Abstract
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.
Highlights
G protein-coupled receptors (GPCRs) represent a large family of integral membrane proteins and their primary function is to transduce extracellular stimuli (e.g ligand binding) into intracellular signals[1]
Β-arrestin recruitment leads to receptor internalization, which generally proceeds by clathrin-coated pits or other mechanisms of endocytosis[14,15,16], in which the receptors are translocated away from the plasma membrane to lager intracellular vesicular or endosomal structures
Initial total internal reflection fluorescence (TIRF) microscopy experiments in this study showed that receptor-ligand complexes of the fluorescent antagonists 1a and 1b bound to dopamine D2S or D2L receptors stably expressed in CHO cells (10 nM, for 1 h at 37 °C) were visible under TIRF illumination as individual, freely diffusing, diffraction limited spots
Summary
G protein-coupled receptors (GPCRs) represent a large family of integral membrane proteins and their primary function is to transduce extracellular stimuli (e.g ligand binding) into intracellular signals[1]. These receptors play fundamental roles in many physiological and pharmacological processes and serve as important drug targets which are addressed by more than 30% of the current drugs on the market[2]. Fluorescence microscopy facilitates a novel way to study ligand binding and the subsequent internalization process of GPCRs in living cells, using fluorescence-based probes such as fluorescent ligands[30]. Total internal reflection fluorescence (TIRF) microscopy has proved useful to study internalization because of its ability to selectively detect both fluorescent molecules situated within or in close proximity (~100 nm) to the plasma membrane with high spatial and temporal resolution[18, 31, 32]
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