RESUMO
The neuropeptide galanin R1 receptor (GalR1) was tagged at its C terminus with EGFP (GalR1-EGFP) to study receptor localization and trafficking. In PC12 and HEK293 cells, functional GalR1-EGFP was expressed on the plasma membrane and internalized into cytoplasmic vesicles after galanin stimulation. The internalization was blocked by 0.4 M sucrose and by silencing of clathrin with siRNA methodology. Internalized GalR1-EGFP and LysoTracker, a lysosomal marker, overlapped in intracellular vesicles after prolonged galanin stimulation. This colocalization was strongly reduced after site-directed mutagenesis of the motif YXXØ on the C terminus of GalR1 (where Ø is a bulky hydrophobic residue and X any amino acid). Taken together, these data suggest that GalR1 is internalized via the clathrin-dependent, endocytic pathway and then, to a large extent, delivered to lysosomes for degradation through the lysosome-targeting signal YXXØ.
