Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane.

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 microCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

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.

Molecular cloning and expression of a type-two somatostatin receptor in goldfish brain and pituitary.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, a SRIF receptor cDNA was cloned and sequenced from goldfish brain using PCR and cDNA library screening. The cDNA encodes a 380-amino acid goldfish type-two SRIF receptor (designated as sst(2)), with seven putative transmembrane domains (TMD) and YANSCANP motif in the seventh TMD, a signature sequence for the mammalian SRIF receptor (sst) family. In addition, the amino acid sequence of the receptor has 61-62% homology to mammalian sst(2), 41-47% homology to other mammalian sst subtypes and 41-43% homology to recently identified fish sst(1) and sst(3) receptors. Both SRIF-14 and [Pro(2)]SRIF-14, two of the native goldfish SRIF forms, but not a putative goldfish SRIF-28, significantly inhibited forskolin-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) release in COS-7 cells transiently expressing goldfish sst(2), suggesting functional coupling of the receptor to adenylate cyclase. None of the three peptides affected inositol phosphate production in the same receptor expression system. Northern blot showed that mRNA for the sst(2) receptor is widely distributed in goldfish brain, and highly expressed in the pituitary. The decrease in pituitary sst(2) mRNA levels following estradiol implantation suggests the presence of a negative feedback mechanism on sst(2) gene expression.

Differential effects of the charge variants of human follicle-stimulating hormone.

FSH is synthesized and secreted by the anterior pituitary gland in multiple molecular forms; the release of these isoforms depends on the endocrine status of the donor at the time of sample collection. In the present study, we analysed the possibility that the FSH charge isoforms may exert differential effects at the target cell. Seven FSH isoform mixes were isolated from pooled anterior pituitary glycoprotein extracts by high resolution chromatofocusing, followed by affinity chromatography, which removed nearly 90% of the LH that co-eluted with the FSH isoforms during chromatofocusing. The isoforms (isoform I, pH >7.10; II, pH range 6.60-6.20; III, pH 5. 47-5.10; IV, pH 5.03-4.60; V, pH 4.76-4.12; VI, pH 4.05-3.82 and VII, pH <3.80) were then tested for their capacity to stimulate cAMP release, androgen aromatization and tissue-type plasminogen activator (tPA) enzyme activity and cytochrome P450 aromatase, tPA and inhibin alpha-subunit mRNA production by rat granulosa cells in culture. cAMP and oestradiol production were determined by RIA, tPA enzyme activity by SDS-PAGE and zymography and all mRNAs by northern blot hybridization analysis and semiquantitative RT-PCR. All isoforms, with the exception of isoform I, stimulated synthesis and release of cAMP, oestrogen and tPA enzyme activity in a dose-dependent manner; the potency of the less acidic isoforms (pH 6. 60-4.60) was greater than that exhibited by the more acidic/sialylated analogs (pH 4.76 to <3.80; potencies II>III>IV>V>VII>VI). A similar trend was observed in terms of cytochrome P450 aromatase and tPA mRNA production. In contrast, when FSH-stimulated production of alpha-inhibin mRNA was analysed, isoforms V-VII were significantly more potent (two- to threefold) than the less acidic/sialylated counterparts (II-IV). In contrast to isoforms II-VII (which behaved as FSH agonists), isoform I (elution pH >7.10) completely blocked P450 aromatase and tPA mRNA expression, without altering that of a constitutively expressed gene (glyceraldehyde-3-phosphate dehydrogenase). These results show for the first time that the naturally occurring human FSH isoforms may exhibit differential or even unique effects at the target cell level.

A mathematical model quantifying GnRH-induced LH secretion from gonadotropes.

A mathematical model is developed to investigate the rate of release of luteinizing hormone (LH) from pituitary gonadotropes in response to short pulses of gonadotropin-releasing hormone (GnRH). The model includes binding of the hormone to its receptor, dimerization, interaction with a G protein, production of inositol 1,4, 5-trisphosphate, release of Ca(2+) from the endoplasmic reticulum, entrance of Ca(2+) into the cytosol via voltage-gated membrane channels, pumping of Ca(2+) out of the cytosol via membrane and endoplasmic reticulum pumps, and release of LH. Cytosolic Ca(2+) dynamics are simplified (i.e., oscillations are not included in the model), and it is assumed that there is only one pool of releasable LH. Despite these and other simplifications, the model explains the qualitative features of LH release in response to GnRH pulses of various durations and different concentrations in the presence and absence of external Ca(2+).

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.

Molecular cloning and expression of two type one somatostatin receptors in goldfish brain.

Somatostatin (SRIF or SS) exerts diverse inhibitory actions through binding to specific receptors. In this study, two SRIF receptor complementary DNAs (cDNAs) were cloned and sequenced from goldfish brain using PCR and cDNA library screening. The two cDNAs share 92% similarity in nucleotide sequence and 98% similarity in the deduced amino acid sequences and are presumably derived from duplicate genes, as goldfish are tetraploid. Two cDNAs encode two 367-amino acid goldfish type one SRIF receptors (designated as sst1A and sst1B, respectively), with seven putative transmembrane domains (TMD) and YANSCANP motif in the 7th TMD, a signature sequence for mammalian SRIF receptor (sst) family. In addition, the amino acid sequences of two receptors have 76% and 75% similarity to human or rat sst1, respectively, and 39-55% similarities to other mammalian sst subtypes (sst2-5), suggesting that the two receptors could be the goldfish homologs of mammalian sst1. The difference between goldfish and mammalian sst1 is mainly reflected by the extreme divergence in their extracellular N termini. Both SRIF-14 and [Pro2]SRIF-14, two of the native goldfish SRIF forms, significantly inhibited forskolin-stimulated cAMP release in COS-7 cells transiently expressing goldfish sSt1A or sst1B, suggesting functional coupling of the two receptors to adenylate cyclase. Northern blot and RT-PCR showed that messenger RNAs (mRNAs) for both receptors are widely distributed throughout goldfish brain, whereas only one receptor mRNA is expressed in the pituitary. RT-PCR analysis also detected sst1 receptor mRNAs in several peripheral tissues. These findings provide fundamental information for studying the mechanism of SRIF actions in vertebrates and structural analysis of mammalian sst receptors.

Simultaneous and independent visualization of the gonadotropin-releasing hormone receptor and its ligand: evidence for independent processing and recycling in living cells.

The first step in GnRH signaling is binding by the peptide to its plasma membrane receptor (GnRHR). The receptor is a member of the seven transmembrane G protein-coupled class but lacks the characteristic C-terminal cytoplasmic tail, making it among the smallest receptors in this superfamily. It has been known since 1980 that agonist occupancy of the GnRHR results in patching, capping, and internalization, although it has not been possible to localize the unoccupied GnRHR, because elaboration of receptor antisera has not been easy to achieve. The recent production of a green fluorescent protein (GFP) conjugate of the GnRHR ("rGnRHR-C-tail-GFP") that is expressed in cells, targeted to the plasma membrane, binds GnRH analogs and couples to G proteins has made it possible to monitor movement of the unoccupied receptor by confocal microscopy. In the present study, we used this probe, along with Texas Red conjugates of a GnRH agonist, to examine simultaneous processing of the receptor and its ligands. The preparation of the GFP GnRHR chimera has been described. A Texas Red conjugate was made from the GnRH agonist D-Lys6-Pro9-des-Gly10EA-GnRH by standard procedures. Bioactivity of this conjugate was confirmed. Confocal fluorescence images of living GGH3 cells showed that the agonist binds the GFP-GnRH receptor construct on the cell membrane and causes the internalization of vesicles delimited by a membrane. Shortly after internalization, the agonist separates from receptor inside the vesicle, although it is still enclosed in membranes containing free receptor. As the vesicles approach the perinuclear space, the separation between receptor and agonist is more pronounced. Free receptor appears at the cell membrane after the internalization of agonist has been completed. The protein synthesis inhibitor, cycloheximide (1 mM) did not inhibit this process, suggesting that the free receptor results from the recycling of previously internalized vesicles rather than from newly synthesized receptor. These studies show visual evidence for recycling of the GnRH receptor in cultured cells.

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.

The role of protein kinases A and C pathways in the regulation of mitogen-activated protein kinase activation in response to gonadotropin-releasing hormone receptor activation.

There is convincing evidence that mitogen-activated protein kinase (MAPK) activation is coupled to both receptor tyrosine kinase and G protein-coupled receptors. The presence of the epidermal growth factor (EGF) receptor and the GnRH receptor on the surface of GGH(3)1' cells makes this cell line a good model for the assessment of MAPK activation by receptor tyrosine kinases and G protein-coupled receptors. In this study, to assess the activated and total (i.e. activated plus inactivated) MAPK, the phosphorylation state of p44 and p42 MAPKs was examined using antisera that distinguish phospho-p44/42 MAPK (Thr202/Tyr204) from p44/42 MAPK (phosphorylation state independent). The data show that both EGF (200 ng/ml) and Buserelin (a GnRH agonist; 10 ng/ml) provoke rapid activation of MAPK (within 5 and 15 min, respectively) after binding to their receptors. The role of protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways in mediating MAPK activation was also assessed. Both phorbol ester (phorbol 12-myristate 13-acetate; 10 ng/ml) and (Bu)2cAMP (1 mM) trigger the phosphorylation of MAPK, suggesting potential roles for PKC and PKA signaling events in MAPK activation in GGH(3)1' cells. Treatment of PKC-depleted cells with Buserelin activated MAPK, suggesting involvement of PKC-independent signal transduction pathways in MAPK activation in response to GnRH. Similarly, treatment of PKC-depleted cells with forskolin (50 microM) or cholera toxin (100 ng/ml) stimulated MAPK activation, whereas pertussis toxin (100 ng/ml) had no measurable effect. To further assess the role of PKA in response to EGF and Buserelin, cells were treated with EGF (200 ng/ml) for 3 min or with Buserelin (10 ng/ml) for 10 min after pretreatment with 3-isobutyl-1-methylxanthine (0.5 mM), forskolin (50 microM), or (Bu)2cAMP (1 mM) for 15 min. The results show that MAPK can be activated in a PKA-dependent manner in GGH(3)1' cells. Consistent with previous reports, the current data support the view that MAPK activation can be achieved via both PKC- and PKA-dependent signaling pathways triggered by the GnRH receptor that couples to G(q/11) and Gs alpha-subunit proteins. In contrast, G(i/o)alpha does not appear to participate in MAPK activation in GGH(3)1' cells.

Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: involvement of multiple signal transduction pathways.

Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.

Gonadotropin-releasing hormone receptor concentration differentially regulates intracellular signaling pathways in GGH3 cells.

Pituitary cell lines (GGH3) expressing the GnRH receptor (GnRHR) were used to investigate the effect of GnRHR concentration on the ability of a GnRH agonist to activate second messenger systems. Four different strategies were utilized to generate cells expressing functionally different concentrations of receptors: (1) transient transfection with different concentrations of wild type GnRHR into GH3 cells, (2) utilization of two cell lines derived from a common stably transfected line expressing high (4,209 +/- 535 receptors/cell) or low (1,031 +/- 36 receptors/cell) concentrations of GnRHR, (3) co-incubation of GGH3-1' cells with a GnRH agonist (Buserelin) and a GnRH antagonist to compete for binding sites, and (4) photo-affinity binding to GnRHR with a GnRH antagonist to change effective receptor concentration. A range of receptor concentrations (1,000-8,000 receptors/cell) were generated by these techniques. Inositol phosphate (IP) and cAMP accumulation were quantified to assess the effect of receptor concentration on receptor-effector coupling. Under all four paradigms, the efficacy and potency of Buserelin stimulated IP production was dependent on receptor concentration. In contrast, Buserelin stimulated cAMP release was relatively unchanged at varying concentrations of GnRHR. This suggests that the cellular concentration of GnRHR affects the induction of cell signaling pathways. These results demonstrate that a single ligand-receptor-complex can differentially activate second messenger systems and present a mechanism by which multiple physiological endpoints can be differentially regulated by a single hormone/receptor interaction.

Structure-activity relationships of G protein-coupled receptors.

The primary function of cell-surface receptors is to discriminate the specific signaling molecule or ligand from a large array of chemically diverse extracellular substances and to activate an effector signaling cascade that triggers an intracellular response and eventually a biological effect. G protein-coupled cell-surface receptors (GPCRs) mediate their intracellular actions through the activation of guanine nucleotide-binding signal-transducing proteins (G proteins), which form a diverse family of regulatory GTPases that, in the GTP-bound state, bind and activate downstream membrane-localized effectors. Hundreds of GPCRs signal through one or more of these G proteins in response to a large variety of stimuli including photons, neurotransmitters, and hormones of variable molecular structure. The mechanisms by which these ligands provoke activation of the receptor/G-protein system are highly complex and multifactorial. Knowledge and mapping of the structural determinants and requirements for optimal GPCR function are of paramount importance, not only for a better and more detailed understanding of the molecular basis of ligand action and receptor function in normal and abnormal conditions, but also for a rational design of early diagnostic and therapeutic tools that may allow exogenous regulation of receptor and G protein function in disease processes.

Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone.

A central question in endocrinology is how a single ligand interacting with a single receptor can mediate multiple responses. GnRH interaction with receptor offers a prime example, leading to the regulation of synthesis and release of at least three molecules, regulation of target cell responsiveness and receptor number. The present study suggests a molecular model consistent with extant data that provides a mechanism by which this may occur and, further, which allows for coordinate regulation.

Mutations at the consensus phosphorylation sites in the third intracellular loop of the rat gonadotropin-releasing hormone receptor: effects on receptor ligand binding and signal transduction.

In this study, site-directed mutagenesis of potential phosphorylation sites (Thr238, Ser253, and Thr264) for protein kinase C and C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rat GnRH receptor (rGnRHR) was performed to assess the significance of these regions in the function of the GnRHR. Mutation at one or all of the three potential phosphorylation sites had differential effects on receptor ligand binding. Mutation of Ser253 or Thr264 to Ala did not significantly affect the receptor-binding affinity but decreased the number of measurable binding sites. Mutation of Thr238 to Ala or triple mutation of Thr238, Ser253, and Thr264 impaired or abolished receptor-binding affinity. Mutations of the potential phosphorylation sites affected receptor-mediated inositol phospholipid (IP) production and correlated with alterations in receptor binding after mutation, but they did not significantly affect receptor-mediated cAMP production or cAMP-mediated prolactin release. In addition, mutation of Ser253 or Thr264 to Ala did not affect the GnRH-provoked desensitization in terms of GnRH agonist-stimulated IP production. Deletion of the C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rGnRHR, including a potential phosphorylation site (Thr264), abolished the receptor-binding affinity and receptor-mediated signal transduction. Replacement of the deleted C-terminal portion with a C-terminal portion (Ala-Ala-Arg-Thr-Leu-Ser) of the third intracellular loop of the Gq/11-coupled rat M1 muscarinic acetylcholine receptor did not restore receptor function. These results suggest that the potential phosphorylation sites or the region around the phosphorylation site of the third intracellular loop of the GnRHR is important for the structural integrity and expression of the receptor but that phosphorylation at these sites is not required for desensitization.

Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH and cyclic adenosine monophosphate.

GnRH appears to regulate messenger RNA levels and synthesis of its own receptor (GnRHR). In this study, we examined the regulation of GnRHR gene transcription by GnRH and cAMP in the GGH3 cell line (GH3 cells stably expressing GnRHR). Transient transfection of GGH3 cells with luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of mouse GnRHR gene resulted in an increase in reporter gene (GnRHR-Luc) activity (11- to 22-fold) compared with the promoterless vector. GnRH or a GnRH agonist (Buserelin) significantly stimulated the GnRHR-Luc activity in a dose-dependent manner. Time-course studies using 10(-7) M Buserelin revealed that GnRHR-Luc activity increased progressively from 1.5-6 h, with a peak at 6 h. The increase in GnRHR-Luc activity was lower at 12 and 24 h. Both cholera toxin and dBcAMP significantly stimulated GnRHR-Luc activity. Pretreatment with dBcAMP also enhanced the extent of stimulation of GnRHR-Luc activity in response to Buserelin. Pertussis toxin did not induce basal or Buserelin-stimulated GnRHR-Luc activity. Treatment of GGH3 cells with 10(-9) or 10(-7) M Buserelin for 6 h was sufficient to stimulate a significant increase in cAMP release. An adenylate cyclase inhibitor SQ 22536 did not affect the basal GnRHR-Luc activity but significantly reduced Buserelin-activated GnRHR-Luc activity. These results suggest that GnRH and cAMP activate transcriptional activity of the GnRHR gene and that GnRH activates GnRHR transcriptional activity, in part, through the cAMP pathway. Progressive 5'-deletion analysis revealed that basal and Buserelin- or dBcAMP-stimulated GnRHR-Luc activity were consistently retained after 5'-deletion at position -456, -381, or -331 relative to the major transcription start site but were significantly decreased after subsequent truncation of the promoter from -331 to -255 relative to the major transcription start site. However, the -255 construct still retained responsiveness to Buserelin and dBcAMP, and the relative activity remained similar under both stimulation conditions. These results suggest that elements located between -331 and -255 necessary for transcriptional activity of the GnRHR gene in GGH3 cells, and that the response elements on the mouse GnRHR gene for both GnRH and cAMP reside at two different sites: between -331 and -255 and between -255 and +62.