A single domain intrabody as a novel tool to bias the subcellular trafficking of the follicle-stimulating hormone receptor
Pauline RaynaudJ GourdonVinesh JugnarainL. BerthetFrédéric Jean‐AlphonseOcéane VaugrenteCamille GauthierAmandine ValletPriyanka AnujanDelphine NaquinThomas BouloChristophe GauthierDanièle KlettYves CombarnousAylin C. HanyalogluÉric ReiterGilles BruneauPascale Crépieux
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Intracellular variable fragments from heavy-chain only antibodies of camelids (intra-VHH) have been successfully used for their stabilizing properties to solve the 3D structure of active G protein-coupled receptors (GPCRs) bound to their cognate transducers. They also provide tools to link a given conformation of a GPCR to the signalling network engaged, thus allowing extensive structure/activity studies. Recently, they have been instrumental in tracking active GPCRs in various subcellular compartments. Here, we report the isolation and characterization of iPRC2, an intra-VHH recognizing the 1st and 3rd intracellular loops of the FSHR, but not of the luteinizing hormone/choriogonadotropin receptor close relative. Its expression in the cell decreases the cAMP production in response to hormone binding, and requires G𝛼s for optimal interaction with the receptor. Importantly, iPRC2 increases the FSHR accumulation in the early endosomes, and consequently, diminishes its recycling to the cell surface. Hence, in contrast to previously described intra-VHH that disclose active GPCR intracellular location, iPRC2 provokes per se a location bias, through its ability to reroute the FSHR. Thus, it is an innovative tool to examine the functional consequences of GPCR accumulation in various sub-cellular compartments.Keywords:
Follicle-stimulating hormone receptor
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In this report, we review our present effort in the field of molecular reproductive endocrinology: to identify a small molecular weight follicle stimulating hormone (FSH) agonistic molecule. To achieve this goal we require a number of molecular tools. We have cloned and expressed the human gonadotrophin, FSH and the human FSH receptor and developed a reliable high throughput assay. We have also proposed a model to explain FSH receptor activation and from that model, begun to create small molecules predicted to induce FSH signal transduction without binding to the extracellular domain of the membrane protein. In this report, we summarize our efforts to date and discuss our future research efforts in this area.
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We had previously reported the region (20–30) from follicle stimulating hormone receptor as being an immunodominant epitope and the smallest reported peptide capable of inhibiting hormone binding. We now report it to be an effective antagonist of ligand‐induced cAMP signalling as well. The region (20–30) of follicle stimulating hormone receptor has three charged residues, namely, E 22 , D 26 and R 29 that are specific to follicle stimulating hormone receptor and are conserved in mammals. This study aimed to verify whether the charged residues contribute to the activity of the follicle stimulating hormone receptor peptide (20–30). This was done using analogs of follicle stimulating hormone receptor peptide (20–30), each having an alanine substitution for a corresponding charged residue. The analog peptides displayed a loss of activity and could not inhibit hormone binding or the subsequent signal transduction. The ability of follicle stimulating hormone receptor peptide (20–30) to bind antipeptide antibodies against follicle stimulating hormone receptor peptide (9–30) was either decreased or abolished with the alanine substituted analog peptides of follicle stimulating hormone receptor peptide (20–30). The loss of function led us to verify whether there was a conformational change as well. CD spectral analysis did not reveal a significant change. These observations indicate that the charged aminoacids present in follicle stimulating hormone receptor peptide (20–30) are crucial for the observed follicle stimulating hormone antagonistic activity. This information could form the basis for the design of novel compounds capable of functioning as follicle stimulating hormone antagonists.
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Follicle-stimulating hormone (FSH) is essential for folliculogenesis, acting through the follicle-stimulating hormone receptor (FSHR) that is present on the membrane of granulosa cells. Polymorphisms in the FSHR gene may lead to an altered pattern of receptor expression on the cell surface or to changes in affinity for FSH. The aim of this prospective study was to detect any association between the follicle-stimulating hormone receptor (FSHR) gene Ala307Thr polymorphism (rs6165) and ovarian reserve, ovarian response or clinical results in IVF/ICSI treatment.This prospective cohort study included 450 women who underwent IVF/ICSI cycles. DNA was extracted from peripheral blood, and the Ala307Thr FSHR polymorphism (rs6165) was genotyped using the TaqMan SNP genotyping assay. Participants were divided into three groups according to their Ala307Thr FSHR genotype: Thr/Thr (n:141), Thr/Ala (n=213) and Ala/Ala (n=96). The results were tested for associations with age, anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), total dose of r-FSH, follicle size, number of retrieved oocytes, and clinical outcome of IVF/ICSI cycles. The statistical analyses were performed using Fisher's exact test and the Kruskal‒Wallis test.An association between the genotype of the FSHR (Ala307Thr) polymorphism and the dose of r-FSH was observed. Patients with the Ala/Ala genotype received a higher r-FSH dose than patients with the Ala/Thr (p=0.0002) and Thr/Thr (p=0.02) genotypes. No other correlation was observed.The Ala/Ala genotype was associated with the use of higher doses of recombinant FSH (r-FSH), suggesting that homozygosis of this allelic variant (Ala) provides lower sensitivity to r-FSH.
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