Human Follicle-stimulating Hormone Receptor Research Articles

Human Follicle Stimulating Hormone Receptor Lipid Raft Residency is Hormone and Caveolin Dependent

Human follicle stimulating hormone (hFSH) is a gonadotropin responsible for the maturation of ovarian follicles in women and production of sperm in men. The receptor (hFSHR) is a G protein‐coupled receptor (GPCR); a class of receptors found embedded in the cell membrane to receive the hormone resulting in initiation of a complex downstream signaling cascade. Through previous research in the Cohen lab, hFSHR has been shown to be located within compartments of the cell membrane known as lipid rafts. Lipid rafts are microdomains of the membrane containing a higher concentration of cholesterol, glycosphingolipids, and a protein known as caveolin. Current research is focused on evaluating the mechanisms and effects of hFSHR lipid raft residency. Using discontinuous sucrose gradients we have shown that hFSH stimulation of an HEK293 cell line stably transfected with hFSHR cDNA results in alteration of hFSHR lipid raft residency in a time dependent manner.To better understand the mechanism and effect of hFSHR lipid raft residency the same cells were treated with a peptide mimetic of transmembrane domain 4 of hFSHR. This peptide includes a sequence consistent with a motif shown in other proteins to interact with caveolin (ϕXϕXXXXϕXXϕ where ϕ is an aromatic amino acid). The putative caveolin interaction motif of hFSHR was synthesized along with the HIV‐TAT leader sequence to allow passage through the membrane. A second peptide where the aromatic amino acids were replaced with leucine was also used. Cells treated with the wild type peptide showed elevated basal signaling in the absence of hFSH, an effect not observed with the mutant peptide.Together, these results suggest that hFSHR residency in lipid rafts is dependent on hormone treatment and interaction with caveolin. Through studying hFSHR lipid raft residency and signaling we hope to identify ways to control the receptor for improved methods for contraception and treatment of infertility.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A004: Developing ovarian cancer xenograft in mouse model to see FSHR-mediated anticancer effect of Moringa oleifera root extract

Abstract Introduction: Sex hormones play an important role in ovarian cancer as novel redirected T-cell-based immunotherapy targeting human follicle-stimulating hormone receptor (FSHR)is tried. Moringa olifera root extract has shown efficacy in cancer cell as well as in hormonal milieu of ovary and reproductive organ. Hence, the aim of the present study was to study the anticancer effect of Moringa oleifera on human OAW 42 & MCF-7 cell line through its putative FSHR antagonistic role. Methods: The colorimetric MTT metabolic activity assay was used to see cell viability. Western blot analysis was performed to check its effect in apoptosis. Clonogenic assay was carried out to see the effect of Moringa root extract on cancer cell proliferation. Cell cycle analysis was done based on incorporation of Propidium Iodide into DNA (BD Cycletest™ Plus DNA Kit). Analyses of the samples were carried out flow cytometrically using BD LSRFortessaTM SORP (San Jose, CA) cell analyzer and BD FACSDiva v8.0.1 software.On day 35, mice were sacrificed and histopathologic analysis of the ovaries was carried out following standard protocol followed by IHC for detection of FSHR. Result: The dose-dependent analysis on viability of OAW-42 and MCF-7 cells by MTT assay showed 50% lethal toxicity (IC-50) of MRE (Moringa root extract) to be 250µg/ml and 200µg/ml, respectively, with synergestic effect of Moringa extract with both cisplatin and paclitaxol in OAW-42 cell line. Moringa extract at its IC-50 value showed visible effect in case of cleaved caspase-9. In clonogenic assay approximately 40% reduction of colony formation was observed. Flow cytometric data showed that after 24 hours and 48 hours of treatment with MOL root extract at its IC-50 dose, DNA content progressively increased in the sub G0 phase. However, approximately 3% upregulation and 11.4% downgulation of PI intensity at G0/G1 phase was also observed. Profuse expression of FSHR and its downstream target cMyc in the stroma of carcinoma control mice compared to control or Moringa-treated mice. Conclusion: We can conclude that MRE is a promising candidate for treatment of ovarian cancer by an FSHR-mediated pathway either alone or in combination with traditional chemotherapeutic drugs like cisplatin and paclitaxol at a sublethal dose of their toxicity. Citation Format: Chinmay Kumar Basu. Developing ovarian cancer xenograft in mouse model to see FSHR-mediated anticancer effect of Moringa oleifera root extract [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A004.

The Impact of Genetic Variation and Gene Expression Level of The Follicle-Stimulating Hormone Receptor on Ovarian Reserve.

Objective Ovarian reserve is defined as the capacity of the ovary to provide fertile oocytes. Diminished ovarian reserve (DOR) is a disorder in which ovaries are prone to go through early menopause. Where this loss of function occurs before the age of 40, it results in the premature ovarian failure (POF) disease. Throughout folliculogenesis, the follicle-stimulating hormone receptor (FSHR) starts a signaling cascade in the granulosa cells where its inactivation leads to the arrest of follicle maturation and therefore adversely affects ovarian reserve. The aim of this study was to investigate the association of genetic variation (polymorphisms and inactivating mutations) of FSHR with POF and DOR. Materials and Methods This case-control study comprised 84 POF, 52 DOR and 80 fertile Iranian women. To determine the presence of the 566C>T mutation and the -29G>A polymorphism in FSHR, PCR-RFLP method was used. SSCP-sequencing was used to identify any allelic variants in exon 10. The expression of human FSHR at the transcript level was also compared between DOR and fertile controls by real time-polymerase chain reaction (PCR). Results The 566C>T polymorphism was normal in all the cases. All genotypes of -29G>A and 919G>A (exon 10) polymorphisms were observed. Statistically significant differences were seen in the genotypic distribution of both polymorphisms when comparing the control group with the DOR patient group. A decrease was observed in FSHR expression of DOR patients compared with the control group but was not significant. ConclusionWe conclude that the -29G>A and 919G>A polymorphisms in FSHR may be associated with DOR. Although these polymorphisms had significant differences at the genic level, no significant variation was found at the transcript level.

Comparative pharmacology of a new recombinant FSH expressed by a human cell line.

Recombinant FSH proteins are important therapeutic agents for the treatment of infertility, including follitropin alfa expressed in Chinese Hamster Ovary (CHO) cells and, more recently, follitropin delta expressed in the human cell line PER.C6. These recombinant FSH proteins have distinct glycosylation, and have distinct pharmacokinetic and pharmacodynamic profiles in women. Comparative experiments demonstrated that follitropin delta and follitropin alfa displayed the same in vitro potency at the human FSH receptor, but varied in their pharmacokinetics in mouse and rat. While follitropin delta clearance from serum depended in part on the hepatic asialoglycoprotein receptor (ASGPR), follitropin alfa clearance was unaffected by ASGPR inhibition in rat or genetic ablation in mice. The distinct properties of follitropin delta and follitropin alfa are likely to contribute to the differing pharmacokinetic and pharmacodynamic profiles observed in women and to influence their efficacy in therapeutic protocols for the treatment of infertility.

Mutation of Follicle Ftimulating Hormone Receptor Putative Caveolin Binding Motif Results in Altered Signaling

The human follicle stimulating hormone receptor (hFSHR) is a glycoprotein hormone receptor belonging to the g protein‐coupled receptor family. It is important in both male and female reproductive processes; defects in hFSHR can lead to infertility, delayed puberty, reduced muscle bulk, and osteoporosis. Work in other labs has shown that GPCRs can be localized to microdomains located within the cell membrane called lipid rafts. These regions are highly resistant to detergents because of the high concentration of sphingolipids and cholesterol. Also within these domains, an intracellular protein, caveolin, is present. Our lab has shown that hFSHR also localizes to lipid raft domains. We hypothesize that interaction with caveolin is important in the raft residency of hFSHR through a putative caveolin binding motif (CBM). The interaction between caveolin and hFSHR in these microdomains may regulate intracellular pathways activated by the receptor.To test our hypothesis, we have constructed CBM mutants and are creating stable cell lines expressing the mutant receptors to study the effects of mutating the key phenylalanine residues of the CBM. Interestingly, wild type FSHR presents as a doublet on a western blot while the mutants only have a single band. We theorize that one of these bands may be a glycosylation variant however further investigation is required. The mutated receptors qualitatively show normal p44‐MAPK signaling in response to FSH, indicating that they are reaching the cell surface and are capable of binding hormone and coupling to signaling partners. We have observed a subtle difference in downstream PKA targets between the mutant and wild type hFSHR, suggesting that there might be a difference in the ability to activate downstream targets. Studying the interaction between hFSHR and caveolin has the potential to develop new contraceptive antagonists and alternative agonists to regulate fertility.

Follicle-Stimulating Hormone Receptor as a Target in the Redirected T-cell Therapy for Cancer.

Adoptive transfer of T cells engineered to express chimeric immunoreceptors is an effective strategy to treat hematologic cancers; however, the use of this type of therapy for solid cancers, such as ovarian cancer, remains challenging because a safe and effective immunotherapeutic target has not yet been identified. Here, we constructed and evaluated a novel redirected T-cell-based immunotherapy targeting human follicle-stimulating hormone receptor (FSHR), a highly conserved molecule in vertebrate animals with expression limited to gonadal tissues, ovarian cancer, and cancer-associated vasculature. Receptor ligand-based anti-FSHR immunoreceptors were constructed that contained small binding fragments from the ligand for FSHR, FSH, fused to T-cell transmembrane and T-cell signaling domains. Human T cells transduced to express anti-FSHR immunoreceptors were specifically immunoreactive against FSHR-expressing human and mouse ovarian cancer cell lines in an MHC-nonrestricted manner and mediated effective lysis of FHSR-expressing tumor cells, but not FSHR-deficient targets, in vitro. Similarly, the outgrowth of human ovarian cancer xenografts in immunodeficient mice was significantly inhibited by the adoptive transfer of FSHR-redirected T cells. Our experimental observations show that FSHR is a promising immunotherapeutic target for ovarian cancer and support further exploration of FSHR-targeted immune therapy approaches for patients with cancer.

206. Follicle-Stimulating Hormone Receptor as a Target in the Redirected T-Cell Therapy for Cancer

Adoptive transfer of T-cells engineered to express chimeric immune receptors is an effective strategy to treat hematological cancers, however, such therapy for solid cancers such as ovarian cancer remains challenging because a safe and effective immunotherapeutic target has not yet been identified. Here, we constructed and evaluated a novel redirected T-cell-based immunotherapy targeting human follicle-stimulating hormone receptor (FSHR), a highly conserved molecule in vertebrate animals with expression limited to gonadal tissues, ovarian cancer and cancer-associated vasculature. Receptor ligand-based anti-FSHR immune receptors were constructed that contained small binding fragments from the ligand for FSHR, follicle stimulating hormone (FSH), fused to T-cell transmembrane and T-cell signaling domains. Human T-cells transduced to express anti-FSHR immune receptors were specifically immunoreactive against FSHR-expressing human and mouse ovarian cancer cell lines in a MHC-non-restricted manner, and produced more than one Th1 type of proinflammatory cytokine. This observation is of special interest, as it provides the opportunity to address potential, but unpredicted, toxicity issues associated with targeting of FSHR in preclinical in vivo studies. Moreover, anti-FSHR-IR T-cells specifically upregulated surface expression of the activation marker CD69 in the presence of FSHR positive targets, but not FSHR-deficient the 293T cell line. Additionally, specific activation of anti-FSHR-IR T-cells by FSHR expressed on tumor cell surface led to efficient tumor cell killing in vitro. Furthermore, to assess the anti-tumor effect of anti-FSHR T-cells, we established human OvCa xenografts in NSG mice by subcutaneous flank injection of CaOV3 cancer cell line followed by two intravenous redirected T-cell administrations. As shown by caliper measurements, FSHR-redirected T-cells elicited anti-tumor potential with suppressed outgrowth of established tumor. Tumor growth was statistically reduced in comparison to control, GFP engineered T-cell treated group or PBS treated animals. These data suggest that systemic delivery of anti-FSHR T-cells to mice bearing FSHR-expressing tumors may result in tumor trafficking, activation, and lysis at location of tumor.In summary, we have successfully exploited for the first time a novel therapeutic antigen, the FSHR protein, expressed on tumor cell surface, to deliver T-cell based immune-therapy of cancer. Given its highly restricted expression in cancer, cancer-associated vessels and gonadal tissues, the rationale for FSHR targeting is strong and supports further exploration of FSHR-targeted immune therapy approaches for cancer patients.

Single-molecule analyses of fully functional fluorescent protein-tagged follitropin receptor reveal homodimerization and specific heterodimerization with lutropin receptor.

We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Ectopic expression of follicle-stimulating hormone receptors in thyroid tumors.

IntroductionIn normal conditions follicle-stimulating hormone receptors (FSHR) are expressed in the ovary and the testis. They can also be expressed in gonadal tumors. However, recently we have found FSHR immunostaining in pituitary adenomas, adrenal tumors and neuroendocrine tumors (carcinoids). The aim of this study was to determine whether the same occurs in thyroid tumors.Material and methodsThirty-six samples of surgically excised thyroids were examined. Follicle-stimulating hormone receptors immunostaining was performed on paraffin sections using the rabbit anti-human FSHR polyclonal antibody raised against a 1-190 amino acid sequence from the human FSHR (sc-13935, Santa Cruz).ResultsNormal thyroid follicles do not show immunopositivity for FSHR. The same concerns the majority of benign lesions, diagnosed as hyperplasia nodularis or thyroid adenomas. However, positive FSHR immunostaining in some follicles was observed. In all but one thyroid cancer (15 papillary, 10 follicular cancers and one case of anaplastic thyroid cancer) 10–100% of tumor cells exhibit positive FSHR immunostaining. In about 40% of samples FSHR immunoreactivity can be observed also in the endothelia of intrathyroidal blood vessels. This immunopositivity was more frequent in the samples of thyroid cancers (13/27) than in benign lesions (2/9).ConclusionsEctopic positive FSHR immunostaining is also present in thyroid cancers, and, to a lesser degree, in benign lesions but not in the normal thyroid epithelium.

Abstract 7: Assessment of FSH receptor expression in the major molecular subtypes of breast cancer

Abstract Recent data from our laboratory show that the follicle-stimulating hormone receptor (FSHR) is expressed on the endothelial surface of tumor peripheral blood vessels in different types of tumors including breast cancer. The main goal of the present study was to determine differences in the endothelial FSHR expression level among the breast cancer types, and within the same tumor type, among tumor stages. We have also compared the FSHR expression with the receptor status of three most important markers for breast cancer diagnosis and therapy (estrogen receptor, progesterone receptor, and HER-2/neu). By using a highly specific monoclonal antibody raised against the human FSHR, we determined the density of FSHR-positive vessels in a band with a width of 10 mm (centered on the demarcation line between the tumor and the normal breast tissue) on the tissue sections by standard peroxidase-immunohistochemistry and immunofluorescent confocal microscopy. We examined 35 patients of Luminal A, 13 patients of Luminal B, 19 patients of HER-2 positive, and 16 patients of Triple negative/basal breast cancer. All four groups expressed almost similar densities of FSHR stained blood vessels with 36.7 ± 4.0/mm2 in Luminal A, 31.4 ± 5.2/mm2 in Luminal B, 39.6 ± 6.0/mm2 in HER-2 positive tumors, and 38.4 ± 8.0/mm2 in triple negative/basal group. A positive correlation has been observed between the percentage of FSHR-positive vessels and total blood vessels in the tumor periphery (r = 0.551; p = 7e-08). We have also noticed a tendency (not reaching statistical significance) towards an overall decrease in the extent of the vascular bed in elder patients as compared to younger patients. No correlation was found between the percentage of FSHR-positive vessels and the expression of receptors for estrogen, progesterone, and HER-2/neu. In conclusion, our data show that: (i) FSHR is robustly expressed in breast cancer, and (ii) the main types of breast cancers and stages do not differ significantly in terms of vascular FSHR expression. These facts strongly increase FSHR potential relevance as a clinical target for breast cancer imaging and for therapy, especially for triple negative/basal breast cancers, a group with only chemotherapy options. Citation Format: François Planeix, Mohammad-Ahsan Siraj, François-Clément Bidard, Jean-Yves Pierga, Xavier Sastre, Martine Antoine, Nicolae Ghinea. Assessment of FSH receptor expression in the major molecular subtypes of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2014-7

Human follicle stimulating hormone receptor signaling is membrane cholesterol and internalization dependent (609.16)

Human follicle stimulating hormone (hFSH) is a dimeric glycoprotein secreted by the gonadotropic cells of the anterior pituitary that is essential for reproduction. Its receptor, (hFSHR), is a G protein‐coupled receptor expressed on the surface of granulosa cells in females and Sertoli cells in males. FSH stimulates the maturation of ovarian follicles in females and triggers sperm production in males. Based on previous research in our lab, we proposed that hFSHR resides in lipid rafts and/or caveolae on the cell membrane and that its signaling is cholesterol and internalization dependent. To test this hypothesis, we treated HEK‐293 cells stably transfected with human FSHR cDNA with the cholesterol depleting compound methyl‐beta‐cyclodextrin (MBCD) or the anti‐fungal compound Nystatin which alters the selective permeability of the cell membrane by sequestering cholesterol. Western blotting was used to measure the phosphorylation of map kinases p38 and p42/44 (ERK 1/2). MBCD did not alter FSH dependent activation of p44/42 or p38, but a decrease in activation of both signaling components was observed when the cells were treated with Nystatin. Both treatments decreased cAMP dependent activation of the cAMP response element‐binding protein. These results suggest that FSHR signaling is dependent not on cholesterol alone, but also on internalization, which is blocked by Nystatin. This adds to an increasing body of evidence for multivalent hFSHR signaling with the possibility of selective modulation of the pathways.

Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH

Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35-37, 38-40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats--a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine.

Follicle-stimulating hormone receptor (FSHR), which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein (FSHR-57aa) as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a(+)-FSHR-57aa plasmid was constructed and expressed in Escherichia coli strain BL21 Star™ (DE3) and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 (16 weeks) after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Correction: In Silico Study on Binding Specificity of Gonadotropins and Their Receptors: Design of a Novel and Selective Peptidomimetic for Human Follicle Stimulating Hormone Receptor.

[This corrects the article DOI: 10.1371/journal.pone.0064475.].

Expression and purification of the extracellular domain of the human follicle‐stimulating hormone receptor using Escherichia coli

Although much is known about the structure and biological functions of follicle-stimulating hormone (FSH) receptor (FSHR), the interaction of FSHR and FSH has been challenging to characterize due to the limited quantity of active FSHR protein produced by simple methods. The goal of this study was to express and purify the extracellular domain (ECD) of human FSHR (hFSHR). Total RNA was isolated from normal human ovary tissue. cDNA for hFSHR ECD were amplified and subsequently ligated into the pET32a(+) vector. The plasmid vector construct was confirmed by polymerase chain reaction and sequencing. Expression in Escherichia coli Rosetta (DE3) pLysS strain was induced by isopropyl-thio-β-D-thiogalactoside, and the recombinant products were purified by immuno-affinity chromatography using an Ni-NTA and High-Q column. The recombinant protein was confirmed by western blotting. Following induction, E. coli expressed a recombinant protein of approximately 65 kDa in size, whereas the non-induced E. coli did not express the recombinant protein. The recombinant fragments purified using a High-Q column demonstrated a single band and an abundant yield. The recombinant protein was soluble and specifically recognized by an antibody for hFSHR. Additionally, four mutation sites were detected that resulted in amino acid shifts at position 112 Asn/Thr, 197 Glu/Ala, 198 Leu/Val and 307 Ala/Thr. The recombinant hFSHR ECD protein was expressed and purified. This method could be easily scaled for increased production and may facilitate additional applications utilizing FSHR in assisted reproductive technology, a contraceptive FSH vaccine and FSHR-targeted therapeutic agents used to treat ovarian cancer.

Identification of two novel, alternatively spliced mRNA transcripts of the human follicle‐stimulating hormone receptor

Glycoprotein hormone receptors contain large extracellular domains encoded by multiple exons that can be alternatively spliced. Using human ovarian surface epithelium, we cloned two new splice variants of the human follicle-stimulating hormone receptor (FSH-R) gene, hFSH-R2 and hFSH-R3. The hFSH-R2 splice variant differed from the full-length FSH-R mRNA by the deletion of exon 10 and inclusion of two small exons after exon 9 whereas the hFSH-R3 splice variant retained only exons 1-6 of the full-length transcript. Both variants were expressed at low levels, but were detected in cells from follicular fluid derived from 30 different subjects. Transfection of these two variants individually into KGN cells, an ovarian cancer cell line that expresses wild-type FSH-R, reduced FSH-mediated phosphorylation of ERK(1/2), Akt, and p38/MAPK. Furthermore, in vitro co-expression of either hFSH-R2 or hFSH-R3 and full-length FSH-R in HEK293T cells reduced signal transduction through full-length FSH-R. Further studies are needed to fully elucidate the functions of these receptor isoforms.

In Silico Study on Binding Specificity of Gonadotropins and Their Receptors: Design of a Novel and Selective Peptidomimetic for Human Follicle Stimulating Hormone Receptor

Gonadotropins bind to specific receptors in spite of sharing a high level of sequence and structural similarity. This specific binding is crucial for maintaining the reproductive health of an organism. In this study, residues that dictate the receptor binding specificity of the gonadotropins (FSH and LH) have been identified using combination of in silico methods. Docking studies (ZDOCK), based on the systematic replacement of these residues, confirmed its importance in receptor binding. An interesting observation is that the relative positioning of the residues conferring binding specificity varied for the gonadotropin-receptor complexes. This spatial difference of the key residues could be exploited for design of specific modulators. Based on the identified residues, we have rationally designed a peptidomimetic (FSHP) that displays good binding affinity and specificity for hFSHR. FSHP was developed by screening 3.9 million compounds using pharmacophore-shape similarity followed by fragment-based approach. It was observed that FSHP and hFSHâ can share the same receptor binding site thereby mimicking the native hFSHR-FSH interactions. FSHP also displayed higher binding affinity to hFSHR as compared to two reported hFSHR antagonists. MD simulation studies on hFSHR-FSHP complex revealed that FSHP is conformationally rigid and the intermolecular interactions are maintained during the course of simulation.

Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation

The present study aims to determine the role of the specific residues of the extracellular loops (ELs) of the FSH receptor (FSHR) in hormone binding and receptor activation. By substituting the sequences of each of the ELs of human FSHR with those of the luteinizing hormone/choriogonadotropin receptor (LH/CGR), we generated three mutant constructs where the three ELs were individually replaced. A fourth construct had all the three substituted ELs. The receptor expression and hormone binding ability of the mutants were comparable to that of the wild type. Hormone-induced signaling and internalization were lower in the EL2 substitution mutant (EL2M). In this mutant, the EL2 of FSHR was substituted with the corresponding loop of LH/CGR. Interestingly, homology modeling revealed a change in the orientation of EL2 in the mutant receptor. Thus, disruption of EL2 affected overall receptor function, suggesting the role of FSHR specific residues of the loop in ligand mediated signaling.

A Multifaceted Investigation of the Localization of the Human Follicle Stimulating Hormone Receptor

Follicle stimulating hormone (FSH) is responsible for follicular maturation in females and production of sperm‐stabilizing proteins in males. FSH receptor (hFSHR) is a G‐protein coupled receptor located in granulosa cells in the ovaries and Sertoli cells in the testes. It is believed that hFSHR is located in lipid raft microdomains of the plasma membrane. Lipid rafts are areas of the cell membrane that demonstrate increased rigidity, resistance to detergents, and have a high percent composition of cholesterol and sphingolipids. It is proposed that hFSHR is associated with a specific class of lipid rafts known as caveolae; hFSHR contains a caveolin binding motif in its 4th transmembrane domain. To verify if hFSHR resides in lipid rafts, sucrose gradient fractionation was performed and analyzed by western blot. hFSHR was observed to reside in the fractions associated with buoyant membrane components. To determine if hFSHR interacts with caveolin, four mutant receptors were created by altering the putative caveolin binding motif. Understanding hFSHR localization in the plasma membrane will lead to a greater understanding of its signaling pathways and the molecular basis of reproduction.

Decreased Degradation of Internalized Follicle-Stimulating Hormone Caused by Mutation of Aspartic Acid 6.30550 in a Protein Kinase-CK2 Consensus Sequence in the Third Intracellular Loop of Human Follicle-Stimulating Hormone Receptor1

A naturally occurring mutation in follicle-stimulating hormone receptor (FSHR) gene has been reported: an amino acid change to glycine occurs at a conserved aspartic acid 550 (D550, D567, D6.30(567)). This residue is contained in a protein kinase-CK2 consensus site present in human FSHR (hFSHR) intracellular loop 3 (iL3). Because CK2 has been reported to play a role in trafficking of some receptors, the potential roles for CK2 and D550 in FSHR function were evaluated by generating a D550A mutation in the hFSHR. The hFSHR-D550A binds hormone similarly to WT-hFSHR when expressed in HEK293T cells. Western blot analyses showed lower levels of mature hFSHR-D550A. Maximal cAMP production of both hFSHR-D550A as well as the naturally occurring mutation hFSHR-D550G was diminished, but constitutive activity was not observed. Unexpectedly, when (125)I-hFSH bound to hFSHR-D550A or hFSHR-D550G, intracellular accumulation of radiolabeled FSH was observed. Both sucrose and dominant-negative dynamin blocked internalization of radiolabeled FSH and its commensurate intracellular accumulation. Accumulation of radiolabeled FSH in cells transfected with hFSHR-D550A is due to a defect in degradation of hFSH as measured in pulse chase studies, and confocal microscopy imaging revealed that FSH accumulated in large intracellular structures. CK2 kinase activity is not required for proper degradation of internalized FSH because inhibition of CK2 kinase activity in cells expressing hFSHR did not uncouple degradation of internalized radiolabeled FSH. Additionally, the CK2 consensus site in FSHR iL3 is not required for binding because CK2alpha coimmunoprecipitated with hFSHR-D550A. Thus, mutation of D550 uncouples the link between internalization and degradation of hFSH.