Fluorescent small-molecule agonists as follicle-stimulating hormone receptor imaging tools.
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ABSTRACT:
SUBMITTER: Hoogendoorn S
PROVIDER: S-EPMC8341919 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) with pivotal roles in reproduction. One key mechanism dictating the signal activity of GPCRs is membrane trafficking. After binding its hormone FSH, FSHR undergoes internalization to very early endosomes (VEEs) for its acute signaling and sorting to a rapid recycling pathway. The VEE is a heterogeneous compartment containing the Adaptor Protein Phosphotyrosine Interacting with Pleckstrin homology Domain and Leucine Zipper 1 (APPL1) with distinct functions in regulating endosomal Gαs/cAMP signaling and rapid recycling. Low molecular weight (LMW) allosteric FSHR ligands were developed for use in assisted reproductive technology yet could also provide novel pharmacological tools to study FSHR. Given the critical nature of receptor internalization and endosomal signaling for FSHR activity, we assessed whether these compounds exhibit differential abilities to alter receptor endosomal trafficking and signaling within the VEE. Two chemically distinct LMW agonists (benzamide, termed B3 and thiazolidinone, termed T1) were employed. T1 was able to induce a greater level of cAMP than FSH and B3. As cAMP signaling drives gonadotrophin hormone receptor recycling, rapid exocytic events were evaluated at single event resolution. Strikingly, T1 was able to induce a 3-fold increase in recycling events compared to FSH and two-fold more compared to B3. As T1-induced internalization was only marginally greater, the dramatic increase in recycling and cAMP signaling may be due to additional mechanisms. All compounds exhibited a similar requirement for receptor internalization to increase cAMP and proportion of FSHR endosomes with active Gαs, suggesting regulation of cAMP signaling induced by T1 may be altered. APPL1 plays a central role for GPCRs targeted to the VEE, and indeed, loss of APPL1 inhibited FSH-induced recycling and increased endosomal cAMP signaling. While T1-induced FSHR recycling was APPL1-dependent, its elevated cAMP signaling was only partially increased following APPL1 knockdown. Unexpectedly, B3 altered the dependence of FSHR to APPL1 in an opposing manner, whereby its endosomal signaling was negatively regulated by APPL1, while B3-induced FSHR recycling was APPL1-independent. Overall, FSHR allosteric compounds have the potential to re-program FSHR activity via altering engagement with VEE machinery and also suggests that these two distinct functions of APPL1 can potentially be selected pharmacologically.
| S-EPMC7734412 | biostudies-literature
Project description:A thienopyr(im)idine (Org41841) activates the luteinizing hormone (LH) receptor but does not compete with the natural ligand binding site and does not show agonistic action on the follicle-stimulating hormone receptor (hFSHR) at sub-millimolar concentrations. When this drug is preincubated at sub-micromolar concentrations with host cells expressing the hFSHR, and then washed out, binding analysis and assessment of receptor-effector coupling show that it increases plasma membrane expression of the hFSHR. Real-time PCR shows that this effect did not result from increased hFSHR mRNA accumulation. It is possible that Org41841 behaves as a pharmacoperone, a drug which increases the percentage of newly synthesized receptor routing to the membrane. Like pharmacoperones for other receptors, this drug was able to rescue a particular mutant hFSHR (A(189)V) associated with misrouting and endoplasmic reticulum retention, although other mutants could not be rescued. This is potentially the first member of the pharmacoperone drug class which binds at a site that is distinctive from the ligand binding site.
| S-EPMC2630403 | biostudies-literature
Project description:Selective overexpression of follicle-stimulating hormone receptor (FSHR) inside the vascular endothelium of tumors has been confirmed to play critical roles in angiogenesis, tumor invasion, and metastases. The expression level of FSHR correlates strongly with the response of tumors to antiangiogenic therapies. In this study, an immunoPET tracer was developed for imaging of FSHR in different cancer types. A monoclonal antibody (FSHR-mAb) against FSHR was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) and used for subsequent (64)Cu-labeling. NOTA-FSHR-mAb preserved FSHR specificity/affinity, confirmed by flow cytometry measurements. (64)Cu-labeling was successfully conducted with decent yields (?25%) and high specific activity (0.93 GBq/mg). The uptake of (64)Cu-NOTA-FSHR-mAb was 3.6 ± 0.8, 13.2 ± 0.7, and 14.6 ± 0.4 %ID/g in FSHR-positive CAOV-3 tumors at 4, 24, and 48 h postinjection, respectively (n = 3), significantly higher (p < 0.05) than that in FSHR-negative SKOV-3 tumors (2.3 ± 1.2, 8.0 ± 0.9, and 9.1 ± 1.3 %ID/g at 4, 24, and 48 h postinjection, respectively (n = 3)) except at 4 h p.i. FSHR-relevant uptake of (64)Cu-NOTA-FSHR-mAb was also readily observed in other tumor types (e.g., triple-negative breast tumor MDA-MB-231 or prostate tumor PC-3). Histology studies showed universal FSHR expression in microvasculature of these four tumor types and also prominent expression in tumor cells of CAOV-3, PC-3, and MDA-MB-231. Correlations between tumor FSHR level and uptake of (64)Cu-NOTA-FSHR-mAb were witnessed in this study. FSHR-specific uptake of (64)Cu-NOTA-FSHR mAb in different tumors enables its applicability for future cancer theranostic applications and simultaneously establishes FSHR as a promising clinical target for cancer.
| S-EPMC4351720 | biostudies-literature
Project description:Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, β-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or β-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for β-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or β-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.
| S-EPMC8469697 | biostudies-literature
Project description:Background and aimThe follicle-stimulating hormone (FSH) gene is an essential regulator of fertility in livestock. This study aims to provide information on the genetic makeup of Madrasin cattle experiencing hypofunction by the FSH profile and FSH receptors (FSHR) polymorphism.Materials and methodsBlood samples were collected from the Bangkalan regency in Indonesia. DNA was isolated and purified following the extraction protocol of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism.ResultsOur results showed that the FSH gene had a band length of 310 bp and produce two alleles (A and B) with restriction enzymes at 250 bp, 230 bp, and 145 bp. Furthermore, the FSHR gene had a band length of 303 bp and produced two homozygous genotypes: GG at bp 239 and CC at bp 188.ConclusionBased on these differences, there was no change in allele frequency and genotype between Madura and Madrasin cattle due to crossbreeding with Limousin cattle. Thus, further detailed investigations of Madrasin cattle are required to elucidate the profile of the LH and LHR genes.
| S-EPMC7311873 | biostudies-literature
Project description:FSH glycosylation varies in two functionally important aspects: microheterogeneity, resulting from oligosaccharide structure variation, and macroheterogeneity, arising from partial FSHβ subunit glycosylation. Although advances in mass spectrometry permit extensive characterization of FSH glycan populations, microheterogeneity remains difficult to illustrate, and comparisons between different studies are challenging because no standard format exists for rendering oligosaccharide structures. FSH microheterogeneity is illustrated using a consistent glycan diagram format to illustrate the large array of structures associated with one hormone. This is extended to commercially available recombinant FSH preparations, which exhibit greatly reduced microheterogeneity at three of four glycosylation sites. Macroheterogeneity is demonstrated by electrophoretic mobility shifts due to the absence of FSHβ glycans that can be assessed by Western blotting of immunopurified FSH. Initially, macroheterogeneity was hoped to matter more than microheterogeneity. However, it now appears that both forms of carbohydrate heterogeneity have to be taken into consideration. FSH glycosylation can reduce its apparent affinity for its cognate receptor by delaying initial interaction with the receptor and limiting access to all of the available binding sites. This is followed by impaired cellular signaling responses that may be related to reduced receptor occupancy or biased signaling. To resolve these alternatives, well-characterized FSH glycoform preparations are necessary.
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Project description:The thyroid-stimulating hormone receptor (TSHR) is the essential molecule for thyroid growth and thyroid hormone production. Since it is also a key autoantigen in Graves' disease and is involved in thyroid cancer pathophysiology, the targeting of the TSHR offers a logical model for disease control.We review the structure and function of the TSHR and the progress in both small molecule ligands and TSHR antibodies for their therapeutic potential.Stabilization of a preferential conformation for the TSHR by allosteric ligands and TSHR antibodies with selective modulation of the signaling pathways is now possible. These tools may be the next generation of therapeutics for controlling the pathophysiological consequences mediated by the effects of the TSHR in the thyroid and other extrathyroidal tissues.
| S-EPMC4484771 | biostudies-literature
Project description:Follicle-stimulating hormone (FSH), a pituitary glycoprotein hormone, is an integral component of the endocrine axis that regulates gonadal function and fertility. To transmit its signal, FSH must bind to its receptor (FSHR) located on Sertoli cells of the testis and granulosa cells of the ovary. Thus, both the magnitude and the target of hormone response are controlled by mechanisms that determine FSHR levels and cell-specific expression, which are supported by transcription of its gene. The present review examines the status of FSHR/Fshr gene regulation, emphasizing the importance of distal sequences in FSHR/Fshr transcription, new insights gained from the influx of genomics data and bioinformatics, and emerging trends that offer direction in deciphering the FSHR/Fshr regulatory landscape.
| S-EPMC4480823 | biostudies-literature
Project description:Results from experiments using mouse models suggest that the role of follicle-stimulating hormone (FSH) in spermatogenesis is the regulation of Sertoli cell proliferation and, ultimately, the size and spermatogenic capacity of the testis. The regulation of the expression of the FSH receptor (FSHR) gene is very cell specific and plays an initial role in the ultimate response of the Sertoli cells to FSH. The extreme cell specificity and the importance of the FSH response to spermatogenesis have led to an extensive characterization of the promoter of the FSHR gene. Several widely expressed transcription factors - including USF 1 and 2, GATA-1, and SF-1 and potential elements such as an E2F site and an Inr region - have been shown to contribute to the maximal transcription of the transfected FSHR gene. However, these experiments have failed to provide clues as to the cell-specific expression of the FSHR gene. In both cell transfections and in transgenic mice, the promoter can direct expression of transgenes promiscuously. The rodent FSHR promoter contains conserved CpG dinucleotides that were shown to be methylated in nonexpressing cells and tissue but unmethylated in Sertoli cells. The methylated CpG sites could interfere with the binding of general transcription factors and/or lead to a repressive chromatin structure in the nonexpressing cells. While yet-undiscovered cell-specific factors may play a role in the expression of the FSHR gene, repression and activation of local chromatin structure are likely to be involved.
| S-EPMC1496959 | biostudies-literature
Project description:Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor-null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of Lhr-/- mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high-dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution.
| S-EPMC5919831 | biostudies-literature