PROKR2 Mutations in Patients with Short Stature Who Have Isolated Growth Hormone Deficiency and Multiple Pituitary Hormone Deficiency

Objective: Recent reports have indicated the role of the prokineticin receptor 2 gene (PROKR2) in the etiology of pituitary hormone deficiencies, suggesting a potential role for the PROK2 pathway in pituitary development, in addition to its role in gonadotropin releasing hormone-expressing neuron development. Here, we present the clinical and molecular findings of four patients with PROKR2 mutations. Methods: Next-generation targeted sequencing was used to screen 25 genes in 59 unrelated patients with multiple pituitary hormone deficiency (MPHD), isolated growth hormone (GH) deficiency, or idiopathic short stature. Results: Two different, very rare PROKR2 missense alterations classified as pathogenic (NM_144773.4:c.518T>G; NP_658986.1:p. (Leu173Arg)) and likely pathogenic (NM_144773.4:c.254G>A; NP_658986.1:p.(Arg85His)) were identified in four patients in heterozygous form. Patient 1 and Patient 2 presented with short stature and were diagnosed as GH deficiency. Patient 3 and Patient 4 presented with central hypothyroidism and cryptorchidism and were diagnosed as MPHD. No other pathogenic alterations were detected in the remaining 24 genes related to short stature, MPHD, and hypogonadotropic hypogonadism. Segregation analysis revealed asymptomatic or mildly affected carriers in the families. Conclusion: PROKR2 dominance should be kept in mind as a very rare cause of GH deficiency and MPHD. Expressional variation or lack of penetrance may imply oligogenic inheritance or other environmental modifiers in individuals who are heterozygous carriers.

Biallelic PROKR2 variants and congenital hypogonadotropic hypogonadism: a case report and a literature review.

Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder that causes gonadotropin-releasing hormone (GnRH) deficiency and sexual immaturity. CHH may accompany an abnormal sense of smell (Kallmann syndrome, KS) or no such manifestation (normosmic-CHH). This unusual combination of manifestations is explained by the fact that GnRH neurons originate in the olfactory placode and migrate to the forebrain during embryogenesis. We describe the case of a 31-year-old man with normosmic-CHH, who also had obesity, type 2 diabetes and intellectual disability. He was noticed to have sexual immaturity (small testes with no pubic hair) at age 20 years, when diabetic ketoacidosis developed. Basal and GnRH-stimulated levels of LH (1.0→12.0 IU/L) and FSH (1.9→6.1 IU/L) were detectable but low. The results of the T&T olfactometer and the Alinamin test were definitely normal, with an anatomically normal olfactory system on MRI. Sequencing of 22 CHH-related genes was performed, and compound heterozygous PROKR2 variants were identified: one was a previously known loss-of-function variant (p.Trp178Ser) and the other was a nonsense variant (p.Trp212*). Through a literature review, we found 22 patients (including our patient) with CHH due to biallelic PROKR2 variants, which led us to recognize that most of the patients (86%) were diagnosed with KS. Clinical observations in this study indicate that, even though they have CHH, biallelic PROKR2 variant carriers may have a normal olfactory system as well as presumably normal migration of GnRH neurons. This suggests that the PROK2-PROKR2 pathway affects the function of GnRH neurons after their migration.

Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress.

G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.

[The significance of "A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis" published in Nature for acupuncture research].

October 2021, Nature published an original research article entitled A neuroanatomical basis for electroacupuncture to drive the vagal-adrenal axis, which draws great attention and arouses extensive discussion in the acupuncture field. Based on previous findings, this study demonstrates that the abundant innervation of PROKR2-Cre neurons in deep fascia tissues mediates the anti-inflammatory effect induced by low-intensity electroacupuncture stimulation at "Zusanli"（ST36） or "Shousanli"(LI10) via the "vagal-adrenal axis". This study is one of milestones in the field of acupuncture basic research and represents a great achievement generated by multi-discipline integration of acupuncture and neuro-immunology. It reveals partial contributing factors involved in acupuncture's effect and the relative specificity of the neuroanatomical basis of acupoints in the context of immune modulation. This study is both very informative and instructive for the innovation and clinical translation of future acupuncture research. Acupuncture researchers are recommended to attach great importance to this study in terms of its research strategy,methods and findings.

Digenic inheritance of PROKR2 and WDR11 mutations in Kallmann syndrome—A case report and literature review / 中华内分泌代谢杂志

Kallmann syndrome(KS) is a subtype of idiopathic hypogonadotropic hypogonadism(IHH), characterized by delayed puberty, undeveloped secondary sexual characters, accompanied by anosmia, or hyposmia. At present, more than 40 genes are related to the pathogenesis of IHH, and new gene loci have been found continuously. At the same time, digenic gene mutation or oligogenic mutation is considered to be an important pathogenic mechanism of IHH. The clinical phenotype of KS/IHH caused by different gene mutations is complex and diverse, and the response to treatment is also variable. This paper presents the clinical data and treatment of a case of KS caused by a compound double heterozygous mutation of WDR11 and PROKR2 genes. PROKR2 gene is a classic KS pathogenic gene, while the WDR11 gene is a relatively new type of KS pathogenic gene. Included with this case report is a literature review of characteristics of cases with WDR11 gene mutation.

Analysis of PROKR2 gene mutation in patients with hypogonadotropic hypogonadism / 中华内科杂志

To investigate the clinical and genetic characteristics of patients with idiopathic hypogonadotropic hypogonadism (IHH), the clinical data of 23 patients with IHH were retrospectively analyzed. Gene analyses were accomplished with whole-exome sequencing (WES) and Sanger sequencing. Functional prediction of mutation sites was conducted using two bioinformatics platforms, SIFT and Polyphen. Among the 23 patients with IHH, 9 patients carried prokinin 2 (PROKR2) gene mutations including 4 missense mutations (p.W178S, p.Y113H, p.A103V, p.R164Q), and 1 frameshift mutation (p.D42delinsDED), the remaining 14 cases were found negative in gene sequencing. Functional prediction showed that the above mutations may affect protein function suggestive of a pathogenic role of PROKR2 mutation in the patients. There were no significant differences in the levels of follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol between the IHH patients with PROKR2 gene mutation and those without. PROKR2 gene mutation might associated with IHH, and the mutations reported in the present study could enrich the pathogenic spectrum of genes.

Comparison of Clinical Characteristics and Spermatogenesis in CHH Patients Caused by PROKR2 and FGFR1 Mutations.

A retrospective study was conducted to investigate the effect of gonadotropin or pulsatile gonadotropin-releasing hormone (GnRH) therapy on spermatogenesis in congenital hypogonadotropic hypogonadism (CHH) patients with PROKR2 (prokineticin receptor 2) or FGFR1 (fibroblast growth factor receptor 1) mutations. Clinical features, gonadotropin levels, testicular volume (TV), and sperm concentration in response to gonadotropin and pulsatile GnRH therapy were compared between groups with PROKR2 and FGFR1 mutations. Twelve patients with PROKR2 gene mutation and fourteen patients with FGFR1 gene mutation were included. The incidence of cryptorchidism in PROKR2 and FGFR1 groups was 16.7% and 50%, respectively (p = 0.110). The baseline TV in the PROKR2 group was larger than that in FGFR1 group (2.0 vs. 1.63, p = 0.047). The initial LH, FSH, and testosterone levels were similar between the two groups. Based on the analysis of achieving spermatogenesis using Kaplan-Meier and log-rank tests, the PROKR2 group demonstrated shorter period of seminal spermatozoa appearance than the FGFR1 group (χ2 = 8.297, p = 0.004); the median duration of achieving spermatogenesis in the PROKR2 and FGFR1 groups was 9 and 16 months, respectively. The PROKR2 mutation group exhibited shorter required time to achieve different sperm concentration thresholds (5, 10, and 15 million/mL) than the FGFR1 mutation group (p = 0.012, 0.024, and 0.040). In conclusion, the PROKR2 group achieved spermatogenesis easily than the FGFR1 group, possibly due to the lower prevalence of cryptorchidism and larger baseline testicular volume in the PROKR2 group.

Identification of MMACHC and PROKR2 mutations causing coexistent cobalamin C disease and Kallmann syndrome in a young woman.

Cobalamin C (cblC) disease and Kallmann syndrome (KS) are rare hereditary diseases. To date, no report has described the coexistence of those two genetic disorders in the same patient, or an association between them. We report the case of a 23-year-old woman with cblC defect and KS. She first presented mild memory problems in puberty, which worsened in adulthood to progressive memory loss accompanied by slow and unsteady walking, slow response, inattention, cognitive impairment, insomnia, no sense of smell, and the lack of spontaneous puberty. Laboratory tests revealed gonadotropin deficiency, a low estrogen level, and remarkably elevated serum homocysteine and serum and urine organic acid levels. Whole-exome sequencing detected compound heterozygous variants in MMACHC [c.398_399del (p.Gln133Argfs*4) and c.482G > A (p.Arg161Gln)] and heterozygous variants in PROKR2 [c.337T > C (p.Tyr113His)]. Thus, clinical and genetic examinations confirmed the cblC disease and KS diagnoses. This report on coexisting cblC disease and KS caused by different pathogenic genes in a single patient enriches the clinical research on these two rare genetic diseases.

Molecular screening of PROKR2 gene in girls with idiopathic central precocious puberty.

BACKGROUND: Prokineticin receptor 2 (PROKR2) loss of function mutations have been described as cause of hypogonadotropic hypogonadism. In 2017, a first case of central precocious puberty (CPP) caused by PROKR2 heterozygous gain of function mutation was described in a 3.5 years-old girl. No other cases have been reported yet. This study performs a molecular screening in girls with early onset CPP (breast budding before 6 years of age) to identify possible alterations in PROKR2. METHODS: We analysed DNA of 31 girls with idiopathic CPP diagnosed via basal LH levels > 0.3 IU/L or peak-LH > 5 IU/L after stimulation, without any MKRN3 mutations. The Fisher exact test was used to compare polymorphism allele frequency to corresponding ones in genome aggregation database (gnomAD). RESULTS: No rare variants were identified. Five polymorphisms were found (rs6076809, rs8116897, rS3746684, rs3746682, rs3746683). All except one (i.e. rs3746682) had a minor allele frequency (MAF) similar to that reported in literature. rs3746682 presented a MAF higher than that described in the gnomAD (0.84 in our cohort vs 0.25 from gnomAD). CONCLUSIONS: As for other G protein-coupled receptors (i.e. GPR54), mutations in PROKR2 do not seem to be a frequent cause of CPP in girls.

Clinical lessons learned in constitutional hypopituitarism from two decades of experience in a large international cohort.

CONTEXT: The international GENHYPOPIT network collects phenotypical data and screens genetic causes of non-acquired hypopituitarism. AIMS: To describe main phenotype patterns and their evolution through life. DESIGN: Patients were screened according to their phenotype for coding sequence variations in 8 genes: HESX1, LHX3, LHX4, PROP1, POU1F1, TBX19, OTX2 and PROKR2. RESULTS: Among 1213 patients (1143 index cases), the age of diagnosis of hypopituitarism was congenital (24%), in childhood (28%), at puberty (32%), in adulthood (7.2%) or not available (8.8%). Noteworthy, pituitary hormonal deficiencies kept on evolving during adulthood in 49 of patients. Growth Hormone deficiency (GHD) affected 85.8% of patients and was often the first diagnosed deficiency. AdrenoCorticoTropic Hormone deficiency rarely preceded GHD, but usually followed it by over 10 years. Pituitary Magnetic Resonance Imaging (MRI) abnormalities were common (79.7%), with 39.4% pituitary stalk interruption syndrome (PSIS). The most frequently associated extrapituitary malformations were ophthalmological abnormalities (16.1%). Prevalence of identified mutations was 7.3% of index cases (84/1143) and 29.5% in familial cases (n = 146). Genetic analysis in 449 patients without extrapituitary phenotype revealed 36 PROP1, 2 POU1F1 and 17 TBX19 mutations. CONCLUSION: This large international cohort highlights atypical phenotypic presentation of constitutional hypopituitarism, such as post pubertal presentation or adult progression of hormonal deficiencies. These results justify long-term follow-up, and the need for systematic evaluation of associated abnormalities. Genetic defects were rarely identified, mainly PROP1 mutations in pure endocrine phenotypes.

Vascular endothelial growth factor (VEGF) and Endocrine gland-VEGF (EG-VEGF) are down regulated in head and neck cancer.

OBJECTIVE: To characterise the role of VEGF, EG-VEGF and its receptors in the development and progression of HNC. DESIGN: Human serum and tissues samples were collected from healthy, epulis and HNC patients and used for ELISA assays and immunohistochemistry studies, respectively. SETTING: Ibn Rochd Hospital of Casablanca (Morocco), INSERM and University of Grenoble Alpes (France). PARTICIPANTS: We used serum from 64 patients with head and neck cancers and from 71 controls without general pathology. Tissues samples were collected from seven patients with OSCC and from seven patients with Epulis. MAIN OUTCOME MEASURES: We compared circulating VEGF and EG-VEGF in normal and HNC patients and determined the expression, localisation and quantification of VEGF, EG-VEGF and its receptors; PROKR1 and PROKR2 as well as Ki67, CD31 and CD34 in OSCC and Epulis patients. RESULTS: Both EG-VEGF and VEGF circulating levels were significantly decreased in the HNC (P < .01). OSCC patients expressed less EG-VEGF and VEGF proteins, higher PROKR1 and PROKR2 with no change in CD31 and CD34 levels. A significant increase in Ki67 was observed in OSCC. CONCLUSIONS: We demonstrated that circulating VEGF and EG-VEGF are downregulated in HNC patients and in OSCC tissue. EG-VEGF receptors were increased in OSCC, along with a stabilisation of two key markers of angiogenesis. These findings strongly suggest that downregulation of angiogenesis in HNC might explain its moderate metastatic feature.

The PROK2/PROKR2 signaling pathway is required for the migration of most olfactory bulb interneurons.

Neural stem cells in the subventricular zone (SVZ) of the lateral ventricle generate new interneurons, which migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB). The PROK2 (prokineticin 2) and PROKR2 (prokineticin receptor 2) signaling pathway has been identified to cause human Kallmann syndrome, a developmental disease that associates hypogonadism with anosmia (OB developmental defects). However, the identities and properties of PROK2+ and PROKR2+ cells in the SVZ-RMS-OB remain largely unknown. Here we examine the expression patterns of Prok2 and Prokr2 in the SVZ-RMS-OB using Prok2EGFP transgenic and Prokr2LacZ/+ knockin mice. Our results show that Prokr2 is expressed in postmitotic immature interneurons in the SVZ-RMS-OB. Prok2 is not expressed in the SVZ, but a few PROK2+ cells are found in the medial part of the RMS; they are not neural progenitors or migrating neuroblasts. In the OB, Prok2 is expressed in a subset of granule cells and tufted cells, but no coexpression of Prok2 and Prokr2 in the OB cells is observed. In Prok2 and Prokr2 mutant mice, severe tangential and radial migration defects of neuroblasts in the SVZ-RMS-OB result in loss of ~75% of GABAergic interneurons in the OB. These analyses demonstrate that PROK2/PROKR2 signaling is crucial for the tangential and radial migration of OB interneurons.

A heterozygous microdeletion of 20p12.2-3 encompassing PROKR2 and BMP2 in a patient with congenital hypopituitarism and growth hormone deficiency.

Congenital growth hormone deficiency is a rare disorder with an incidence of approximately 1 in 4,000 live births. Pituitary development is under the control of a multitude of spatiotemporally regulated signaling molecules and transcription factors. Mutations in the genes encoding these molecules can result in hypopituitarism but for the majority of children with congenital hypopituitarism, the aetiology of their disease remains unknown. The proband is a 5-year-old girl who presented with neonatal hypoglycaemia and prolonged jaundice. No definitive endocrine cause of hypoglycaemia was identified in the neonatal period. She was born of normal size at 42 weeks but demonstrated growth failure with a progressive reduction in height to -3.2 SD by age 4.5 years and failed a growth hormone stimulation test with a peak growth hormone of 4.2 mcg/L. MRI of the pituitary gland demonstrated a hypoplastic anterior lobe and ectopic posterior lobe. Array CGH demonstrated an inherited 0.2 Mb gain at 1q21.1 and a de novo 4.8 Mb heterozygous deletion at 20p12.2-3. The deletion contained 17 protein coding genes including PROKR2 and BMP2, both of which are expressed during embryological development of the pituitary gland. PROKR2 mutations have been associated with hypopituitarism but a heterozygous deletion of this gene with hypopituitarism is a novel observation. In conclusion, congenital hypopituitarism can be present in individuals with a 20p12.3 deletion, observed with incomplete penetrance. Array CGH may be a useful investigation in select cases of early onset growth hormone deficiency, and patients with deletions within this region should be evaluated for pituitary hormone deficiencies.

Loss of ZBTB20 impairs circadian output and leads to unimodal behavioral rhythms.

Many animals display morning and evening bimodal activities in the day/night cycle. However, little is known regarding the potential components involved in the regulation of bimodal behavioral rhythms in mammals. Here, we identified that the zinc finger protein gene Zbtb20 plays a crucial role in the regulation of bimodal activities in mice. Depletion of Zbtb20 in nerve system resulted in the loss of early evening activity, but the increase of morning activity. We found that Zbtb20-deficient mice exhibited a pronounced decrease in the expression of Prokr2 and resembled phenotypes of Prok2 and Prokr2-knockout mice. Injection of adeno-associated virus-double-floxed Prokr2 in suprachiasmatic nucleus could partly restore evening activity in Nestin-Cre; Zbtb20fl/fl (NS-ZB20KO) mice. Furthermore, loss of Zbtb20 in Foxg1 loci, but intact in the suprachiasmatic nucleus, was not responsible for the unimodal activity of NS-ZB20KO mice. Our study provides evidence that ZBTB20-mediated PROKR2 signaling is critical for the evening behavioral rhythms.

Mutation analyses in pedigrees and sporadic cases of ethnic Han Chinese Kallmann syndrome patients.

Kallmann syndrome, a form of idiopathic hypogonadotropic hypogonadism, is characterized by developmental abnormalities of the reproductive system and abnormal olfaction. Despite association of certain genes with idiopathic hypogonadotropic hypogonadism, the genetic inheritance and expression are complex and incompletely known. In the present study, seven Kallmann syndrome pedigrees in an ethnic Han Chinese population were screened for genetic mutations. The exons and intron-exon boundaries of 19 idiopathic hypogonadotropic hypogonadism (idiopathic hypogonadotropic hypogonadism)-related genes in seven Chinese Kallmann syndrome pedigrees were sequenced. Detected mutations were also tested in 70 sporadic Kallmann syndrome cases and 200 Chinese healthy controls. In pedigrees 1, 2, and 7, the secondary sex characteristics were poorly developed and the patients' sense of smell was severely or completely lost. We detected a genetic mutation in five of the seven pedigrees: homozygous KAL1 p.R191ter (pedigree 1); homozygous KAL1 p.C13ter (pedigree 2; a novel mutation); heterozygous FGFR1 p.R250W (pedigree 3); and homozygous PROKR2 p.Y113H (pedigrees 4 and 5). No genetic change of the assayed genes was detected in pedigrees 6 and 7. Among the 70 sporadic cases, we detected one homozygous and one heterozygous PROKR2 p.Y113H mutation. This mutation was also detected heterozygously in 2/200 normal controls and its pathogenicity is likely questionable. The genetics and genotype-phenotype relationships in Kallmann syndrome are complicated. Classical monogenic inheritance does not explain the full range of genetic inheritance of Kallmann syndrome patients. Because of stochastic nature of genetic mutations, exome analyses of Kallmann syndrome patients may provide novel insights.

FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies.

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca(2) (+) pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition.

Combined pituitary hormone deficiency with unique pituitary dysplasia and morning glory syndrome related to a heterozygous PROKR2 mutation.

Recent reports have indicated the role of the prokineticin receptor 2 gene (PROKR2) in the etiology of congenital hypopituitarism, including septo-optic dysplasia and Kallmann syndrome. In the present study, using next-generation targeted sequencing, we identified a novel heterozygous PROKR2 variant (c.742C>T; p.R248W) in a female patient who had combined pituitary hormone deficiency (CPHD), morning glory syndrome and a severely malformed pituitary gland. No other mutation was present in 27 genes related to hypogonadotropic hypogonadism, pituitary hormone deficiency and optic nerve malformation. The substituted amino acid was located on the third intracellular loop of the PROKR2 protein, which is a G protein-coupled receptor. Computational analyses with two programs (SIFT and PolyPhen-2) showed that the substitution was deleterious to PROKR2 function. The p.R248W mutation was transmitted from the patient's mother, who had a slightly delayed menarche. Collectively, we provide further genetic evidence linking heterozygous PROKR2 mutations and the development of CPHD.

Effects of Gelsemium sempervirens L. on pathway-focused gene expression profiling in neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium sempervirens L. is a traditional medicinal plant mainly distributed in the southeastern of the United States, employed in phytotheraphy and homeopathy as nervous system relaxant to treat various types of anxiety, pain, headache and other ailments. Although animal models showed its effectiveness, the mechanisms by which it might operate on the nervous system are largely unknown. This study investigated for the first time by a real-time PCR technique (RT-PCR Array) the gene expression of a panel of human neurotransmitter receptors and regulators, involved in neuronal excitatory signaling, on a neurocyte cell line. MATERIALS AND METHODS: Human SH-SY5Y neuroblastoma cells were exposed for 24h to Gelsemium sempervirens at 2c and 9c dilutions (i.e. 2 and 9-fold centesimal dilutions from mother tincture) and the gene expression profile compared to that of cells treated with control vehicle solutions. RESULTS: Exposure to the Gelsemium sempervirens 2c dilution, containing a nanomolar concentration of active principle gelsemine, induced a down-regulation of most genes of this array. In particular, the treated cells showed a statistically significant decrease of the prokineticin receptor 2, whose ligand is a neuropeptide involved in nociception, anxiety and depression-like behavior. CONCLUSIONS: Overall, the results indicate a negative modulation trend in neuronal excitatory signaling, which can suggest new working hypotheses on the anxiolytic and analgesic action of this plant.

Functional analysis of the distal region of the third intracellular loop of PROKR2.

Mutations in the G-protein-coupled receptor PROKR2 have been identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) manifesting with delayed puberty and infertility. Recently, the homozygous mutation V274D was identified in a man displaying KS with an apparent reversal of hypogonadism. The affected amino acid, valine 274, is located at the junction region of the third intracellular loop (IL3) and the sixth transmembrane domain (TM6). In this study, we first studied the effect of V274D and related mutations (V274A, V274T, and V274R) on the signaling activity and cell surface expression of PROKR2. Our data indicate that a charged amino acid substitution at residue 274 of PROKR2 results in low cell surface expression and loss-of-function. Furthermore, we studied the effects of two clusters of basic amino acids located at the proximal region of Val274 on the cell surface expression and function of PROKR2. The deletion of RRK (270-272) resulted in undetectable cell surface expression, whereas RKR (264-266)-deleted PROKR2 was expressed normally on the cell surface but showed loss-of-function due to a deficiency in G-protein coupling. Our data indicate that the distal region of the IL3 of PROKR2 may differentially influence receptor trafficking and G-protein coupling.

PROK2/PROKR2 Signaling and Kallmann Syndrome.

Kallmann syndrome (KS) is a developmental disease that associates hypogonadism and a deficiency of the sense of smell. The reproductive phenotype of KS results from the primary interruption of the olfactory, vomeronasal, and terminal nerve fibers in the frontonasal region, which in turn disrupts the embryonic migration of neuroendocrine gonadotropin-releasing hormone (GnRH) synthesizing cells from the nose to the brain. This is a highly heterogeneous genetic disease, and mutations in any of the nine genes identified so far have been found in approximately 30% of the KS patients. PROKR2 and PROK2, which encode the G protein-coupled prokineticin receptor-2 and its ligand prokineticin-2, respectively, are two of these genes. Homozygous knockout mice for the orthologous genes exhibit a phenotype reminiscent of the KS features, but biallelic mutations in PROKR2 or PROK2 (autosomal recessive mode of disease transmission) have been found only in a minority of the patients, whereas most patients carrying mutations in these genes are heterozygotes. The mutations, mainly missense mutations, have deleterious effects on PROKR2 signaling in transfected cells, ranging from defective cell surface-targeting of the receptor to defective coupling to G proteins or impaired receptor-ligand interaction, but the same mutations have also been found in apparently unaffected individuals, which suggests a digenic/oligogenic mode of inheritance of the disease in heterozygous patients. This non-Mendelian mode of inheritance has so far been confirmed only in a few patients. However, it may account for the unusually high proportion of KS sporadic cases compared to familial cases.