Molecular origin of idiopathic central hypogonadism: variable impairment of two signal-transduction pathways due to PROKR2 mutations
Domenico Vladimiro Libri1, Marco Bonomi1,2, Marta Busnelli3, Gunnar Kleinau4, Csilla Krausz5, Paolo Beck-Peccoz1, Antonio Agostino Sinisi6, Luca Persani1,2 & ICH study group of the Italian Societies for Endocrinology and Paediatric Endocrinology1,2
ICH is a heterogeneous disease mainly due to functional defects of GnRH neurons. Recent studies have described the importance of prokineticin pathway in the pathogenesis of these defects. In a series of 166 ICH patients we found germline mutations affecting this pathway in about 10% of the cases. PROKR2 gene analysis revealed 3 novel (V158I, T260M, V334M) and 4 already known (L173R, R268C, V274D, V331M) mutations affecting different transmembrane and intracellular regions of this G protein coupled receptor (GPCR). For the first time, we evaluate the functional effects of these mutations on the two different PROKR2-dependent signalling pathways: IP3-Ca2+ (via Gq coupling) and cAMP (via Gs coupling) by HTRF-BRET and RIA techniques. At FACS analysis, the expression levels in CHO-K1 cells were similar to the WT in the case of V158I, V331M, V334M and reduced by 50% in the case of the other mutants. Interestingly, functional studies showed highly variable degrees of impairment in both signalling pathways for most of the mutations. For example, T260M, R268C and V331M mutants showed no changes in cAMP EC50 while the IP3 signalling appeared strongly affected with an increase of tenfolds in the EC50 versus WT. In contrast, R173C and V274D presented no virtual response in terms of cAMP accumulation, whereas R173C had an IP3 response similar to the WT and V274D presented a tenfold increase of IP3 EC50. Differently, V334M showed a similar fivefold increase of EC50 for both cAMP and IP3. In conclusion, our study shows the importance to evaluate both PROKR2-dependent signalling pathways since single missense mutations can variably affect the two cascades, thus suggesting that the integrity of both cAMP- and IP3-dependent signals should be required for a physiological function of GnRH neuron. These studies open novel perspectives for the understanding of the structure-function relationships of this class of GPCRs.