Figure 4.

Internalized kisspeptin undergoes rapid processing. CHO-KISS1R cells were treated with 0.05 nM ¹²⁵I-KP10 at 37°C for 1 hour to achieve significant internalization. After removal of free and cell surface ¹²⁵I-KP10, the cells were incubated at 37°C for the times indicated. After this incubation, the culture medium was collected, and ¹²⁵I-KP10 that was released from the cells into the medium during the incubation was separated by TCA precipitation to distinguish intact from degraded ¹²⁵I-KP10. Cell surface-bound ¹²⁵I-KP10 was released by an acidic wash and collected; ¹²⁵I-KP10 that remained internalized intracellularly was collected after cell lysis by 0.5 M NaOH. The cell surface bound, internalized, and extracellular intact and degraded radioligand was then quantified by counting in a Beckman γ-counter. A representative plot from 3 independent experiments, each done in triplicate, is shown. Mean ± SEM percentage at each time point is shown.