title: 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 trafficking--instead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increase on mutant receptor recycled back to the plasma membrane.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA. sbianco@med.miami.edu
authors.forename: Suzy D C
authors.initials: SD
authors.lastname: Bianco
authors.forename: Lauren
authors.initials: L
authors.lastname: Vandepas
authors.forename: Mayrin
authors.initials: M
authors.lastname: Correa-Medina
authors.forename: Balázs
authors.initials: B
authors.lastname: Gereben
authors.forename: Abir
authors.initials: A
authors.lastname: Mukherjee
authors.forename: Wendy
authors.initials: W
authors.lastname: Kuohung
authors.forename: Rona
authors.initials: R
authors.lastname: Carroll
authors.forename: Milena G
authors.initials: MG
authors.lastname: Teles
authors.forename: Ana Claudia
authors.initials: AC
authors.lastname: Latronico
authors.forename: Ursula B
authors.initials: UB
authors.lastname: Kaiser
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation; D008247:Lysosomes / Q000378:metabolism; D009154:Mutation; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D000074502:Receptors, Kisspeptin-1
chemical_list: C118688:KISS1R protein, human; D043562:Receptors, G-Protein-Coupled; D000074502:Receptors, Kisspeptin-1