Time-Resolved Tracking of Separately Internalized Neuropeptide Y₂ Receptors by Two-Color Pulse-Chase

Internalization and intracellular trafficking of G protein-coupled receptors (GPCR) plays an important role in the signal transduction. These processes are often highly dynamic and take place rapidly. In the past 10 years, it became obvious that internalized GPCRs are also capable of signaling via arrestin or heterotrimeric G proteins within the endosomal compartment. Real-time imaging of receptors in living cells can help to evaluate the temporal and spatial localization. We achieved a two-color pulse-chase labeling approach, which allowed the tracking of the human neuropeptide Y₂ receptor (hY₂R) in the same cell at different times. The ability to visualize the internalization pathway of two separately labeled and separately stimulated subsets of hY₂R in a time-resolved manner revealed a rapid trafficking. Fusion of the two hY₂R subsets was already observed 10 min after stimulation in the early endosomal compartment without subsequent separation of the fused receptor populations. The results demonstrate that the cells do not discriminate between receptors that were stimulated and internalized at different time points.