Figure 3.

KISS1R undergoes rapid ligand-dependent and -independent internalization and recycling. A, CHO-KISS1R cells were incubated with 0.05 nM ¹²⁵I-KP10 at 37°C in the presence or absence of 5 μM PAO. Cell surface and internalized radioligand were separated by acidic washes after various incubation times as indicated. The ratio of internalized to surface radioligand was plotted by linear regression. The graph shown is a representative plot from 1 of 3 independent experiments with similar results, and the t_1/2 values shown are the means ± SEM from the 3 experiments. B, HEK-myc-KISS1R cells were incubated with or without 10⁻⁷ M KP10 at 37°C for 20 minutes. Some cells were then washed 5 times to remove KP10 and reincubated in KP10-free medium for an additional 20 minutes. Cells were fixed, and cell surface myc-KISS1R was detected using an HRP-anti-myc antibody. Data shown are means ± SEM from 3 independent experiments. *, P < .05; ***, P < .001. C, CHO-KISS1R cells were treated with 0.25% trypsin for 30 minutes at 4°C and then incubated at 37°C for the times indicated. The amount of cell surface KISS1R was measured by incubation with 0.05 nM ¹²⁵I-KP10 plus 1 nM cold KP10 at 4°C for 1 hour. Means ± SEM are shown from a representative experiment, performed with triplicate samples, of 3 independent experiments, each with similar results. Statistical analysis was performed using one-way ANOVA followed by a post hoc Bonferroni multiple comparison test. *, P < .05; **, P < .001; ***, P < .0001 compared with time 0.