Fig. 1.

hY₂R recycles back to the membrane, while the peptide is degraded. a Scheme of hY₂R internalization and recycling experiment quantifying cell surface receptors. b Live cell images of HEK293 cells transiently transfected with HA-Y₂R-eYFP. Receptor (green) localization was determined by fluorescence microscopy prior (w/o) and after stimulation with 1 μM of the endogenous ligand NPY at 37 °C, subsequent washing and incubation in ligand free medium supplemented with 100 μg/ml CHX in a 60 min recycling period. c Quantification of cell surface fluorescence intensity using Image J. Cell surface receptors before stimulation is set to 100% (w/o, black bar). Stimulation with 1 μM NPY reduced the amount of membrane receptors (white bar), which increased again after the recycling period (light grey). However, NH₄Cl- treated cells displayed no reappearance of hY₂R back to the membrane (dark grey). d Live cell images of HEK293-HA-hY₂R-eYFP cells stained with 1 μM LysoTracker®Blue (blue). Subsequent incubation with 100 nM TAMRA-NPY (red) for 60 min at 37 °C leads to a rapid co-localization of hY₂R and peptide (yellow) in early endosomes (EE1A, blue) after immunostaining (e). The receptor was separated during a 60 min recycling period and transported back to the membrane, whereas TAMRA-NPY was co-localized with the lysosomal marker (light blue). Scale bar: 10 μm, experiments represent data n ≥ 3; significance was determined by one-way ANOVA, Tukey post test, ns: not significant, ***: P < 0.0001