Fig. 5.

Membrane trafficking of KISS1R. COS-7 cells expressing wild-type (WT) or Arg386Pro KISS1R were equilibrated with ¹²⁵I-kisspeptin on ice before incubation at 37 C for 0–120 min as indicated. Membrane and internalized ¹²⁵I-kisspeptin were extracted and counted as described in the Materials and Methods. A, Rate of membrane trafficking (internalization and recycling) in WT and Arg386Pro KISS1R is represented as the relative distribution of receptors in the membrane and internalized at each time-point. Left panel, relative percent of receptor in the membrane; right panel, relative percent of internalized receptors. Results are the mean ± se of seven independent experiments. Open bars, WT KISS1R; solid bars, Arg386Pro KISS1R. B, Total KISS1R trafficking with detection of recycled receptors. Total receptors in membrane (left panel) or internalized (right panel) fractions are shown as cpm/mg protein of the mean ± se of triplicates in this representative experiment, which was repeated at least three times with comparable results. Open bars, WT KISS1R; solid bars, Arg386Pro KISS1R. ***, P < 0.0002 by Student's t test vs. WT receptor at 120 min. C, Ratio of (total) internalized KISS1R with detection of recycled receptors: Time-course of stimulation. Bars represent the combined ratio derived from three independent experiments. Ratio in each experiment was calculated by dividing the mean of triplicate measurements of Arg386Pro KISS1R by that of triplicate measurements from WT receptor. *, P < 0.05 by ANOVA followed by the Dunnett's test vs. time zero. D and E, Measurement of KISS1R trafficking without detection of recycled receptors: Effect of removal of radioligand. In D and E, unbound ¹²⁵I-kisspeptin was removed at the end of equilibration on ice but before incubation at 37 C, so that only receptors bound to radioligand at time zero could be detected and tracked thereafter. D, The (total) receptors are represented as the mean ± se of triplicate samples from a representative experiment shown as cpm/mg of protein, replicated at least three times with comparable results. Open bars, WT KISS1R; solid bars, Arg386Pro KISS1R. Left panel, total membrane receptors; right panel, total internalized receptors. E, Combined ratio of Arg386Pro:WT KISS1R. Bars represent the average ratio derived from three independent time-course experiments. The ratio of each experiment was calculated by dividing the mean of triplicate samples of Arg386Pro KISS1R by that of triplicate samples of WT KISS1R from the same experiment. Left panel (open bars), ratio in the membrane; right panel (solid bars), ratio of internalized receptors. F, KISS1R trafficking without detection of recycled receptors: Effect of pretreatment with monensin. Thirty minutes of incubation with monensin (recycling inhibitor) before the addition of ¹²⁵I-kisspeptin prevented the reinsertion of recycled receptors into the membrane. Results are the mean ± se of triplicate samples from a representative experiment, shown as the ratio of Arg386Pro over WT KISS1R. Left panel (open bars), ratio in the membrane; right panel (solid bars), ratio of internalized receptors.