR remain unchanged while hSSTR5 displayed agonist induced internalization. Upon activation with formoterol, β₂AR internalized as indicated by the loss of β₂AR and colocalization with hSSTR5 at cell surface which is accompanied with the increased intracellular expression. Combined treatment with SST and formoterol displayed colocalization between hSSTR5 and β₂AR. In all representative photographs DAPI in blue color indicates nuclear staining. Scale bar = 10 μm. (B) BD LSRII flow cytometer configured with a 488 nm and 561 nm laser was used for the experiments. Cy3- (β₂AR expression, panels b, d, f and h) was monitored using the 561 nm laser and a 610/20 emission filter whereas 488 nm laser was used to detect SSTR5 expression (FITC, panels a, c, e and g) and the emission was detected using a 530/30 filter. Cells were processed as described in materials and methods section. Note the left side shifts in the FITC and Cy3 expression (Panels c and f) indicating agonist dependent receptor specific internalization. No significant change in the expression pattern for the receptors was observed upon combined activation with both the ligands (Panels g and h).