Postendocytotic traffic of the galanin R1 receptor:: A lysosomal signal motif on the cytoplasmic terminus

The neuropeptide galanin R1 receptor (GaIR1) was tagged at its C terminus with EGFP (GaIR1-EGFP) to study receptor localization and trafficking. In PC12 and HEK293 cells, functional GaIR1-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 GaIR1-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 YXXO on the C terminus of GaIR1 (where 0 is a bulky hydrophobic residue and X any amino acid). Taken together, these data suggest that GaIR1 is internalized via the clathrin-dependent, endocytic pathway and then, to a large extent, delivered to lysosomes for degradation through the lysosome-targeting signal YXXO.