Inhibition of Human Type I Gonadotropin-Releasing Hormone Receptor (GnRHR) Function by Expression of a Human Type II GnRHR Gene Fragment
http://www.100md.com
内分泌学杂志 2005年第6期
Human Reproductive Sciences Unit, Medical Research Council, EH16 4SB Edinburgh, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. Adam J. Pawson, Human Reproductive Sciences Unit, Medical Research Council, The University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom. E-mail: a.pawson@hrsu.mrc.ac.uk.
Abstract
Humans possess only one functional GnRH receptor, the type I GnRH receptor (GnRHR-I). A type II GnRH receptor (GnRHR-II) gene homolog exists, but it is disrupted by a frame shift and premature stop codon, suggesting that a conventional receptor is not translated from this gene. However, the gene remains transcriptionally active and displays alternative splicing. We identified a putative translational start site 117 bp downstream of the premature stop codon. Use of this start codon encodes a protein (designated as the GnRHR-II-reliquum) corresponding to the domains from the cytoplasmic end of transmembrane domain-5 to the carboxyl terminus of the putative full-length receptor. Immunocytochemistry revealed that GnRHR-II-reliquum expression appeared to be localized throughout the cytoplasm. Transient cotransfection of GnRHR-I and GnRHR-II-reliquum constructs into COS-7 cells resulted in reduced expression of the GnRHR-I at the cell surface and impaired signaling via the GnRHR-I as revealed by reduction of GnRH-induced inositol phosphate accumulation. This inhibitory effect was specific and dependent on the degree of GnRHR-II-reliquum coexpressed. Immunoblot analysis revealed that the total cell GnRHR-I complement, i.e. both cell-surface and nascent intracellular receptors, was markedly reduced by coexpression of the GnRHR-II-reliquum. Treatments with cell-permeable agents that blocked either de novo protein synthesis (cycloheximide) or proteinase-mediated degradation (leupeptin and phenylmethylsulfonyl fluoride) failed to alter the inhibitory effect of GnRHR-II-reliquum coexpression, suggesting that the inhibitory effect is exerted at the nucleus/endoplasmic reticulum or Golgi apparatus level, possibly by perturbing normal processing of GnRHR-I from these sites. We suggest that the GnRHR-II-reliquum plays a modulatory role in GnRHR-I expression.
Introduction
GNRH IS A HYPOTHALAMIC DECAPEPTIDE that acts via a rhodopsin-like heptahelical G protein-coupled receptor (GPCR) on anterior pituitary gonadotropes, stimulating the release of both FSH and LH and thus controlling reproduction. The GnRH receptor (GnRHR) couples to phospholipase C? activation via the Gq/11 family of G proteins (1, 2, 3). Rhodopsin-like GPCRs are characterized structurally by their seven transmembrane-spanning helices, linked by extracellular loops and intracellular loops. These receptors have an extracellular amino terminal domain, which may be glycosylated, and a cytoplasmic carboxyl terminal tail, which may be palmitoylated to allow membrane anchorage. In general, the extracellular domains and/or transmembrane regions are involved in the formation of the ligand-binding pocket, whereas the cytoplasmic regions present sites for interactions with G proteins and other intracellular regulatory proteins (1, 4, 5).
Mammalian type I GnRHRs (GnRHR-Is) are unique among the rhodopsin-like GPCR superfamily because they completely lack a cytoplasmic carboxyl terminal tail (4, 5). In many GPCRs, this region has been demonstrated to play a crucial regulatory role in agonist-induced receptor phosphorylation, uncoupling, desensitization, internalization, and resensitization (6, 7). In contrast to mammalian GnRHR-Is but in common with other GPCRs, the cloned monkey type II GnRHRs (GnRHR-IIs) and all nonmammalian GnRHRs have a carboxyl terminal tail (4, 5, 8, 9, 10, 11, 12, 13). We recently cloned the functional marmoset GnRHR-II that is highly selective for the evolutionarily conserved GnRH-II ligand (14) and reported a disrupted gene homolog for the human and sheep GnRHR-II (15, 16). Humans may be unusual with respect to GnRH control of reproductive endocrinology in that they possess two distinct GnRH precursor genes, on chromosomes 8p11-p21 and 20p13, which encode GnRHR-I and GnRHR-II, but have only one conventional GnRHR subtype (i.e. GnRHR-I) encoded on chromosome 4. The disrupted human GnRHR-II gene homolog exists on chromosome 1q21. The human gene contains a frame shift in coding exon 1 and a premature stop codon in exon 2 (Fig. 1). The gene also overlaps two flanking genes but remains transcriptionally active and displays alternative splicing (15, 17). Disruption or silencing of the GnRHR-II gene has been noted in the human and chimpanzee (15), bovine (5), sheep (16), and rat (18, 19) genomes. In contrast, the marmoset (14), African green and rhesus monkeys (13), several nonmammalian vertebrates (20), and possibly pig (15) have retained the presence of a functional GnRHR-II (18).
FIG. 1. Nucleotide and deduced amino acid sequence of the human GnRHR-II. The full GnRHR-II sequence is shown. The position of the frame shift in exon 1 is indicated by the arrow and X. The premature stop codon in exon 2 is indicated in bold italics and asterisk. The putative translation start codon 117 bp downstream of the premature stop codon is indicated in bold. The encoded GnRHR-II-reliquum sequence is underlined.
To date, all studies focusing on the functional relevance of the human GnRHR-II gene have failed to produce direct evidence of the transcription and translation of a functional full-length seven transmembrane-spanning G protein-coupled GnRHR-II (5). The fact that the gene remains transcriptionally active led us to hypothesize that the various transcripts that are produced may encode functional modulatory proteins (15). It is presently unclear whether alternative splicing of GnRHR-I and GnRHR-II gene transcripts play a physiological role. Certainly alternatively spliced receptor isoforms have been characterized for GnRHRs (15, 21, 22, 23) and implicated in altered GnRHR function (22, 23).
In the present study, we expressed a fragment (the GnRHR-II-reliquum) of the human GnRHR-II gene (Fig. 2) and examined its effect on the function on the human GnRHR-I. The existence of processed mRNA transcripts, which correspond to the open-reading frame encoding the GnRHR-II-reliquum, have been previously demonstrated by RT-PCR (15, 24). Thus, our results point to a potential modulatory role for the disrupted human GnRHR-II gene because coexpression of the gene fragment causes a selective perturbation of GnRHR-I expression and signaling.
FIG. 2. Generating and subcloning the GnRHR-II-reliquum. The structure of the full-length GnRHR-II gene coding region is shown. Exons are indicated as boxes. TMD-1 to -7 (numbered black boxes) and the extracellular amino-terminal (N) and cytoplasmic carboxyl-terminal tail (C) of the putative GnRHR-II are indicated. The position of the frame shift (FS), in-frame premature stop codon (TGA between TMD-4 and -5), and putative translation start site (ATG in TMD-5) 117 bp downstream of the premature stop codon are indicated. The GnRHR-II-reliquum open-reading frame was PCR amplified using the indicated oligonucleotide primers (E and X) and subcloned into pcDNA1/Amp (Invitrogen) and N terminal-pFLAG-CMV2 (Sigma) mammalian expression vectors, as described in Materials and Methods. The corresponding GnRHR-II-reliquum protein domains are depicted schematically.
Materials and Methods
DNA constructs and materials
The hemagglutinin (HA)-tagged human GnRHR-I was generated by a PCR-based method as previously described (25, 26) and subcloned into pcDNA1/Amp (Invitrogen, Paisley, UK). The GnRHR-II sequence was cloned from cDNAs from a range of human tissues and cell lines by RT-PCR as described previously (15, 24). The GnRHR-II-reliquum open-reading frame was then PCR amplified using flanking sense and antisense primers, with EcoRI and XhoI restriction site (underlined) linkers, respectively [sense (E): 5'-GCG GCG GAA TTC GCC ATG GCC TAC TGC TAT AGC-3'; antisense (X): 5'-GCG GCG CTC GAG TCA GAT AGA TGT TAT AGA-3'] (Fig. 2). The PCR product was cloned into the EcoRI/XhoI sites of pcDNA1/Amp (Invitrogen), and subsequently subcloned into the EcoRI/XbaI sites of N-terminal-pFLAG-CMV2 (Sigma, Poole, UK). Plasmid DNA for transient transfection was prepared using Maxi-Prep columns (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
Cell culture and transient transfection
COS-7 cells were cultured as previously described (25) and transiently transfected using the SuperFect (QIAGEN) method according to the manufacturer’s instructions. Pretreatments with cycloheximide (CHX; 10 μg/ml; Sigma), leupeptin (10 μg/ml; Sigma), and phenylmethylsulfonyl fluoride (PMSF; 1 mM; Sigma) were performed at 37 C for 3 h before assays.
Immunocytochemistry and confocal laser microscopy
Confocal laser microscopy was performed on an LSM510 laser-scanning microscope (Carl Zeiss, G?ttingen, Germany). COS-7 cells transiently cotransfected with GnRHR-I and GnRHR-II-reliquum constructs were plated on poly-L-lysine-coated 8-well chamber slides (Nunc-Nalgene, Hereford, UK) at a density of 60,000 cells/chamber. Cell monolayers were washed twice with ice-cold PBS (with Ca2+/Mg2+) and then fixed in 200 μl of 100% methanol at –20 C for 10 min. When permeabilization was required, fixed cell monolayers were washed in PBS and incubated for 30 min in a Nonidet P-40-based cell permeabilization solution [PBS, 10% fetal calf serum (FCS), 1% BSA, 0.2% Nonidet P-40] at room temperature. After permeabilization the fixed cells were blocked in a PBS-based blocking solution (PBS, 10% FCS, 1% BSA) for 1 h at room temperature or 16 h at 4 C. To visualize GnRHR-I or GnRHR-II-reliquum proteins, the cells were incubated for 2 h at room temperature with antisera against both the HA-epitope (anti-HA 12CA5 monoclonal antibody-fluorescein conjugate (Roche Molecular Biochemicals, Mannheim, Germany) for GnRHR-I) and the FLAG-epitope (anti-FLAG M2 monoclonal antibody-Cy3 conjugate (Sigma) for GnRHR-II-reliquum) at a dilution of 1:200. Cell monolayers were washed three times in PBS and then mounted in Permafluor fixative (Immunotech, Westbrooke, ME).
Receptor binding assays
Whole-cell receptor binding assays used the 125I-[His5, D-Tyr6]GnRH analog (27). Transiently transfected COS-7 cells in 12-well culture plates were washed once with ice-cold HEPES/DMEM/10% FCS, and incubated for 5 h on ice in HEPES/DMEM/10% FCS with 105 cpm/well 125I-[His5, D-Tyr6]GnRH and varying concentrations of unlabeled GnRH-I. Cell monolayers were rapidly washed twice in ice-cold PBS and solubilized in 0.1 M NaOH, before the amount of bound radioligand was assessed in a 1261 Multi--counter. Nonspecific binding, consistently found to be less than 10% of total binding, was determined using vector-transfected (pcDNA1/Amp) COS-7 cells and was subtracted from total binding to give specific binding.
Total inositol phosphate assays
GnRH stimulation of total inositol phosphate production was as described (26). Briefly, transiently transfected COS-7 cells were incubated with inositol-free DMEM containing 1% dialyzed heat-inactivated FCS and 0.5 μCi/well myo-[3H]inositol (Amersham Pharmacia Biotech, Little Chalfont, UK) for 48 h. Medium was removed and the cells washed with 1 ml buffer (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM glucose, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml BSA) containing 10 mM LiCl and incubated for 1 h at 37 C in 0.5 ml buffer containing 10 mM LiCl and GnRH agonist at the specified concentration. Reactions were terminated by the removal of agonist and the addition of 1 ml ice-cold 10 mM formic acid with incubation for 30 min at 4 C. Total [3H]inositol phosphates were separated from the formic acid cell extracts on AG 1-X8 anion exchange resin (Bio-Rad Laboratories, Hemel Hempstead, UK) and eluted with a 1 M ammonium formate/0.1 M formic acid solution. The associated radioactivity was determined by liquid scintillation counting.
Immunoprecipitation and Western blotting
HA-tagged GnRHR-I protein was immunoprecipitated (28) from cell lysates by overnight incubation with 30% preconjugated HA-agarose slurry (Santa Cruz Biotechnology, Santa Cruz, CA) and washed. The immunoprecipitates were resolved by SDS-PAGE and electrotransferred to polyvinyl difluoride membrane (NEN Life Science Products, Boston, MA). GnRHR-I protein was detected by using antimouse 12CA5 antisera (Roche Molecular Biochemicals) and visualized by enzyme-linked chemifluorescence (Amersham Pharmacia Biotech) and quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Results
A human GnRHR-II gene homolog exists, but it is disrupted by a frame shift and premature stop codon (Fig. 1), suggesting that a conventional GnRHR-II system is unlikely to exist in humans. However, this does not discount the possibility that the gene may yet encode functional proteins. Indeed, we previously demonstrated that the GnRHR-II gene remains transcriptionally active and displays alternative splicing (15). Furthermore, immunocytochemical studies using an antiserum (designated ZGHR-II-5) generated to the conserved extracellular loop (ECL)-3 domain of the human GnRHR-II showed specific staining in the human and sheep pituitary. This staining was found to colocalize with LH-positive cells, suggesting the expression of proteins encoded by the GnRHR-II gene in pituitary gonadotropes (14). Silencing of the sheep GnRHR-II has been previously demonstrated (16).
In the present study, we identify a putative translational start site 117 bp downstream of the premature stop codon (Fig. 1). This is the first start codon occurring in-frame after the premature stop codon. Use of this translation start site encodes a protein (the GnRHR-II-reliquum) corresponding to the domains from the cytoplasmic end of transmembrane domain (TMD)-5, intracellular loop-3, TMD-6, ECL-3, TMD-7, and the carboxyl-terminal tail of the putative human GnRHR-II (Fig. 2). We demonstrated previously that processed mRNA transcripts that encode the GnRHR-II-reliquum are present in many human tissues and cell lines (15, 24).
The open-reading frame encoding the GnRHR-II-reliquum was subcloned into mammalian expression vectors (Figs. 1 and 2). To ascertain the expression and subcellular localization of the GnRHR-II-reliquum, immunocytochemical studies were performed. Confocal fluorescence microscopy images of permeabilized COS-7 cells transiently transfected with either the FLAG-tagged GnRHR-II-reliquum (Fig. 3A) or HA-tagged GnRHR-I (Fig. 3B) demonstrated that they were expressed and revealed a similar general cellular distribution of both proteins. GnRHR-II-reliquum expression appeared to be localized throughout the cytoplasm (Fig. 3, D–G, J, M, and P). When coexpressed, both proteins showed a high degree of colocalization at sites within the cell (Fig. 3, G–R).
FIG. 3. Subcellular localization of the GnRHR-II-reliquum and GnRHR-I. Immunofluorescence studies were carried out with transfected COS-7 cells grown in 8-well chamber glass slides (Lab-Tek, Christchurch, New Zealand) as described in Materials and Methods. Cells were transiently transfected with either (A–D) FLAG-tagged GnRHR-II-reliquum or HA-tagged GnRHR-I cDNA constructs or both (G-X), methanol fixed, permeabilized (A–R) or left unpermeabilized (S–X), and incubated with anti-FLAG M2 monoclonal antibody-Cy3 conjugate (Sigma) and/or anti-HA 12CA5 monoclonal antibody-fluorescein conjugate (Roche Molecular Biochemicals) directed against the amino-terminal epitope tags present in the FLAG-tagged GnRHR-II-reliquum and HA-tagged GnRHR-I constructs, respectively. Fluorescence images were obtained by confocal laser microscopy as described. Images in the panels correspond to: FLAG-tagged GnRHR-II-reliquum expressed alone (A), HA-tagged GnRHR-I expressed alone (B), and mock transfected cells stained with both anti-FLAG M2 monoclonal antibody-Cy3 conjugate (Sigma) and anti-HA 12CA5 monoclonal antibody-fluorescein conjugate (Roche Molecular Biochemicals) as a negative control (C); FLAG-tagged GnRHR-II-reliquum expressed alone (D), corresponding phase-contrast image (E), and merged image (F); coexpressed FLAG-tagged GnRHR-II-reliquum (G, J, M, P, S, and V) and HA-tagged GnRHR-I (H, K, N, Q, T, and W), with the corresponding merged images to demonstrate their colocalization (I, L, O, R, U, and X).
We next investigated whether the GnRHR-II-reliquum was capable of cell-surface plasma membrane insertion. Confocal microscopy of nonpermeabilized COS-7 cells coexpressing the FLAG-tagged GnRHR-II-reliquum (Fig. 3, S and V) and HA-tagged GnRHR-I (Fig. 3, T and W) failed to detect any significant fluorescence at the cell periphery for the former, in contrast to the latter. Therefore, whereas the GnRHR-II- reliquum is clearly expressed nascently, it appears not to be significantly inserted into the cell plasma membrane.
In line with previous literature demonstrating the inhibitory effect of human GnRHR-I and bullfrog GnRHR-III gene splice variants on GnRHR function (22, 23), we sought to investigate the effect of GnRHR-II-reliquum expression on GnRHR-I function. Transient cotransfection of GnRHR-I and GnRHR-II-reliquum constructs in COS-7 cells, resulted in a reduced expression of the GnRHR-I at the cell surface (Fig. 4A). This inhibitory effect was directly related to the amount of GnRHR-II-reliquum cDNA cotransfected with GnRHR-I. Thus, when increasing amounts of GnRHR-II-reliquum cDNA (0.4–1.6 μg/well) were cotransfected with a constant amount of GnRHR-I cDNA (0.4 μg/well), a progressive reduction in plasma membrane expression of GnRHR-I was observed (Fig. 4A). Cotransfection with the highest amount of GnRHR-II-reliquum cDNA (1.6 μg/well) did not significantly alter the affinity of GnRH-I ligand for GnRHR-I yet profoundly reduced the GnRHR-I expression at the cell surface as revealed by decreased maximal GnRH binding (Fig. 4B).
FIG. 4. Inhibition of GnRHR-I cell-surface expression by coexpression of GnRHR-II-reliquum in COS-7 cells. A, COS-7 cells were transfected with constant amounts of GnRHR-I cDNA (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0 to 1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Specific binding was measured as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments performed in triplicate. B, COS-7 cells were transfected with GnRHR-I and GnRHR-II-reliquum cDNA (0.4 and 1.6 μg/well, respectively) and incubated with increasing concentrations of cold GnRH-I ligand in the presence of 125I-[His5, D-Tyr6]GnRH as described in Materials and Methods. Data represent the mean ± SEM of three individual experiments performed in duplicate.
Concomitant with the GnRHR-II-reliquum-mediated inhibition of GnRHR-I expression, we noted a dose-dependent inhibitory effect on the extent of GnRH-I-induced inositol phosphate accumulation in cells expressing the GnRHR-I. Thus, when increasing amounts of GnRHR-II-reliquum cDNA (0.4–1.6 μg/well) were cotransfected with a constant amount of GnRHR-I cDNA (0.4 μg/well), a progressive reduction of GnRH-I agonist-induced inositol phosphate accumulation was observed (Fig. 5A). Cotransfection of the highest amount (1.6 μg/well) of GnRHR-II-reliquum cDNA also did not alter the EC50 of GnRH-induced inositol phosphate accumulation but did reduce the maximal level of the GnRH response (Fig. 5B).
FIG. 5. Inhibition of GnRHR-I signaling by coexpressed GnRHR-II-reliquum in COS-7 cells. A, COS-7 cells were transfected with constant amounts of GnRHR-I cDNA (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0 to 1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Total inositol phosphate accumulation in response to 1 μM GnRH-I was measured as described in Materials and Methods. Data represent the mean ± SEM of three individual experiments performed in triplicate. B, COS-7 cells were transfected with GnRHR-I and GnRHR-II-reliquum cDNA (0.4 and 1.6 μg/well, respectively) and incubated with increasing concentrations of cold GnRH-I ligand and total inositol phosphate accumulation measured as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments performed in duplicate.
To test the specificity of the inhibitory effect of the GnRHR-II-reliquum on GnRHR-I expression, we employed the rat TRH receptor (TRHR), and a nonmammalian GnRHR-I (the chicken GnRHR-I), both of which are Gq/11-coupled receptors and possess carboxyl-terminal cytoplasmic tail domains, unlike the uniquely tailless mammalian GnRHR-Is. When increasing amounts of GnRHR-II-reliquum cDNA (0.4–1.6 μg/well) were cotransfected with a constant amount of either rat TRHR or chicken GnRHR-I cDNA (0.4 μg/well), no progressive reduction of TRH or GnRH-I agonist-induced inositol phosphate accumulation was observed (Fig. 6, A–C).
FIG. 6. Specificity of the GnRHR-II-reliquum inhibitory effect. COS-7 cells were transfected with constant amounts of human GnRHR-I (A), rat TRHR (B), and chicken GnRHR-I cDNA (C) (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0 to 1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Total inositol phosphate (InsP) accumulation in response to 1 μM GnRH-I (A and C) and 1 μM TRH (B) was measured as described in Materials and Methods. D and E, COS-7 cells were transfected with GnRHR-I (0.4 μg/well) and either GnRHR-II-reliquum cDNA (1.6 μg/well) or rat TRHR cDNA (1.6 μg/well) and incubated with increasing concentrations of cold GnRH-I ligand (D only), and total inositol phosphate accumulation and binding (Bo, maximal binding only) was measured as described in Materials and Methods. All data are representative of three individual experiments performed in duplicate.
The possibility that the increased GnRHR-II-reliquum expression inhibits GnRH-induced inositol phosphate accumulation or GnRHR-I expression in a more general way, such as by hijacking available Gq/11-coupling units or competition for cellular translational capacity, was also addressed. Figure 6 (A–C) demonstrates that the inhibition of GnRH-induced inositol phosphate accumulation by GnRHR-II-reliquum is not due to lack of available Gq/11-coupling units because no inhibitory effect was seen for the rat TRHR or chicken GnRHR-I, both themselves Gq/11-coupled receptors. Furthermore, when human GnRHR-I was coexpressed with rat TRHR instead of GnRHR-II-reliquum, no inhibition of either GnRH-induced inositol phosphate accumulation or GnRH binding was observed at the human GnRHR-I, in contrast to the inhibition produced by coexpression of GnRHR-II-reliquum (Fig. 6, D and E).
When taken together, the above data suggest that the GnRHR-II-reliquum-induced reduction of GnRH-I agonist-induced inositol phosphate accumulation appears to be as a direct consequence of the specifically reduced expression of the GnRHR-I at the cell surface, rather than any interference in Gq/11-coupling capacity of the GnRHR-I or competition for cellular translational machinery.
Immunoprecipitation and Western blot analysis revealed that the total cellular expression of GnRHR-I was also markedly reduced by coexpression of the GnRHR-II-reliquum (Fig. 7A). The reduction in both cell-surface and nascent intracellular GnRHR-I levels suggested that the inhibition might be occurring at a site close to the site of de novo synthesis of GnRHR-I, perhaps at the nucleus/endoplasmic reticulum. To investigate the mechanism of this inhibition, we subjected COS-7 cells coexpressing GnRHR-I and GnRHR-II-reliquum to treatment with cell permeable agents to block de novo protein synthesis (CHX) or proteinase-mediated degradation (leupeptin and PMSF). However, these treatments failed to alter the inhibitory effect of GnRHR-II-reliquum coexpression on GnRHR-I levels, as demonstrated by immunoprecipitation and Western blot analysis (Fig. 7B) and GnRH-I agonist-induced inositol phosphate accumulation assays (Fig. 8). These data therefore suggest that the inhibitory effect is being exerted at the nucleus/endoplasmic reticulum or Golgi apparatus level, possibly by preventing or diverting the normal processing of GnRHR-I from these sites or enhancing GnRHR-I degradation via proteinase- or proteosome-mediated pathways that are not inhibited by leupeptin or PMSF.
FIG. 7. GnRHR-II-reliquum coexpression reduces both cell-surface and nascent GnRHR-I expression levels. A and B, COS-7 cells were transfected with GnRHR-I and GnRHR-II-reliquum cDNA (2 and 8 μg per 100-mm dish, respectively), subjected to pretreatments with CHX, leupeptin, and PMSF (B only). HA-tagged GnRHR-I was immunoprecipitated (IP) from cell lysates and subjected to Western blot (IB) analysis as described in Materials and Methods. Each bar represents the mean ± SEM of at least three individual experiments.
FIG. 8. No effect of CHX, leupeptin, or PMSF pretreatments on GnRHR-II-reliquum inhibition of GnRHR-I-mediated inositol phosphate (InsP) production. COS-7 cells were transfected with constant amounts of GnRHR-I cDNA (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0–1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Pretreatments with CHX, leupeptin, and PMSF were performed as described, and total inositol phosphate accumulation in response to 1 μM GnRH-I was measured as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments.
Discussion
Alternative splicing of GPCR gene transcripts has long been thought to be a source of structural diversity and to provide a mechanism for the functional regulation of GPCRs. Some of these splice variants may themselves function as receptors in their own right and may bind ligands and effectively couple to a cognate G protein and diverse downstream signaling pathways. Others may serve a regulatory/modulatory role and interact to form complexes with the wild-type/full-length receptor to alter expression, function, and signal transduction. The formation of these complexes would probably occur through interhelical interactions between the individual helices spanning the membrane (22, 29, 30, 31). Previous studies of GnRHR-I and GnRHR-III alternative splice variants have reported their inhibitory activity on full-length GnRHR function (22, 23). In the present study, we demonstrated the inhibition of human GnRHR-I expression by a transiently coexpressed fragment of the human GnRHR-II gene homolog (i.e. the GnRHR-II-reliquum). Furthermore, we observed that this inhibition was specific, augmented by increasing amounts of cotransfected GnRHR-II-reliquum cDNA and was not altered by treatments that either inhibited de novo protein synthesis or proteinase-mediated degradation.
Our previous finding that the disrupted human GnRHR-II gene homolog remains transcriptionally active and displays alternative splicing (15) suggested that the gene may encode a functional entity. The complexities and potential roles of the disrupted human GnRHR-II gene homolog have been extensively debated and reviewed in the past few years (5, 18, 19, 20, 31, 32, 33). A recent review on the subject (31) discusses emerging evidence for the existence of a functional type II receptor for the cognate GnRH-II ligand in humans and suggests the interesting possibility that protein fragments derived from the disrupted human GnRHR-II gene homolog may associate with one another or with the human GnRHR-I to produce a functional GnRH-II-responsive receptor. A number of studies reported that GnRH-II has more potent antiproliferative effects than GnRH-I in various human tumor-derived cell lines (34, 35, 36), and such findings may support the conclusion that a functional GnRHR-II is present in humans. However, it is equally plausible that the effects observed in these studies are a result of altered agonist pharmacology at the GnRHR-I, leading to the activation of antiproliferative signal transduction pathways (37). Such ligand-selective signaling is dependent on intracellular environmental modulation of GnRHR-I ligand pharmacology, as has been recently demonstrated (38, 39). In their review, Neill et al. (31) elude to the same human GnRHR-II fragment sequence that we identify in the present study as the GnRHR-II-reliquum. It is worth noting that preliminary studies in our laboratory have shown no alteration in the ligand pharmacology of GnRH-I and GnRH-II (IC50 3 and 30 nM, respectively) at the human GnRHR-I when coexpressed with GnRHR-II-reliquum (data not shown).
The study by Grosse et al. (22) reported that coexpression of a truncated isoform of the human GnRHR-I leads to inhibition of signaling mediated by the full-length GnRHR-I. Similar findings were reported for the bullfrog GnRHR (23). Grosse et al. (22) also reported that a truncated GnRHR-I protein corresponding to TMDs-1–5, when coexpressed with a fragment of GnRHR-I corresponding to TMD-6 and -7, lead to a GnRH-induced inositol phosphate accumulation in COS-7 cells. These data suggested that the two receptor fragments were capable of interacting in a membrane environment to produce a functional GnRHR that was capable of coupling to Gq/11 and activating inositol phosphate accumulation (22). It is interesting to note that the C-terminal protein fragment (i.e. corresponding to TMD-6 and -7) used in the study by Grosse et al. (22) corresponds to approximately the same domains that constitute the GnRHR-II-reliquum.
There are several lines of evidence to suggest the endogenous expression of a protein fragment corresponding to TMD-6 and -7 of the human GnRHR-II (i.e. the GnRHR-II-reliquum). Immunocytochemical studies using an antiserum (ZGRH-II-5) generated to the conserved ECL-3 domain of the human GnRHR-II showed specific staining in the human pituitary (14). Similar specific staining was obtained when the same ECL-3 antiserum was used on sheep pituitary sections (14). Furthermore, the staining was found to colocalize with LH-positive cells, suggesting expression in human and sheep gonadotropes (14). An antiserum (ZGRH-II-8) directed at ECL-2 (the domain immediately preceding the putative start site identified here) failed to detect any immunoreactivity in sheep gonadotropes, in which the ECL-3 domain could be detected. The sheep GnRHR-II gene has been silenced by disruptions similar to those that have been reported to occur in the human GnRHR-II gene (15, 16). We have demonstrated that processed mRNA transcripts, which encode a protein fragment corresponding to the GnRHR-II-reliquum, are produced in various human tissues and cell lines (15, 24).
The presence of a putative start codon downstream of the premature stop codon suggests that synthesis of a GnRHR-II-reliquum is conceivable, should this be used as a translational start site. Indeed, any mRNA transcript derived from the putative GnRHR-II gene, in which intron 2 has been spliced out (Fig. 2), would lead to translation and synthesis of the GnRHR-II-reliquum, should the downstream start site be used by the translation machinery. Such transcripts (i.e. in which intron 2 has been spliced out) are now known to be present in various human tissues and cell lines (15, 24). Furthermore, it is intriguing to note that the putative start codon identified here occurs in the context of a partial Kozak consensus sequence (GCC ATG G). The full Kozak consensus required for initiation of translation was originally identified as GCC G/ACC ATG G (40). However, more recently it has emerged that the Kozak consensus cannot be reliably used as a criterion for the prediction of protein expression from a particular open reading frame and that the selection of translational initiation site in eukaryotic mRNAs involves many complex mechanisms (41, 42, 43). The role or importance, if any, of the partial Kozak consensus sequence in directing expression of the GnRHR-II-reliquum remains to be investigated.
In the present study, we did not detect significant cell-surface staining of FLAG-tagged GnRHR-II-reliquum using an anti-FLAG M2 monoclonal antibody-Cy3 conjugate in nonpermeabilized cells (Fig. 3). We were, however, able to demonstrate cytoplasmic staining of nascent GnRHR-II-reliquum in permeabilized COS-7 cells using the same antibody (Fig. 3). Such cytoplasmic staining would include FLAG-tagged GnRHR-II-reliquum residing in intracellular membranes such as vesicles and the endoplasmic reticulum. Because the GnRHR-II-reliquum corresponds to the C-terminal region of the putative GnRHR-II, from the cytoplasmic end of TMD-5 to the carboxyl terminus (Fig. 2), the FLAG epitope at the amino-terminal end of this fragment is most likely situated on the cytosolic side of the plasma membrane. This scenario would preclude the detection of FLAG-tagged GnRHR-II-reliquum at the cell surface in nonpermeabilized cells. Given that the C-terminal protein fragment (i.e. corresponding to TMD-6 and -7) used in the study by Grosse et al. (22) was apparently able to interact with the TMD-1 through -5 fragment to produce a functional receptor, we suggest that the GnRHR-II-reliquum may similarly interact, via interhelical interactions, with the GnRHR-I in the membrane environment. This scenario may therefore provide a mechanism for the inhibition of GnRHR-I expression reported in the present study. Thus, after its synthesis at the ribosome, the GnRHR-I protein is inserted into membranes of the endoplasmic reticulum. In the absence of coexpressed GnRHR-II-reliquum, the GnRHR-I is processed in the usual way, from the endoplasmic reticulum to the Golgi apparatus and onward to the plasma membrane. However, a physical interaction between GnRHR-I and GnRHR-II-reliquum may lead to an unstable or misfolded receptor/complex that would be prevented from onward processing and/or undergo defective intracellular transport from the endoplasmic reticulum, or enhanced degradation, thus accounting for the overall decreased cellular expression of GnRHR-I. Finally, it was interesting to observe that the GnRHR-II-reliquum exerted no inhibitory action on the chicken GnRHR-I (Fig. 6). One may speculate that the presence of the carboxyl-terminal cytoplasmic tail in the chicken GnRHR-I (and indeed all nonmammalian GnRHRs) may preclude a direct physical association with the GnRHR-II- reliquum.
It is becoming increasingly apparent that a conventional GnRHR-II system is not present in humans, although the exact nature of a specific GnRH-II-responsive system in humans, should it exist, remains elusive. Taken together, our studies suggest that a protein corresponding to TMD-6 and -7 of the human putative GnRHR-II is expressed in the gonadotrope with the GnRHR-I, pointing to a possible role for the disrupted human GnRHR-II gene in modulating the function of the pituitary human GnRHR-I, perhaps in a manner similar to that described here. Clearly further work is required to ascertain the precise nature of potentially functional protein products of the disrupted yet transcriptionally active human GnRHR-II gene. However, in the present study, we have clearly demonstrated that a putative human GnRHR-II-reliquum can perturb the human GnRHR-I functionally in a controlled environment.
Acknowledgments
We are grateful to Robin Sellar and Nicola Miller for their excellent technical assistance.
References
Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499
Naor Z, Benard O, Seger R 2000 Activation of MAPK cascades by G-protein-coupled receptors: the case of gonadotropin-releasing hormone receptor. Trends Endocrinol Metab 11:91–99
Kraus S, Naor Z, Seger R 2001 Intracellular signaling pathways mediated by the gonadotropin-releasing hormone (GnRH) receptor. Arch Med Res 32:499–509
Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205
Millar RP, Lu Z-L, Pawson AJ, Flanagan CA, Morgan K, Maudsley S 2004 Gonadotropin-releasing hormone receptors. Endocr Rev 25:235–275
Ferguson SS, Barak LS, Zhang J, Caron MG 1996 G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74:1095–1110
Ferguson SS 2001 Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24
Tensen C, Okuzawa K, Blomenrohr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140
Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc Natl Acad Sci USA 96:2526–2531
Sun YM, Flanagan CA, Illing N, Ott TR, Sellar R, Fromme BJ, Hapgood J, Sharp P, Sealfon SC, Millar RP 2001 A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants. J Biol Chem 276:7754–7761
Wang L, Bogerd J, Choi HS, Seong JY, Soh JM, Chun SY, Blomenr?r M, Troskie BE, Millar RP, Kwon HB 2001 Three distinct types of gonadotropin-releasing hormone receptor characterized in the bullfrog. Proc Natl Acad Sci USA 98:361–366
Bogerd J, Diepenbroek WB, Hund E, Van Oosterhout F, Teves AC, Leurs R, Blomenrohr M 2002 Two gonadotropin-releasing hormone receptors in the african catfish: no differences in ligand selectivity, but differences in tissue distribution. Endocrinology 143:4673–4682
Neill JD, Duck LW, Sellers JC, Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282:1012–1018
Millar RP, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641
Morgan K, Conklin D, Pawson AJ, Sellar R, Ott TR, Millar RP 2003 A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q. 12. Endocrinology 144:423–436
Gault PM, Morgan K, Pawson AJ, Millar RP, Lincoln GA 2004 Sheep exhibit novel variations in the organization of the mammalian type II gonadotropin-releasing hormone receptor gene. Endocrinology 145:2362–2374
Faurholm B, Millar RP, Katz AA 2001 The genes encoding the type II gonadotropin-releasing hormone receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci. Genomics 78:15–18
Millar RP 2003 GnRH II and type II GnRH receptors. Trends Endocrinol Metab 14:35–43
Pawson AJ, Morgan K, Maudsley SR, Millar RP 2003 Type II gonadotropin-releasing hormone (GnRH-II) in reproductive biology. Reproduction 126:271–278
Morgan K, Millar RP 2004 Evolution of GnRH ligand precursors and GnRH receptors in protochordate and vertebrate species. Gen Comp Endocrinol 139:191–197
Kottler ML, Bergametti F, Carre MC, Morice S, Decoret E, Lagarde JP, Starzec A, Counis R 1999 Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene. Eur J Endocrinol 140:561–569
Grosse R, Schoneberg T, Schultz G, Gudermann T 1997 Inhibition of gonadotropin-releasing hormone receptor signaling by expression of a splice variant of the human receptor. Mol Endocrinol 11:1305–1318
Wang L, Oh DY, Bogerd J, Choi HS, Ahn RS, Seong JY, Kwon HB 2001 Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling. Endocrinology 142:4015–4025
Millar R, Conklin D, Lofton-Day C, Hutchinson E, Troskie B, Illing N, Sealfon SC, Hapgood J 1999 A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript. J Endocrinol 162:117–126
Pawson AJ, Katz A, Sun YM, Lopes J, Illing N, Millar RP, Davidson JS 1998 Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail. J Endocrinol 156:R9–R12
Pawson AJ, Maudsley SR, Lopes J, Katz AA, Sun YM, Davidson JS, Millar RP 2003 Multiple determinants for rapid agonist-induced internalization of a nonmammalian gonadotropin-releasing hormone receptor: a putative palmitoylation site and threonine doublet within the carboxyl-terminal tail are critical. Endocrinology 144:3860–3871
Flanagan CA, Fromme BJ, Davidson JS, Millar RP 1998 A high affinity gonadotropin-releasing hormone (GnRH) tracer, radioiodinated at position 6, facilitates analysis of mutant GnRH receptors. Endocrinology 139:4115–4119
Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM 2000 The ?(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 275:9572–9580
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Trong IL, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745
Suel GM, Lockless SW, Wall MA, Ranganathan R 2003 Evolutionarily conserved networks of residues mediate allosteric communication in proteins. Nat Struct Biol 10:59–69
Neill JD, Musgrove LC, Duck LW 2004 Newly recognized GnRH receptors: function and relative role. Trends Endocrinol Metab 15:383–392
Pawson AJ, Morgan K, Maudsley S, Millar RP, The type II GnRH receptor and the "SECIS element" of its pseudogene. Proc 2nd Cooloquium of the DFG-Priority Programme Selenoproteins, Charite, Berlin, Germany, 2003
Neill JD 2002 GnRH and GnRH receptor genes in the human genome. Endocrinology 143:737–743
Grundker C, Gunthert AR, Millar RP, Emons G 2002 Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J Clin Endocrinol Metab 87:1427–1430
Grundker C, Emons G 2003 Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer. Reprod Biol Endocrinol 1:65
Grundker C, Schlotawa L, Viereck V, Eicke N, Horst A, Kairies B, Emons G 2004 Antiproliferative effects of the GnRH antagonist cetrorelix and of GnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor. Eur J Endocrinol 151:141–149
Maudsley S, Davidson L, Pawson AJ, Chan R, Lopez de Maturana R, Millar RP 2004 GnRH antagonists promote pro-apoptotic signaling in peripheral tissues by activating a G-i-coupling state of the type I GnRH receptor. Cancer Res 64:7533–7544
Caunt CJ, Hislop JN, Kelly E, Matharu AL, Green LD, Sedgley KR, Finch AR, McArdle CA 2004 Regulation of gonadotropin-releasing hormone receptors by protein kinase C: inside out signalling and evidence for multiple active conformations. Endocrinology 145:3594–3602
Millar RP, Pawson AJ 2004 Outside-in and inside-out signalling: the new concept that selectivity of ligand binding at the gonadotropin-releasing hormone receptor is modulated by the intracellular environment. Endocrinology 145:3590–3593[Free Full Text]
Kozak M 1986 Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292
Kozak M 2002 Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1–34
Kozak M 2003 Alternative ways to think about mRNA sequences and proteins that appear to promote internal initiation of translation. Gene 318:1–23
Kapp LD, Lorsch JR 2004 The molecular mechanics of eukaryotic translation. Annu Rev Biochem 73:657–704(Adam J. Pawson, Stuart Ma)
Address all correspondence and requests for reprints to: Dr. Adam J. Pawson, Human Reproductive Sciences Unit, Medical Research Council, The University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom. E-mail: a.pawson@hrsu.mrc.ac.uk.
Abstract
Humans possess only one functional GnRH receptor, the type I GnRH receptor (GnRHR-I). A type II GnRH receptor (GnRHR-II) gene homolog exists, but it is disrupted by a frame shift and premature stop codon, suggesting that a conventional receptor is not translated from this gene. However, the gene remains transcriptionally active and displays alternative splicing. We identified a putative translational start site 117 bp downstream of the premature stop codon. Use of this start codon encodes a protein (designated as the GnRHR-II-reliquum) corresponding to the domains from the cytoplasmic end of transmembrane domain-5 to the carboxyl terminus of the putative full-length receptor. Immunocytochemistry revealed that GnRHR-II-reliquum expression appeared to be localized throughout the cytoplasm. Transient cotransfection of GnRHR-I and GnRHR-II-reliquum constructs into COS-7 cells resulted in reduced expression of the GnRHR-I at the cell surface and impaired signaling via the GnRHR-I as revealed by reduction of GnRH-induced inositol phosphate accumulation. This inhibitory effect was specific and dependent on the degree of GnRHR-II-reliquum coexpressed. Immunoblot analysis revealed that the total cell GnRHR-I complement, i.e. both cell-surface and nascent intracellular receptors, was markedly reduced by coexpression of the GnRHR-II-reliquum. Treatments with cell-permeable agents that blocked either de novo protein synthesis (cycloheximide) or proteinase-mediated degradation (leupeptin and phenylmethylsulfonyl fluoride) failed to alter the inhibitory effect of GnRHR-II-reliquum coexpression, suggesting that the inhibitory effect is exerted at the nucleus/endoplasmic reticulum or Golgi apparatus level, possibly by perturbing normal processing of GnRHR-I from these sites. We suggest that the GnRHR-II-reliquum plays a modulatory role in GnRHR-I expression.
Introduction
GNRH IS A HYPOTHALAMIC DECAPEPTIDE that acts via a rhodopsin-like heptahelical G protein-coupled receptor (GPCR) on anterior pituitary gonadotropes, stimulating the release of both FSH and LH and thus controlling reproduction. The GnRH receptor (GnRHR) couples to phospholipase C? activation via the Gq/11 family of G proteins (1, 2, 3). Rhodopsin-like GPCRs are characterized structurally by their seven transmembrane-spanning helices, linked by extracellular loops and intracellular loops. These receptors have an extracellular amino terminal domain, which may be glycosylated, and a cytoplasmic carboxyl terminal tail, which may be palmitoylated to allow membrane anchorage. In general, the extracellular domains and/or transmembrane regions are involved in the formation of the ligand-binding pocket, whereas the cytoplasmic regions present sites for interactions with G proteins and other intracellular regulatory proteins (1, 4, 5).
Mammalian type I GnRHRs (GnRHR-Is) are unique among the rhodopsin-like GPCR superfamily because they completely lack a cytoplasmic carboxyl terminal tail (4, 5). In many GPCRs, this region has been demonstrated to play a crucial regulatory role in agonist-induced receptor phosphorylation, uncoupling, desensitization, internalization, and resensitization (6, 7). In contrast to mammalian GnRHR-Is but in common with other GPCRs, the cloned monkey type II GnRHRs (GnRHR-IIs) and all nonmammalian GnRHRs have a carboxyl terminal tail (4, 5, 8, 9, 10, 11, 12, 13). We recently cloned the functional marmoset GnRHR-II that is highly selective for the evolutionarily conserved GnRH-II ligand (14) and reported a disrupted gene homolog for the human and sheep GnRHR-II (15, 16). Humans may be unusual with respect to GnRH control of reproductive endocrinology in that they possess two distinct GnRH precursor genes, on chromosomes 8p11-p21 and 20p13, which encode GnRHR-I and GnRHR-II, but have only one conventional GnRHR subtype (i.e. GnRHR-I) encoded on chromosome 4. The disrupted human GnRHR-II gene homolog exists on chromosome 1q21. The human gene contains a frame shift in coding exon 1 and a premature stop codon in exon 2 (Fig. 1). The gene also overlaps two flanking genes but remains transcriptionally active and displays alternative splicing (15, 17). Disruption or silencing of the GnRHR-II gene has been noted in the human and chimpanzee (15), bovine (5), sheep (16), and rat (18, 19) genomes. In contrast, the marmoset (14), African green and rhesus monkeys (13), several nonmammalian vertebrates (20), and possibly pig (15) have retained the presence of a functional GnRHR-II (18).
FIG. 1. Nucleotide and deduced amino acid sequence of the human GnRHR-II. The full GnRHR-II sequence is shown. The position of the frame shift in exon 1 is indicated by the arrow and X. The premature stop codon in exon 2 is indicated in bold italics and asterisk. The putative translation start codon 117 bp downstream of the premature stop codon is indicated in bold. The encoded GnRHR-II-reliquum sequence is underlined.
To date, all studies focusing on the functional relevance of the human GnRHR-II gene have failed to produce direct evidence of the transcription and translation of a functional full-length seven transmembrane-spanning G protein-coupled GnRHR-II (5). The fact that the gene remains transcriptionally active led us to hypothesize that the various transcripts that are produced may encode functional modulatory proteins (15). It is presently unclear whether alternative splicing of GnRHR-I and GnRHR-II gene transcripts play a physiological role. Certainly alternatively spliced receptor isoforms have been characterized for GnRHRs (15, 21, 22, 23) and implicated in altered GnRHR function (22, 23).
In the present study, we expressed a fragment (the GnRHR-II-reliquum) of the human GnRHR-II gene (Fig. 2) and examined its effect on the function on the human GnRHR-I. The existence of processed mRNA transcripts, which correspond to the open-reading frame encoding the GnRHR-II-reliquum, have been previously demonstrated by RT-PCR (15, 24). Thus, our results point to a potential modulatory role for the disrupted human GnRHR-II gene because coexpression of the gene fragment causes a selective perturbation of GnRHR-I expression and signaling.
FIG. 2. Generating and subcloning the GnRHR-II-reliquum. The structure of the full-length GnRHR-II gene coding region is shown. Exons are indicated as boxes. TMD-1 to -7 (numbered black boxes) and the extracellular amino-terminal (N) and cytoplasmic carboxyl-terminal tail (C) of the putative GnRHR-II are indicated. The position of the frame shift (FS), in-frame premature stop codon (TGA between TMD-4 and -5), and putative translation start site (ATG in TMD-5) 117 bp downstream of the premature stop codon are indicated. The GnRHR-II-reliquum open-reading frame was PCR amplified using the indicated oligonucleotide primers (E and X) and subcloned into pcDNA1/Amp (Invitrogen) and N terminal-pFLAG-CMV2 (Sigma) mammalian expression vectors, as described in Materials and Methods. The corresponding GnRHR-II-reliquum protein domains are depicted schematically.
Materials and Methods
DNA constructs and materials
The hemagglutinin (HA)-tagged human GnRHR-I was generated by a PCR-based method as previously described (25, 26) and subcloned into pcDNA1/Amp (Invitrogen, Paisley, UK). The GnRHR-II sequence was cloned from cDNAs from a range of human tissues and cell lines by RT-PCR as described previously (15, 24). The GnRHR-II-reliquum open-reading frame was then PCR amplified using flanking sense and antisense primers, with EcoRI and XhoI restriction site (underlined) linkers, respectively [sense (E): 5'-GCG GCG GAA TTC GCC ATG GCC TAC TGC TAT AGC-3'; antisense (X): 5'-GCG GCG CTC GAG TCA GAT AGA TGT TAT AGA-3'] (Fig. 2). The PCR product was cloned into the EcoRI/XhoI sites of pcDNA1/Amp (Invitrogen), and subsequently subcloned into the EcoRI/XbaI sites of N-terminal-pFLAG-CMV2 (Sigma, Poole, UK). Plasmid DNA for transient transfection was prepared using Maxi-Prep columns (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
Cell culture and transient transfection
COS-7 cells were cultured as previously described (25) and transiently transfected using the SuperFect (QIAGEN) method according to the manufacturer’s instructions. Pretreatments with cycloheximide (CHX; 10 μg/ml; Sigma), leupeptin (10 μg/ml; Sigma), and phenylmethylsulfonyl fluoride (PMSF; 1 mM; Sigma) were performed at 37 C for 3 h before assays.
Immunocytochemistry and confocal laser microscopy
Confocal laser microscopy was performed on an LSM510 laser-scanning microscope (Carl Zeiss, G?ttingen, Germany). COS-7 cells transiently cotransfected with GnRHR-I and GnRHR-II-reliquum constructs were plated on poly-L-lysine-coated 8-well chamber slides (Nunc-Nalgene, Hereford, UK) at a density of 60,000 cells/chamber. Cell monolayers were washed twice with ice-cold PBS (with Ca2+/Mg2+) and then fixed in 200 μl of 100% methanol at –20 C for 10 min. When permeabilization was required, fixed cell monolayers were washed in PBS and incubated for 30 min in a Nonidet P-40-based cell permeabilization solution [PBS, 10% fetal calf serum (FCS), 1% BSA, 0.2% Nonidet P-40] at room temperature. After permeabilization the fixed cells were blocked in a PBS-based blocking solution (PBS, 10% FCS, 1% BSA) for 1 h at room temperature or 16 h at 4 C. To visualize GnRHR-I or GnRHR-II-reliquum proteins, the cells were incubated for 2 h at room temperature with antisera against both the HA-epitope (anti-HA 12CA5 monoclonal antibody-fluorescein conjugate (Roche Molecular Biochemicals, Mannheim, Germany) for GnRHR-I) and the FLAG-epitope (anti-FLAG M2 monoclonal antibody-Cy3 conjugate (Sigma) for GnRHR-II-reliquum) at a dilution of 1:200. Cell monolayers were washed three times in PBS and then mounted in Permafluor fixative (Immunotech, Westbrooke, ME).
Receptor binding assays
Whole-cell receptor binding assays used the 125I-[His5, D-Tyr6]GnRH analog (27). Transiently transfected COS-7 cells in 12-well culture plates were washed once with ice-cold HEPES/DMEM/10% FCS, and incubated for 5 h on ice in HEPES/DMEM/10% FCS with 105 cpm/well 125I-[His5, D-Tyr6]GnRH and varying concentrations of unlabeled GnRH-I. Cell monolayers were rapidly washed twice in ice-cold PBS and solubilized in 0.1 M NaOH, before the amount of bound radioligand was assessed in a 1261 Multi--counter. Nonspecific binding, consistently found to be less than 10% of total binding, was determined using vector-transfected (pcDNA1/Amp) COS-7 cells and was subtracted from total binding to give specific binding.
Total inositol phosphate assays
GnRH stimulation of total inositol phosphate production was as described (26). Briefly, transiently transfected COS-7 cells were incubated with inositol-free DMEM containing 1% dialyzed heat-inactivated FCS and 0.5 μCi/well myo-[3H]inositol (Amersham Pharmacia Biotech, Little Chalfont, UK) for 48 h. Medium was removed and the cells washed with 1 ml buffer (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM glucose, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml BSA) containing 10 mM LiCl and incubated for 1 h at 37 C in 0.5 ml buffer containing 10 mM LiCl and GnRH agonist at the specified concentration. Reactions were terminated by the removal of agonist and the addition of 1 ml ice-cold 10 mM formic acid with incubation for 30 min at 4 C. Total [3H]inositol phosphates were separated from the formic acid cell extracts on AG 1-X8 anion exchange resin (Bio-Rad Laboratories, Hemel Hempstead, UK) and eluted with a 1 M ammonium formate/0.1 M formic acid solution. The associated radioactivity was determined by liquid scintillation counting.
Immunoprecipitation and Western blotting
HA-tagged GnRHR-I protein was immunoprecipitated (28) from cell lysates by overnight incubation with 30% preconjugated HA-agarose slurry (Santa Cruz Biotechnology, Santa Cruz, CA) and washed. The immunoprecipitates were resolved by SDS-PAGE and electrotransferred to polyvinyl difluoride membrane (NEN Life Science Products, Boston, MA). GnRHR-I protein was detected by using antimouse 12CA5 antisera (Roche Molecular Biochemicals) and visualized by enzyme-linked chemifluorescence (Amersham Pharmacia Biotech) and quantified using a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Results
A human GnRHR-II gene homolog exists, but it is disrupted by a frame shift and premature stop codon (Fig. 1), suggesting that a conventional GnRHR-II system is unlikely to exist in humans. However, this does not discount the possibility that the gene may yet encode functional proteins. Indeed, we previously demonstrated that the GnRHR-II gene remains transcriptionally active and displays alternative splicing (15). Furthermore, immunocytochemical studies using an antiserum (designated ZGHR-II-5) generated to the conserved extracellular loop (ECL)-3 domain of the human GnRHR-II showed specific staining in the human and sheep pituitary. This staining was found to colocalize with LH-positive cells, suggesting the expression of proteins encoded by the GnRHR-II gene in pituitary gonadotropes (14). Silencing of the sheep GnRHR-II has been previously demonstrated (16).
In the present study, we identify a putative translational start site 117 bp downstream of the premature stop codon (Fig. 1). This is the first start codon occurring in-frame after the premature stop codon. Use of this translation start site encodes a protein (the GnRHR-II-reliquum) corresponding to the domains from the cytoplasmic end of transmembrane domain (TMD)-5, intracellular loop-3, TMD-6, ECL-3, TMD-7, and the carboxyl-terminal tail of the putative human GnRHR-II (Fig. 2). We demonstrated previously that processed mRNA transcripts that encode the GnRHR-II-reliquum are present in many human tissues and cell lines (15, 24).
The open-reading frame encoding the GnRHR-II-reliquum was subcloned into mammalian expression vectors (Figs. 1 and 2). To ascertain the expression and subcellular localization of the GnRHR-II-reliquum, immunocytochemical studies were performed. Confocal fluorescence microscopy images of permeabilized COS-7 cells transiently transfected with either the FLAG-tagged GnRHR-II-reliquum (Fig. 3A) or HA-tagged GnRHR-I (Fig. 3B) demonstrated that they were expressed and revealed a similar general cellular distribution of both proteins. GnRHR-II-reliquum expression appeared to be localized throughout the cytoplasm (Fig. 3, D–G, J, M, and P). When coexpressed, both proteins showed a high degree of colocalization at sites within the cell (Fig. 3, G–R).
FIG. 3. Subcellular localization of the GnRHR-II-reliquum and GnRHR-I. Immunofluorescence studies were carried out with transfected COS-7 cells grown in 8-well chamber glass slides (Lab-Tek, Christchurch, New Zealand) as described in Materials and Methods. Cells were transiently transfected with either (A–D) FLAG-tagged GnRHR-II-reliquum or HA-tagged GnRHR-I cDNA constructs or both (G-X), methanol fixed, permeabilized (A–R) or left unpermeabilized (S–X), and incubated with anti-FLAG M2 monoclonal antibody-Cy3 conjugate (Sigma) and/or anti-HA 12CA5 monoclonal antibody-fluorescein conjugate (Roche Molecular Biochemicals) directed against the amino-terminal epitope tags present in the FLAG-tagged GnRHR-II-reliquum and HA-tagged GnRHR-I constructs, respectively. Fluorescence images were obtained by confocal laser microscopy as described. Images in the panels correspond to: FLAG-tagged GnRHR-II-reliquum expressed alone (A), HA-tagged GnRHR-I expressed alone (B), and mock transfected cells stained with both anti-FLAG M2 monoclonal antibody-Cy3 conjugate (Sigma) and anti-HA 12CA5 monoclonal antibody-fluorescein conjugate (Roche Molecular Biochemicals) as a negative control (C); FLAG-tagged GnRHR-II-reliquum expressed alone (D), corresponding phase-contrast image (E), and merged image (F); coexpressed FLAG-tagged GnRHR-II-reliquum (G, J, M, P, S, and V) and HA-tagged GnRHR-I (H, K, N, Q, T, and W), with the corresponding merged images to demonstrate their colocalization (I, L, O, R, U, and X).
We next investigated whether the GnRHR-II-reliquum was capable of cell-surface plasma membrane insertion. Confocal microscopy of nonpermeabilized COS-7 cells coexpressing the FLAG-tagged GnRHR-II-reliquum (Fig. 3, S and V) and HA-tagged GnRHR-I (Fig. 3, T and W) failed to detect any significant fluorescence at the cell periphery for the former, in contrast to the latter. Therefore, whereas the GnRHR-II- reliquum is clearly expressed nascently, it appears not to be significantly inserted into the cell plasma membrane.
In line with previous literature demonstrating the inhibitory effect of human GnRHR-I and bullfrog GnRHR-III gene splice variants on GnRHR function (22, 23), we sought to investigate the effect of GnRHR-II-reliquum expression on GnRHR-I function. Transient cotransfection of GnRHR-I and GnRHR-II-reliquum constructs in COS-7 cells, resulted in a reduced expression of the GnRHR-I at the cell surface (Fig. 4A). This inhibitory effect was directly related to the amount of GnRHR-II-reliquum cDNA cotransfected with GnRHR-I. Thus, when increasing amounts of GnRHR-II-reliquum cDNA (0.4–1.6 μg/well) were cotransfected with a constant amount of GnRHR-I cDNA (0.4 μg/well), a progressive reduction in plasma membrane expression of GnRHR-I was observed (Fig. 4A). Cotransfection with the highest amount of GnRHR-II-reliquum cDNA (1.6 μg/well) did not significantly alter the affinity of GnRH-I ligand for GnRHR-I yet profoundly reduced the GnRHR-I expression at the cell surface as revealed by decreased maximal GnRH binding (Fig. 4B).
FIG. 4. Inhibition of GnRHR-I cell-surface expression by coexpression of GnRHR-II-reliquum in COS-7 cells. A, COS-7 cells were transfected with constant amounts of GnRHR-I cDNA (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0 to 1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Specific binding was measured as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments performed in triplicate. B, COS-7 cells were transfected with GnRHR-I and GnRHR-II-reliquum cDNA (0.4 and 1.6 μg/well, respectively) and incubated with increasing concentrations of cold GnRH-I ligand in the presence of 125I-[His5, D-Tyr6]GnRH as described in Materials and Methods. Data represent the mean ± SEM of three individual experiments performed in duplicate.
Concomitant with the GnRHR-II-reliquum-mediated inhibition of GnRHR-I expression, we noted a dose-dependent inhibitory effect on the extent of GnRH-I-induced inositol phosphate accumulation in cells expressing the GnRHR-I. Thus, when increasing amounts of GnRHR-II-reliquum cDNA (0.4–1.6 μg/well) were cotransfected with a constant amount of GnRHR-I cDNA (0.4 μg/well), a progressive reduction of GnRH-I agonist-induced inositol phosphate accumulation was observed (Fig. 5A). Cotransfection of the highest amount (1.6 μg/well) of GnRHR-II-reliquum cDNA also did not alter the EC50 of GnRH-induced inositol phosphate accumulation but did reduce the maximal level of the GnRH response (Fig. 5B).
FIG. 5. Inhibition of GnRHR-I signaling by coexpressed GnRHR-II-reliquum in COS-7 cells. A, COS-7 cells were transfected with constant amounts of GnRHR-I cDNA (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0 to 1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Total inositol phosphate accumulation in response to 1 μM GnRH-I was measured as described in Materials and Methods. Data represent the mean ± SEM of three individual experiments performed in triplicate. B, COS-7 cells were transfected with GnRHR-I and GnRHR-II-reliquum cDNA (0.4 and 1.6 μg/well, respectively) and incubated with increasing concentrations of cold GnRH-I ligand and total inositol phosphate accumulation measured as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments performed in duplicate.
To test the specificity of the inhibitory effect of the GnRHR-II-reliquum on GnRHR-I expression, we employed the rat TRH receptor (TRHR), and a nonmammalian GnRHR-I (the chicken GnRHR-I), both of which are Gq/11-coupled receptors and possess carboxyl-terminal cytoplasmic tail domains, unlike the uniquely tailless mammalian GnRHR-Is. When increasing amounts of GnRHR-II-reliquum cDNA (0.4–1.6 μg/well) were cotransfected with a constant amount of either rat TRHR or chicken GnRHR-I cDNA (0.4 μg/well), no progressive reduction of TRH or GnRH-I agonist-induced inositol phosphate accumulation was observed (Fig. 6, A–C).
FIG. 6. Specificity of the GnRHR-II-reliquum inhibitory effect. COS-7 cells were transfected with constant amounts of human GnRHR-I (A), rat TRHR (B), and chicken GnRHR-I cDNA (C) (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0 to 1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Total inositol phosphate (InsP) accumulation in response to 1 μM GnRH-I (A and C) and 1 μM TRH (B) was measured as described in Materials and Methods. D and E, COS-7 cells were transfected with GnRHR-I (0.4 μg/well) and either GnRHR-II-reliquum cDNA (1.6 μg/well) or rat TRHR cDNA (1.6 μg/well) and incubated with increasing concentrations of cold GnRH-I ligand (D only), and total inositol phosphate accumulation and binding (Bo, maximal binding only) was measured as described in Materials and Methods. All data are representative of three individual experiments performed in duplicate.
The possibility that the increased GnRHR-II-reliquum expression inhibits GnRH-induced inositol phosphate accumulation or GnRHR-I expression in a more general way, such as by hijacking available Gq/11-coupling units or competition for cellular translational capacity, was also addressed. Figure 6 (A–C) demonstrates that the inhibition of GnRH-induced inositol phosphate accumulation by GnRHR-II-reliquum is not due to lack of available Gq/11-coupling units because no inhibitory effect was seen for the rat TRHR or chicken GnRHR-I, both themselves Gq/11-coupled receptors. Furthermore, when human GnRHR-I was coexpressed with rat TRHR instead of GnRHR-II-reliquum, no inhibition of either GnRH-induced inositol phosphate accumulation or GnRH binding was observed at the human GnRHR-I, in contrast to the inhibition produced by coexpression of GnRHR-II-reliquum (Fig. 6, D and E).
When taken together, the above data suggest that the GnRHR-II-reliquum-induced reduction of GnRH-I agonist-induced inositol phosphate accumulation appears to be as a direct consequence of the specifically reduced expression of the GnRHR-I at the cell surface, rather than any interference in Gq/11-coupling capacity of the GnRHR-I or competition for cellular translational machinery.
Immunoprecipitation and Western blot analysis revealed that the total cellular expression of GnRHR-I was also markedly reduced by coexpression of the GnRHR-II-reliquum (Fig. 7A). The reduction in both cell-surface and nascent intracellular GnRHR-I levels suggested that the inhibition might be occurring at a site close to the site of de novo synthesis of GnRHR-I, perhaps at the nucleus/endoplasmic reticulum. To investigate the mechanism of this inhibition, we subjected COS-7 cells coexpressing GnRHR-I and GnRHR-II-reliquum to treatment with cell permeable agents to block de novo protein synthesis (CHX) or proteinase-mediated degradation (leupeptin and PMSF). However, these treatments failed to alter the inhibitory effect of GnRHR-II-reliquum coexpression on GnRHR-I levels, as demonstrated by immunoprecipitation and Western blot analysis (Fig. 7B) and GnRH-I agonist-induced inositol phosphate accumulation assays (Fig. 8). These data therefore suggest that the inhibitory effect is being exerted at the nucleus/endoplasmic reticulum or Golgi apparatus level, possibly by preventing or diverting the normal processing of GnRHR-I from these sites or enhancing GnRHR-I degradation via proteinase- or proteosome-mediated pathways that are not inhibited by leupeptin or PMSF.
FIG. 7. GnRHR-II-reliquum coexpression reduces both cell-surface and nascent GnRHR-I expression levels. A and B, COS-7 cells were transfected with GnRHR-I and GnRHR-II-reliquum cDNA (2 and 8 μg per 100-mm dish, respectively), subjected to pretreatments with CHX, leupeptin, and PMSF (B only). HA-tagged GnRHR-I was immunoprecipitated (IP) from cell lysates and subjected to Western blot (IB) analysis as described in Materials and Methods. Each bar represents the mean ± SEM of at least three individual experiments.
FIG. 8. No effect of CHX, leupeptin, or PMSF pretreatments on GnRHR-II-reliquum inhibition of GnRHR-I-mediated inositol phosphate (InsP) production. COS-7 cells were transfected with constant amounts of GnRHR-I cDNA (0.4 μg/well) and increasing amounts of GnRHR-II-reliquum cDNA (0–1.6 μg/well) supplemented with vector cDNA to keep the total amount of plasmid DNA per well constant. Pretreatments with CHX, leupeptin, and PMSF were performed as described, and total inositol phosphate accumulation in response to 1 μM GnRH-I was measured as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments.
Discussion
Alternative splicing of GPCR gene transcripts has long been thought to be a source of structural diversity and to provide a mechanism for the functional regulation of GPCRs. Some of these splice variants may themselves function as receptors in their own right and may bind ligands and effectively couple to a cognate G protein and diverse downstream signaling pathways. Others may serve a regulatory/modulatory role and interact to form complexes with the wild-type/full-length receptor to alter expression, function, and signal transduction. The formation of these complexes would probably occur through interhelical interactions between the individual helices spanning the membrane (22, 29, 30, 31). Previous studies of GnRHR-I and GnRHR-III alternative splice variants have reported their inhibitory activity on full-length GnRHR function (22, 23). In the present study, we demonstrated the inhibition of human GnRHR-I expression by a transiently coexpressed fragment of the human GnRHR-II gene homolog (i.e. the GnRHR-II-reliquum). Furthermore, we observed that this inhibition was specific, augmented by increasing amounts of cotransfected GnRHR-II-reliquum cDNA and was not altered by treatments that either inhibited de novo protein synthesis or proteinase-mediated degradation.
Our previous finding that the disrupted human GnRHR-II gene homolog remains transcriptionally active and displays alternative splicing (15) suggested that the gene may encode a functional entity. The complexities and potential roles of the disrupted human GnRHR-II gene homolog have been extensively debated and reviewed in the past few years (5, 18, 19, 20, 31, 32, 33). A recent review on the subject (31) discusses emerging evidence for the existence of a functional type II receptor for the cognate GnRH-II ligand in humans and suggests the interesting possibility that protein fragments derived from the disrupted human GnRHR-II gene homolog may associate with one another or with the human GnRHR-I to produce a functional GnRH-II-responsive receptor. A number of studies reported that GnRH-II has more potent antiproliferative effects than GnRH-I in various human tumor-derived cell lines (34, 35, 36), and such findings may support the conclusion that a functional GnRHR-II is present in humans. However, it is equally plausible that the effects observed in these studies are a result of altered agonist pharmacology at the GnRHR-I, leading to the activation of antiproliferative signal transduction pathways (37). Such ligand-selective signaling is dependent on intracellular environmental modulation of GnRHR-I ligand pharmacology, as has been recently demonstrated (38, 39). In their review, Neill et al. (31) elude to the same human GnRHR-II fragment sequence that we identify in the present study as the GnRHR-II-reliquum. It is worth noting that preliminary studies in our laboratory have shown no alteration in the ligand pharmacology of GnRH-I and GnRH-II (IC50 3 and 30 nM, respectively) at the human GnRHR-I when coexpressed with GnRHR-II-reliquum (data not shown).
The study by Grosse et al. (22) reported that coexpression of a truncated isoform of the human GnRHR-I leads to inhibition of signaling mediated by the full-length GnRHR-I. Similar findings were reported for the bullfrog GnRHR (23). Grosse et al. (22) also reported that a truncated GnRHR-I protein corresponding to TMDs-1–5, when coexpressed with a fragment of GnRHR-I corresponding to TMD-6 and -7, lead to a GnRH-induced inositol phosphate accumulation in COS-7 cells. These data suggested that the two receptor fragments were capable of interacting in a membrane environment to produce a functional GnRHR that was capable of coupling to Gq/11 and activating inositol phosphate accumulation (22). It is interesting to note that the C-terminal protein fragment (i.e. corresponding to TMD-6 and -7) used in the study by Grosse et al. (22) corresponds to approximately the same domains that constitute the GnRHR-II-reliquum.
There are several lines of evidence to suggest the endogenous expression of a protein fragment corresponding to TMD-6 and -7 of the human GnRHR-II (i.e. the GnRHR-II-reliquum). Immunocytochemical studies using an antiserum (ZGRH-II-5) generated to the conserved ECL-3 domain of the human GnRHR-II showed specific staining in the human pituitary (14). Similar specific staining was obtained when the same ECL-3 antiserum was used on sheep pituitary sections (14). Furthermore, the staining was found to colocalize with LH-positive cells, suggesting expression in human and sheep gonadotropes (14). An antiserum (ZGRH-II-8) directed at ECL-2 (the domain immediately preceding the putative start site identified here) failed to detect any immunoreactivity in sheep gonadotropes, in which the ECL-3 domain could be detected. The sheep GnRHR-II gene has been silenced by disruptions similar to those that have been reported to occur in the human GnRHR-II gene (15, 16). We have demonstrated that processed mRNA transcripts, which encode a protein fragment corresponding to the GnRHR-II-reliquum, are produced in various human tissues and cell lines (15, 24).
The presence of a putative start codon downstream of the premature stop codon suggests that synthesis of a GnRHR-II-reliquum is conceivable, should this be used as a translational start site. Indeed, any mRNA transcript derived from the putative GnRHR-II gene, in which intron 2 has been spliced out (Fig. 2), would lead to translation and synthesis of the GnRHR-II-reliquum, should the downstream start site be used by the translation machinery. Such transcripts (i.e. in which intron 2 has been spliced out) are now known to be present in various human tissues and cell lines (15, 24). Furthermore, it is intriguing to note that the putative start codon identified here occurs in the context of a partial Kozak consensus sequence (GCC ATG G). The full Kozak consensus required for initiation of translation was originally identified as GCC G/ACC ATG G (40). However, more recently it has emerged that the Kozak consensus cannot be reliably used as a criterion for the prediction of protein expression from a particular open reading frame and that the selection of translational initiation site in eukaryotic mRNAs involves many complex mechanisms (41, 42, 43). The role or importance, if any, of the partial Kozak consensus sequence in directing expression of the GnRHR-II-reliquum remains to be investigated.
In the present study, we did not detect significant cell-surface staining of FLAG-tagged GnRHR-II-reliquum using an anti-FLAG M2 monoclonal antibody-Cy3 conjugate in nonpermeabilized cells (Fig. 3). We were, however, able to demonstrate cytoplasmic staining of nascent GnRHR-II-reliquum in permeabilized COS-7 cells using the same antibody (Fig. 3). Such cytoplasmic staining would include FLAG-tagged GnRHR-II-reliquum residing in intracellular membranes such as vesicles and the endoplasmic reticulum. Because the GnRHR-II-reliquum corresponds to the C-terminal region of the putative GnRHR-II, from the cytoplasmic end of TMD-5 to the carboxyl terminus (Fig. 2), the FLAG epitope at the amino-terminal end of this fragment is most likely situated on the cytosolic side of the plasma membrane. This scenario would preclude the detection of FLAG-tagged GnRHR-II-reliquum at the cell surface in nonpermeabilized cells. Given that the C-terminal protein fragment (i.e. corresponding to TMD-6 and -7) used in the study by Grosse et al. (22) was apparently able to interact with the TMD-1 through -5 fragment to produce a functional receptor, we suggest that the GnRHR-II-reliquum may similarly interact, via interhelical interactions, with the GnRHR-I in the membrane environment. This scenario may therefore provide a mechanism for the inhibition of GnRHR-I expression reported in the present study. Thus, after its synthesis at the ribosome, the GnRHR-I protein is inserted into membranes of the endoplasmic reticulum. In the absence of coexpressed GnRHR-II-reliquum, the GnRHR-I is processed in the usual way, from the endoplasmic reticulum to the Golgi apparatus and onward to the plasma membrane. However, a physical interaction between GnRHR-I and GnRHR-II-reliquum may lead to an unstable or misfolded receptor/complex that would be prevented from onward processing and/or undergo defective intracellular transport from the endoplasmic reticulum, or enhanced degradation, thus accounting for the overall decreased cellular expression of GnRHR-I. Finally, it was interesting to observe that the GnRHR-II-reliquum exerted no inhibitory action on the chicken GnRHR-I (Fig. 6). One may speculate that the presence of the carboxyl-terminal cytoplasmic tail in the chicken GnRHR-I (and indeed all nonmammalian GnRHRs) may preclude a direct physical association with the GnRHR-II- reliquum.
It is becoming increasingly apparent that a conventional GnRHR-II system is not present in humans, although the exact nature of a specific GnRH-II-responsive system in humans, should it exist, remains elusive. Taken together, our studies suggest that a protein corresponding to TMD-6 and -7 of the human putative GnRHR-II is expressed in the gonadotrope with the GnRHR-I, pointing to a possible role for the disrupted human GnRHR-II gene in modulating the function of the pituitary human GnRHR-I, perhaps in a manner similar to that described here. Clearly further work is required to ascertain the precise nature of potentially functional protein products of the disrupted yet transcriptionally active human GnRHR-II gene. However, in the present study, we have clearly demonstrated that a putative human GnRHR-II-reliquum can perturb the human GnRHR-I functionally in a controlled environment.
Acknowledgments
We are grateful to Robin Sellar and Nicola Miller for their excellent technical assistance.
References
Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499
Naor Z, Benard O, Seger R 2000 Activation of MAPK cascades by G-protein-coupled receptors: the case of gonadotropin-releasing hormone receptor. Trends Endocrinol Metab 11:91–99
Kraus S, Naor Z, Seger R 2001 Intracellular signaling pathways mediated by the gonadotropin-releasing hormone (GnRH) receptor. Arch Med Res 32:499–509
Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205
Millar RP, Lu Z-L, Pawson AJ, Flanagan CA, Morgan K, Maudsley S 2004 Gonadotropin-releasing hormone receptors. Endocr Rev 25:235–275
Ferguson SS, Barak LS, Zhang J, Caron MG 1996 G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74:1095–1110
Ferguson SS 2001 Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24
Tensen C, Okuzawa K, Blomenrohr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140
Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc Natl Acad Sci USA 96:2526–2531
Sun YM, Flanagan CA, Illing N, Ott TR, Sellar R, Fromme BJ, Hapgood J, Sharp P, Sealfon SC, Millar RP 2001 A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants. J Biol Chem 276:7754–7761
Wang L, Bogerd J, Choi HS, Seong JY, Soh JM, Chun SY, Blomenr?r M, Troskie BE, Millar RP, Kwon HB 2001 Three distinct types of gonadotropin-releasing hormone receptor characterized in the bullfrog. Proc Natl Acad Sci USA 98:361–366
Bogerd J, Diepenbroek WB, Hund E, Van Oosterhout F, Teves AC, Leurs R, Blomenrohr M 2002 Two gonadotropin-releasing hormone receptors in the african catfish: no differences in ligand selectivity, but differences in tissue distribution. Endocrinology 143:4673–4682
Neill JD, Duck LW, Sellers JC, Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282:1012–1018
Millar RP, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641
Morgan K, Conklin D, Pawson AJ, Sellar R, Ott TR, Millar RP 2003 A transcriptionally active human type II gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the antisense orientation on chromosome 1q. 12. Endocrinology 144:423–436
Gault PM, Morgan K, Pawson AJ, Millar RP, Lincoln GA 2004 Sheep exhibit novel variations in the organization of the mammalian type II gonadotropin-releasing hormone receptor gene. Endocrinology 145:2362–2374
Faurholm B, Millar RP, Katz AA 2001 The genes encoding the type II gonadotropin-releasing hormone receptor and the ribonucleoprotein RBM8A in humans overlap in two genomic loci. Genomics 78:15–18
Millar RP 2003 GnRH II and type II GnRH receptors. Trends Endocrinol Metab 14:35–43
Pawson AJ, Morgan K, Maudsley SR, Millar RP 2003 Type II gonadotropin-releasing hormone (GnRH-II) in reproductive biology. Reproduction 126:271–278
Morgan K, Millar RP 2004 Evolution of GnRH ligand precursors and GnRH receptors in protochordate and vertebrate species. Gen Comp Endocrinol 139:191–197
Kottler ML, Bergametti F, Carre MC, Morice S, Decoret E, Lagarde JP, Starzec A, Counis R 1999 Tissue-specific pattern of variant transcripts of the human gonadotropin-releasing hormone receptor gene. Eur J Endocrinol 140:561–569
Grosse R, Schoneberg T, Schultz G, Gudermann T 1997 Inhibition of gonadotropin-releasing hormone receptor signaling by expression of a splice variant of the human receptor. Mol Endocrinol 11:1305–1318
Wang L, Oh DY, Bogerd J, Choi HS, Ahn RS, Seong JY, Kwon HB 2001 Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling. Endocrinology 142:4015–4025
Millar R, Conklin D, Lofton-Day C, Hutchinson E, Troskie B, Illing N, Sealfon SC, Hapgood J 1999 A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript. J Endocrinol 162:117–126
Pawson AJ, Katz A, Sun YM, Lopes J, Illing N, Millar RP, Davidson JS 1998 Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail. J Endocrinol 156:R9–R12
Pawson AJ, Maudsley SR, Lopes J, Katz AA, Sun YM, Davidson JS, Millar RP 2003 Multiple determinants for rapid agonist-induced internalization of a nonmammalian gonadotropin-releasing hormone receptor: a putative palmitoylation site and threonine doublet within the carboxyl-terminal tail are critical. Endocrinology 144:3860–3871
Flanagan CA, Fromme BJ, Davidson JS, Millar RP 1998 A high affinity gonadotropin-releasing hormone (GnRH) tracer, radioiodinated at position 6, facilitates analysis of mutant GnRH receptors. Endocrinology 139:4115–4119
Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM 2000 The ?(2)-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 275:9572–9580
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Trong IL, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745
Suel GM, Lockless SW, Wall MA, Ranganathan R 2003 Evolutionarily conserved networks of residues mediate allosteric communication in proteins. Nat Struct Biol 10:59–69
Neill JD, Musgrove LC, Duck LW 2004 Newly recognized GnRH receptors: function and relative role. Trends Endocrinol Metab 15:383–392
Pawson AJ, Morgan K, Maudsley S, Millar RP, The type II GnRH receptor and the "SECIS element" of its pseudogene. Proc 2nd Cooloquium of the DFG-Priority Programme Selenoproteins, Charite, Berlin, Germany, 2003
Neill JD 2002 GnRH and GnRH receptor genes in the human genome. Endocrinology 143:737–743
Grundker C, Gunthert AR, Millar RP, Emons G 2002 Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J Clin Endocrinol Metab 87:1427–1430
Grundker C, Emons G 2003 Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer. Reprod Biol Endocrinol 1:65
Grundker C, Schlotawa L, Viereck V, Eicke N, Horst A, Kairies B, Emons G 2004 Antiproliferative effects of the GnRH antagonist cetrorelix and of GnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor. Eur J Endocrinol 151:141–149
Maudsley S, Davidson L, Pawson AJ, Chan R, Lopez de Maturana R, Millar RP 2004 GnRH antagonists promote pro-apoptotic signaling in peripheral tissues by activating a G-i-coupling state of the type I GnRH receptor. Cancer Res 64:7533–7544
Caunt CJ, Hislop JN, Kelly E, Matharu AL, Green LD, Sedgley KR, Finch AR, McArdle CA 2004 Regulation of gonadotropin-releasing hormone receptors by protein kinase C: inside out signalling and evidence for multiple active conformations. Endocrinology 145:3594–3602
Millar RP, Pawson AJ 2004 Outside-in and inside-out signalling: the new concept that selectivity of ligand binding at the gonadotropin-releasing hormone receptor is modulated by the intracellular environment. Endocrinology 145:3590–3593[Free Full Text]
Kozak M 1986 Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292
Kozak M 2002 Pushing the limits of the scanning mechanism for initiation of translation. Gene 299:1–34
Kozak M 2003 Alternative ways to think about mRNA sequences and proteins that appear to promote internal initiation of translation. Gene 318:1–23
Kapp LD, Lorsch JR 2004 The molecular mechanics of eukaryotic translation. Annu Rev Biochem 73:657–704(Adam J. Pawson, Stuart Ma)