TACR3 mutations disrupt NK3R function through distinct mechanisms in GnRH-deficient patients.
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.
['Noel|Sekoni D|SD|', 'Abreu|Ana Paula|AP|', 'Xu|Shuyun|S|', 'Muyide|Titilayo|T|', 'Gianetti|Elena|E|', 'Tusset|Cintia|C|', 'Carroll|Jessica|J|', 'Latronico|Ana Claudia|AC|', 'Seminara|Stephanie B|SB|', 'Carroll|Rona S|RS|', 'Kaiser|Ursula B|UB|']
D000818:Animals D001665:Binding Sites D001667:Binding, Competitive D015153:Blotting, Western D019556:COS Cells D002462:Cell Membrane D048049:Extracellular Signal-Regulated MAP Kinases D031541:Fluorescence Resonance Energy Transfer D043802:GTP-Binding Protein alpha Subunits, Gq-G11 D007987:Gonadotropin-Releasing Hormone D057809:HEK293 Cells D006801:Humans D007295:Inositol Phosphates D008164:Luminescent Proteins D008856:Microscopy, Fluorescence D020125:Mutation, Missense D015287:Neurokinin B D010766:Phosphorylation D055503:Protein Multimerization D018042:Receptors, Neurokinin-3 D015398:Signal Transduction
neurokinin B GPCR hypogonadotropic hypogonadism tachykinins