title: RF9 Acts as a KISS1R Agonist In Vivo and In Vitro.
abstract: RF9, a reported antagonist of the mammalian gonadotropin-inhibitory hormone receptor, stimulates gonadotropin secretion in mammals. Recent studies have suggested that the stimulatory effect of RF9 on gonadotropin secretion relies on intact kisspeptin receptor (KISS1R) signaling, but the underlying mechanisms remain to be elucidated. Using Chinese Hamster Ovary cells stably transfected with KISS1R, we show that RF9 binds specifically to KISS1R, with a Kd of 1.6 × 10(-5)M, and stimulates an increase in intracellular calcium and inositol phosphate accumulation in a KISS1R-dependent manner, with EC50 values of 3.0 × 10(-6)M and 1.6 × 10(-7)M, respectively. RF9 also stimulated ERK phosphorylation, with a time course similar to that of kisspeptin-10. RFRP-3, the putative endogenous ligand for NPFFR1, did not stimulate inositol phosphate accumulation or pERK, nor did it alter responses to of kisspeptin-10 or RF9. In agreement with these in vitro data, we found that RF9 stimulated a robust LH increase in Npffr1(-/-) mice, similar to that in wild-type littermates, whereas the stimulatory effect of RF9 was markedly reduced in Kiss1r(-/-) and double Kiss1r(-/-)/Npfrr1(-/-) mice. The stimulatory effect of RF9 on LH secretion was restored by the selective rescue of Kiss1r expression in GnRH neurons, in Kiss1r(-/-T) mice. Taken together, our study demonstrates that RF9 acts primarily as a KISS1R agonist, but not as an allosteric modulator, to stimulate LH secretion. Our findings raise questions regarding the utility of RF9 for assessing NPFF1R function and de-emphasize a predominant role of this signaling system in central regulation of reproduction.
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Le
authors.initials: L
authors.lastname: Min
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Silvia
authors.initials: S
authors.lastname: Leon
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Huan
authors.initials: H
authors.lastname: Li
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Leonor
authors.initials: L
authors.lastname: Pinilla
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Rona S
authors.initials: RS
authors.lastname: Carroll
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Manuel
authors.initials: M
authors.lastname: Tena-Sempere
authors.affiliation: Division of Endocrinology, Diabetes and Hypertension (L.M., H.L., R.S.C., U.B.K.), Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Department of Cell Biology, Physiology and Immunology (S.L., L.P., M.T.-S.), University of Córdoba; CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, and Instituto Maimónides de Investigación Biomédica de Córdoba/Hospital Universitario Reina Sofia, Córdoba, 14004 Spain; and FiDiPro Program, Department of Physiology (M.T.-S.), University of Turku, FIN-20520 Turku, Finland.
authors.forename: Ursula B
authors.initials: UB
authors.lastname: Kaiser
mesh_terms: D000218:Adamantane / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003412:Cricetulus; D004151:Dipeptides / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D007457:Iodine Radioisotopes; D059648:Kisspeptins / Q000378:metabolism; D007986:Luteinizing Hormone / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism; D009479:Neuropeptides / Q000494:pharmacology*; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000187:drug effects* / Q000235:genetics; D000074502:Receptors, Kisspeptin-1; D018013:Receptors, Neuropeptide / Q000187:drug effects* / Q000235:genetics
chemical_list: D004151:Dipeptides; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; C106628:KISS1 protein, human; C118688:KISS1R protein, human; C492248:Kiss1r protein, mouse; D059648:Kisspeptins; D009479:Neuropeptides; C416987:RFamide peptide; D043562:Receptors, G-Protein-Coupled; D000074502:Receptors, Kisspeptin-1; D018013:Receptors, Neuropeptide; C508361:adamantylcarbonyl-arginyl-phenylalaninamide; C083929:neuropeptide FF receptor; D007986:Luteinizing Hormone; D048049:Extracellular Signal-Regulated MAP Kinases; D000218:Adamantane; D002118:Calcium