Molecular screening of a large cohort of Moroccan patients with congenital hypopituitarism.

BACKGROUND/OBJECTIVES: Congenital hypopituitarism is a rare disease which, for most patients, has no identified molecular cause. We aimed to document the molecular basis of growth retardation in a Moroccan cohort. DESIGN/PATIENTS: 80 index cases [54 with isolated growth hormone deficiency (IGHD), 26 with combined pituitary hormone deficiency (CPHD)] were screened for molecular defects in GH1 (including LCR-GH1), GHRHR, GHSR, GHRH, PROP1, POU1F1, HESX1, LHX3, LHX4 and SOX3. RESULTS: Five different deleterious mutations were identified in 14 patients from eight families. In the IGHD group, three genes were found to be involved: GH1, GHRHR and GHSR. In the CPHD group, PROP1 was the only mutated gene. In addition, two heterozygous variations whose deleterious effect remains to be demonstrated were identified (in GH1 and LHX4), and two polymorphisms (missense variations) were detected (in LHX3 and in GHSR). The prevalence of mutations in this Moroccan GHD cohort was 10% (8/80), 11·1% (6/54) in the IGHD group and 7·7% (2/26) in the CPHD group. CONCLUSION: This is the first molecular screening of congenital GHD in a Moroccan population and, like other studies, mutations were preferentially identified in familial cases (75%); mutations in genes such as POU1F1, HESX1, SOX3, LHX3 and LHX4 are extremely rare. The p.R73C PROP1 mutation was the most frequent mutation in CPHD; this should be the first one to screen in this population. Our results should contribute to a better diagnosis and management of this heterogeneous disease condition.

[G-protein-coupled receptors targeting: the allosteric approach]. / Ciblage thérapeutique des récepteurs couplés aux protéines G - La voie allostérique.

G-protein-coupled receptors (GPCR) are a major family of drug targets. Essentially all drugs targeting these receptors on the market compete with the endogenous ligand (agonists or antagonists) for binding the receptor. Recently, non-competitive compounds binding to distinct sites from the cognate ligand were documented in various classes of these receptors. These compounds, called allosteric modulators, generally endowed of a better selectivity are able to modulate specifically the endogenous signaling of the receptor. To better understand the promising potential of this class of GPCRs targeting compounds, this review highlights the properties of allosteric modulators, the strategies used to identify them and the challenges associated with the development of these compounds.

The GhsrQ343X allele favors the storage of fat by acting on nutrient partitioning.

The Growth Hormone Secretagogue Receptor (GHSR) mediates key properties of the gut hormone ghrelin on metabolism and behavior. Nevertheless, most recent observations also support that the GHSR is a constitutively active G protein-coupled receptor endowed of a sophisticated tuning involving a balance of endogenous ligands. Demonstrating the feasibility of shifting GHSR canonical signaling in vivo, we previously reported that a model with enhanced sensitivity to ghrelin (GhsrQ343X mutant rats) developed fat accumulation and glucose intolerance. Herein, we investigated the contribution of energy homeostasis to the onset of this phenotype, as well as behavioral responses to feeding or pharmacological challenges, by comparing GhsrM/M rats to wild-type littermate rats 1) as freely behaving animals and 2) in feeding and locomotor paradigms. Herein, GhsrM/M rats showed enhanced locomotor response to a GHSR agonist while locomotor or anorexigenic responses to amphetamine or cabergoline (dopamine receptor 2 agonist), respectively, were preserved. Ad libitum fed GhsrM/M rats consumed and conditioned for sucrose similarly to littermate control rats. In calorie-restricted conditions, GhsrM/M rats retained food anticipatory activity and maintained better their body weight and glycemia. Importantly, prior to fat accumulation, male GhsrM/M rats preferentially used carbohydrates as fuel substrate without alterations of energy intake, energy expenditure or physical activity and showed alterations of the GHSR system (i.e. enhanced ratio of GHSR hormones LEAP2:acyl-ghrelin and increased Ghsr expression in the hypothalamus). Overall, the present study provides proof of concept that shifted GHSR signaling can specifically alter nutrient partitioning resulting in modified balance of carbohydrate/lipid utilization.

Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature.

The growth hormone (GH) secretagogue receptor (GHSR) was cloned as the target of a family of synthetic molecules endowed with GH release properties. As shown recently through in vitro means, this receptor displays a constitutive activity whose clinical relevance is unknown. Although pharmacological studies have demonstrated that its endogenous ligand--ghrelin--stimulates, through the GHSR, GH secretion and appetite, the physiological importance of the GHSR-dependent pathways remains an open question that gives rise to much controversy. We report the identification of a GHSR missense mutation that segregates with short stature within 2 unrelated families. This mutation, which results in decreased cell-surface expression of the receptor, selectively impairs the constitutive activity of the GHSR, while preserving its ability to respond to ghrelin. This first description, to our knowledge, of a functionally significant GHSR mutation, which unveils the critical importance of the GHSR-associated constitutive activity, discloses an unusual pathogenic mechanism of growth failure in humans.

Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetite.

The ability of the gut hormone ghrelin to promote positive energy balance is mediated by the growth hormone secretagogue receptor (GHSR). GHSR is a G protein-coupled receptor (GPCR) that is found centrally and peripherally and that can signal in a ligand-independent manner basally or when heterodimerized with other GPCRs. However, current Ghsr knockout models cannot dissect ghrelin-dependent and ghrelin-independent signaling, precluding assessment of the physiological importance of these signaling pathways. An animal model carrying a Ghsr mutation that preserves GHSR cell surface abundance, but selectively alters GHSR signaling, would be a useful tool to decipher GHSR signaling in vivo. We used rats with the Ghsr(Q343X) mutation (Ghsr(M/M)), which is predicted to delete the distal part of the GHSR carboxyl-terminal tail, a domain critical for the signal termination processes of receptor internalization and ß-arrestin recruitment. In cells, the GHSR-Q343X mutant showed enhanced ligand-induced G protein-dependent signaling and blunted activity of processes involved in GPCR signal termination. Ghsr(M/M)rats displayed enhanced responses to submaximal doses of ghrelin or GHSR agonist. Moreover, Ghsr(M/M)rats had a more stable body weight under caloric restriction, a condition that increases endogenous ghrelin tone, whereas under standard housing conditions,Ghsr(M/M)rats showed increased body weight and adiposity and reduced glucose tolerance. Overall, our data stress the physiological role of the distal domain of GHSR carboxyl terminus as a suppressor of ghrelin sensitivity, and we propose using the Ghsr(M/M)rat as a physiological model of gain of function in Ghsr to identify treatments for obesity-related conditions.

Stimulation of human trophoblast invasion by placental growth hormone.

A critical step in establishment of human pregnancy is the invasion of the uterus wall by the extravillous cytotrophoblast (EVCT), a process regulated by multiple autocrine and paracrine factors. Hormones belonging to the GH/prolactin family are expressed at the maternofetal interface. Because they are involved in cell motility in various models, we examined the possible regulatory role of human placental GH (hPGH) in EVCT invasiveness. By using an in vitro invasion model, we found that EVCT isolated from first-trimester chorionic villi and cultured on Matrigel secreted hPGH and expressed human GH receptor (hGHR). These data were confirmed by in situ immunohistochemistry. EVCT expressed the full-length and truncated forms of hGHR, and the Janus kinase-2/signal transducer and activator of transcription factor-5 signaling pathway was activated in EVCT by hPGH treatment. Strong hPGH and hGHR expression was observed when EVCT invaded Matrigel and moved through the pores of the filter on which they were cultured. hPGH stimulated EVCT invasiveness, and this effect was inhibited by a Janus kinase-2 inhibitor. Interestingly, hPGH was more efficient than pituitary GH in stimulating EVCT invasiveness. These results offer the first evidence for a placental role of hPGH and suggest an autocrine/paracrine role of hPGH in the regulation of trophoblast invasion.

Heterozygous nonsense mutation in exon 3 of the growth hormone receptor (GHR) in severe GH insensitivity (Laron syndrome) and the issue of the origin and function of the GHRd3 isoform.

Mutations in the GH receptor gene (GHR) cause congenital GH insensitivity, a genetic disorder characterized by severe growth retardation associated with high serum concentration of GH and low serum levels of IGF-I. Molecular defects have been identified in all GHR-coding exons, except exon 3, a sequence that encodes part of the extracellular domain of the receptor. In humans, GHR transcripts exist in two isoforms differing by the retention (GHRfl) or exclusion (GHRd3) of this particular exon. As shown recently, such a dimorphic expression pattern, of unknown significance, could result from a retrovirus-mediated deletion event involving exon 3. This model for the generation of those two isoforms, however, leaves open the possibility that GHRd3 transcripts also arise from GHRfl alleles through alternative splicing. Here we report the identification of the first mutation in exon 3 of the GHR (W16X) in a patient with GH insensitivity and who also carries another nonsense mutation in exon 4. Intrafamilial correlation analyses of genotypes (presence of normal or mutant GHRfl and/or GHRd3 alleles), GHR expression patterns, and phenotypes provided direct evidence against an alternative splicing of exon 3. In particular, this exon was retained into transcripts originating from the GHRfl-W16X allele in both the patient and his mother. These observations, given the normal phenotype of the heterozygous parents, revealed also that a single copy of either GHRfl or GHRd3 is sufficient for normal growth.

Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.

The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: ß(2)AR agonists - the ß(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.

Recessive isolated growth hormone deficiency and mutations in the ghrelin receptor.

CONTEXT: Both GH releasing- and orexigenic properties of the gut-to-brain hormone ghrelin are mediated by the GH secretagogue receptor (GHSR). Recently in several patients, a missense mutation (p.A204E) resulting in a complete loss of GHSR constitutive activity has been implicated in short stature with dominant transmission. OBJECTIVE: The objective of the study was to describe the phenotype associated with partial isolated GH deficiency of a young patient born to unrelated parents and identify the molecular basis of his disease. RESULTS: The growth delay (-3.0 sd) was associated with recurrent episodes of abdominal pain, vomiting, ketosis, hypoglycemia, and a low body mass index. GHSR sequencing revealed that the patient was compound heterozygous for two new defects: 1) an early occurring transition predicting a premature stop codon (c.6G>A, p.W2X) inherited from his unaffected father, therefore strongly arguing against haploinsufficiency as a disease mechanism, and 2) a missense mutation (c.709A>T, p.R237W) inherited from his healthy mother, involving an evolutionary invariant residue from the third intracellular loop. In vitro experiments showed that the p.R237W mutation would result in a partial loss of constitutive activity of the receptor, whereas both its ability to respond to ghrelin and its cell surface expression are preserved. CONCLUSION: These data, which describe the first case of recessive partial isolated GH deficiency due to GHSR mutations and emphasize the physiological importance of the GHSR in somatic growth, are discussed in light of the dominantly expressed p.A204E mutation.

Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1.

Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation.