Molecular Human Reproduction, Vol. 8, No. 4, 311-317,
April 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
Functional characterization of the human FSH receptor with an inactivating Ala189Val mutation
1 Department of Physiology, University of Turku, FIN-20520, 2 Department of Urology and 3 Department of Medical Genetics, University of Helsinki, Finland and 4 INSERM EPI-120, Bâtiment Grégory Pincus, 94275 Le Kremlin Bicêtre, France
Abstract
An Ala189Val mutation of the human FSH receptor (FSHR) has been found to cause hypergonadotrophic ovarian failure with arrest of follicular maturation in women, and suppressed spermatogenesis in men. We have now characterized the molecular mechanisms of the receptor inactivation. Wild-type and mutant FSHR cDNAs were expressed in monkey kidney (COS-7) cells and murine granulosa tumour (KK-1) cells. Similar steady-state levels of FSHR mRNA were found in COS-7 and KK-1 cells transfected with both types of FSHR cDNA. Conspicuously, immunofluorescence and confocal microscopy studies revealed that whereas the wild-type receptor could be readily detected on the plasma membrane, most of the mutated protein was intracellularly sequestered. Ligand binding studies confirmed the greatly reduced cell surface expression of the mutant FSHR. A low level of mutated receptors were expressed at the cell surface, as shown by ligand binding and cAMP response. The capacity of these receptors to evoke another second messenger response, that of inositol trisphosphate (IP3), was almost totally lost. This finding may be related to the clinical picture of the patients, i.e. blockade of follicular maturation. There is a highly conserved stretch of five amino acids (Ala-Phe-Asn-Gly-Thr) in the region of the mutation in all glycoprotein hormone receptors. We therefore created the same Ala to Val transition in the human LHR and studied its functional consequences. Similar functional alterations, i.e. intracellular sequestration and attenuated signal transduction, were found, as with mutated FSHR. Hence, this particular mutation in the conserved extracellular region of glycoprotein hormone receptors induces a conformational change that suppresses cell membrane targeting of the mutated receptor, probably through altered intracellular folding.
FSH receptor/LH receptor/mutation/signal transduction
Introduction
We have previously identified an inactivating mutation in the human FSH receptor (FSHR) gene that causes hypergonadotrophic arrest of follicular maturation in women (Aittomäki et al., 1995
) and oligozoospermia of variable degrees in men (Tapanainen et al., 1997). The most conspicuous feature of the ovaries of patients with the FSHR mutation, in comparison with ovarian dysgenesis due to other causes, is the ample presence of follicles, usually in the primordial stage, but occasionally in more mature stages of development (Aittomäki et al., 1996). The phenotype of women with the Ala189Val mutation is more severe than that of women expressing partially inactivating forms of FSHR (Beau et al., 1998; Touraine et al., 1999; Dohertyet al., 2001). Although it is possible that some of the FSHR function could be left in the Ala189Val mutation, this must be minimal in light of the similarity of the phenotype with inactivating FSHß mutation in women and mouse models with disrupted FSHß and FSHR genes (Themmen and Huhtaniemi, 2000). Characterization of the inactivating FSHR mutations and corresponding animal models have changed the concept of FSH action. FSH no longer seems to be obligatory for male fertility, in contrast with female fertility where normal ovarian function is critically dependent on normal FSH action (Levallet et al., 1999; Themmen and Huhtaniemi, 2000). In the present study we have identified the level(s) at which the receptor function is affected and we show that the highly conserved residue which is mutated in the FSHR is also critical for cell membrane targeting of the LHR.
Materials and methods
Expression plasmids and mutagenesis
Construction of the wild-type (wt) and mutant (mut) human (h)FSHR expression plasmids is described elsewhere (Aittomäki et al., 1995). The full-length human LHR (hLHR) cDNA (a gift from Dr L.Dunkel, University of Helsinki, Finland) was inserted into the pSG5 expression vector (Stratagene, La Jolla, CA, USA). Mutagenesis of the LHR cDNA was carried out using the overlapping PCR method (Ausubel et al., 1994). The sequences of the mutation primers were 5'-GTCATGTATTCAATGGGACG-3' and 5'-CCCATTGAATACATGACTTTGTA CTTCTTC-3' (the mutated residue is shown in bold). The resulting mutated human LHR cDNA fragment was subcloned into the pSG5 expression vector using restriction enzymes Eco47III and BsgI. The constructs were verified by automatic sequencing (Perkin Elmer Inc., Foster City, CA, USA).
Transfections
Monkey kidney (COS-7) and murine granulosa tumour (KK-1) (Kananen et al., 1995) cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1:1) (Gibco BRL, Life Technologies, Glasgow, UK) supplemented with 10% fetal calf serum (Bioclear, Berks, UK) and 0.1 g/l gentamicin (Biological Industries, Bet-HaEmek, Israel). The COS-7 cells were transfected using the calcium phosphate precipitation method, as described elsewhere (Zhang et al., 1997). Briefly, cells were plated at a density of 0.5x106 per 9 cm diameter plate 1 day before transfection. A total of 20 µg of the expression plasmid (wt or mut) was used for transfection together with 3 µg of the ß-galactosidase expression plasmid, and the precipitates were left on cells overnight without glycerol shock. The cells were harvested and used for analyses 2–3 days after transfection. Transfection efficiencies in the transient transfections were monitored by ß-galactosidase expression, and the average coefficient of variation was 12%.
The KK-1 cells were stably transfected using the lipofection method (Paukku et al., 1997). A total of 6 µg of the expression plasmid (wt or mut) was co-transfected together with 0.6 µg of the neomycin resistance plasmid pPGKneobpA (Southern and Berg, 1982) in 9 cm diameter plates. Three days after transfection, the cells were split into selection medium containing 600 mg/l G-418 sulphate (Promega, Madison, WI, USA). After 3 weeks of selection, resistant colonies were picked, expanded and screened for FSHR mRNA expression. The stably transfected KK-1 cells were subsequently cultured in the presence of 400 mg/l G-418.
Isolation of RNA and Northern analysis
RNA was extracted using the single-step method (Chomzynski and Sacchi, 1987), and 30 µg of total RNA was size-fractioned in 1.2% agarose gel, transferred to a nylon membrane (Hybond-N; Amersham Pharmacia Biotech, International Plc, UK) and subjected to hybridization. The [32P]-labelled cRNA probe was synthesized using the Riboprobe synthesis II kit (Promega), [
-32P]-UTP (specific activity ~30 TBq/mmol; Amersham) and pGEM-4Z containing a HindIII fragment (bases 169–778) of the human FSHR cDNA as template. Prehybridization, hybridization and washings were carried out under stringent conditions, as described earlier (Rannikko et al., 1995). The filters were exposed to X-ray film (Kodak XAR-5; Eastman Kodak Co., Rochester, NY, USA) at room temperature overnight. Densitometric values of hybridization signal were normalized by the amount of 28S ribosomal RNA transferred per lane, as estimated by ethidium bromide staining. Clones with similar levels of expression were chosen for further studies (w1 and m1).
Immunofluorescence and confocal microscopy
The indirect immunofluorescence study was performed as previously described (Touraine et al., 1999) using the FSHR323 monoclonal antibody (Vannier et al., 1996) which recognizes an epitope localized in the extracellular domain (amino acids 172–358) of the FSHR. The size of the main receptor species found in the human ovary by the antibody is 87 kDa, and that of a minor species is 81 kDa. Detailed characterization of the antibody can be found elsewhere (Vannier et al., 1996).
Briefly, COS-7 cells transfected with wt or the Ala189Val-mutated FSHR were used in the intact state, or permeabilized with saponin before being incubated with the antibody. A cy3-labelled rabbit anti-mouse immunoglobulin G was used as the secondary antibody. The labelling was then analysed using a Zeiss microscope (Axiovert 135M) in conjunction with a confocal LSM 410 laser scanning unit (Carl Zeiss, Thornwood, NY, USA).
[125I]iodo-rhFSH and [125I]iodo-HCG binding experiments
Recombinant human FSH (rhFSH; Org 32489, Oss, The Netherlands) and highly purified human urinary HCG (CR-127, NIDDH, NIH, Bethesda, MD, USA) were radioiodinated with Na[125I]iodine (IMS 300; Amersham) using a solid-phase lactoperoxidase method (Karonen et al., 1975), to specific activities of 3800 and 35 000 cpm/ng respectively, as determined according to Catt et al. (Catt et al., 1976). The cells were first washed twice with cold phosphate-buffered saline (PBS) and scraped off into Dulbecco's PBS (D-PBS) containing 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MD, USA). Harvested cells were pelleted by centrifugation at 250 g. The pelleted cells were washed twice and reconstituted in D-PBS/BSA, and 100 µl aliquots of the cells were incubated overnight at room temperature in the presence of 100 000 cpm of [125I]iodo-HCG or [125I]iodo-rhFSH in the presence or absence of 50 IU of unlabelled HCG (Pregnyl; Organon, The Netherlands) or 1.5 IU of rhFSH (Org 32489) respectively, in a total volume of 250 µl. After incubation, the cells were washed with 4 ml of ice-cold D-PBS, centrifuged at 1500 g for 10 min, and the radioactivity in the cell pellets was counted in a 
-spectrometer. For Scatchard analysis, aliquots of intact cells were incubated at room temperature with increasing concentrations of [125I]iodo-HCG or [125I]iodo-rhFSH (up to 500 000 cpm/tube) as described above, and the binding data were converted to Scatchard plots.
cAMP, inositol trisphosphate (IP3) and progesterone assays
For cAMP measurements, the cells were plated at a density of 50 000 per well (24-well plates), 24 h before stimulations. The cells were stimulated for 3 h in a medium containing 0.2 mmol/l 3-isobutyl-1-methylxantine (MIX; Aldrich-Chemie, Steinheim, Germany) with increasing concentrations of rhFSH (Organon, 0–1000 IU/l) or HCG (CR-127, 0–400 µg/l). All stimulations were performed in quadruplicate. At the end of the incubation, the media were collected for measurement of extracellular cAMP using a standard radioimmunoassay method (Harper and Brooker, 1975).
For IP3 measurements, the cells were cultured in 6-well plates at a concentration of 5x105 cells per well in inositol-free DMEM/F12. After 24 h incubation, the cells were washed twice with HEPES-3 buffer (10 mmol/l HEPES, 58 mmol/l NaCl, 0.3 mmol/l NaH2PO4, 3.4 mmol/l sodium acetate, 5 mmol/l KCl, 0.6 mmol/l MgSO4, 1.5 mmol/l CaCl2, 2 mmol/l D-glucose and 0.1% fatty-acid-free BSA, pH 7.2) and stimulated with increasing concentrations of rhFSH (0–5000 IU/l) or HCG (0–5000 µg/l) for 20 min in the presence of LiCl. Accumulation of IP3 was terminated, and the substance extracted, by keeping the cells on ice with 10% ice-cold perchloric acid, followed immediately by neutralization with 1.5 mol/l KOH in 60 mmol/l HEPES, pH 7.2. Production of IP3 was determined by the Inositol-1,4,5-Triphosphate [3H]-Radioreceptor Assay Kit (NEN® Research Products Du Pont de Nemours & Co., Boston, MA, USA) according to the manufacturer's instructions. The sensitivity of the system was found to be ~1 pmol per assay tube.
For progesterone measurements, the cells were incubated for 48 h with FSH (0–1000 IU/l) and the progesterone concentration was measured using radioimmunoassay, as described previously (Vuorento et al., 1989).
Each experiment presented was repeated at least twice with similar results.
Results
Northern hybridization
The FSHR mRNA levels were measured in stably transfected KK-1 cells in order to rule out defective transcription or decreased mRNA stability of the mutated gene as a cause of the decreased receptor levels. Northern analysis revealed specific hybridization to an mRNA species of ~2.5 kb in size (Figure 1). The clones that showed similar hybridization signals were chosen for further analysis. Hence, the similar steady-state levels of the wt and mut FSHR mRNAs provide evidence that the C566T point mutation does not markedly affect either transcription or mRNA stability of the FSHR gene. This finding was corroborated by similar data on transiently transfected COS-7 cells (result not shown).
|
Immunofluorescence and confocal microscopy
Cell surface expression of the wt and mut receptors was analysed on COS-7 cells transiently transfected with cDNA of either receptor type. For this purpose, intact or permeabilized cells were incubated with FSHR323 monoclonal antibody, which recognizes the extracellular domain of the receptor (see Materials and methods). Confocal microscopic analysis of permeabilized cells (Figure 2a,c) showed a strong intracellular staining for both types of receptors. However, when the cells were not permeabilized (Figure 2b,d), cell surface labelling was only observed in cells expressing the wt receptor. This finding indicates defective cell surface targeting of the Ala189Val FSHR mutant. The similar levels of staining with both receptor forms provide further evidence that synthesis or stability of receptor protein is not compromised by the mutation.
Ligand binding experiments
We attempted to ascertain if similar amounts of the FSHR proteins, as observed by immunofluorescence, result in similar amounts of functional wt and mut hFSHR proteins, i.e. receptor molecules capable of ligand binding and signal transduction. In the original paper describing the FSHR mutation (Aittomäki et al., 1995), we observed dramatically decreased ligand binding of the mutated receptor. Scatchard analysis was performed to determine the affinities and number of binding sites of FSHR in stably transfected KK-1 cells. While the equilibrium dissociation constant (Kd) of the mutated receptor remained unaltered (wt hFSHR 780 pmol/l; mut hFSHR 820 pmol/l), the amount of mutated receptors detected at the cell surface was only 18 ± 4.8% of that detected in cells transfected with wt hFSHR cDNA (Figure 3A). Bmax was ~37 000 receptors per cell for wt FSHR and ~8000 receptors per cell for mut FSHR. Similar results were obtained using different stably transfected KK-1 clones (results not shown), thus suggesting that the changes observed were due to the mutation, and not to clonal variation.
|
The five amino acid sequence around the mutated Ala189 is identical in human FSH, LH and TSH receptors (189Ala-Phe-Asn-Gly-Thr193 in FSHR) (Libert et al., 1989
; Loosfelt et al., 1989). We therefore used targeted mutagenesis to create the same mutation (C566T transition in the FSHR gene) in the human LHR gene. Both wt and mut LHR expression plasmids were transiently expressed in COS-7 cells and studied for ligand binding properties of the recombinant LHR proteins. Scatchard analysis revealed the same phenomenon as with human FSHR, i.e. while the mutation did not change the affinity of the LHR (Kd for the wt hLHR = 300 pmol/l, for the mut hLHR = 310 pmol/l), it dramatically decreased the receptor number on the cell surface to only 4.5 ± 1.9% of that in cells transfected with the wt LHR (Figure 3B
). Bmax was ~27 000 receptors per cell for wt LHR and ~1000 receptors per cell for mut LHR. From this result, we can conclude that this region of the LHR is also critical in determining behaviour of the receptor protein, probably through a specific conformational structure.
Stimulation experiments
An important question is whether the mutation totally abolishes the ligand-induced signal transduction and downstream cellular responses, thus making the gonad totally resistant to FSH, even to very high doses. We therefore measured the cAMP, IP3 and steroidogenic responses to ligand stimulation with both gonadotrophin receptors and their homologous mutants in KK-1 cells. The results showed that while FSH stimulation led to ~27-fold stimulation of cAMP accumulation in cells transfected with wt hFSHR, only ~8-fold stimulation was observed in cells expressing mut hFSHR (Figure 4A). The EC50 value was 20 IU/l for wt FSHR and 40 IU/l for mut FSHR.
|
IP3 production in response to FSH showed a more dramatic difference between the two receptor forms. While a 6-fold stimulation of IP3 was observed in cells transfected with wt FSHR, only ~1.5-fold stimulation of IP3 accumulation was observed in those transfected with mut hFSHR (Figure 4B
). The EC50 value was 70 IU/l for wt FSHR and 500 IU/l for mut FSHR. As a downstream event in FSH action, progesterone production was measured. The result concurs with the cAMP responses, showing ~23-fold stimulation of progesterone production in the wt hFSHR transfected cells, but only ~7-fold stimulation in cells expressing mut hFSHR (Figure 4C). The EC50 value for wt FSHR was 10 IU/l and for mut FSHR, 20 IU/l.
We also studied whether the same mutation would be inhibitory to LHR signal transduction. Maximal cAMP stimulation in transiently transfected COS cells with wt LHR was ~11-fold, and 3-fold in cells expressing mut hLHR (Figure 5A), with EC50 values of 10 and 60 µg/l respectively. Stimulation with HCG led to ~5.5- and ~1.5-fold stimulation of IP3 accumulation in the wt and mut hLHR transfected cells respectively (Figure 5B). The EC50 value of this response for wt LHR was 50 µg/l, and 500 µg/l for mut LHR.
|
Discussion
With respect to the inactivating Ala189Val hFSHR mutation (Aittomäki et al., 1995), Aittomäki et al. performed a detailed study on clinical features of the hypergonadotrophic ovarian failure caused either by the inactivating FSHR mutation (termed FSH resistant ovary; FSHRO) or some other cause (termed ovarian dysgenesis) (Aittomäki et al., 1996). Since follicles arrested at early stages of maturation were only found in the FSHRO patients, it was suggested that some residual activity for the mutated FSHR may remain. Resolving this question is important, since treatment of infertility of the FSHRO patients could be possible by ovarian stimulation with massive doses of FSH. In fact, this is the case with another FSHR mutation recently described. Beau et al. found a patient compound heterozygous for two FSHR mutations: Ile160Thr with 10% residual activity (as measured by cAMP stimulation in transfected COS-7 cells) and Arg573Cys with 24% residual activity (Beau et al., 1998). Their patient presented with primary amenorrhoea, high FSH and follicles up to 5 mm in size. When she was treated with exogenous FSH, follicle growth and estradiol production could be enhanced, although not to normal levels.
Our results show that some function of the Ala189Val FSHR mutant may remain in vitro, when the mutated receptor is transfected into COS-7 or KK-1 cells. cAMP and progesterone production could be stimulated by FSH in cells expressing the mutant hFSHR. Taking into account the reduced receptor number on cells expressing the mutated receptor, the amount of cAMP produced per receptor on the cell surface was roughly unaltered. Production of the other second messenger, IP3, was more severely blunted, being barely detectable. In our stimulation studies, the concentration of FSH observed in the FSHRO patients (60 IU/l) (Aittomäki et al., 1996) would cause >5-fold stimulation in cAMP production, but have no effect on IP3 production. In healthy women, the serum FSH concentration ranges between 1–10 IU/l, which would cause ~2- to 10-fold stimulation of cAMP and progesterone in cells transfected with wt FSHR, and up to 3-fold stimulation of IP3 production. Therefore, the major effect of the FSHR mutation in FSHRO patients may lie in the lack of the FSH-stimulated IP3 response.
The loss of the IP3 response in KK-1 cells expressing mut FSHR, in the face of a partially conserved cAMP response, provides an example that mutations of gonadotrophin receptors may affect the second messenger systems to different extents. With respect to LHR, a recent finding on an activating somatic mutation in Leydig cell tumours showed activation of both the cAMP and IP3 pathways (Liu et al., 1999), whereas in male-limited gonadotrophin-independent precocious puberty, the usual phenotype with activating LHR mutations, only the cAMP pathway is activated (Shenker et al., 1993).
The above conclusions assume that the mutated receptor is expressed in the granulosa cells of the FSHR patients at the same level as in transfected cells, which may not be true. Since ovarian biopsy material for receptor measurements is not available from the patients, this question remains open. It is likely that the ovarian expression of the mutated receptor in vivo is even lower, since the endogenous FSHR promoter is much weaker than the powerful viral promoter used in the expression constructs of transfection studies (Sprengel et al., 1990; Minegishi et al., 1991; Huhtaniemi et al., 1992; Kelton et al., 1992; Levallet et al., 2000). The residual FSHR protein in the gonads of patients may therefore be much lower than that demonstrated by the present cell transfections, and the actual receptor inactivation may therefore be more complete. In accordance, our preliminary findings on FSHRO patients with the Ala189Val mutation show no response to massive doses of FSH (300–900 IU/day for 10 days; T.Vaskivuo, K.Aittomäki, I.Huhtaniemi and J.Tapanainen, unpublished observation). Moreover, the residual FSHR activity in these patients must be very low since their clinical picture is identical to that of animal models totally devoid of FSH action (Halpin et al., 1986; Ojeda and Urbanski, 1994; Kendall et al., 1995; Kumar et al., 1997; Dierich et al., 1998; Abel et al., 2000), and with three women reported to have inactivating mutations in the FSHß gene (Matthews et al., 1993; Layman et al., 1997; Matthews and Chatterjee, 1997). Furthermore, the clinical picture of men homozygous for the inactivating FSHR mutation is identical to that of FSHß knock-out mice, i.e. reduced testis size and variable reduction of spermatogenesis and fertility (Tapanainen et al., 1997). By contrast, in partial loss of function mutations of the FSHR, follicular maturation can proceed up to the small antral stages (Beau et al., 1998; Touraine et al., 1999).
The FSHR mutation studied here is in the amino terminal extracellular domain of the receptor, in an area with high homology between the three glycoprotein hormone receptors, as well as between the gonadotrophin receptors of different species (Loosfelt et al., 1989; Sprengel et al., 1990; Minegishi et al., 1991; Kelton et al., 1992; Yarney et al., 1993; Layman et al., 1997; Matthews and Chatterjee, 1997), thus allowing us to study the effect of the same mutation on the human LHR. Ligand binding studies showed that this mutation affects both gonadotrophin receptors in a similar fashion. It did not change the affinity of the human LHR for the ligand, but dramatically reduced its cell surface expression, as observed for FSHR. In-vitro stimulation tests showed that the mutation reduced HCG-induced cAMP and IP3 production, as was also observed for FSHR. RNA analysis of transiently and stably transfected cells with wt and mutant hFSHR constructs revealed that the mutation did not change the steady-state mRNA levels. Moreover, similar levels of immunoreactive FSHR protein were detected in immunofluorescence studies, suggesting that translation and/or degradation are not significantly affected by the mutation.
In conclusion, we have analysed further the signal transduction properties of the mut human FSHR causing FSHRO syndrome in women. Our results suggest that, whereas some residual activity for the mutated receptor may remain with the two signal transducers studied, cAMP and IP3, the latter seems to be more affected. We also extended our studies to the closely related human LHR and conclude that the alanine in the highly conserved five-amino acid stretch (Ala-Phe-Asn-Gly-Thr), encompassing the site of the Ala189Val FSHR mutation, may not be important for direct receptor–ligand interaction. However, it appears to be crucial for proper folding and trafficking of the receptor, and for proper signal transduction.
|
Acknowledgements
This study was supported by grants from The Academy of Finland, and The Sigrid Jusélius Foundation.
Notes
5 To whom correspondence should be addressed. E-mail: ilpo.huhtaniemi{at}utu.fi 
References
Abel, M.H., Wootton, A.N., Wilkins, V., Huhtaniemi, I., Knight, P. and Charlton, H.M. (2000) The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction.Endocrinology, 105, 633–641.
Aittomäki, K.J., Lucena, L.D., Pakarinen, P., Sistonen, P., Tapanainen, J., Gromoll, J., Kaskikari, R., Sankila, E., Lehväslaiho, H., Engel, A.R., Nieschlag, E., Huhtaniemi, I. and de la Chapelle, A. (1995) Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell, 82, 959–968.[Web of Science][Medline]
Aittomäki, K., Herva, R., Stenman, U., Juntunen, K., Ylöstalo, P., Hovatta, O. and de la Chapelle, A. (1996) Clinical features of primary ovarian failure caused by a point mutation in the follicle-stimulating hormone receptor gene. J. Clin. Endocrinol. Metab., 81, 3722–3726.[Abstract]
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (eds) (1994) Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York, NY, USA, pp. 857–859.
Beau, I., Touraine, P., Meduri, G., Gougeon, A., Desroches, A., Matuchansky, C., Milgrom, E., Kuttenn, F. and Misrahi, M. (1998) A novel phenotype related to partial loss of function mutations of the follicle-stimulating hormone receptor. J. Clin. Invest., 102, 1352–1359.[Web of Science][Medline]
Catt, K.J., Ketelslegers, M.L. and Dufau, M.L. (1976) Receptors for gonadotropic hormones. In Blecher, M. (ed.) Methods in Receptor Research, 1st edn. Marcel Dekker, New York, pp. 175–250.
Chomzynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156–159.[Web of Science][Medline]
Dierich, A., Sairam, M.R., Monaco, L., Fimia, G.M., Gansmuller, A., LeMeur, M. and Sassone-Corsi, P. (1998) Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc. Natl Acad. Sci. USA, 95, 13612–13617.
Doherty, E., Pakarinen, P., Tiitinen, A., Kiilavuori, A., Huhtaniemi, I., Forrest, S. and Aittomäki, K. (2002) A novel mutation in the follicle-stimulating hormone receptor inhibits signal transduction and results in primary ovarian failure. J. Clin. Endocr. Metab., in press.
Halpin, D.M.G., Jones, A., Fink, G. and Charlton, H.M. (1986) Postnatal ovarian follicle development in hypogonadal (hpg) and normal mice and associated changes in the hypothalamic–pituitary–ovarian axis. J. Reprod. Fertil., 77, 287–296.
Harper, J. and Brooker, G. (1975) Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'-O-acetylation by acetic anhydride in aqueous solution. J. Cycl. Nucleotide Res., 1, 207–218.[Web of Science][Medline]
Huhtaniemi, I.T., Eskola, V., Pakarinen, P., Matikainen, T. and Sprengel, R. (1992) The murine LH and FSH receptor genes: mapping of transcription initiation sites and putative promoter sequences. Mol. Cell. Endocrinol., 88, 55–66.[Web of Science][Medline]
Kananen, K., Markkula, M., Rainio, E., Su, J., Hsueh, A.J.W. and Huhtaniemi, I.T. (1995) Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin -subunit promoter/Simian virus T-antigen fusion gene: characterization of ovarian tumours and establishment of gonadotropin-responsive granulosa cell lines. Mol. Endocrinol., 9, 616–627.
Karonen, S., Mörsky, P., Sirén, M. and Seuderling, U. (1975) An enzymatic solid-phase method for trace iodination of proteins and peptides with 125iodine. Anal. Biochem., 67, 1–10.[Web of Science][Medline]
Kelton, C.A., Cheng, S.V.Y., Nugent, N.P., Schweickhardt, R.L., Rosenthal, J.L., Overton, S.A., Wands, G.D., Kuzeja, J.B., Luchette, C.A. and Chappel, S.C. (1992) The cloning of the human follicle stimulating hormone receptor and its expression in COS-7, CHO, and Y-1 cells. Mol. Cell. Endocrinol., 89, 141–151.[Web of Science][Medline]
Kendall, S.K., Samuelson, L.C., Saunders, T.L., Wood, R.I. and Camper, S.A. (1995) Targeted disruption of the pituitary glycoprotein hormone a-subunit produces hypogonadal and hypothyroid mice. Genes Dev., 9, 2007–2019.
Kumar, T.R., Wang, Y., Lu, N. and Matzuk, M.M. (1997) Follicle stimulating hormone is required for ovarian follicle maturation but not for spermatogenesis. Nature Genet., 15, 201–204.[Web of Science][Medline]
Layman, L.C., Lee, E.J., Peak, D.B., Namnoum, A.B., Vu, K.V., van Lingen, B.L., Gray, M.R., McDonough, P.G., Reindollar, R.H. and Jameson, J.L. (1997) Delayed puberty and hypogonadism caused by mutations in the follicle-stimulating hormone beta-subunit gene. N. Engl. J. Med., 37, 607–611.
Levallet, J., Pakarinen, P. and Huhtaniemi, I.T. (1999) Follicle-stimulating hormone ligand and receptor mutations, and gonadal dysfunction. Arch. Med. Res., 30, 486–494.[Web of Science][Medline]
Levallet, J., Koskimies, P., Rahman, N. and Huhtaniemi, I. (2000) The promoter of murine follicle-stimulating hormone receptor; functional characterization and regulation by transcription factor SF-1. Mol. Endocrinol., 15, 80–92.
Libert, F., Lefort, A., Gerard, C., Parmentier, M., Perret, J., Ludgate, M., Dumont, J. and Vassart, G. (1989) Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem. Biophys. Res. Commun., 165, 1250–1255.[Web of Science][Medline]
Liu, G., Duranteau, L., Carel, J.C., Monroe, J., Doyle, D.A. and Shenker, A. (1999) Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N. Engl. J. Med., 341, 1731–1734.
Loosfelt, H., Misrahi, M., Atger, M., Salesse, R., Thi, M.T.V., Jolivet, A., Guiochon-Mantel, A., Sar, S., Jallal, B., Garnier, J. and Milgrom, E. (1989) Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domainScience, 245, 525–528.
Matthews, C. and Chatterjee, V.K. (1997) Isolated deficiency of follicle-stimulating hormone re-revisited. N. Engl. J. Med., 337, 642.
Matthews, C.H., Gorgato, S., Beck-Peccoz, P., Adams, M., Tone, Y., Gambino, G., Casagrande, S., Tedeschini, G., Benedetti, A. and Chatterjee, V.K. (1993) Primary amenorrhea and infertility due to a mutation in the beta-subunit of follicle-stimulating hormone. Nature Genet., 5, 83–86.[Web of Science][Medline]
Minegishi, T., Nakamura, K., Takakura, Y., Ibuki, Y. and Igarashi, M. (1991) Cloning and sequencing of fuman FSH receptor cDNA. Biochem. Biophys. Res. Comm., 3, 1125–1130.
Ojeda, S.R. and Urbanski, H.F. (1994) Puberty in the rat. In Knobil, E. and Neil, J.D. (eds) The Physiology of Reproduction, 2nd edn. Raven Press, New York, pp. 363–409.
Paukku, T., Lauraeus, S., Huhtaniemi, I. and Kinnunen, P.K.J. (1997) Novel cationic liposomes for DNA-transfection with high efficiency and low toxicity. Chem. Phys. Lipids, 87, 23–29.[Web of Science][Medline]
Rannikko, A.S., Zhang, F. and Huhtaniemi, I.T. (1995) Ontogeny of follicle-stimulating hormone receptor gene expression in the rat testis and ovary. Mol. Cell. Endocrinol., 107, 199–208.[Web of Science][Medline]
Shenker, A., Laue, L., Kosugi, S., Merendino, J.J., Minegishi, T. and Cutler, G.B. (1993) A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious pubertyNature, 365, 652–654.[Medline]
Southern, P.J. and Berg, P. (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. App. Gen., 1, 327–341.
Sprengel, R., Braun, T., Nikolics, K., Segaloff, D.L. and Seeburg, P.H. (1990) The testicular receptor for follicle-stimulating hormone: structure and functional expression of cloned cDNA. Mol. Endocrinol., 4, 525–530.
Tapanainen, J.S., Aittomäki, K., Min, J., Vaskivuo, T. and Huhtaniemi, I.T. (1997) Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nature Genet., 15, 205–206.[Web of Science][Medline]
Themmen, A.P.N. and Huhtaniemi, I.T. (2000) Mutations of gonadotropins and gonadotropin receptors: Elucidating the physiology and pathophysiology of pituitary–gonadal function. Endocr. Rev., 21, 551–583.
Touraine, P., Beau, I., Gougeon, S., Meduri, G., Desroches, A., Pichard, C., Detoeuf, M., Paniel, B., Prieur, M., Zorn, J.R., Milgrom, E., Kuttenn, F. and Misrahi, M. (1999) New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype. Mol. Endocrinol., 13, 1844–1854.
Vannier, B., Loosfelt, H., Meduri, G., Pichon, C. and Milgrom, E. (1996) Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor. Biochemistry, 35, 1358–1366.[Medline]
Vuorento, T., Lahti, A., Hovatta, O. and Huhtaniemi, I. (1989) Daily measurements of salivary progesterone reveal high rate of anovulation in healthy students. Scand. J. Clin. Lab. Invest., 49, 395–401.[Web of Science][Medline]
Yarney, T.A., Sairam, M.R., Khan, H., Ravindranath, N., Payne, S. and Seidah, N.G. (1993) Molecular cloning and expression of the ovine testicular follicle stimulating hormone receptor. Mol. Cell. Endocrinol., 93, 219–226.[Web of Science][Medline]
Zhang, F., Rannikko, A.S., Manna, P.R., Fraser, H.M. and Huhtaniemi, I.T. (1997) Cloning and functional expression of the luteinizing hormone receptor cDNA from the marmoset monkey testis; absence of sequences encoding exon 10 in other species. Endocrinology, 138, 2481–2490.
Submitted on April 26, 2001; resubmitted on October 19, 2001; accepted on January 11, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Gerasimova, M. N. Thanasoula, D. Zattas, E. Seli, D. Sakkas, and M. D. Lalioti Identification and in Vitro Characterization of Follicle Stimulating Hormone (FSH) Receptor Variants Associated with Abnormal Ovarian Response to FSH J. Clin. Endocrinol. Metab., February 1, 2010; 95(2): 529 - 536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Wang, L. Li, F. Ni, J. Song, J. Wang, Y. Mu, X. Ma, and Y. Cao Mutational analysis of SAL-Like 4 (SALL4) in Han Chinese women with premature ovarian failure Mol. Hum. Reprod., September 1, 2009; 15(9): 557 - 562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Allan, P. Lim, M. Robson, J. Spaliviero, and D. J. Handelsman Transgenic mutant D567G but not wild-type human FSH receptor overexpression provides FSH-independent and promiscuous glycoprotein hormone Sertoli cell signaling Am J Physiol Endocrinol Metab, May 1, 2009; 296(5): E1022 - E1028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Agrawal and R. R. Dighe Critical Involvement of the Hinge Region of the Follicle-stimulating Hormone Receptor in the Activation of the Receptor J. Biol. Chem., January 30, 2009; 284(5): 2636 - 2647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ghadami, S.A. Salama, N. Khatoon, R. Chilvers, M. Nagamani, P.J. Chedrese, and A. Al-Hendy Toward gene therapy of primary ovarian failure: adenovirus expressing human FSH receptor corrects the Finnish C566T mutation Mol. Hum. Reprod., January 1, 2008; 14(1): 9 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. V. Bogatcheva, A. Ferlin, S. Feng, A. Truong, L. Gianesello, C. Foresta, and A. I. Agoulnik T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane Am J Physiol Endocrinol Metab, January 1, 2007; 292(1): E138 - E144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P N Themmen An update of the pathophysiology of human gonadotrophin subunit and receptor gene mutations and polymorphisms Reproduction, September 1, 2005; 130(3): 263 - 274. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Meduri, P. Touraine, I. Beau, O. Lahuna, A. Desroches, M. C. Vacher-Lavenu, F. Kuttenn, and M. Misrahi Delayed Puberty and Primary Amenorrhea Associated with a Novel Mutation of the Human Follicle-Stimulating Hormone Receptor: Clinical, Histological, and Molecular Studies J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3491 - 3498. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Allen, J. C. Achermann, P. Pakarinen, T. J. Kotlar, I. T. Huhtaniemi, J. L. Jameson, T. D. Cheetham, and S. G. Ball A novel loss of function mutation in exon 10 of the FSH receptor gene causing hypergonadotrophic hypogonadism: clinical and molecular characteristics Hum. Reprod., February 1, 2003; 18(2): 251 - 256. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||