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a Department of Physiology, University of Turku, 20520 Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit and a hormone-specific ß subunit, and, acting in concert, they promote growth and differentiation of developing gonads, control gametogenesis, and regulate gonadal endocrine function [1, 2]. The actions of gonadotropins are targeted to testicular and ovarian somatic cells through specific cell-surface receptors. These receptors, namely the LH receptor (LHR) and the FSH receptor (FSHR), are members of the G protein-coupled receptor superfamily, showing the distinctive carboxy-terminal cluster of seven transmembrane-spanning domains [3, 4]. However, at variance with most other members of this group, they present an unusually large extracellular domain, responsible for specific, high-affinity binding [5–7]. Upon receptor activation, different signaling pathways are involved in the transduction of the pleiotropic biological actions of gonadotropins, cAMP being the main intracellular second messenger [3, 4, 8, 9]. Unlike most other G protein-coupled receptor genes, which are small and intronless, those of the gonadotropin receptors are large and provided with a complex exon-intron structure [3, 4]. The LHR gene is composed of 11 exons and 10 introns, while the FSHR gene contains 10 exons and 9 introns. The extracellular domain is encoded mostly by exons 1 to 10 in the LHR and by exons 1 to 9 in the FSHR, whereas in both genes, the last exon codes for the transmembrane and intracellular regions, as well as for a short part of the extracellular domain, next to the plasma membrane. Such a complex genomic organization favors extensive alternative splicing during the processing of the primary transcripts. In fact, despite the presence of single forms of functional receptors in target cells, in all species so far tested the gonadotropin receptor genes are expressed as multiple mRNA transcripts ([3, 4, 10], and references therein). The functional role of this alternative splicing remains to be fully characterized. However, recent reports point out the relevance of this phenomenon in the generation of gonadotropin receptor isoforms with ability to modulate holoreceptor function [11, 12].
Recently, new insight has been gained into the structure-function relationship of gonadotropin receptors based on comparative analyses of gonadotropin receptors from different species [8, 13–19], site-directed mutagenesis (reviewed in [10]), behavior of chimeric receptors [7, 20–22], and evaluation of the functional impact of several naturally occurring mutations in gonadotropin receptor genes (reviewed in [23, 24]). So far, most efforts in this field have focused on the LHR. To further expand our understanding on the structure-function relationship of the FSHR, we decided to characterize the entire coding region of mouse (m) FSHR cDNA and carried out the functional expression of several splice variants of the mFSHR. In addition, we evaluated the impact on mFSHR function of a C566T transition in exon 7 of the coding sequence, which is analogous to the inactivating mutation occurring in the human FSHR gene and responsible for a form of hypergonadotropic ovarian failure [25].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The complete coding region of mFSHR cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from mouse testicular RNA. Total RNA was isolated from 129/Sv mouse testes using the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method, as described previously [26]. For RT-PCR reactions, several primers were designed based on the analysis of conserved sequences of rat, human, porcine, and ovine FSHR cDNAs, as well as on the partially characterized mFSHR cDNA sequence [14, 16, 27–30]. After testing, primer pairs yielding proper amplification patterns, as predicted from the FSHR cDNAs of other species, were selected for further analyses (Table 1).
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For amplification of the cDNA sequence encoding the entire mFSHR, RT and PCR reactions were run in two separate steps. First, 4 µg total testicular RNA was heat-denatured and reverse-transcribed by incubation at 42°C for 2 h with 12.5 U avian myeloblastosis virus (AMV)-RT (Promega, Madison, WI), 20 U ribonuclease inhibitor RNasin (Promega), 200 nM deoxy-NTP mixture, and 1 nM r3'A antisense primer in a final volume of 30 µl of single-strength AMV-RT buffer. The reaction was terminated by heating at 96°C for 5 min and cooling on ice. The reverse-transcribed single-strand cDNA was extracted using phenol-chloroform, precipitated overnight at -20°C with absolute ethanol and 3 M sodium acetate, pH 5.2, and dissolved in 20 µl sterile water. In a second step, the generated cDNA was amplified in two sequential rounds of PCR. In the first round, 5 µl of reverse-transcribed cDNA was amplified in 50 µl of single-strength PCR buffer in the presence of 2.5 U Taq-DNA polymerase (Promega), 200 nM deoxy-NTP mixture, and the primer pair (1 nM) m5'A-r3'A (-130 to +2201 base pairs [bp]) encompassing the complete coding region of mFSHR cDNA. The PCR reaction consisted of a first denaturing cycle at 97°C for 5 min, followed by 25 cycles of amplification defined by denaturation at 96°C for 1.5 min, annealing at 55°C for 1.5 min, and extension at 72°C for 3 min. A final extension cycle of 72°C for 10 min was included. In the second round, nested-PCR amplification of overlapping partial fragments of mFSHR cDNA was carried out. Aliquots (5 µl) of the first PCR reaction were amplified under conditions similar to those described above, using the following primer pairs (1 nM): m5'B-r/m10Aas (-60 to +873 bp), m7-r10B (+545 to +1177 bp), and r/m10As-r3'B (+847 to +2131 bp). The generated cDNA fragments were resolved in 1.5% agarose gels, and the products with expected sizes were cut and isolated using the Qiaex II gel extraction kit (Qiagen, Chatsworth, CA). It is noteworthy that PCR amplification using the primer pair m5'B-r/m10Aas, i.e., encompassing exons 1–9 of the mFSHR, persistently gave rise to 4 major DNA fragments, which were isolated and processed separately. Finally, the DNA fragments were subcloned into p-GEM5f T-vector (Promega) and sequenced from both strands using a fluorescent dye termination reaction (Prism Ready Reaction Dye Terminator Cycle Sequencing kit) and an automated sequencer (Perkin-Elmer, Foster City, CA). Analyses of nucleotide sequence and predicted amino acid composition of mFSHR were carried out using the DNASTAR program (DNASTAR, Madison, WI). To avoid PCR-derived sequence errors, positive alignment of at least three independent sequences was requested for any given area of the mFSHR cDNA.
Construction of mFSHR cDNA Expression Vectors
For construction of full-length cDNA expression vector, two overlapping fragments, encoding the extracellular and transmembrane/intracellular domains of the mFSHR, were generated by PCR (see previous section) using the primer pairs EcoRI/m5'B-r/m10Aas and m7-r3'B, respectively. The entire coding cDNA sequence of mFSHR was engineered by fusing EcoRI/m5'B-r/m10Aas and m7-r3'B overlapping fragments at the unique XbaI site (at 772 nucleotides). The generated cDNA was subcloned at the EcoRI/BamHI sites of the pSG5 eukaryotic expression vector (Stratagene, La Jolla, CA). For comparative functional analysis, expression plasmids for the human and rat FSHR and human LHR were used. The full-length rat FSHR cDNA (donated by R. Sprengel, University of Heidelberg, Germany) and the human LHR cDNA (a gift from L. Dunkel, University of Helsinki, Finland) were inserted into pSG5 expression vector. The construction of pSG5/human FSHR expression plasmid is described in detail elsewhere [25].
As described above, PCR amplification of mFSHR cDNA using primers flanking the extracellular domain, besides generation of the product with expected size, resulted in multiple DNA fragments with lower molecular sizes. Systematic subcloning and sequencing revealed that these fragments corresponded to alternatively spliced mFSHR variants, selectively lacking exons 2, 2 and 5, 5 and 6, and 2, 5, and 6 of the coding sequence (see Results). To evaluate the potential functional role of such variants, expression vectors were generated using an approach similar to that used for the complete cDNA coding sequence. Alternately spliced fragments were first generated by RT-PCR using the EcoRI/m5'B-r/m10Aas primer pair, and then fused to the m7-r3'B overlapping DNA product at the XbaI site. The resulting cDNAs were subcloned at the EcoRI/BamHI sites of the pSG5 expression vector.
For construction of the C566T mutant mFSHR expression vector, site-directed mutagenesis was carried out using the overlapping PCR-based method. In the first PCR reaction, two overlapping fragments were generated using the cloned mFSHR cDNA as template and the primer pairs EcoRI/m5'B-mut7as and mut7s-r/m10Aas. The sequences of the mutated primers are presented in Table 1. The PCR reaction consisted of a first denaturing cycle at 97°C for 5 min, followed by 20 cycles of amplification defined by denaturation at 96°C for 1.5 min, annealing at 55°C for 1.5 min, and extension at 72°C for 3 min, with a final extension cycle of 72°C for 15 min. The generated DNA products were isolated and used as template for the second PCR round, with the primer pair EcoRI/m5'B-r/m10Aas. The resulting single DNA fragment was isolated, subcloned, and sequenced as described above. Correct clones carrying the C566T transition were selected. For construction of the mutant mFSHR expression vector, the mutated EcoRI/m5'B-r/m10Aas product was fused to the m7-r3'B overlapping fragment at the XbaI site (see above), and the resulting cDNA was subcloned at the EcoRI/BamHI sites of the pSG5 expression vector.
Transfections
For expression studies, human embryonic kidney (HEK 293) cells and mouse ovarian (KK-1) cells were used. The origin of the parental KK-1 cell line has been described in detail previously [31]. In this cell line, increasing number of passages in culture induces the loss of endogenous gonadotropin receptors, and for the present experiments a batch of KK-1 cells lacking the FSHRs but with a high steroidogenic capacity was selected. The cells were maintained in Dulbecco's Modified Eagle's medium (DMEM)-Ham's F-12 medium (1:1; Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum, 0.1 g/L gentamicin (Biological Industries, KB Haemek, Israel), and 2.5 mg/L of fungizone (Life Technologies) at 37°C in a humidified atmosphere of 95% O2:5% CO2. Transient transfection of HEK 293 cells was carried out using the electroporation method. Briefly, cells (8 x 106 per cuvette, in serum-free DMEM-Ham's F-12 medium) were electroshocked using Genepulser (Bio-Rad, Richmond, CA) at 250 V and 960 µF, in the presence of 15–20 µg of the expression plasmid under analysis and 5 µg of ß-galactosidase expression vector, to monitor transfection efficiency [32]. After electroporation, cells were plated in 10-cm dishes and allowed to grow in DMEM-Ham's F-12 complete medium for 36–48 h before ligand-binding and functional assays were performed.
Ovarian KK-1 cells were transfected using lipofectamine (Life Technologies) according to the manufacturer's instructions. For transient transfections, 1.5 x 106 cells were seeded in 10-cm dishes, grown to up to 70–80% confluency in complete medium, and transfected with 8–10 µg of expression plasmids using 8 ml of serum-free Opti-MEM medium (Life Technologies) and 75 µg of lipofectamine. As a control for transfection efficiency, 1 µg of ß-galactosidase expression vector was routinely included in the transfection mixture. After 4–6 h, the medium was replaced with fresh DMEM-Ham's F-12 complete medium, and the cells were allowed to grow for 36–48 h before use for functional analyses. For generation of clonal cell lines stably expressing the mFSHR, ovarian KK-1 cells were cotransfected with 8–10 µg of full-length mFSHR expression vector and 1 µg of the neomycin resistance pcDNA3.1 (+) vector (Invitrogen, San Diego, CA), using lipofectamine as described above. Two days after transfection, the cells were split into selection medium containing 450 mg/L G418 (geneticin; Life Technologies). After 4 wk of selection, resistant clones were picked, expanded, and screened for FSHR expression as estimated by FSH binding and stimulated cAMP responses. Clones showing higher receptor contents were selected for further experiments (see Results).
Ligand-Binding Assays
Recombinant human FSH (rhFSH, 32489; Organon, Oss, The Netherlands) and highly purified human hCG (CR-127, NIDDK, Rockville, MD) were iodinated, using a solid-phase lactoperoxidase method [33], to specific activities of 16 000 cpm/ng and 36 000 cpm/ng, respectively, as estimated according to the method of Catt et al. [34]. For binding measurements on intact cells, transfected cells were washed twice with ice-cold PBS and scraped into Dulbecco's PBS containing 0.1% BSA (Sigma Chemical Co., St. Louis, MO). The cells were pelleted by centrifugation at 1500 rpm, washed twice, and resuspended in Dulbecco's PBS-BSA. For single-point binding measurements, 100-µl aliquots of cell suspension (~3 x 105 cells) were incubated in triplicate in the presence of a saturating concentration of [125I]iodo-rhFSH (100 000 cpm) or [125I]iodo-hCG (150 000 cpm). To assess nonspecific binding, additional duplicates were incubated in presence of > 1000-fold excess of unlabeled rhFSH (2.0 IU) or hCG (50 IU; Pregnyl; Organon), respectively. For competition studies, 100-µl aliquots of the same cell suspension were incubated in triplicate with increasing amounts of unlabeled rhFSH (0–10 000 mIU) or hCG (CR-127; 0–10 000 ng) in the presence of 100 000 cpm [125I]iodo-rhFSH. Nonspecific binding was evaluated in matched samples in the presence of > 1000-fold molar excess of unlabeled rhFSH (2.0 IU). For Scatchard analysis, similar aliquots of cells were incubated in the presence of increasing concentrations of [125I]iodo-rhFSH (50 000–500 000 cpm/tube), the nonspecific binding being assessed as described above. In all binding assays, the incubation time was 16 h at room temperature (22–24°C). Bound and free hormones were separated by 15-fold dilution with ice-cold Dulbecco's PBS-BSA followed by centrifugation of the samples at 2500 x rpm for 10 min, and the radioactivity bound to cell pellets was counted in a 
-spectrometer.
Binding measurements on solubilized cells were carried out as described previously [35], with minor modifications. Transfected cells were washed twice in buffer B (150 mM NaCl, 20 mM Hepes, pH 7.4) supplemented with the protease inhibitors leupeptin (20 µM; Sigma) and PMSF (1 mM; Sigma). The cells were then solubilized by suspension in buffer B containing protease inhibitors, 1% Nonidet P-40 (Sigma), and 20% glycerol, followed by incubation on ice for 30 min with vortexing of the samples every 5 min. The extracts were centrifuged at 12 000 rpm for 30 min at 4°C, and the supernatants were used for binding assays as described above. The incubation time was 16 h at 4°C. Bound and free [125I]iodo-rhFSH were separated using -globulin/polyethyleneglycol precipitation method, as described elsewhere [35]. All binding assays were repeated at least twice, using cells from independent transfections.
Cyclic AMP, Inositol Trisphosphate (IP3), and Progesterone Assays
Signal transduction efficiency of the recombinant receptors was assessed by measuring cAMP, IP3, and progesterone responses in transfected cells after stimulation with increasing doses of rhFSH. All stimulation experiments were repeated at least three times, using cells from independent transfections. For cAMP assays, transfected cells were seeded at a density of 75 000 cells per well (24-well plate) 18 h before stimulation. The cells were washed twice with DMEM/Ham's F-12 medium containing 0.1% BSA and incubated in 1 ml of medium containing 0.2 mM 3-isobutyl-1-methylxanthine (Sigma) for 1–3 h in the presence of increasing concentrations of rhFSH (0.1 to 103 mIU). All stimulations were performed in quadruplicate. At the end of the incubation period, media were collected for measurement of extracellular cAMP using a standard RIA method [36].
For IP3 measurements, transfected cells were plated at a density of 4 x 105 cells per well (6-well plate) and labeled with [3H]inositol (NEN Life Science Products Inc., Boston, MA) by incubation for 30–36 h in 0.1% BSA-supplemented DMEM/Ham's F-12 medium containing 2 µCi/ml [3H]inositol. Before stimulation, the cells were washed twice with PBS and incubated in culture medium containing 12.5 mM LiCl for 15 min. Thereafter, the cells were stimulated for 30 min with increasing concentrations of rhFSH (1 to 104 mIU). All stimulations were performed in quadruplicate. The reactions were terminated by addition of ice-cold perchloric acid to a final concentration of 2.5%. After 15-min incubation on ice, cell debris was scraped off, brief vortexing was performed, and centrifugation was carried out at 3000 rpm for 10 min at 4°C. The supernatants were collected and equilibrated with 1.5 M KOH, and the samples were applied to AG-1 anion exchange resin columns (Bio-Rad). The columns were washed with 7.5 ml of water to remove unbound materials, and IP3 was selectively eluted with 5 ml of 0.1 M formic acid and 1.0 M ammonium formate. For IP3 measurement, the eluates were collected, and 0.5 ml of each fraction was mixed with 5 ml of scintillation liquid and counted for radioactivity in a beta spectrometer.
For progesterone measurements, transfected KK-1 cells were seeded at a density of 125 000 cells per well (24-well plate) 18 h before stimulation. The cells were washed twice with DMEM/Ham's F-12 medium containing 0.1% BSA and incubated for 12 h in the presence of increasing concentrations of rhFSH (0.1 to 103 mIU). All stimulations were performed in quadruplicate. At the end of the incubation period, media were collected for measurement of progesterone, using specific RIA as described before [37].
RNA Isolation from Transfected Cells and RT-PCR Analysis
To monitor transcription efficiency of expression vectors under analysis, total RNA was isolated from transfected cells using the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method [26], and a region of mFSHR mRNA, encoding the extracellular domain, was amplified by RT-PCR. This pattern of amplification allows clear discrimination between full-length and various exon-lacking species. The RT and PCR reactions were run sequentially in the same assay tube. Four micrograms of total RNA was reverse-transcribed using AMV-RT and the oligoprimer r/m10Aas. The reverse-transcribed cDNA was further amplified by PCR using the primer pair m5'B-r/m10Aas. Fifty microliters of RT-PCR reaction contained 2.5 U Taq-DNA polymerase, 200 nM deoxy-NTP mixture, and 1 nM of each oligoprimer. The reaction was started at 50°C for 15 min, followed by a period of 3 min at 97°C, and then run in PCR for 30 cycles (1.5 min at 96°C, 1.5 min at 55°C, and 3 min at 72°C), with a final cycle at 72°C for 15 min. This number of cycles was optimized to ensure amplification of the messages in the exponential phase of PCR. The generated cDNA fragments were resolved in 1.5% agarose gels, and their molecular sizes determined by comparison with size markers run together with the DNA products. To avoid potential plasmid DNA amplification, RNA samples were treated with RQ1 RNase-free DNase and extracted with phenol-chloroform prior RT-PCR amplification. In addition, in all tests, RT-PCR of total RNA from mock-transfected cells was performed, and liquid controls and reactions without RT were included.
Statistics
The results are presented as mean ± SEM, when appropriate. When relevant, statistically significant differences between groups were determined by one-way ANOVA, followed by Duncan's test; p 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overlapping partial fragments of mFSHR cDNA were amplified by RT-PCR using 129/Sv mouse testicular RNA and primers complementary to the rat or the partially characterized mFSHR sequence [14, 29, 30]. The DNA products generated were sequenced and aligned to produce the complete mFSHR cDNA coding region. Sequence analysis revealed an open reading frame for the cloned cDNA of 2076 bp (Fig. 1), with 93%, 85%, and 83–82% homology with rat, human, and bovine and ovine FSHRs, respectively. The deduced amino acid sequence of the mouse receptor, as well as comparison between mouse, rat, human, bovine, and ovine FSHRs, is presented in Figure 2. The predicted translated sequence of mFSHR includes a putative 17-amino acid signal peptide and a mature 675-amino acid receptor protein. In terms of receptor structure, the extracellular domain is 348 amino acids long and contains 3 potential sites for N-linked glycosylation; the transmembrane region is formed by 264 amino acids organized into 7 transmembrane-spanning domains, and the cytoplasmic tail is formed by 63 amino acids. At the protein level, the overall sequence homology was 94% with rat, 87% with human, and 85–84% with bovine and ovine FSHRs.
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Amplification of mFSHR cDNA using primers flanking the extracellular domain, besides generation of the expected size product, resulted in multiple DNA fragments with lower molecular sizes. Subcloning and sequencing of such species demonstrated that these fragments corresponded to alternatively spliced mFSHR variants. Assuming that the structural organization of the mouse gene is similar to that of the rat and human FSHR genes [38, 39], such variants arise by the selective splicing of exons 2, 2 and 5, 5 and 6, and 2, 5, and 6 of the coding sequence. The structure of some of these transcripts was described previously [30]. These findings, together with the persistent amplification of the aforementioned truncated PCR products using different primer pairs around the extracellular domain (data not shown), rule out the possibility of a PCR artifact. Furthermore, the strategy of amplification, using an antisense primer for RT located in the 3' untranslated region followed by two rounds of nested PCR, suggests that, in fact, the mFSHR splice variants described here represent complete receptor species selectively lacking the regions encoded by the missing exon(s).
Functional Expression of mFSHR cDNA and Its Splice Variants
To test the functionality of the cloned holoreceptor, an expression plasmid (pSG5/mFSHR) was generated by inserting the entire coding region of mFSHR into the eukaryotic expression vector pSG5. HEK 293 cells and ovarian KK-1 cells were transiently transfected, and the recombinant mFSHR was studied by means of radioligand-binding and functional assays. HEK 293 cells transfected with pSG5/mFSHR expression plasmid showed high levels of specific binding to [125I]iodo-rhFSH. In contrast, no specific binding was detected in cells transfected with pSG5 vector alone (data not shown). Incubation of HEK 293 cells expressing the full-length mFSHR with increasing concentrations of [125I]iodo-rhFSH resulted in a dose-dependent increase in specific binding of labeled rhFSH. Scatchard analysis of the data, fitted using a single-site model, demonstrated for the mFSHR high-affinity [125I]iodo-rhFSH binding, with a Kd value of 1.65·10-9 M (Fig. 3, top). This is similar to that measured for the human FSHR ([14], and present results). In addition, as the binding data were apparently curvilinear, Scatchard analysis was performed also using a two-site model (Fig. 3, top, inset). This method of analysis further confirmed that the recombinant mouse and human FSHRs behave similarly in terms of binding affinity. Finally, [125I]iodo-rhFSH binding to the mouse receptor was dose-dependently displaced by increasing amounts of unlabeled rhFSH, but not hCG, thus confirming binding specificity (Fig. 3, bottom).
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The functional capacity of the cloned mFSHR was further assessed by studying its ability to generate cAMP, IP3, and progesterone responses in transfected cells after stimulation with increasing doses of rhFSH (0.1 to 104 mIU/ml). Treatment of pSG5/mFSHR-transfected HEK 293 cells with rhFSH resulted in a dose-dependent increase in cAMP production, with a 35-fold increase over the basal rate of production in response to 103 mIU rhFSH (Fig. 4, top). This pattern of response was similar to those evoked by human and rat recombinant FSHRs. In addition, HEK 293 cells expressing the mFSHR displayed a dose-dependent increase in IP3 production after stimulation with increasing doses of rhFSH, with a 4.5-fold increase over basal values at 104 mIU rhFSH (Fig. 4, middle panel). Again, this response was similar to those shown by cells expressing human and rat FSHRs. Finally, the cloned receptor was able to evoke biological responses in a homologous cell system, as incubation of pSG5/mFSHR-transfected ovarian KK-1 cells with rhFSH resulted in a significant increase (p 0.01) in progesterone production for all doses tested, although the pattern of response was biphasic with maximum progesterone release after 100 mIU rhFSH stimulation. No responses were observed in mock-transfected cells (Fig. 4, bottom).
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The cloned mFSHR splice variants, selectively lacking exons 2, 2 and 5, 5 and 6, and 2, 5, and 6, were functionally characterized following an approach similar to that described above. The expression plasmids (namely pSG5/-2, pSG5/-2,5, pSG5/-5,6, and pSG5/-2,5,6) were generated by subcloning the coding region of the alternate transcripts into the pSG5 vector, and transient transfections of HEK 293 and ovarian KK-1 cells were carried out. Radioligand-receptor assays demonstrated that, in contrast to observations in cells expressing the mouse holoreceptor, no specific binding to [125I]iodo-rhFSH was detected in HEK 293 cells transfected with the different mFSHR variants, either in intact or in solubilized cell preparations (Table 2). This occurred despite successful transcription of the transfected plasmids as estimated by RT-PCR (Fig. 5). In addition, cells expressing the different mFSHR variants showed no specific binding to [125I]iodo-hCG, either in intact cells or in solubilized cell preparations (Table 2). The latter assessment was made in order to exclude the possibility that removal of discrete regions of the extracellular domain in the different mFSHR variants might have affected the binding specificity. Finally, exposure to rhFSH (0.1 to 103 mIU) of KK-1 cells transfected with expression plasmids corresponding to the different mFSHR variants failed to evoke cAMP production (Fig. 6) or progesterone secretion (data not shown).
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Cotransfection Studies
The potential involvement of the cloned mFSHR splice variants in the modulation of mouse holoreceptor function was further assessed in the context of an ovarian cell line stably expressing the full-length mFSHR. KK-1 cells were stably transfected with pSG5/mFSHR plasmid, and a clonal cell line (KK-1/clone12) expressing high levels of functional receptors was selected. Individual transient transfections of these cells with the different mFSHR variants failed to modify the pattern of response to increasing doses of rhFSH (0.1 to 103 mIU) as estimated in terms of cAMP production (data not shown). Similarly, cotransfection of a "cocktail" of all the mFSHR splice variants into clonal KK-1 cells stably expressing the full-length mFSHR did not induce changes in the amount or affinity of binding to [125I]iodo-rhFSH (Fig. 7, top); neither did it result in changes in the pattern of cAMP production in response to increasing concentrations of rhFSH (Fig. 7, bottom panels). This occurred in spite of successful transcription of the cocktail of expression plasmids (Fig. 8). Finally, transient transfection of the expression vector pSG5/mFSHR, encoding the functional mouse holoreceptor, into clonal KK-1 cells stably expressing the full-length mFSHR resulted in increased rhFSH-induced cAMP accumulation, thus showing that the cotransfection system used is able to unravel potential changes in net receptor function if plasmids encoding functional receptor isoforms are transfected (Fig. 7, bottom panels).
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Functional Expression of C566T Mutant mFSHR cDNA
Finally, the impact on mFSHR function of a C566T transition in exon 7 of the coding sequence was evaluated. Site-directed mutagenesis was used to generate a mutated expression plasmid (pSG5/mFSHRmut), and HEK 293 cells were selected for transient transfections. Radioligand-receptor assays demonstrated a marked decrease in total ligand binding for the mutated mFSHR. Scatchard analysis of the binding data showed, however, that the equilibrium association constant (Kd) of the mutated mFSHR was similar to that of the mouse holoreceptor (Kd mFSHRmut = 1.67 nM vs. Kd mFSHR = 1.64 nM) (Fig. 9, top). This was also the case for the analogous mutation in the human FSHR, responsible for a form of hereditary hypergonadotropic ovarian failure [25]. The functionality of the mutated mFSHR was further tested by assessing its ability to elicit cAMP and IP3 production in response to rhFSH stimulation. Treatment of HEK 293 cells expressing the functional mFSHR with increasing doses of rhFSH resulted in dose-dependent increases in cAMP and IP3 production, with maximal 30- and 4-fold increases over basal levels, respectively. The C566T substitution in mFSHR cDNA severely impaired the ability of the cognate receptor to evoke cAMP and IP3 responses after stimulation. However, subtle differences were noted between the two signaling pathways, as a residual 2.5-fold increase in cAMP production was observed after stimulation with high doses (103 mIU) of rhFSH, but no response in terms of IP3 production was detected (Fig. 9, middle and bottom panels).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Functional analysis demonstrated that the cloned receptor is biologically active when expressed in mammalian cells. HEK 293 cells expressing the recombinant mFSHR showed specific, high-affinity binding to the cognate ligand and displayed dose-dependent cAMP and IP3 responses to rhFSH stimulation. In addition, rhFSH elicited progesterone production in steroidogenic KK-1 cells transfected with the full-length mFSHR cDNA. Comparative analysis showed that the pattern of cAMP and IP3 production after rhFSH stimulation is similar between human, rat, and mouse receptors. In this sense, some controversy exists concerning species-specific differences in FSHR coupling to the inositol phosphate (IP) pathway. While the ability of rodent gonadotropin receptors to elicit IP3 production after stimulation has been demonstrated ([8, 46], and present results), it has been reported that unlike the human LHR, the cognate FSHR is able to induce only marginal IP responses [21]. These results are in contrast to those presented here (Fig. 4, bottom) and data obtained using a KK-1 cell line stably transfected with the human FSHR cDNA (unpublished results). Furthermore, evidence for the dual coupling of human FSHR to cAMP-PKA and PKC pathways has been presented recently [47]. Differences in cell lines and methods for IP measurement can offer explanations for this controversy.
A hallmark of gonadotropin receptor expression is the complex pattern of alternative splicing of the cognate messages. The exon-intron structure of gonadotropin receptor genes favors the formation of multiple mRNA species, even though only single forms of functional receptors have been so far identified in gonadotropin target cells. A form of alternative splicing, termed cassette-exon exclusion, has been reported for gonadotropin receptors from several species. Considering the similar intron phasing in the gonadotropin receptor genes, this mechanism allows exon deletion without frame shift, thus opening up the possibility of generating putative functional receptor isoforms. For the LHR, transcripts lacking one or several exons of the extracellular domain have been identified in rats, humans, and sheep [15, 48, 49]. Exon-skipping is also common for the FSHR in various species, and exon-lacking mRNA variants of human, sheep, horse, and donkey receptors have been reported [18, 50, 51]. In addition, splice variants, selectively lacking exons 2, 2 and 5, and 2, 5, and 6, were previously shown for the mFSHR [30]. The present study confirms such findings and extends the characterization of the splicing events of mFSHR: a new variant, lacking exons 5 and 6 of mFSHR coding sequence, was identified, and functional expression of all the cloned alternately spliced mFSHR transcripts was carried out. In this sense, despite extensive evidence for exon-lacking FSHR transcripts in numerous species, no data on the functional expression and physiological significance of such variants are available.
Two approaches were used to assess the functional capacity of the cloned mFSHR splice variants. First, the exon-skipped transcripts, composed of the entire cDNA sequence except for the missing exon(s), were individually expressed in HEK 293 and ovarian KK-1 cells, and receptor function was evaluated in terms of ligand binding and rhFSH-stimulated cAMP and progesterone responses. Radioligand-receptor assays demonstrated that no specific binding sites for [125I]iodo-rhFSH were present in transfected HEK 293 cells, either in the cell surface or trapped intracellularly, for any of the alternate transcripts under analysis. This was found despite successful transcription of the transfected expression plasmids (Fig. 5). In addition, specific binding to [125I]iodo-hCG was tested in the same paradigms in order to evaluate whether removal of the areas of the extracellular domain encoded by the missing exons results in loss of binding specificity. It is worth noting that the inhibitory binding determinants responsible for preventing hCG binding to the FSHR were mapped to parts of exons 5 and 6 [20]. However, no specific [125I]iodo-hCG binding was detected in transfected HEK 293 cells. Likewise, even high doses of rhFSH failed to induce any detectable cAMP and progesterone responses in transfected KK-1 cells, thus strongly suggesting that a potential functional role of the cloned receptor variants in targeting FSH actions is unlikely. Such a lack of function may arise from disruption of the binding domain or inappropriate folding/processing of the receptor variants. As intracellular trapping of the FSHR during synthesis has been reported to alter the ability of the nascent receptor for ligand binding [52], the mechanism(s) for the lack of function of the receptor variants cannot be outlined on the basis of the present results. Moreover, as we did not quantify expression of the full-length and alternately spliced receptor isoforms at the protein level, we cannot rule out the possibility of inadequate translation of the mRNAs encoding the different receptor variants.
In a second approach, the potential modulatory role of the cloned variants on mouse holoreceptor function was assessed in the context of an ovarian KK-1 cell line stably expressing the functional mFSHR. In this sense, inhibition and stimulation of gonadotropin receptor function by alternately spliced receptor variants have been reported previously [11, 12]. However, our cotransfection studies demonstrated that neither the number nor the binding affinity of mFSHR was altered by coexpression of the cloned receptor splice variants. Likewise, functional studies indicated that the pattern of cAMP response to increasing doses of rhFSH was not affected by transfection of the single variants or cotransfection of a cocktail of all the cloned transcripts. Taken together, despite the lack of quantification of receptor protein in cells expressing the different mFSHR variants, our results strongly indicate that such transcripts do not encode functional receptor isoforms. These variants may arise in the context of the complex alternative splicing events of FSHR gene expression and constitute nonfunctional messages. Similarly, a very recent study indicated the lack of function of two different splice variants of the rat FSHR gene [53]. Nevertheless, exon-skipping is a common feature in the processing of the FSHR gene in numerous species, and it is evident that more extensive analyses are needed to reveal the functional role, if any, of such a phenomenon.
In recent years, identification of mutations in gonadotropin receptor genes has not only helped to establish the molecular basis of some forms of infertility but has also extended our knowledge on basic aspects of the receptor structure and function. Both activating and inactivating mutations have been described in LHR and FSHR genes [23, 24]. Concerning the FSHR, the first inactivating mutation for the human gene was identified in connection with hereditary hypergonadotropic ovarian failure. It consisted of a C to T transition in position 566 of exon 7 of the FSHR coding sequence, predicting an Ala to Val substitution [25]. Interestingly, this mutation was located in a stretch of five amino acids with perfect conservation among species, including the mouse. This sequence is also present in the human LHR and TSHR and contains a conserved consensus site for N-linked glycosylation. In the present study, we evaluated the functional impact of the same C566T transition on mFSHR function in order to confirm the relevance of this area for receptor functionality. Expression analysis demonstrated for the mutant mFSHR a marked reduction of ligand binding, with unaltered affinity, as well as severely impaired cAMP and IP3 responses to rhFSH stimulation, i.e., a phenotype similar to that of the mutant human FSHR (A. Rannikko, I.T. Huhtaniemi, unpublished results). It is worth noting that a marginal 2.5-fold increase in cAMP was detected for the mutant mFSHR after stimulation with high doses of rhFSH, whereas no response in terms of IP3 was observed. Differences in assay sensitivities between cAMP and IP3 measurements likely explain the differential responses. However, on the basis of recent findings for the rat LHR [54], the possibility also exists that receptor signaling may be differentially affected by the mutation under analysis. Overall, our data indicate that the integrity of the mutated region is highly important for proper receptor function. In line with these findings, the introduction of an analogous Ala to Val substitution similarly disrupted the functionality of human LHR (P. Pakarinen, I.T. Huhtaniemi, unpublished results). In accordance, a second inactivating mutation was recently discovered in the same area of the human FSHR [24]. Since our results suggest that the impact of C566T mutation in the FSHR is similar in the mouse and human, the possibility arises of developing animal models for studies on the pathophysiology and therapeutic strategies of hereditary forms of ovarian failure.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Ilpo Huhtaniemi, Department of Physiology, University of Turku, Kiinamyllynkaty 10, 20520 Turku, Finland. FAX: 358 2 2502610; ilpo.huhtaniemi{at}utu.fi
3 Current address: Department of Physiology, University of Córdoba, Avda Menéndez Pidal s/n, 14004 Córdoba, Spain.
Accepted: February 1, 1999.
Received: September 29, 1998.
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