KISS1R Intracellular Trafficking and Degradation: Effect of the Arg386Pro Disease-Associated Mutation

Abstract

The goal of this study was to investigate how the Arg386Pro mutation prolongs KiSS-1 receptor (KISS1R) responsiveness to kisspeptin, contributing to human central precocious puberty. Confocal imaging showed colocalization of wild-type (WT) KISS1R with a membrane marker, which persisted for up to 5 h of stimulation. Conversely, no colocalization with a lysosome marker was detected. Also, overnight treatment with a lysosome inhibitor did not affect WT KISS1R protein, whereas overnight treatment with a proteasome inhibitor increased protein levels by 24-fold. WT and Arg386Pro KISS1R showed time-dependent internalization upon stimulation. However, both receptors were recycled back to the membrane. The Arg386Pro mutation did not affect the relative distribution of KISS1R in membrane and internalized fractions when compared to WT KISS1R for up to 120 min of stimulation, demonstrating that this mutation does not affect KISS1R trafficking rate. Nonetheless, total Arg386Pro KISS1R was substantially increased compared with WT after 120 min of kisspeptin stimulation. This net increase was eliminated by blockade of detection of recycled receptors, demonstrating that recycled receptors account for the increased responsiveness of this mutant to kisspeptin. We therefore conclude the following: 1) WT KISS1R is degraded by proteasomes rather than lysosomes; 2) WT and Arg386Pro KISS1R are internalized upon stimulation, but most of the internalized receptors are recycled back to the membrane rather than degraded; 3) the Arg386Pro mutation does not affect the rate of KISS1R traffickinginstead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increaseonmutantreceptorrecycledbacktotheplasmamembrane

Effects and Therapeutic Potentials of Kisspeptin Analogs: Regulation of the Hypothalamic-Pituitary-Gonadal Axis

“…3 ) [63] . This hypothesis requires remaining KISS1R on the cell surface after chronic exposure to Kp agonist analogs, and it may be supported by previous findings suggesting intracellular KISS1R pools and dynamic receptor recycling even after chronic exposure to the ligand [75,76] . In addition, this continuous stimulation of GnRH neurons and subsequent nonpulsatile low-level leakage may disrupt Kp-independent GnRH release as suggested by Chan et al [77] .…”

“…Thus, under the continuous administration of TAK-683 it is plausible that GnRH neurons become insensitive to endogenous Kp pulses. Investigations in mouse GnRH neurons and KISS1R-expressing CHO cells suggest that KISS1R desensitization occurs within 12 h. A potential mechanism of KISS1R desensitization may involve receptor internalization and subsequent reduction in cell surface KISS1R, uncoupling to G proteins, altered downstream signaling, or ubiquitination [69,70] . In in vivo experiments, continuous Kp-10 infusion in monkeys induced abrupt elevations in LH levels for a few hours [59] .…”

MOLECULAR EVOLUTION OF GPCRS: Kisspeptin/kisspeptin receptors

“…The altered flexibility of the C-terminal tail modifies its interaction with partner proteins (Chevrier et al 2013). In this regard, it is interesting to note that, in a case of precocious puberty, an Arg386Pro mutation located in the C-terminal region induces a sustained response to Kiss activation (Teles et al 2008, Bianco et al 2011. This PRR repeat pattern is not found in non-mammalian vertebrate KissR proteins (Supplementary Figure 1), which indicates an evolutionary variability in signal transduction and/or intracellular trafficking mechanisms.…”

Role of Kisspeptine in Regulation of Reproductive Function

“…Поэтому в отличие от мутаций, связанных с усилением функции, во многих сопряженных с G-белком рецепторах, которые вызывают конститутивную активацию рецеп-торов, мутации р.R386P снижают степень де-сенситизации мутантного KISS1R на поверх-ности клеток после связывания с лигандом и генерации импульсов. Действительно, мута-ция р.R386P приводит к снижению деградации KISS1R, в результате чего увеличивается ко-личество рецепторов на плазматической мем-бране [79]. Несомненно, сам KISS1 является еще одним очевидным кандидатным геном для объяснения механизмов преждевременного полового созревания.…”

“…Определенные особенности анатомических изменений и экс-прессии нейропептидов расцениваются как ас-социированные с ППР [79]. Экспрессия ГнРГ, ре-цепторов ГнРГ, TGF-α, KISS1, KISS1R и GRM1A была исследована в гамартомах у пациентов с наличием или отсутствием преждевременно-го полового развития [82].…”