The Heterozygous R295S NK3R Mutation Identified in a Patient with GnRH Deficiency has Dominant Negative Effects on WT NK3R by Impairing G Protein Catalytic Activation

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 134-163-GnRH & Gonadotroph Biology & Signaling
Bench to Bedside
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-150
Sekoni D. Noel*1, Ana Paula Abreu2, Shuyun Xu3, Titilayo Muyide2, Jessica Carroll4, Elena Gianetti5, Ana Claudia Latronico6 and Stephanie Beth Seminara*7
1Brigham & Women's Hospital/Harvard Med School, Boston, MA, 2Brigham and Women's Hospital, Boston, MA, 3Brigham & Womens Hosp, Boston, MA, 4Brigham and Women's Hospital, 5Univ of Pisa, Pisa, Italy, 6Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, 7Massachusetts General Hospital, Boston, MA
Mutations in TAC3, the gene encoding neurokinin B (NKB), and in TACR3, encoding its G protein-coupled receptor, NK3R, have been implicated in causing idiopathic hypogonadotropic hypogonadism (IHH) in humans. We previously reported a heterozygous R295S mutation, located in the third intracellular loop (ICL3) of NK3R, in a patient with IHH. We investigated the mechanism through which this mutation affects NK3R function to cause IHH. Ligand binding and inositol phosphate (IP) assays performed in transiently transfected COS-7 cells demonstrated that R295S bound 125I-NKB, albeit with a slight reduction in maximal binding compared to wild type NK3R, but failed to respond to NKB with any appreciable IP accumulation. Western blot analysis demonstrated that R295S had a modest but significant decrease in cellular receptor protein levels, consistent with the reduced maximal binding.  However, the receptor levels and ligand binding capacity were not congruent with the near-total loss of IP signaling by R295S. To further explore the R295S signaling defect, we determined whether the R295S NK3R could interact with Gq protein.  Co-immunoprecipitation studies demonstrated that R295S complexed with Gq similarly to WT NK3R. Therefore, we next examined whether the R295S mutation impaired the catalytic activation of Gq following NKB stimulation, using a Fluorescence Resonance Energy Transfer (FRET)-based approach.  COS-7 cells transiently co-expressing Gαq–CFP, Gβ–YFP and WT NK3R produced a basal FRET signal ratio that decreased following NKB treatment, reflecting the dissociation of Gαq–CFP and Gβ–YFP from the receptor and from each other following receptor activation, leading to activation of downstream signaling cascades.  In contrast, COS-7 cells expressing Gαq–CFP and Gβ–YFP together with R295S failed to show a decrease in FRET ratios following NKB stimulation. These results indicate that, unlike WT NK3R, the Gα and Gβ subunits failed to dissociate from R295S following NKB stimulation, demonstrating impaired catalytic activation. Because the R295S mutant was identified in the heterozygous state, we tested for a potential dominant negative effect on WT receptor function using IP assays and FRET. Co-transfection of WT and R295S receptors in COS-7 cells significantly impaired NKB-stimulated IP accumulation compared to cells expressing WT NK3R alone.  In addition, FRET ratios were not reduced following NKB treatment in cells expressing both WT and R295S receptors, further supporting a dominant negative effect. Collectively, our data indicate that the R295S mutation impairs NK3R signaling by disrupting ligand-stimulated activation of Gq by the R295S mutant itself as well as by WT NK3R, suggesting receptor-receptor interactions and/or cross-talk.  Our studies also indicate roles for R295 in the coupling and activation of G proteins by NK3R and for ICL3 in NK3R signaling.

Nothing to Disclose: SDN, APA, SX, TM, JC, EG, ACL, SBS

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