A functional spectrum of PROKR2 mutations identified in isolated hypogonadotropic hypogonadism

Abstract

Isolated hypogonadotropic hypogonadism (IHH) is a rare disease with hypogonadism and infertility caused by the defects in embryonic migration of hypothalamic gonadotropin-releasing hormone (GnRH) neurons, hypothalamic GnRH secretion or GnRH signal transduction. PROKR2 gene, encoding a G-protein coupled receptor PROKR2, is one of the most frequently mutated genes identified in IHH patients. However, the functional consequences of several PROKR2 mutants remain elusive. In this study, we systematically analyzed the Gαq, Gαs and ERK1/2 signaling of 23 IHH-associated PROKR2 mutations which are yet to be functionally characterized. We demonstrate that blockage of Gαq, instead of MAPK/ERK pathway, inhibited PROK2-induced migration of PROKR2-expressing cells, implying that PROKR2-related IHH results primarily due to Gαq signaling pathway disruption. Combined with previous reports, we categorized a total of 63 IHH-associated PROKR2 mutations into four distinct groups according Gαq pathway functionality: (i) neutral (N, >80% activity); (ii) low pathogenicity (L, 50-80% activity); (iii) medium pathogenicity (M, 20-50% activity) and (iv) high pathogenicity (H, <20% activity). We further compared the cell-based functional results with in silico mutational prediction programs. Our results indicated that while Sorting Intolerant from Tolerant predictions were accurate for transmembrane region mutations, mutations localized in the intracellular and extracellular domains were accurately predicted by the Combined Annotation Dependent Depletion prediction tool. Our results thus provide a functional database that can be used to guide diagnosis and appropriate genetic counseling in IHH patients with PROKR2 mutations.

Figure 1

Gαq signaling is required for the PROK2-induced migration of PROKR2-expressing cells. (A) PROK2 dose-dependently induced the migration of CHO cells stably expressing PROKR2 as assayed with transwell experiments. Left, representative images of migrated cells without PROK2 treatment (control) and with 1 nM PROK2. Right, the statistical data of three independent experiments. (B–E) The effects of PROKR2 antagonist A457 (0.1 μM), Gαq signaling pathway inhibitor YM-254890 (1 μM), PKC inhibitor Gö6983 (10 μM) and MAPK/ERK signaling pathway inhibitor SL327 (10 μM) on the migration of PROKR2-expressing cells induced by PROK2 (1 nM). Each value was the mean ± SEM of three independent experiments, ns, not significant; ^*P ˂ 0.05; ^***P ˂ 0.001; ^****P ˂ 0.0001, unpaired t test.

Figure 2

Schematic diagram of all IHH-related PROKR2 mutations according the Gαq signaling pathway. The green dots represent neutral (N, AUC > 80% of WT PROKR2); the blue dots represent low pathogenicity (L, AUC 50–80% of WT PROKR2); the purple dots represent medium pathogenicity (M, AUC 20–50% of WT PROKR2) and the red dots represent high pathogenicity (H, AUC < 20% of WT PROKR2).