Human Gonadotropin-releasing Hormone Receptor Gene Research Articles

A corazonin G protein-coupled receptor gene in the tick Ixodes scapularis yields two splice variants, each coding for a specific corazonin receptor

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G16. CRZ-Ra turned out to be selective for I. scapularis corazonin (EC50 = 10−8 M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC50 = 4 x 10−8 M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene.

Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells

GnRH, acts via the GnRH receptor (GnRHR), plays a pivotal role in human reproduction by stimulating the synthesis and secretion of gonadotropins from pituitary gonadotropes. Studies have also suggested that it has other extra-pituitary functions. To date, the transcriptional regulation of human GnRHR gene in the brain remains largely unknown. Recently, the human cerebellar medulloblastoma cell line TE-671 is found to express GnRH. We report here for the first time that GnRHR is also expressed in this neuronal cell line. Treatment with GnRHR agonist stimulated the phosphorylation of both ERK1/2 and JNK in the cells. Moreover, transient transfection of various human GnRHR promoter-luciferase constructs into the cells identified an upstream promoter region located between -2197 and -1018. Important cis-acting regulatory elements were found at -1300/-1018 and -2197/- 1900, as deletion of either region caused a dramatic decrease in the promoter activity. An upstream GnRHR promoter element was identified to be important for basal transcription in the human neuronal TE-671 cells, in contrast to the previous finding that a downstream promoter is responsible for the gonadotrope-specific expression. Furthermore, we showed that antide (GnRHR antagonist) significantly stimulated the GnRHR promoter activity and inhibition of protein kinase C (PKC) pathway by staurosporine could also up-regulate the promoter activity in dose- and time-dependent manners. Taken together, these data suggest that activation of the GnRHR by interacting with GnRH may transcriptionally down-regulate itself via the PKC pathway in human neuronal cells.

Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans

In human beings, two forms of GnRH, termed GnRH-I and GnRH-II, encoded by separate genes have been identified. Although these hormones share comparable cDNA and genomic structures, their tissue distribution and regulation of gene expression are significantly dissimilar. The actions of GnRH are mediated by the GnRH receptor, which belongs to a member of the rhodopsin-like G protein-coupled receptor superfamily. However, to date, only one conventional GnRH receptor subtype (type I GnRH receptor) uniquely lacking a carboxyl-terminal tail has been found in the human body. Studies on the transcriptional regulation of the human GnRH receptor gene have indicated that tissue-specific gene expression is mediated by differential promoter usage in various cell types. Functionally, there is growing evidence showing that both GnRH-I and GnRH-II are potentially important autocrine and/or paracrine regulators in some extrapituitary compartments. Recent cloning of a second GnRH receptor subtype (type II GnRH receptor) in nonhuman primates revealed that it is structurally and functionally distinct from the mammalian type I receptor. However, the human type II receptor gene homolog carries a frameshift and a premature stop codon, suggesting that a full-length type II receptor does not exist in humans.

An activator protein 1-like motif mediates 17beta-estradiol repression of gonadotropin-releasing hormone receptor promoter via an estrogen receptor alpha-dependent mechanism in ovarian and breast cancer cells.

Although it is recognized that estrogen is one of the most important regulators of GnRH receptor (GnRHR) gene expression, the mechanism underlying the regulation at the transcriptional level is unknown. In the present study, we demonstrated that 17beta-estradiol (E2) repressed human GnRHR promoter via an activator protein 1-like motif and estrogen receptor-alpha, of which the DNA-binding domain and the ligand-binding domain were indispensable for the repression. Interestingly, the same cis-acting motif was also found to be important for both the basal activity and phorbol 12-myristate 13-acetate responsiveness of the GnRHR promoter. EMSAs indicated that multiple transcription factors including c-Jun and c-Fos bound to the activator protein 1-like site and that their DNA binding activity was not significantly affected by E2 treatment. In addition, we demonstrated that the E2 repression could be antagonized by phorbol 12-myristate 13-acetate, which stimulated c-Jun phosphorylation on serine 63, a process that is a prerequisite for recruitment of the transcriptional coactivator cAMP response element binding protein (CREB)-binding protein (CBP). Concomitantly, we found that overexpression of CBP could reverse the suppression in a dose-dependent manner. Taken together, our data indicate that E2-activated estrogen receptor-alpha represses human GnRHR gene transcription via an indirect mechanism involving CBP and possibly other transcriptional regulators.

Two Inr elements are important for mediating the activity of the proximal promoter of the human gonadotropin-releasing hormone receptor gene.

Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3' CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5' and 3' deletion mutant analysis. The promoter element drove a 13.1 +/- 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived alphaT3-1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36-150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our studies suggested that these two promoters share common protein factors to regulate transcription initiations at two different regions. Additional mechanisms are needed to modulate the efficiencies of individual promoters for developmental and/or tissue-specific regulations.

Oct-1 is involved in the transcriptional repression of the gonadotropin-releasing hormone receptor gene.

Previous deletion analysis of the 5'-flanking region of human GnRH receptor (GnRHR) gene has revealed a powerful negative regulatory element (NRE) located between nucleotide -1017 and -771. In the present study, we demonstrated that this NRE could repress the homologous promoter, irrespective of its position and completely abolish the activity of a heterologous thymidine kinase promoter in an orientation-dependent manner. Progressive 3'-deletion analysis revealed that most of the silencing activity of the NRE resided in a putative octamer regulatory sequence (5'AAGCAAACT3'), which alone could repress the promoter activities by 69-90% in ovarian OVCAR-3, placental JEG-3, and gonadotrope-derived alphaT3-1 cells. Mutation of the AAAC residues of the octamer sequence completely removed its silencing activity. Interestingly, conversion of the octamer sequence into that of the rodent GnRHR promoter (5'AAGCAAAGT3') did not attenuate its silencing effect, indicating that the repressive role of the octamer sequence is evolutionarily conserved. EMSAs showed that common DNA-protein complexes of the same mobility were formed with nuclear extracts from the reproductive cells and gonadotropes, and a consensus octamer transcription factor-1 (Oct-1) binding sequence could dose dependently inhibit the complex formation. Antibody supershift and Southwestern blot assays confirmed that the protein binding to the octamer sequence was the ubiquitously expressed transcription factor Oct-1. Overexpression of Oct-1 augmented the silencing activity of the octamer sequence in alphaT3-1 cells. Taken together, our results clearly indicate a role of Oct-1 in the transcriptional repression of the human GnRHR gene.

Characterization of a new upstream GnRH receptor promoter in human ovarian granulosa-luteal cells.

GnRH has been implicated as an important local autocrine and paracrine factor in regulating ovarian function. However, to date, the transcriptional regulation of GnRH receptor (GnRHR) gene in human ovary remains poorly understood. Here we report the characterization of a new upstream promoter for the GnRHR gene in human granulosa-luteal cells. Using progressive deletion analysis, a region between nucleotide -1300 and -1018 (relative to the translation start site) was shown to exhibit the highest promoter activities in two immortalized human granulosa-luteal cell lines, SVOG-4o and SVOG-4m. Two putative CCAAT/enhancer binding protein (C/EBP) motifs and one GATA motif were identified within this region. Mutational studies showed that these three motifs cooperated synergistically to regulate GnRHR gene transcription in the granulosa cells but not in other cell types including human ovarian carcinoma OVCAR-3, human embryonic kidney-293 (HEK-293) and mouse pituitary gonadotrope-derived alphaT3-1 cells. Surprisingly, by competitive EMSAs, we found that an Oct-1 consensus sequence was able to inhibit protein complex formation with the distal C/EBP motif, suggesting a possible cross-talk between the Oct-1 transcription factor and this C/EBP motif. Taken together, our results strongly indicate a role of the C/EBP and GATA motifs in regulating GnRHR gene transcription in human granulosa-luteal cells and further suggest that tissue-specific expression of human GnRHR gene is mediated by differential promoter usage.

Gonadotropin-releasing hormone receptor: cloning, expression and transcriptional regulation

In summary, isolation of GnRH receptor cDNA, its gene, and identification of regulatory elements in the flanking region of the gene have added to our knowledge regarding the tissue-specific expression of the GnRH receptor gene, and the mechanisms that mediate and influence its transcriptional regulation. However, the interactions of the different regulatory factors (nuclear factors) and the effects of these interactions on the regulation of the GnRH receptor gene remain unclear. Due to existence of multiple promoters and transcriptional start sites in human GnRH receptor gene and the lack of a human gonadotrope cell line, the precise promoter and transcriptional start sites in human pituitary, extra-pituitary tissues and tumors have not yet been identified.

Differential Role of PR-A and -B Isoforms in Transcription Regulation of Human GnRH Receptor Gene

Differential Role of PR-A and -B Isoforms in Transcription Regulation of Human GnRH Receptor Gene

Differential role of PR-A and -B isoforms in transcription regulation of human GnRH receptor gene.

The presence of progesterone response element (PRE) in the 5'-flanking region of the human GnRH receptor (GnRHR) suggests the possible regulation of this gene by progesterone (P). In the present study, we examined the effects of P in transcriptional regulation of human GnRHR gene expression at the pituitary and placenta levels since the GnRHR has been detected in both tissues. By the use of transient transfection assays, a differential regulation of human GnRHR promoter activity by P was observed. P treatment resulted in a decrease in promoter activity in the pituitary alphaT3-1 cells, suggesting a P-mediated inhibitory action. Interestingly, P is found to have a stimulatory role at the placental expression of this gene. Addition of RU486 to, or inhibition of endogenous P production by, the placental JEG-3 cells leads to a decrease in promoter activity, which is reversed by the replacement of P. Further studies have identified a putative PRE, namely human GR-PRE (located between -535 and -521, related to translation start site), that may be responsible for the P action since the mutation of these motifs reversed the P-mediated effects. The binding of PR to this element is confirmed by antibody supershift assays. The physiological effects of P are mediated through two PR isoforms, namely PR-A and PR-B. In the present study, overexpression of human PR-A resulted in a decrease in human promoter activity in both pituitary and placental cells. Interestingly, overexpression of PR-B exhibits a cell-dependent transcriptional activity, whereby it functions as a transcription activator in the placenta but as a transcription repressor in the pituitary. In summary, our results demonstrated a differential usage of PR-A and PR-B in transcriptional regulation of human GnRHR gene expression by P at the pituitary and placenta levels.

Human gonadotropin-releasing hormone receptor gene transcription: up-regulation by 3′,5′-cyclic adenosine monophosphate/protein kinase A pathway

Transient transfection of mouse gonadotrope-derived (αT3-1) cells with a 2297 bp human GnRHR promoter-luciferase construct (p2300-LucF) showed a dose- and time-dependent increase in the human gonodotropin-releasing hormone receptor (GnRHR) promoter activity after forskolin treatment. An average of 4.8-fold increase in promoter activity was observed after 12 h of 10 μM forskolin treatment. This effect was mimicked by administrating of cholera toxin, cAMP analog or pituitary adenylate cyclase activating polypeptide 38 (PACAP). A specific adenylate cyclase (AC) inhibitor (ACI) or protein kinase A (PKA) inhibitor (PKAI) pretreatment reversed the forskolin- and PACAP-induced increase in the human GnRHR promoter activity. These results not only confirm the stimulatory effect of Cyclic adenosine monophosphate (cAMP) in human GnRHR promoter activation, but also suggest that hormones or neurotransmitters that activate adenylate cyclase in pituitary gonadotropes may increase the expression of human GnRHR gene in transcriptional level. Progressive 5′ deletion assays identified a 412 bp fragment (−577 to 167) in the human GnRHR 5′-flanking region that is essential in maintaining the basal responsiveness to cAMP. Mutagenesis coupled with functional studies have identified two putative AP-1/CREB binding sites, namely hGR-AP/CRE-1 and hGR-AP/CRE-2 that participated in mediating the cAMP-stimulatory effect. Mutation of the putative hGR-AP/CRE-1 and hGR-CRE-2 resulted in a 38 and 32% decrease in the forskolin-induced stimulation. However, mutation of both binding sites did not completely abolish the cAMP-stimulatory effect, suggesting that multiple transcription factor binding sites were involved in full response in cAMP stimulation. The binding of CREB to these motifs was confirmed by gel mobility shift assay and antibody supershift assay.

Interplay of pituitary adenylate cyclase-activating polypeptide with a silencer element to regulate the upstream promoter of the human gonadotropin-releasing hormone receptor gene

Multiple transcription start sites were identified in the human gonadotropin releasing hormone receptor (hGnRHR) gene. Recently, an upstream promoter residing at −1727/−1674, in vicinity of a CAP site at −1673, was characterized. In this report, we elucidated the underlying mechanisms for the regulation of this promoter. Functionally, this promoter was constitutively suppressed by a silencer element (−1673/−1351) situated immediately downstream to it. On the other hand, pituitary adenylate cyclase-activating polypeptide (PACAP), via the cAMP pathway, was found to be the extracellular cue to control the upstream promoter. Following PACAP-27, PACAP-38 (30 nM) and forskolin (25 μM) treatment, there were significant increases in the reporter gene activities. By deletion analysis, the region residing at −1727 to −1577, containing the distal promoter and 97 bp of the silencer was subsequently found to be responsible for PACAP/cAMP induction. To localize the PACAP-dependent cis-acting element(s) within the silencer, block replacement scanning mutation was performed and a hGnRHR gene PACAP-responsive element (GPRE) was identified at −1676/−1648. The actions of PACAPs and forskolin on the GPRE were further evidenced by gel mobility shift assays. There was an increase in protein binding onto this element only after peptide treatment. As GnRH receptor number on gonadotrope cell surface is a key factor in regulating gonadotropin release, the present study provides an insight into the interplay between PACAP and GnRH receptors on pituitary gonadotropes to control human reproductive functions.

Functional mapping of a placenta-specific upstream promoter for human gonadotropin-releasing hormone receptor gene.

GnRH has been showed to regulate hCG expression and secretion from the placenta through a GnRH receptor (GnRHR)-mediated process. Recently, we have reported the isolation of human GnRHR full-length complementary DNA from the human placental cells including choriocarcinoma JEG-3 cells, immortalized extravillous trophoblasts, and primary cultures of trophoblasts. Despite these observations, the molecular mechanism that controls the transcription regulation of the GnRHR gene expression in the placenta remains unknown. Here we described the identification of an upstream placenta-specific promoter located between nucleotide (nt) -1737 and -1346 (relative to the translation start site) for the human GnRHR gene. Using transient transfection studies, this upstream promoter has been shown to determine the placental cell-specific expression of this gene. Primer extension studies further confirmed the utilization of this promoter in JEG-3 cells in vivo. By mutagenesis coupled to functional studies, we have identified four putative transcription factor-binding sites, namely human glucocorticoid receptor (hGR)-Oct-1 (nt -1718 to -1710), hGR-cAMP response element (CRE; nt -1649 to -1641), hGR-GATA (nt -1602 to -1597), and hGR-activating protein-1 (nt -1518 to -1511), that are essential to the expression of this gene. Mutations of these cis-acting motifs reduced the promoter activity. The CRE and GATA motifs were subsequently shown to be placenta specific, as mutations of these motifs caused a dramatic loss in promoter activities in the placental JEG-3 cells, but not in the ovarian carcinoma OVCAR-3, monkey kidney COS-1, and human embryonic kidney 293 cells. Gel mobility assays confirmed the binding of nuclear proteins Oct-1, CRE-binding protein, GATA-2, GATA-3, c-Fos, and c-Jun from JEG-3 cells to these four elements.

Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: role of protein kinase C and activating protein 1.

Clinical applications of GnRH agonists (GnRHa) are based primarily on the decrease in gonadotropin release after down-regulation of the GnRH receptor (GnRHR) by continuous GnRHa administration. However, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR gene after prolonged GnRH treatment remain poorly understood. In the present study GnRHa-mediated regulation of human GnRHR gene transcription was studied by transiently transfecting the mouse gonadotrope-derived (alphaT3-1) cells with a 2297-bp human GnRHR promoter-luciferase construct (p2300-LucF). A dose- and time-dependent decrease in human GnRHR promoter activity was observed after GnRHa treatment. An average 71% decrease in promoter activity was observed after 24-h treatment with 0.1 microM GnRHa, which was blocked by cotreatment of the GnRH antagonist, antide. This effect was mimicked by phorbol 12-myristate 13-acetate (TPA) administration. In addition, the GnRHa- and TPA-mediated decrease in the human GnRHR promoter activity was reversed by a specific protein kinase C (PKC) inhibitor, GF109203X, or depletion of PKC by TPA pretreatment. These findings indicate that the activation of the PKC pathway is important in regulating the human GnRHR gene expression. By progressive 5'-deletion studies, we have identified a 248-bp DNA fragment (-1018 to -771, relative to the translation start site) at the 5'-flanking region of the human GnRHR gene that is responsible for the GnRHa-mediated down-regulation of human GnRHR promoter activity. Analysis of this sequence reveals the existence of two putative activating protein-1 (AP-1) sites with 87% homology to the consensus sequence (5'-TGA(G/C)T(C/A)A-3'), located at -1000 to -994 (5'-TTAGACA-3', in complementary orientation) and -943 to 937 (5'-TGAATAA-3'). Using competitive gel mobility shift assays, AP-1 binding was observed within this 248-bp region. Site-directed mutation of the putative AP-1-binding site located at -1000 to -994 abolished the GnRHa-induced inhibition. Further competitive GMSA and supershift experiments confirmed the identity of AP-1 binding in this region. By the use of Western blot analysis, a significant increase in c-Jun (100%; P < 0.05) and c-Fos (50%; P < 0.05) protein levels was observed after GnRHa treatment in alphaT3-1 cells. In addition, our data suggested that a change in AP-1 composition, particularly c-Fos, was important in mediating GnRHa-induced inhibition of human GnRHR gene expression. We conclude that activation of the PKC pathway by GnRH is important in controlling human GnRHR gene expression. In addition, the putative AP-1-binding site located at -1000 to -994 of the human GnRHR5'-flanking region has been functionally identified to be involved in mediating this down-regulatory effect.

Polymorphisms at the 5' end of the human gonadotropin-releasing hormone receptor gene are not associated with the timing of menarche in Japanese girls

We read with interest the article by Kakar (1) and the commentary by Miller (2) in the European Journal of Endocrinology, which showed the diversity and species speci®city of the regulatory elements in the gonadotropin-releasing hormone receptor (GnRHR) gene and reviewed the important role of GnRHR gene regulation in the control of the hypothalamic±pituitary±gonadal axis respectively. Thus we hypothesized that polymorphism(s) in the regulatory region in the GnRHR gene could be a genetic factor that in uences the timing of the onset of puberty. By direct sequencing of the human GnRHR gene following PCR ampli®cation of genomic DNA, we identi®ed seven polymorphisms at the 5 end of the gene (Table 1). Allele patterns obtained by direct sequencing and sequencing followed by subcloning of the region containing all seven polymorphisms suggested that the polymorphisms were tightly linked on the same allele. The allele frequency with a 10 bp insertion (allele I) in the 86 healthy Japanese subjects was 0.46, which was determined by PCR ampli®cation of the region and electrophoresis in a 4% agarose gel. The primer pairs used were as follows: PP2-F, 5-CCTAATGAATCAGTCCATCATTAAC-3; PP2-R, 5-TAGGGGAAAAAGATTAGAGCATATC-3. The length of allele I was 198 bp, whereas that of the wild type (allele D) was 188 bp. Then we genotyped the polymorphism in 196 Japanese girls (aged 18±20) in a school of nursing. Ages at menarche were obtained by means of a questionnaire and the heights were measured at the time of blood sampling. Age at menarche and height for the whole group were (means 6 S.D.) 12.2 6 1.1 years and 157.7 6 5.3 cm respectively. These values were consistent with a previous report on the Japanese population (3). The frequency of allele I was 0.47. However, there were no signi®cant differences in the timing of menarche or the height among the three genotypes (I/I, I/D and D/D; tested by one-way analysis of variance) or among the two homozygous genotypes (I/I and D/D; tested by paired t-test). Thus, this high-frequency polymorphic allele at the 5 end of the GnRHR gene is not likely to be associated with the timing of menarche or with ®nal height in Japanese girls. To date, only one polymorphism in the coding region of the GnRHR gene has been reported for a subset of pituitary adenomas (4). As far as the timing of menarche is concerned, there are no reports studying polymorphisms of the genes related to the hypothalamic±pituitary±gonadal axis, though positive associations with CYP17 gene (5) and vitamin D receptor gene (6) polymorphisms have been reported. In this study, we expected that the polymorphism(s) in the 5 end of the GnRHR gene would be one of the strong candidate regions affecting the timing of menarche and, therefore, ®nal height, but the results excluded this possibility.

Regulation of Human Gonadotropin-Releasing Hormone Receptor Gene Expression in Placental Cells

Regulation of Human Gonadotropin-Releasing Hormone Receptor Gene Expression in Placental Cells

Identification of an Upstream Promoter in the Human Gonadotropin-Releasing Hormone Receptor Gene

Analysis of the human gonadotropin-releasing hormone receptor (hGnRHR) gene 5′ flanking region revealed the presence of multiple TATA, CCAAT, and transcription start sites. In addition, at least three different transcripts (5.0, 2.5, and 1.5 kb) were detected by Northern blot analysis. Taken together, these data indicated the existence of multiple promoter elements in the hGnRHR gene, and these promoters are responsible for the multiplicity of regulation of human reproductive functions. In this report, by progressive 5′ and 3′ deletion (−2197 to −1351, relative to the ATG) and NotI linker scanning mutagenesis coupled to transient transfection into the mouse gonadotrope-derived αT3-1 cell, a distal promoter element was identified at −1705/−1674. The promoter was located immediately 5′ to a previously identified CAP site at −1673 in human pituitary and it drove a 17.6- ± 1.0-fold increase in reporter gene activity. Within the promoter, a pyrimidine-rich initiator element (Inr) (−1682) and a CCAAT box (−1702) were found and mutation of these elements abrogated both protein bindings and promoter activities. By 1- and 2-D SouthWestern blot assays, multiple nuclear factors (40 to 54 kDa) were found to interact specifically with this promoter element. These nuclear factors were also present in other cells, including COS-7, JEG-3, and SKOV-3 cells, and these findings were consistent with functional studies which showed that the promoter is also active in these cells.

Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells

In terms of regulation of gene expression, gonadotropin-releasing hormone receptor (GnRHR) found in extrapituitary tissues has been suggested to be different from that in the pituitary. In the present study, we examined the molecular basis of this difference using the pituitary αT3-1 and ovarian carcinoma OVCAR-3 cells. As a first step, the different expression levels of GnRHR mRNA in the pituitary and ovarian cells were investigated using semi-quantitative RT-PCR. Quantitative analysis showed that the expression level of hGnRHR is a nine-fold higher in primary pituitary tissues than the primary culture of ovarian carcinomas (PCO). In pituitary αT3-1 cells, the expression level of hGnRHR was ten-fold higher than ovarian carcinoma OVCAR-3 cells. The possibility of the differential use of various cell-specific promoters in different cells was addressed by transiently transfecting cells with 3′-deletion clones of human GnRHR promoter. Sequential deletion of the 5′-flanking region of the gene revealed the use of two putative promoters, designated PR1 (-771 to -557) and PR2 (-1351 to -1022), and a negative control region (-1022 to -771), in the pituitary and ovarian cells. The same promoters appeared to be utilized for driving the basal promoter activities in both αT3-1 and OVCAR-3 cells, suggesting that there is no cell-specific promoter usage for the human GnRHR gene. Alternatively, the involvement of different regulatory protein factors was investigated using electrophoretic gel mobility shift assays. When end-labeled PR1 was used as a probe, two unique shifted complexes were identified in OVCAR-3 cells when compared to αT3-1 cells. One unique protein–DNA complex was observed in αT3-1 cells compared to OVCAR-3 cells when incubated with end-labeled PR2 as a probe. These DNA–protein complexes appeared to be specific, as they competed with excess amount of unlabelled competitor PR1 and PR2, respectively. In summary, it is unlikely that the use of a cell-specific promoter contributes to the different characteristics of ovarian GnRHR. Our study demonstrates that one mechanism by which cell-specific expression of human GnRHR is achieved is through the binding of distinct and/or combinations of cell-specific regulatory factors to various promoter elements in the 5′-flanking region of the gene.

Steroidogenic Factor-1 Interacts with a Gonadotrope-Specific Element within the First Exon of the Human Gonadotropin-Releasing Hormone Receptor Gene to Mediate Gonadotrope-Specific Expression

Steroidogenic Factor-1 Interacts with a Gonadotrope-Specific Element within the First Exon of the Human Gonadotropin-Releasing Hormone Receptor Gene to Mediate Gonadotrope-Specific Expression

Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene.

The expression pattern of the GnRH receptor was investigated in a variety of normal and neoplastic human tissues by RT-PCR-Southern blotting. In addition to the full-length cDNA (sb1), we identified two other transcripts: the first (sb2) was characterized by a 128 bp deletion as previously described; the second was an unexpected finding composed of a shorter cDNA (sb3), the sequence of which revealed a 220 bp deletion corresponding in size to exon 2. These three transcripts were found in normal pituitary and pituitary adenomas, and in granulosa tumors, but not in testis, where sb2 was lacking. Only sb1 was expressed in normal, fibrocystic and malignant breast tissue. No transcript with a full-length region was found in endometrium, intestine or lymphocytes. This is the first report that shows that splicing of the gonadotropin-releasing hormone receptor gene is tissue dependent. We also determined the intron-exon nucleotide sequence of the gene and identified an MaeIII polymorphic site in exon 1 created by a silent C453T transition found in 10% of unrelated French whites.