Visualization of ligand-induced dopamine D2S and D2L receptor internalization by TIRF microscopy

G protein-coupled receptors (GPCRs), including the dopamine receptors, represent a group of important pharmacological targets. Upon agonist binding, GPCRs frequently undergo internalization, a process that is known to attenuate functional responses upon prolonged exposure to agonists. In this study, internalization was visualized by means of total internal reflection fluorescence (TIRF) microscopy at a level of discrete single events near the plasma membrane with high spatial resolution. A novel method has been developed to determine the relative extent of internalized fluorescent receptor-ligand complexes by comparative fluorescence quantification in living CHO cells. The procedure entails treatment with the reducing agent sodium borohydride, which converts cyanine-based fluorescent ligands on the membrane surface to a long-lived reduced form. Because the highly polar reducing agent is not able to pass the cell membrane, the fluorescent receptor-ligand complexes located in internalized compartments remain fluorescent under TIRF illumination. We applied the method to investigate differences of the short (D2S) and the long (D2L) isoforms of dopamine D2 receptors in their ability to undergo agonist-induced internalization.