Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 14, Pages 5609-5613Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0801456105
Keywords
degradation; endocytosis; G protein-coupled receptor; green fluorescent protein; internalization
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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.
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