Rat GnRH Receptor Research Articles

Discovery of a Lead Brain-Penetrating Gonadotropin-Releasing Hormone Receptor Antagonist with Saturable Binding in Brain.

We report the synthesis, radiosynthesis and biological characterisation of two gonadotropin‐releasing hormone receptor (GnRH−R) antagonists with nanomolar binding affinity. A small library of GnRH−R antagonists was synthesised in 20–67 % overall yield with the aim of identifying a high‐affinity antagonist capable of crossing the blood–brain barrier. Binding affinity to rat GnRH−R was determined by autoradiography in competitive‐binding studies against [125I]buserelin, and inhibition constants were calculated by using the Cheng–Prusoff equation. The radioligands were obtained in 46–79 % radiochemical yield and >95 % purity and with a molar activity of 19–38 MBq/nmol by direct nucleophilic radiofluorination. Positron emission tomography imaging in rat under baseline conditions in comparison to pretreatment with a receptor‐saturating dose of GnRH antagonist revealed saturable uptake (0.1 %ID/mL) into the brain.

Evaluation by metabolic profiling and in vitro autoradiography of two promising GnRH-receptor ligands for brain SPECT imaging.

The increased expression of gonadotropin releasing hormone receptor (GnRH-R) in brain has been strongly linked to Alzheimer disease. Therefore, the development of radiolabeled imaging agents for GnRH-R is relevant for early diagnosis of Alzheimer disease. We have recently disclosed the discovery of two promising compounds displaying nanomolar-range affinity for the GnRH-R. In the present study, a preclinical evaluation of the compound properties was performed to evaluate their potential as single photon emission computed tomography (SPECT) radiotracers for imaging the GnRH-receptor. The compounds were assessed in vitro by performing serum stability analysis by human and rat serum, metabolic profiling by human liver microsomes, and exploratory rat brain autoradiography. The investigated compounds displayed satisfactory stability against human, rat serum, and liver microsomal metabolism, which favors their potential as SPECT-imaging agents. Additionally, we identified and quantified the formation rate of the metabolites by fragmentation of up to five mass spectrometric stages. The GnRH-R rat brain specificity of these compounds was tested in competition with a known ligand for the receptor and the in vitro autoradiography confirmed that compounds 3 and 4 binds to rat GnRH-R in different rat brain regions.

Evaluation of 111In-Labeled GnRH-I Tracer for SPECT Tumor Imaging

Total synthesis, quality control, and preclinical evaluation of [111In]-DOTA-Triptorelin ([111In]-DOTA-TRP) for diagnostic SPECT imaging have been made. For 111In labeling, the peptide synthesis was followed by conjugation with DOTA using pSCN-Bn-DOTA. The synthesized conjugate prepared at optimized conditions was purified with a reversed-phase semipreparative column using gradient of water: acetonitrile mixture. The molecular mass was confirmed by mass spectroscopy. The conjugated Triptorelin was labeled with 500–550 MBq of 111In chloride (in 0.2 M HCl). The radiochemical purity of the product prepared under optimized conditions was about 98% (RTLC) and >95% (HPLC). The serum stability of the tracer was determined up to 24 h. The 111In-peptide chelate exhibits high stability. The binding affinity of Triptorelin peptide was determined in a binding assay for both human and rat GnRH receptors. For in vivo studies, 111In-peptide was injected intravenously via the tail vein to rats. In vitro radioligand binding assays were performed with GnRHR-expressing human cell lines using [125I]Triptorelin as the standard radioligand. The receptor affinity of the new radioligand was IC50 = 0.35 ± 0.08 nM vs. 0.13±0.01 nM for Triptorelin, and the internalization efficiency was 3.4 ± 0.7% at 1 h and 11.8 ± 1.9% at 4 h.

Preparation of a radiolabeled GnRH-I analogue derivative with 111 In as a new anti-proliferative agent.

The new GnRH-Ιanalogue developed in this paper was based on the D-Trp6 -GnRH-Ι-scaffold, and its potency was increased by the replacement Gly-NH2 by NH-NH2 binding to the Gly at position 10. Triptorelin-Hydrazide analogue was synthesized using solid phase. For 111 In labeling, synthesized peptide was followed by conjugation with DOTA using pSCN-Bn-DOTA. The conjugated Triptorelin-Hydrazide was labeled with 500-550MBq of 111 In-chloride (in 0.2M HCl). At optimized conditions after labeling, radio-chromatography showed radiochemical purity of approximately equal to 98% (RTLC) and greater than 95% (HPLC). The serum stability of the tracer was determined up to 24hr. Binding affinities of Triptorelin-Hydrazide analogue were determined in a binding assay for both human and rat GnRH receptors. For in vivo studies, 111 In-peptide was injected intravenously via the tail vein into rats and significant ovaries uptake consist with reported GnRH receptor mappings. In vitro radioligand binding assays performed with GnRHR-expressing human cell lines using 125 I-Triptorelin as the standard radioligand. The quantities of internalization efficiency and receptor affinity of the new radioligand were IC50 =0.20±0.04nM vs 0.13±0.08nM for Triptorelin and internalization: 3.5±0.9% at 1hr and 12.8±1.8% at 4hr of the internal reference.

Brain penetrant small molecule 18F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats

IntroductionThe gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood–brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [18F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these 18F-labeled compounds in brain of living rats. MethodsWe synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for 18F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo. ResultsA palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The 18F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5–15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the 18F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo. ConclusionsThe novel small molecule 18F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro, and may prove useful for quantitative autoradiographic studies in vitro. The compounds were permeable to the blood–brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain.

Augmentation of gonadotropin-releasing hormone receptor expression in the post-lactational mammary tissues of rats.

Gonadotropin-releasing hormone (GnRH) is a neurohormone of the hypothalamus controlling pituitary gonadotropin secretion and hence gametogenesis. While it has also been believed that GnRH is synthesized and functions in various peripheral tissues, the expression of GnRH receptor (GnRH-R) in peripheral tissues is not well-described. We previously found that annexin A5, which is increased in the pituitary gonadotropes by GnRH, is dramatically increased in rat mammary epithelial cells after weaning, suggesting that local GnRH is responsible for this increase. Annexin A5 is a member of the annexin family of proteins and is thought to be involved in various regulatory mechanisms, including apoptosis. In the present study, we examined GnRH-R expression in the mammary tissues after weaning. Although GnRH-R mRNA was not detected in the mammary tissues during lactation, it was dramatically increased after weaning. Forced weaning at mid-lactation (day 10) also promoted the expression of GnRH-R transcripts in mammary tissues within 2 days. Furthermore, western blotting analysis with anti-GnRH-R showed that the expression of an immuno-positive 60-kDa protein, whose size was equivalent to that of rat GnRH-R, was confirmed to increase after weaning. These findings clarified the induction of GnRH-R in the mammary tissues after weaning and suggest that GnRH is involved in the involution and tissue remodeling of post-lactating rat mammary tissues.

Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. LogP (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. 18F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.

Suppression of the hypothalamic–pituitary–gonadal axis by TAK-385 (relugolix), a novel, investigational, orally active, small molecule gonadotropin-releasing hormone (GnRH) antagonist: Studies in human GnRH receptor knock-in mice

TAK-385 (relugolix) is a novel, non-peptide, orally active gonadotropin-releasing hormone (GnRH) antagonist, which builds on previous work with non-peptide GnRH antagonist TAK-013. TAK-385 possesses higher affinity and more potent antagonistic activity for human and monkey GnRH receptors compared with TAK-013. Both TAK-385 and TAK-013 have low affinity for the rat GnRH receptor, making them difficult to evaluate in rodent models. Here we report the human GnRH receptor knock-in mouse as a humanized model to investigate pharmacological properties of these compounds on gonadal function. Twice-daily oral administration of TAK-013 (10mg/kg) for 4 weeks decreased the weights of testes and ventral prostate in male knock-in mice but not in male wild-type mice, demonstrating the validity of this model to evaluate antagonists for the human GnRH receptor. The same dose of TAK-385 also reduced the prostate weight to castrate levels in male knock-in mice. In female knock-in mice, twice-daily oral administration of TAK-385 (100mg/kg) induced constant diestrous phases within the first week, decreased the uterus weight to ovariectomized levels and downregulated GnRH receptor mRNA in the pituitary after 4 weeks. Gonadal function of TAK-385-treated knock-in mice began to recover after 5 days and almost completely recovered within 14 days after drug withdrawal in both sexes. Our findings demonstrate that TAK-385 acts as an antagonist for human GnRH receptor in vivo and daily oral administration potently, continuously and reversibly suppresses the hypothalamic–pituitary–gonadal axis. TAK-385 may provide useful therapeutic interventions in hormone-dependent diseases including endometriosis, uterine fibroids and prostate cancer.

GATA2-Induced Silencing and LIM-Homeodomain Protein-Induced Activation Are Mediated by a Bi-Functional Response Element in the Rat GnRH Receptor Gene

GATA2 transcription factor and LIM homeodomain proteins Islet1 (ISL1) and LIM homeobox 3 (LHX3) are suspected to be involved in gonadotrope cell fate and maintenance. The GnRH receptor gene (Gnrhr), crucial for gonadotrope function, is expressed in the pituitary gland from embryonic day 13.5 onward, well before LH and FSH β-subunits. This expression pattern together with the presence of WGATAR and TAAT motifs in Gnrhr promoter sequences suggests the involvement of early transcription factors in promoter activation. In this study, using a well-characterized transgenic mouse model, GATA2 was found colocalized with Gnrhr promoter activity in the pituitary. Transient transfection of Gnrhr promoter luciferase fusion constructs together with either GATA2 expression vectors or small interfering RNA in gonadotrope cell lines indicated that GATA2, which typically acts as a trans-activator, unexpectedly repressed Gnrhr promoter activity. Using DNA chromatography affinity and EMSA, we demonstrated that GATA2 operates via a response element containing a peculiar palindromic GATA motif that overlaps a critical TAAT motif involved in LHX3/ISL1 trans-activation. Indeed, despite the inhibitory action of GATA2, this element displayed a clear-cut enhancer activity in gonadotrope cells. Chromatin immunoprecipitation assays indicated that GATA2, LHX3, and ISL1 interact with a Gnrhr promoter fragment encompassing this element. The trans-repressive action of GATA2 on Gnrhr promoter activity is likely balanced or even hindered by trans-activating effects of LIM homeodomain proteins via this novel bifunctional LIM/GATA response element. Such a hierarchical interplay may contribute to finely adjust Gnrhr gene expression in gonadotrope cell lineage during pituitary development as well as in the adult animal.

Identification and Analysis of Two Novel Sites of Rat GnRH Receptor Gene Promoter Activity: The Pineal Gland and Retina

Background and Aims: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary. Methods: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created. Results: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context. Conclusion: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.

Silicon Analogues of the Nonpeptidic GnRH Antagonist AG‐045572: Syntheses, Crystal Structure Analyses, and Pharmacological Characterization

AG-045572 (CMPD1, 1 a) is a nonpeptidic gonadotropin-releasing hormone (GnRH) antagonist that has been investigated for the treatment of sex hormone-related diseases. In the context of systematic studies on sila-substituted drugs, the silicon analogue disila-AG-045572 (1 b) and its derivative 2 were prepared in multi-step syntheses and characterized by elemental analyses (C, H, N), NMR spectroscopic studies (1H, 13C, 29Si), and single-crystal X-ray diffraction. The pharmacological properties of compounds 1 a, 1 b, and 2 were compared in terms of their in vitro potency at cloned human and rat GnRH receptors. Compounds 1 a and 2 were also examined in regard to their pharmacokinetics and in vivo efficacy in both castrated rat (luteinizing hormone (LH) suppression) and intact rat (testosterone suppression) models. The efficacy and pharmacokinetic profiles of 1 a and its silicon-containing analogue 2 appear similar, indicating that replacement of the 5,6,7,8-tetrahydronaphthalene ring system by the 1,3-disilaindane skeleton led to retention of efficacy. Therefore, the silicon compound 2 represents a novel drug prototype for the design of potent, orally available GnRH antagonists suitable for once-daily dosing.

Reporter transgenic mouse models highlight the dual endocrine and neural facet of GnRH receptor function

In the pituitary of mammals, the GnRH receptor (GnRHR) plays crucial roles in the neuroendocrine control of reproductive function. This receptor is specifically expressed by the gonadotrope cells scattered among the five other endocrine cell types constituting the anterior pituitary; it is also expressed in other organs, such as the gonads and brain where its function is not well defined. To gain insight into GnRHR function, distribution, and regulation, several transgenic approaches have been developed using a range of reporter genes under the control of the mouse, rat, or ovine GnRHR gene (Gnrhr) promoters. Comprehensive reviews of the literature, together with recent results obtained in our laboratory, illustrate how these transgenic models highlight the endocrine as well as the neural facet of GnRHR function. In this review, the endocrine aspect will be discussed with regard to the pituitary and gonad function, whereas the neural aspect will be discussed with regard to hippocampal formation and the oculomotor pathway, the latter constituting an unpreviously described site of Gnrhr promoter activity. These approaches should help elucidate the properties of the mammalian GnRH system.

Elevated GnRH receptor expression plus GnRH agonist treatment inhibits the growth of a subset of papillomavirus 18‐immortalized human prostate cells

Human metastatic prostate cancer cell growth can be inhibited by GnRH analogs but effects on virus-immortalized prostate cells have not been investigated. Virus-immortalized prostate cells were stably transfected with rat GnRH receptor cDNA and levels of GnRH binding were correlated with GnRH effects on signaling, cell cycle, growth, exosome production, and apoptosis. High levels of cell surface GnRH receptor occurred in transfected papillomavirus-immortalized WPE-1-NB26 epithelial cells but not in non-tumourigenic RWPE-1, myoepithelial WPMY-1 cells, or SV40-immortalized PNT1A. Endogenous cell surface GnRH receptor was undetectable in non-transfected cells or cancer cell lines LNCaP, PC3, and DU145. GnRH receptor levels correlated with induction of inositol phosphates, elevation of intracellular Ca(2+) , cytoskeletal actin reorganization, modulation of ERK activation and cell growth-inhibition with GnRH agonists. Hoechst 33342 DNA staining-cell sorting indicated accumulation of cells in G2 following agonist treatment. Release of exosomes from transfected WPE-1-NB26 was unaffected by agonists, unlike induction observed in HEK293([SCL60]) cells. Increased PARP cleavage and apoptotic body production were undetectable during growth-inhibition in WPE-1-NB26 cells, contrasting with HEK293([SCL60]) . EGF receptor activation inhibited GnRH-induced ERK activation in WPE-1-NB26 but growth-inhibition was not rescued by EGF or PKC inhibitor Ro320432. Growth of cells expressing low levels of GnRH receptor was not affected by agonists. Engineered high-level GnRH receptor activation inhibits growth of a subset of papillomavirus-immortalized prostate cells. Elucidating mechanisms leading to clone-specific differences in cell surface GnRH receptor levels is a valuable next step in developing strategies to exploit prostate cell anti-proliferation using GnRH agonists.

Synthesis and biological evaluation of piperazinyl heterocyclic antagonists of the gonadotropin releasing hormone (GnRH) receptor

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has resulted in positive clinical results in reproductive tissue disorders such as endometriosis and prostate cancer. Following the recent discovery of orally active GnRH antagonists based on a 4-piperazinylbenzimidazole template, we sought to investigate the properties of heterocyclic isosteres of the benzimidazole template. We report here the synthesis and biological activity of eight novel scaffolds, including imidazopyridines, benzothiazoles and benzoxazoles. The 2-(4-tert-butylphenyl)-8-(piperazin-1-yl)imidazo[1,2-a]pyridine ring system was shown to have nanomolar binding potency at the human and rat GnRH receptors as well as functional antagonism in vitro. Additional structure-activity relationships within this series are reported along with a pharmacokinetic comparison to the benzimidazole-based lead molecule.

Gonadotropin-Releasing Hormone Analog Structural Determinants of Selectivity for Inhibition of Cell Growth: Support for the Concept of Ligand-Induced Selective Signaling

GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.

Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression

A significant proportion of human gonadotropin-releasing hormone receptors (GnRHRs) are normally retained in the endoplasmic reticulum (ER); however, nearly all rat GnRHRs are routed to the plasma membrane. When mutations are introduced into either receptor, considerably more of the proteins are recognized by the quality control system (QCS) as misfolded and retained compared with wild-type (WT) receptor, resulting in decreased signaling in the presence of agonist. Calnexin, a component of the QCS, decreased plasma membrane expression of the GnRHRs, an effect that was mediated by a physical interaction between the receptor and the calnexin. Only the human receptor showed reduced signaling because it had fewer spare receptors compared with the rat GnRHR, allowing calnexin to affect signaling. Calnexin did not affect receptor signaling when K(191) was deleted from the human WT GnRHR. Removal of this amino acid decreases receptor misfolding and increases plasma membrane expression. K(191) is not present in the rat WT GnRHR. A pharmacological chaperone that corrects GnRHR misfolding, increased expression of the human WT GnRHR in the presence of calnexin. Calnexin apparently retains misfolded GnRHRs but routes correctly folded receptors to the plasma membrane. Mutation of a calnexin protein kinase C consensus phosphorylation site promoted increased retention of the human GnRHR, suggesting that calnexin phosphorylation controls the retention mechanism. We conclude that a proportion of the human and the rat WT GnRHR appears to be retained in the ER by calnexin, an effect that decreases GnRHR signaling capacity.

Identification of new gonadotrophin-releasing hormone partial agonists

GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10(-8) to 10(-5) M) of GnRH analogues (c(4-10)[Asp4,DNal6,Dpr10]-GnRH; c(4-10) [Dpr4,DNal6,Asp10]-GnRH; c(4-10)[Cys(4,10),DNal6]-GnRH; c[Eaca1,DNal6]-GnRH; c[Gly1,DNal6]-GnRH; c[betaAla1,DTrp6]-GnRH; c[Dava1,DNal6]-GnRH; c[Gaba1, DNal6]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed. With both human and rat GnRHRs, c[Eaca1,DNal6]-GnRH, c[Gly1,DNal6]-GnRH, c[betaAla1,DTrp6]-GnRH and c[Dava1,DNal6]-GnRH exhibited partial agonist activity (35-87% of the maximal efficacy shown by 10(-6) M GnRH), whereas c[Gaba1,DNal6]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4-10)[Asp4, DNal6,Dpr10]-GnRH and c(4-10)[Dpr4,DNal6,Asp10]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4-10) [Cys(4,10),DNal6]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba1,DNal6]-GnRH stimulation of the human GnRHR, and c[Dava1,DNal6]-GnRH and c[Gaba1, DNal6]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist. The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.

The LIM-Homeodomain Proteins Isl-1 and Lhx3 Act with Steroidogenic Factor 1 to Enhance Gonadotrope-Specific Activity of the Gonadotropin-Releasing Hormone Receptor Gene Promoter

The GnRH receptor (GnRH-R) plays a central role in mammalian reproductive function throughout adulthood. It also appears as an early marker gene of the presumptive gonadotrope lineage in developing pituitary. Here, using transient transfections combined with DNA/protein interaction assays, we have delineated cis-acting elements within the rat GnRH-R gene promoter that represent targets for the LIM-homeodomain (LIM-HD) proteins, Isl-1 and Lhx3. These factors, critical in early pituitary development, are thus also crucial for gonadotrope-specific expression of the GnRH-R gene. In heterologous cells, the expression of Isl-1 and Lhx3, together with steroidogenic factor 1 (SF-1), culminates in the activation of both the rat as well as human GnRH-R promoter, suggesting that this combination is evolutionarily conserved among mammals. The specificity of these LIM-HD factors is attested by the inefficiency of related proteins, including Lhx5 and Lhx9, to activate the GnRH-R gene promoter, as well as by the repressive capacity of a dominant-negative derivative of Lhx3. Accordingly, targeted deletion of the LIM response element decreases promoter activity. In addition, experiments with Gal4-SF-1 fusion proteins suggest that LIM-HD protein activity in gonadotrope cells is dependent upon SF-1 binding. Finally, using a transgenic model that allows monitoring of in vivo promoter activity, we show that the overlapping expression of Isl-1 and Lhx3 in the developing pituitary correlates with promoter activity. Collectively, these data suggest the occurrence of a specific LIM-HD pituitary code and designate the GnRH-R gene as the first identified transcriptional target of Isl-1 in the anterior pituitary.

Regulation of G Protein-coupled Receptor Trafficking by Inefficient Plasma Membrane Expression: MOLECULAR BASIS OF AN EVOLVED STRATEGY

Despite the prevalence of G protein-coupled receptors as transducers of signals from hormones, neurotransmitters, odorants, and light, little is known about mechanisms that regulate their plasma membrane expression (PME), although misfolded receptors are recognized and retained by a cellular quality control system (QCS). Convergent evolution of the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) progressively decreases inositol phosphate production in response to agonist, validated as a measure of PME of receptor. A pharmacological chaperone that optimizes folding also increases PME of human, but not of rat or mouse, GnRHR because a higher percentage of human GnRHRs are misfolded structures due to their failure to form an apparent sulfhydryl bridge, and they are retained by the QCS. Bridge formation is increased by deleting (primate-specific) Lys191. In rat or mouse GnRHR that lacks Lys191, the bridge is non-essential and receptor is efficiently routed to the plasma membrane. Addition of Lys191 alone to the rat sequence did not diminish PME, indicating that other changes are required for its effects. A strategy, based on identification of amino acids that both 1) co-evolved with the Lys191 and 2) were thermodynamically unfavorable substitutions, identified motifs in multiple domains of the human receptor that control the destabilizing influence of Lys191 on a particular Cys bridge, resulting in diminished PME. The data show a novel and underappreciated means of posttranslational control of a G protein-coupled receptor by altering its interaction with the QCS and provide a biochemical explanation of the basis of disease-causing mutations of this receptor.

Distribution, cloning and sequencing of GnRH, its receptor, and effects of gastric acid secretion of GnRH analogue in gastric parietal cells of rats

Our objective was to study the distribution of gonadotropin-releasing hormone (GnRH) and its receptor, cloning and sequencing of GnRH and its receptor gene in cultured gastric parietal cells of rats. The distribution of GnRH and its receptor mRNA were investigated through immunocytochemical ABC methods and in situ hybridization methods in cultured gastric parietal cells of rats. After isolation of the total RNA from the parietal cells, RT-PCR was conducted to obtain GnRH and its receptor cDNA. Then, the products of PCR was purified, digested by the restriction enzyme of Hind III and EcoR I, and DNA fragments of interests were cloned into pUC19 vector. The products of PCR were analyzed by sequencing with Sanger's method after identified by PCR and digestion of restriction enzyme. Gastric parietal cells showed GnRH and its receptor immunoreactivity; positive material was located in cytoplasm other than in nuclei. GnRH and its receptor mRNA hybridized signals were also detected in cytoplasm with negative nuclei. The specific amplified band of GnRH and its receptor sequences were detected through Agarose gel electrophoresis, and GnRH gene sequence is identical to that of GnRH which has been reported in rat hypothalamus and GnRH receptor sequence is identical to that of the pituitary of rat. GnRH analogue (Alarelin) could inhibit the gastric acid secretion both by direct actions on parietal cells and by inhibiting vagous function. Our data suggest that GnRH could be produced by gastric parietal cells of rats and may modulate physiological function of gastric parietal cells of rats through autocrinal and paracrinal way.