title: TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.
abstract: Neurokinin B (NKB) and its G-protein-coupled receptor, NK3R, have been implicated in the neuroendocrine control of GnRH release; however, little is known about the structure-function relationship of this ligand-receptor pair. Moreover, loss-of-function NK3R mutations cause GnRH deficiency in humans. Using missense mutations in NK3R we previously identified in patients with GnRH deficiency, we demonstrate that Y256H and Y315C NK3R mutations in the fifth and sixth transmembrane domains (TM5 and TM6), resulted in reduced whole-cell (79.3±7.2%) or plasma membrane (67.3±7.3%) levels, respectively, compared with wild-type (WT) NK3R, with near complete loss of inositol phosphate (IP) signaling, implicating these domains in receptor trafficking, processing, and/or stability. We further demonstrate in a FRET-based assay that R295S NK3R, in the third intracellular loop (IL3), bound NKB but impaired dissociation of Gq-protein subunits from the receptor compared with WT NK3R, which showed a 10.0 ± 1.3% reduction in FRET ratios following ligand binding, indicating activation of Gq-protein signaling. Interestingly, R295S NK3R, identified in the heterozygous state in a GnRH-deficient patient, also interfered with dissociation of G proteins and IP signaling from wild-type NK3R, indicative of dominant-negative effects. Collectively, our data illustrate roles for TM5 and TM6 in NK3R trafficking and ligand binding and for IL3 in NK3R signaling.
authors.affiliation: 1Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115, USA. ukaiser@partners.org.
authors.forename: Sekoni D
authors.initials: SD
authors.lastname: Noel
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Abreu
authors.forename: Shuyun
authors.initials: S
authors.lastname: Xu
authors.forename: Titilayo
authors.initials: T
authors.lastname: Muyide
authors.forename: Elena
authors.initials: E
authors.lastname: Gianetti
authors.forename: Cintia
authors.initials: C
authors.lastname: Tusset
authors.forename: Jessica
authors.initials: J
authors.lastname: Carroll
authors.forename: Ana Claudia
authors.initials: AC
authors.lastname: Latronico
authors.forename: Stephanie B
authors.initials: SB
authors.lastname: Seminara
authors.forename: Rona S
authors.initials: RS
authors.lastname: Carroll
authors.forename: Ursula B
authors.initials: UB
authors.lastname: Kaiser
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000172:deficiency*; D057809:HEK293 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D020125:Mutation, Missense; D015287:Neurokinin B / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D055503:Protein Multimerization; D018042:Receptors, Neurokinin-3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D007295:Inositol Phosphates; D008164:Luminescent Proteins; D018042:Receptors, Neurokinin-3; D007987:Gonadotropin-Releasing Hormone; D015287:Neurokinin B; D048049:Extracellular Signal-Regulated MAP Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
keywords: GPCR; hypogonadotropic hypogonadism; neurokinin B; tachykinins