SUN-157 Unravelling the Regulation of the Kisspeptin Receptor (kiss1r) Through Its Interaction With G Protein-Coupled Estrogen Receptor (gper)

Abstract Disclosure: M.A. Safar: None. V. Jugnarain: None. E. Reiter: None. S.F. Betz: None. A. Abbara: None. W.S. Dhillo: None. A.C. Hanyaloglu: None. The kisspeptin receptor (KISS1R) and G-protein-coupled estrogen receptor (GPER) are both pivotal in the endocrine regulation of reproductive health. KISS1R is essential for pubertal onset and regulating gonadotropin secretion via GnRH neurons, while GPER mediates non-genomic estrogen signalling and has been shown to dimerize with KISS1R, potentially playing an important role in distinct processes including endocrine feedback. We have previously shown that pretreatment with estrogen potentiates gonadotropin secretion after administration of the potent kisspeptin receptor agonist TAK-448. To date, there is limited data exploring the impact of estrogen and GPER on KISS1R activity activated by distinct KISS1R ligands. To understand the role of the interaction between KISS1R and GPER on KISS1R cell surface expression, signalling and internalization. HEK293 cells were transfected with human KISS1R and/or human GPER. Bioluminescence Resonance Energy Transfer (BRET) assays confirmed that KISS1R and GPER form heteromers, while KISS1R also forms homomers. Heteromer formation was not influenced by KISS1R ligand treatment with either kisspeptin-10 (KP10, 10nM), kisspeptin-54 (KP54, 10nM), or TAK-448 (1nM). Flow cytometry revealed that KISS1R surface expression was reduced when co-expressed with GPER, and this reduction was not reversed upon treatment with KISS1R ligands. KISS1R internalization in response to KP10, KP54, and TAK-448 was analysed between 0 and 60 minutes, with maximum internalization observed 10 minutes post ligand treatment. Intriguingly, there was a different internalization profile observed across all ligands with distinct kinetics and degree of receptor internalized. TAK-448 induced the greatest level of KISS1R internalization (20.5%), however, when receptors were co-expressed, internalization was reduced to 14%. In contrast, the transient KISS1R internalization profile induced by KP54, was sustained in cells co-expressing GPER. To investigate the influence of dimerization on receptor function, we measured intracellular inositol phosphate-1 (IP1) accumulation following ligand treatment. Cells that expressed GPER exhibited a high basal level of IP1. Co-expression of KISS1R and GPER decreased basal levels of IP1, however, there was no further increase following treatment with any of the KISS1R ligands, including pre-treatment with estrogen (50 pg/ml). These findings demonstrate that KISS1R and GPER heteromerization may not only influence KISS1R surface expression and agonist-induced Gq signal activity but could differentially modulate the agonist-induced endocytic trafficking profiles of KISS1R. These data highlight the regulatory role of receptor heterodimerization and trafficking in modulating KISS1R activity, providing insights for its potential use to treat endocrine disorders. Presentation: Sunday, July 13, 2025