TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction.

A. Families used to identify critical interval on chromosome 4 harbouring genetic defect causing nIHH B. Critical region on chromosome 4 showing homozygosity (black) in all affected individuals, with known or predicted genes in region indicated. C. Location of pathogenic mutation in NK3R D. evolutionary conservation of mutated residues.

A. Family used to identify region on chromosome 12 harbouring genetic defect causing nIHH with detail of subset of genes in critical interval B. Location of pathogenic mutation in TAC3. Shaded amino acids represent the tachykinin signature motif. C. Conservation of mutated residue among selected paralogues and orthologues.

Both missense mutations in NK3R result in loss of receptor function. A) Bright field (left) and GFP-fluorescent only (right) identifying TACR3 transfected HEK293 cells. B) Pseudocolour images of 340/380 nm ratio for the same optical field as in (A) before (left) and during (right) the application of 100 nM wt-NKB. Note that only GFP-positive cells respond. C) Representative Ca2+-responses of 3-5 wt- or mutant TACR3 transfected cells to application of different NKB doses as indicated. D) NKB dose responses for wt (black squares, n=93-206), G93D- (red squares, n=112-145) and P353S-mutant (green squares, n=156) TACR3. Peak [Ca2+]-responses recorded as in (C) in at least 5 dishes/construct were averaged over 30s and the data was fit with the logistic function [Ca2+] = (A1-A2)/(1+([NKB]/EC50))+A2 using origin software (Microcal). The resulting fit parameters were: wt-TACR3: A1=45±6 nM, A2=227±6 nM, EC50=6.6±1.3 nM; TACR3-G93D: A1=45±2 nM, A2=91±5 nM, EC50=290±146 nM; TACR3-P353S: A1=47±3 nM, A2=75 nM (fixed), EC50=4.0±2.1 μM. Error bars represent 1SE.

The missense mutations in NKB results in severe loss of agonist function at the NK3R A) Representative Ca2+-responses of 3 wt-TACR3 transfected cells to application of different NKB and NKB-M90T doses as indicated. B) NKB dose responses for NKB (black squares), and NKB-M90T (red circles). Peak [Ca2+]-responses recorded as in (A) in 5 dishes (n=113 cells) were averaged over 30s and the data was fit with the logistic function [Ca2+] = (A1-A2)/(1+([NKB]/EC50))+A2 using origin software (Microcal). The resulting fit parameters were: NKB: A1=40±8 nM, A2=242±11 nM, EC50=5.8±1.5 nM; NKB-M90T: A1=38±4 nM, A2=242 nM (fixed), EC50=52±6 nM. Error bars represent 1SE; responses to similar doses of the two peptides were compared using a paired t-test; i p=3*10−12, ii p=3*10−20, iii p=6*10−7.