CRH-stimulation of cyclic adenosine 5'-monophosphate pathway is partially inhibited by the coexpression of CRH-R1 and CRH-R2alpha.

Corticotropin-releasing hormone (CRH) is one of the major proteins responsible for brain stress regulation. Two well-known receptors have been described: type 1 and type 2alpha, both members of the receptor superfamily of G protein-coupled receptors (GPCR). We investigated receptor regulation when both CRH receptor subtypes are coexpressed in the same mammalian cell line. When both types of receptors are coexpressed, cAMP second messenger production is partially inhibited compared to when receptors are expressed separately. However, neither binding kinetics nor internalization rates are modified by coexpression of these receptors. To our knowledge this is the first demonstration of receptor interaction that results in the modification of CRH-mediated signal transduction pathway. Because CRH-R1 and CRH-R2alpha have overlapping mRNA expression patterns in the brain, these receptors may be coexpressed in neurons, suggesting that receptor interaction may play an important role in the effect evoked by CRH, contributing to the complexity of differential coupling of the CRH receptors in different endocrine and stress behavior responses.

Regulation of follicle-stimulating and luteinizing hormone receptor signaling by.

Follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) belong to the super-family of G protein-coupled receptors (GPCR); GPCRs are negatively regulated by RGS ("regulators of G protein signaling") proteins. In this study we evaluated the effects of RGS3 and RGS10 on FSHR and LHR ligand binding and effector coupling. FSHR and LHR ligand binding were unchanged in the presence of RGS3 or RGS10. However, signaling by FSHR and LHR was diminished by RGS3 but not by RGS10. This constitutes the first demonstration of an interaction between RGS proteins and LH and FSH signaling pathways and identifies a mechanism for negative regulation of RGS3 on FSHR and LHR signaling.

Cyclic adenosine 3',5'-monophosphate (cAMP) and cAMP responsive element-binding protein are involved in the transcriptional regulation of gonadotropin-releasing hormone (GnRH) receptor by GnRH and mitogen-activated protein kinase signal transduction pathway in GGH(3) cells.

Stimulation of mouse GnRH receptor promoter by a GnRH agonist (Buserelin), or by a cAMP analogue, significantly increased reporter (luciferase) activity. Overexpression of Raf-1, ERK1, or ERK2 partially blocked Buserelin-stimulated luciferase activity. In contrast, treatment with a mitogen-activated protein kinase (MAPK) kinase inhibitor (PD 98059) activated basal and Buserelin-stimulated luciferase activity in a dose-dependent manner. Transient transfection of the deleted cAMP response element expression vector followed by pretreatment with PD98059 prior to Buserelin stimulation showed that the transcriptional response was decreased compared to wild-type promoter. A gel-mobility shift assay using a probe containing the cAMP response element showed the presence of two specific protein-DNA complexes that contain one or more members of the cAMP responsive element-binding (CREB) protein family. These results suggest that cAMP and CREB participate in the GnRH activation of GnRH receptor promoter activity and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRH receptor transcriptional activity.

Gonadotropin-releasing hormone receptor microaggregation. Rate monitored by fluorescence resonance energy transfer.

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release and is a therapeutic target for human and animal reproductive diseases. In the present study we have utilized the technique of fluorescence resonance energy transfer to monitor the rate of GnRH receptor-receptor interactions. This technique relies on the observation that the degree of physical intimacy of molecules can be assessed by the tendency of proximal fluorophores to exchange energy. Our data indicate that GnRH agonist, but not antagonist, occupancy of the GnRH receptor promotes physical intimacy (microaggregation) between receptors. The time course indicates that this occurs promptly (<1 min) after occupancy and persists for at least 80 min and within the physiologically relevant range of the releasing hormone. The process measured is not inhibited by 0.1 mm vinblastin, 2 microm cytochalasin D, or 3 mm EGTA, an observation that distinguishes it from macroaggregation (patching, capping, and internalization). These observations, along with reports from other laboratories, are consonant with a growing body of evidence that indicates that microaggregation is an early event following agonist occupancy of the receptor and part of the mechanism by which effector regulation occurs.

Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression.

The mammalian gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) has been a therapeutic target for human and animal medicine. This receptor is a unique G-protein-coupled receptor that lacks the intracellular C-terminal domain commonly associated with this family. Development of highthrough put screens for agents active in humans has been hampered by low expression levels of the hGnRH-R in cellular models. Two sites have attracted the interest of laboratories studying regulation of expression. The chimeric addition of the C-terminal tail from catfish GnRH-R (cfGnRH-R) to the rat GnRH-R significantly augmented receptor expression in GH3 cells. In addition, rodent GnRH-R contains 327 amino acids, but cow, sheep, and human GnRH-R (hGnRH-R) contain 328 residues, the "additional" residue being a Lys 191. Deletion of Lys 191 (del 191) from the hGnRH-R resulted in increased receptor expression levels and decreased internalization rates in both COS-7 and HEK 293 cells. In this study, the combined effect of the addition of the C-tail from cfGnRH-R and deletion of the Lys 191 from the hGnRH-R was compared to expression of the wild-type (WT) or either alteration alone in a transient expression system using primate cells. The altered receptor (hGnRH-R[del 191]-C-tail) showed significantly increased receptor expression at the cell surface compared with the WT or either modification alone. The inositol phosphate response to stimulation was also significantly elevated in response to GnRH agonist. After treatment with a GnRH agonist, the altered receptors showed a slower internalization rate. The homologous steady-state regulation of the WT and the altered receptors was similar, although the response of the altered receptors was significantly decreased. These results suggest that the conformational change in the receptor as a result of the deletion of Lys 191 and the addition of the C-terminus tail substantially increased the steady-state receptor expression and decreased internalization and homologous regulation. Because the effects on expression are greater than additive, it appears that these alterations exert their effects by differing means. These techniques for expression of the hGnRH-R in transfected mammalian cells provide the basis for a therapeutic screen for GnRH analogs, agonists, and antagonists of the hGnRH.

Transcriptional regulation of the gonadotropin-releasing hormone receptor gene is mediated in part by a putative repressor element and by the cyclic adenosine 3',5'-monophosphate response element.

The levels of the GnRH receptor (GnRHR) and its messenger RNA depend on the pattern of administration of GnRH. In this study, internal deletion mutants in a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment of mouse GnRHR gene were used to examine the regulation of GnRHR gene transcription in GGH3 cells. Our results indicate that the mouse GnRHR promoter contains one putative repressor element located at position -343/-335. When this sequence was deleted, the GnRHR promoter activity was significantly increased in both basal and GnRH agonist (Buserelin)-, phorbol ester-, and forskolin-stimulated cells. Gel mobility shift assay showed that the sequence -343/-335 is capable of binding GGH3 nuclear proteins. With deletion of the cAMP response element (-107/-100), basal and Buserelin-stimulated transcription was decreased. The same response was observed after stimulation with forskolin. Stimulation with (Bu)2cAMP did not alter transcription above basal levels. The stimulation with phorbol ester resulted in an attenuated increase in transcriptional activity, suggesting that this sequence of the GnRHR promoter is a cAMP response element. These results suggest that the transcriptional activity of the GnRHR gene is mediated in part by a putative repressor element and by the cAMP response element.

An atypical contiguous gene syndrome: molecular studies in a family with X-linked Kallmann's syndrome and X-linked ichthyosis.

BACKGROUND AND OBJECTIVE: Kallmann's syndrome (KS) is characterized by hypogonadotrophic hypogonadism in association with anosmia or hyposmia. This entity can be associated with X-linked ichthyosis (XLI) in a contiguous gene syndrome. Genetic defects have been demonstrated on the Xp22.3 region explaining the presence of one or both entities in affected individuals. In this report we describe the molecular findings in four patients, pertaining to a three generation family, with KS which was associated with XLI in two of them. MEASUREMENTS: Enzymatic activity of steroid sulphatase was measured in leucocytes. Polymerase chain reaction of the 14 exons of the Kallmann gene (KAL) and of the 5' and 3' extremes of the steroid sulphatase gene was performed in genomic DNA. PCR products of the 14 exons of the KAL gene were purified and sequenced. RESULTS: Absence of steroid sulphatase activity and a complete deletion of the STS gene were demonstrated in both patients with XLI. In all subjects, the 14 KAL gene exons amplified in a normal fashion; no mutation was documented after sequencing all exons. CONCLUSIONS: Although it has been proposed recently that the X-linked form of the disease accounts for the minority of patients with Kallman's syndrome, the pedigree chart of this family demonstrates this inheritance pattern. Various possibilities are mentioned in order to explain the absence of mutation in the KAL gene. The coexistence, in this family, of Kallman's syndrome individuals and patients with Kallman's syndrome and X-linked ichthyosis is discussed.

Renal abnormalities in patients with Kallmann syndrome.

OBJECTIVE: To report experience in patients with Kallmann syndrome (KS) in whom urography was used to establish the type and frequency of renal anomalies associated with the disorder. PATIENTS AND METHODS: Of 19 patients with KS, 15 had the X-linked recessive form of the disease, whereas the remaining four were sporadic. Each patient underwent intravenous pyelography (IVP) using a non-ionic, low osmolarity contrast medium. RESULTS: Of the 19 patients with KS, 10 had kidney abnormalities; four presented with unilateral renal agenesis and six had less severe forms of renal abnormality (renal malrotation in four and bilateral dilatation of the calyces and pelves in two). One of the patients with unilateral renal agenesis carried a deletion in KAL, the gene responsible for the X-linked type of KS. Three of the four patients with renal malrotation had a confirmed X-linked recessive form and one carried a point mutation in KAL. CONCLUSION: These results suggest that kidney abnormalities are more frequent and diverse in patients with KS than previously reported. They also indicate that defects in the KAL gene may contribute to abnormal renal development. However, a review of the literature revealed no close correlation between KAL mutations and kidney anomalies in the X-linked type of disease. Taken together, these data suggest that KAL mutations are not invariably associated with failure of renal development and that additional factors (epigenetic or local) may compensate for defects in the KAL protein.

Contiguous gene syndrome due to deletion of the first three exons of the Kallmann gene and complete deletion of the steroid sulphatase gene.

OBJECTIVE: Large terminal or interstitial deletions of the 22.3 region on the short arm of the X chromosome cause contiguous gene syndromes. Kallmann syndrome (hypogonadotrophic hypogonadism with anosmia or hyposmia) associated with X-linked ichthyosis, due to a contiguous gene syndrome, is an uncommon finding. Genetic defects have been demonstrated in the Xp22.3 region, explaining the presence of one or both entities in affected individuals. In this report we describe the molecular findings of a patient with Kallmann syndrome and X-linked ichthyosis. PATIENT: A 20-year-old subject with hypogonadism, anosmia and generalized ichthyosis was studied endocrinologically, biochemically and molecularly. MEASUREMENTS: Levels of LH, FSH, GH, testosterone, oestradiol and cortisol were determined basally and after specific stimulation tests. Enzymatic activity of steroid sulphatase was measured in leucocytes. Polymerase chain reaction of the 14 exons of the Kallmann gene and of the 5' and 3' extremes of the steroid sulphatase gene was performed in genomic DNA. RESULTS: A partial deletion from exon 1 to exon 3 of the Kallmann gene, as well as a complete deletion of the steroid sulphatase gene were observed. CONCLUSIONS: A patient bearing a contiguous gene syndrome with partial deletion of the Kallmann syndrome gene and complete deletion of the steroid sulphatase gene is described. This is the first time a mutation in the conserved cysteine-rich N-terminal region which corresponds to the whey acidic protein motif of the Kallmann gene has been characterized, thus demonstrating the importance of this specific region for the function of the gene.

A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann's syndrome.

Kallmann's syndrome (KS) is defined by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes regulating GnRH secretion. Genetic defects have been demonstrated in the KAL gene, located on the Xp22.3 region, explaining the X-linked form of the disease. We report molecular findings regarding the KAL gene in 12 unrelated males with X-linked KS. PCR of the 14 exons of the KAL gene was performed on genomic DNA. PCR products of all exons were purified and sequenced. Genetic defects in the KAL gene were found in 7 patients. One exhibits a deletion from exon 3 to exon 5. Six individuals present a previously unidentified missense mutation in exon 11, consisting of a G to A substitution at codon 514 (GAA to AAA). In the remaining 5 individuals, no mutations were observed. We also found three different polymorphic changes. The first one, in exon 2, had not been reported previously. The other two were located at exons 11 and 12. The deletion described, comprises only part (exon 5) of the coding region of the first fibronectin type III-like repeat of the KAL protein. The rest of the deletion comprises part of the conserved cysteine-rich N-terminal region that corresponds to the whey acidic protein motif. The same missense mutation was found in 6 of the 12 patients, indicating the possibility that it derived from a common ancestor or suggesting the presence of a hot spot in this region of the gene.

X-linked ichthyosis in Mexico: high frequency of deletions in the steroid sulfatase encoding gene.

The present study analyzes the frequency of molecular deletions in the steroid sulfatase (STS) encoding gene in a sample of 50 Mexican subjects with biochemical diagnosis of X-linked ichthyosis (XLI). To establish the correct diagnosis, STS activity was determined in leukocytes using 7-(3)H-dehydroepiandrosterone sulfate as the substrate. No amplification of the 3' and 5' ends of the STS gene by PCR was detected in the DNA of 49 patients, whereas only one sample of 50 presented a normal amplification. This report shows a very high frequency of deletions in the human STS encoding gene in a representative sample of the Mexican population, and it defines the characteristics of XLI in patients whose STS gene has a complete deletion as a major molecular defect.