An intracellular VHH targeting the Luteinizing Hormone receptor modulates G protein-dependent signaling and steroidogenesis.

Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor ß-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.

Towards the convergent therapeutic potential of G protein-coupled receptors in autism spectrum disorders.

Autism spectrum disorders (ASDs) are diagnosed in 1/100 children worldwide, based on two core symptoms: deficits in social interaction and communication, and stereotyped behaviours. G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors that transduce extracellular signals to convergent intracellular signalling and downstream cellular responses that are commonly dysregulated in ASD. Despite hundreds of GPCRs being expressed in the brain, only 23 are genetically associated with ASD according to the Simons Foundation Autism Research Initiative (SFARI) gene database: oxytocin OTR; vasopressin V1A and V1B ; metabotropic glutamate mGlu5 and mGlu7 ; GABAB2 ; dopamine D1 , D2 and D3 ; serotoninergic 5-HT1B ; ß2 -adrenoceptor; cholinergic M3 ; adenosine A2A and A3 ; angiotensin AT2 ; cannabinoid CB1 ; chemokine CX3 CR1; orphan GPR37 and GPR85; and olfactory OR1C1, OR2M4, OR2T10 and OR52M1. Here, we review the therapeutic potential of these 23 GPCRs, as well as 5-HT2A and 5-HT7 , for ASD. For each GPCR, we discuss its genetic association, genetic and pharmacological manipulation in animal models, pharmacopoeia for core symptoms of ASD and rank them based on these factors. Among these GPCRs, we highlight D2 , 5-HT2A , CB1 , OTR and V1A as the more promising targets for ASD. We discuss that the dysregulation of GPCRs and their signalling is a convergent pathological mechanism of ASD. Their therapeutic potential has only begun as multiple GPCRs could mitigate ASD.

Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

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, ß-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 ß-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 ß-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 ß-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.

Accurate determination of epitope for antibodies with unknown 3D structures.

MAbTope is a docking-based method for the determination of epitopes. It has been used to successfully determine the epitopes of antibodies with known 3D structures. However, during the antibody discovery process, this structural information is rarely available. Although we already have evidence that homology models of antibodies could be used instead of their 3D structure, the choice of the template, the methodology for homology modeling and the resulting performance still have to be clarified. Here, we show that MAbTope has the same performance when working with homology models of the antibodies as compared to crystallographic structures. Moreover, we show that even low-quality models can be used. We applied MAbTope to determine the epitope of dupilumab, an anti- interleukin 4 receptor alpha subunit therapeutic antibody of unknown 3D structure, that we validated experimentally. Finally, we show how the MAbTope-determined epitopes for a series of antibodies targeting the same protein can be used to predict competitions, and demonstrate the accuracy with an experimentally validated example.3D: three-dimensionalRMSD: root mean square deviationCDR: complementary-determining regionCPU: central processing unitsVH: heavy chain variable regionVL: light chain variable regionscFv: single-chain variable fragmentsVHH: single-chain antibody variable regionIL4Rα: Interleukin 4 receptor alpha chainSPR: surface plasmon resonancePDB: protein data bankHEK293: Human embryonic kidney 293 cellsEDTA: Ethylenediaminetetraacetic acidFBS: Fetal bovine serumANOVA: Analysis of varianceEGFR: Epidermal growth factor receptorPE: PhycoerythrinAPC: AllophycocyaninFSC: forward scatterSSC: side scatterWT: wild typeKeywords: MAbTope, Epitope Mapping, Molecular docking, Antibody modeling, Antibody-antigen docking.

Direct impact of gonadotropins on glucose uptake and storage in preovulatory granulosa cells: Implications in the pathogenesis of polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is often associated with higher levels of LH, and arrested ovarian follicular growth. The direct impact of high LH on FSH mediated metabolic responses in PCOS patients is not clearly understood. METHOD: In order to investigate the impact of FSH and LH on glucose metabolism in preovulatory granulosa cells (GCs), we used [U14C]-2 deoxyglucose, D-[U14C]-glucose or 2-NBD glucose to analyse glucose uptake and its incorporation into glycogen. To reproduce the high androgenic potential in PCOS patients, we administered hCG both in vitro and in vivo. The role of IRS-2/PI3K/Akt2 pathway was studied after knockdown with specific siRNA. Immunoprecipitation and specific assays were used for the assessment of IRS-2, glycogen synthase and protein phosphatase 1. Furthermore, we examined the in vivo effects of hCG on FSH mediated glycogen increase in normal and PCOS rat model. HEK293 cells co-expressing FSHR and LHR were used to demonstrate glucose uptake and BRET change by FSH and hCG. RESULTS: In normal human and rat granulosa cells, FSH is more potent than hCG in stimulating glucose uptake, however glycogen synthesis was significantly upregulated only by FSH through increase in activity of glycogen synthase via IRS-2/PI3K/Akt2 pathway. On the contrary, an impaired FSH-stimulated glucose uptake and glycogen synthesis in granulosa cells of PCOS-patients indicated a selective defect in FSHR activation. Further, in normal human granulosa cells, and in immature rat model, the impact of hCG on FSH responses was such that it inhibited the FSH-mediated glucose uptake as well as glycogen synthesis through inhibition of FSH-stimulated IRS-2 expression. These findings were further validated in HEK293 cells overexpressing Flag-LHR and HA-FSHR, where high hCG inhibited the FSH-stimulated glucose uptake. Notably, an increased BRET change was observed in HEK293 cells expressing FSHR-Rluc8 and LHR-Venus possibly suggesting increased heteromerization of LHR and FSHR in the presence of both hCG and FSH in comparison to FSH or hCG alone. CONCLUSION: Our findings confirm a selective attenuation of metabolic responses to FSH such as glucose uptake and glycogen synthesis by high activation level of LHR leading to the inhibition of IRS-2 pathway, resulting in depleted glycogen stores and follicular growth arrest in PCOS patients.

Methods to Determine Interaction Interfaces Between ß-Arrestins and Their Protein Partners.

ß-arrestins are so-called hub proteins: they make complexes with many different partners, assembling functional complexes, and thereby fulfilling their biological function. The importance of this process in G protein-coupled receptor (GPCR) signalling has been fully demonstrated for many different receptors. For direct interactions, determining the interface regions, on ß-arrestins and on the partners, is crucial for understanding the function of the complex. Indeed, this brings information on which proteins can interact simultaneously with ß-arrestins, or, on the contrary, which partners are exclusive. We present here a method in two steps: protein-protein docking allows finding a limited number of peptides predicted to be involved in the interaction, and then experimental approaches that might be used for validating the prediction.

FSH Receptor Signaling: Complexity of Interactions and Signal Diversity.

FSH is synthesized in the pituitary by gonadotrope cells. By binding to and interacting with its cognate receptor [FSH receptor (FSHR)] in the gonads, this gonadotropin plays a key role in the control of gonadal function and reproduction. Upon activation, the FSHR undergoes conformational changes leading to transduction of intracellular signals, including dissociation of G protein complexes into components and activation of several associated interacting partners, which concertedly regulate downstream effectors. The canonical Gs/cAMP/protein kinase A pathway, considered for a long time as the sole effector of FSHR-mediated signaling, is now viewed as one of several mechanisms employed by this receptor to transduce intracellular signals in response to the FSH stimulus. This complex network of signaling pathways allows for a fine-tuning regulation of the gonadotropic stimulus, where activation/inhibition of its multiple components vary depending on the cell context, cell developmental stage, and concentration of associated receptors and corresponding ligands. Activation of these multiple signaling modules eventually converge to the hormone-integrated biological response, including survival, proliferation and differentiation of target cells, synthesis and secretion of paracrine/autocrine regulators, and, at the molecular level, functional selectivity and differential gene expression. In this mini-review, we discuss the complexity of FSHR-mediated intracellular signals activated in response to ligand stimulation. A better understanding of the signaling pathways involved in FSH action might potentially influence the development of new therapeutic strategies for reproductive disorders.

Antibodies targeting G protein-coupled receptors: Recent advances and therapeutic challenges.

Le STUDIUM conference was held November 24-25, 2016 in Tours, France as a satellite workshop of the 5th meeting of the French GDR 3545 on "G Protein-Coupled Receptors (GPCRs) -From Physiology to Drugs," which was held in Tours during November 22-24, 2016. The conference gathered speakers from academia and industry considered to be world leaders in the molecular pharmacology and signaling of GPCRs, with a particular interest in the development of therapeutic GPCR antibodies (Abs). The main topics were new advances and challenges in the development of antibodies targeting GPCRs and their potential applications to the study of the structure and function of GPCRs, as well as their implication in physiology and pathophysiology. The conference included 2 sessions, with the first dedicated to the recent advances in methodological strategies used for GPCR immunization using thermo-stabilized and purified GPCRs, and the development of various formats of Abs such as monoclonal IgG, single-chain variable fragments and nanobodies (Nbs) by in vitro and in silico approaches. The second session focused on GPCR Nbs as a "hot" field of research on GPCRs. This session started with discussion of the pioneering Nbs developed against GPCRs and their application to structural studies, then transitioned to talks on original ex vivo and in vivo studies on GPCR-selective Nbs showing promising therapeutic applications of Nbs in important physiologic systems, such as the central nervous and the immune systems, as well as in cancer. The conference ended with the consensus that Abs and especially Nbs are opening a new era of research on GPCR structure, pharmacology and pathophysiology.

Profiling of FSHR negative allosteric modulators on LH/CGR reveals biased antagonism with implications in steroidogenesis.

Biased signaling has recently emerged as an interesting means to modulate the function of many G protein-coupled receptors (GPCRs). Previous studies reported two negative allosteric modulators (NAMs) of follicle-stimulating hormone receptor (FSHR), ADX68692 and ADX68693, with differential effects on FSHR-mediated steroidogenesis and ovulation. In this study, we attempted to pharmacologically profile these NAMs on the closely related luteinizing hormone/chorionic gonadotropin hormone receptor (LH/CGR) with regards to its canonical Gs/cAMP pathway as well as to ß-arrestin recruitment in HEK293 cells. The NAMs' effects on cAMP, progesterone and testosterone production were also assessed in murine Leydig tumor cell line (mLTC-1) as well as rat primary Leydig cells. We found that both NAMs strongly antagonized LH/CGR signaling in the different cell models used with ADX68693 being more potent than ADX68692 to inhibit hCG-induced cAMP production in HEK293, mLTC-1 and rat primary Leydig cells as well as ß-arrestin 2 recruitment in HEK293 cells. Interestingly, differential antagonism of the two NAMs on hCG-promoted steroidogenesis in mLTC-1 and rat primary Leydig cells was observed. Indeed, a significant inhibition of testosterone production by the two NAMs was observed in both cell types, whereas progesterone production was only inhibited by ADX68693 in rat primary Leydig cells. In addition, while ADX68693 totally abolished testosterone production, ADX68692 had only a partial effect in both mLTC-1 and rat primary Leydig cells. These observations suggest biased effects of the two NAMs on LH/CGR-dependent pathways controlling steroidogenesis. Interestingly, the pharmacological profiles of the two NAMs with respect to steroidogenesis were found to differ from that previously shown on FSHR. This illustrates the complexity of signaling pathways controlling FSHR- and LH/CGR-mediated steroidogenesis, suggesting differential implication of cAMP and ß-arrestins mediated by FSHR and LH/CGR. Together, our data demonstrate that ADX68692 and ADX68693 are biased NAMs at the LH/CGR in addition to the FSHR. These pharmacological characteristics are important to consider for potential contraceptive and therapeutic applications based on such compounds.

Assessing Gonadotropin Receptor Function by Resonance Energy Transfer-Based Assays.

Gonadotropin receptors belong to the super family of G protein-coupled receptors and mediate the physiological effects of follicle-stimulating hormone (FSHR) and luteinizing hormone (LHR). Their central role in the control of reproductive function has made them the focus of intensive studies. Upon binding to their cognate hormone, they trigger complex signaling and trafficking mechanisms that are tightly regulated in concentration, time, and space. Classical cellular assays often fail to capture all these dynamics. Here, we describe the use of various bioluminescence and fluorescence resonance energy transfer (BRET and FRET) assays to investigate the activation and regulation of FSHR and LHR in real-time, in living cells (i.e., transiently expressed in human embryonic kidney 293 cells). Indeed, the dynamics of hormone-mediated heterotrimeric G protein activation, cyclic adenosine-monophosphate (cAMP) production, calcium release, ß-arrestin 2 recruitment, and receptor internalization/recycling was assessed. Kinetics and dose-response analyses confirmed the expected pharmacological and signaling properties of hFSHR and hLHR but revealed interesting characteristics when considering the two major pathways (cAMP and ß-arrestin 2) of the two receptors assessed by BRET. Indeed, the EC50 values were in picomolar range for cAMP production while nanomolar range was observed for ß-arrestin 2 recruitment as well as receptor internalization. Interestingly, the predicted receptor occupancy indicates that the maximal G protein activation and cAMP response occur at <10% of receptor occupancy whereas >90% of activated receptors is required to achieve full ß-arrestin 2 recruitment and subsequent receptor internalization. The rapid receptor internalization was also followed by a recycling phase. Collectively, our data reveal that ß-arrestin-mediated desensitization, internalization, and the subsequent fast recycling of receptors at the plasma membrane may provide a mechanistic ground to the "spare receptor" paradigm. More generally, the novel tools described here will undoubtedly provide the scientific community investigating gonadotropin receptors with powerful means to decipher their pharmacology and signaling with the prospect of pathophysiological and drug discovery applications.

Novel Role for p110ß PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells.

The organismal roles of the ubiquitously expressed class I PI3K isoform p110ß remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110ß, we document that full inactivation of p110ß leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110ß kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110ß results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110ß was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110ß also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110ß inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110ß inactivation. In line with a crucial role for p110ß in SCs, selective inactivation of p110ß in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110ß and AR have previously been reported to functionally interact.

Unraveling the molecular architecture of a G protein-coupled receptor/ß-arrestin/Erk module complex.

ß-arrestins serve as signaling scaffolds downstream of G protein-coupled receptors, and thus play a crucial role in a plethora of cellular processes. Although it is largely accepted that the ability of ß-arrestins to interact simultaneously with many protein partners is key in G protein-independent signaling of GPCRs, only the precise knowledge of these multimeric arrangements will allow a full understanding of the dynamics of these interactions and their functional consequences. However, current experimental procedures for the determination of the three-dimensional structures of protein-protein complexes are not well adapted to analyze these short-lived, multi-component assemblies. We propose a model of the receptor/ß-arrestin/Erk1 signaling module, which is consistent with most of the available experimental data. Moreover, for the ß-arrestin/Raf1 and the ß-arrestin/ERK interactions, we have used the model to design interfering peptides and shown that they compete with both partners, hereby demonstrating the validity of the predicted interaction regions.

Activation of a GPCR leads to eIF4G phosphorylation at the 5' cap and to IRES-dependent translation.

The control of mRNA translation has been mainly explored in response to activated tyrosine kinase receptors. In contrast, mechanistic details on the translational machinery are far less available in the case of ligand-bound G protein-coupled receptors (GPCRs). In this study, using the FSH receptor (FSH-R) as a model receptor, we demonstrate that part of the translational regulations occurs by phosphorylation of the translation pre-initiation complex scaffold protein, eukaryotic initiation factor 4G (eIF4G), in HEK293 cells stably expressing the FSH-R. This phosphorylation event occurred when eIF4G was bound to the mRNA 5' cap, and probably involves mammalian target of rapamycin. This regulation might contribute to cap-dependent translation in response to FSH. The cap-binding protein eIF4E also had its phosphorylation level enhanced upon FSH stimulation. We also show that FSH-induced signaling not only led to cap-dependent translation but also to internal ribosome entry site (IRES)-dependent translation of some mRNA. These data add detailed information on the molecular bases underlying the regulation of selective mRNA translation by a GPCR, and a topological model recapitulating these mechanisms is proposed.

mRNA-selective translation induced by FSH in primary Sertoli cells.

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m(7)GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5'cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far.

Preferential ß-arrestin signalling at low receptor density revealed by functional characterization of the human FSH receptor A189 V mutation.

The A189 V inactivating mutation of the human FSH receptor (FSHR) leads to subfertility in men and primary ovarian failure in women. This mutation has previously been associated with intracellular retention of the FSHR and impaired cAMP production. Here, we show that the A189 V FSHR stably expressed in HEK293N cells provoked ERK MAP kinases phosphorylation through ß-arrestins, independently of the canonical cAMP/PKA pathway. Interesting, both the A189 V and wild-type (Wt) FSHRs selectively activated cAMP-independent ERK phosphorylation when expressed at low plasma membrane densities. These data indicate that the selective intracellular signalling triggered by the A189 V FSHR resulted from reduced membrane expression rather than by switching receptor coupling. Hence, receptor density at the plasma membrane might control the balance between distinct signal transduction mechanisms. Furthermore, our results help to clarify why mutations of FSHß are more deleterious to human fertility than the FSHR A189 V mutation which preserves parts of receptor signalling repertoire.

Methionine deprivation regulates the S6K1 pathway and protein synthesis in avian QM7 myoblasts without activating the GCN2/eIF2 alpha cascade.

Amino acids modulate mRNA translation through the 70 kDa ribosomal protein S6 kinase (S6K1) and the general control nondepressible 2 protein kinase (GCN2)/eukaryotic initiation factor 2 alpha eIF2 alpha pathways. The aim of the present study was therefore to explore the signaling cascades potentially modulated by methionine availability in quail muscle QM7 myoblasts using media providing all other amino acids. Methionine deprivation caused a lower S6K1 phosphorylation compared with control (Ctl) cells. Supplying the methionine-deprived media with L- and DL-methionine isomers restored S6K1 phosphorylation to the levels observed in Ctl cells. Methionine also regulated downstream S6K1 targets (i.e. ribosomal protein S6 and eukaryotic elongation factor 2), modulated translation preinitiation complex (PIC) assembly, and stimulated protein synthesis. Replacing the lacking methionine with D-methionine or its hydroxyanalog [2-hydroxy-(4-methylthio) butanoic acid] did not restore S6K1 activation or protein synthesis. Conversely, the S6K1 pathway was activated by a methionine precursor, the ketoanalog of methionine. Methionine availability regulated the GCN2/eIF2 alpha pathway. However, our results indicate that methionine deprivation led to lower protein synthesis without activating eIF2 alpha phosphorylation, a process known to limit the formation of the 43S PIC. Using the amino acid alcohol methioninol did not decrease S6K1 phosphorylation or activity and did not alter the regulation of protein synthesis by methionine. These findings suggest that methionine exerts an effect on S6K1 signaling and protein synthesis in avian QM7 myoblasts through a mechanism partly independent of the global regulation via tRNA charging.

Partially deglycosylated equine LH preferentially activates beta-arrestin-dependent signaling at the follicle-stimulating hormone receptor.

Deglycosylated FSH is known to trigger poor Galphas coupling while efficiently binding its receptor. In the present study, we tested the possibility that a deglycosylated equine LH (eLHdg) might be able to selectively activate beta-arrestin-dependent signaling. We compared native eLH to an eLH derivative [i.e. truncated eLHbeta (Delta121-149) combined with asparagine56-deglycosylated eLHalpha (eLHdg)] previously reported as an antagonist of cAMP accumulation at the FSH receptor (FSH-R). We confirmed that, when used in conjunction with FSH, eLHdg acted as an antagonist for cAMP accumulation in HEK-293 cells stably expressing the FSH-R. Furthermore, when used alone at concentrations up to 1 nM, eLHdg had no detectable agonistic activity on cAMP accumulation, protein kinase A activity or cAMP-responsive element-dependent transcriptional activity. At higher concentrations, however, a weak agonistic action was observed with eLHdg, whereas eLH led to robust responses whatever the concentration. Both eLH and eLHdg triggered receptor internalization and led to beta-arrestin recruitment. Both eLH and eLHdg triggered ERK and ribosomal protein (rp) S6 phosphorylation at 1 nM. The depletion of endogenous beta-arrestins had only a partial effect on eLH-induced ERK and rpS6 phosphorylation. In contrast, ERK and rpS6 phosphorylation was completely abolished at all time points in beta-arrestin-depleted cells. Together, these results show that eLHdg has the ability to preferentially activate beta-arrestin-dependent signaling at the FSH-R. This finding provides a new conceptual and experimental framework to revisit the physiological meaning of gonadotropin structural heterogeneity. Importantly, it also opens a field of possibilities for the development of selective modulators of gonadotropin receptors.

FSH-stimulated PTEN activity accounts for the lack of FSH mitogenic effect in prepubertal rat Sertoli cells.

Follicle-stimulating hormone (FSH) controls the proliferation and differentiation of Sertoli cells of the testis. FSH binds a G protein-coupled receptor (GPCR) to stimulate downstream effectors of the phosphoinositide-3 kinase (PI3K)-dependent pathway, without enhancing PI3K activity. To clarify this paradox, we explored the activity of phosphatase and tensin homolog deleted in chromosome 10 (PTEN), the PI3K major regulator, in primary cultures of rat Sertoli cells. We show that, within minutes, FSH increases PTEN neo-synthesis, requiring the proteasomal degradation of an unidentified intermediate, as well as PTEN enzymatic activity. Importantly, introducing an antisense cDNA of PTEN into differentiating Sertoli cells restores FSH-dependent cell proliferation. In conclusion, these results provide a new mechanism of PTEN regulation, which could serve to block entry into S phase of Sertoli cells, while they are proceeding through differentiation in prepubertal animals.

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells.

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.

Dichlorodiphenyltrichloroethane impairs follicle-stimulating hormone receptor-mediated signaling in rat Sertoli cells.

Any toxicant that affects Sertoli cell development can potentially disturb male fertility. So far, the effects of organochlorine compounds have been poorly investigated in male. Here, we studied the effects of dichlorodiphenyltrichloroethane (DDT), an organochloride pesticide, on Sertoli cells. DDT inhibited the cAMP response to follicle-stimulating hormone (FSH), the major endocrine control of Sertoli cell development, and to a beta2-agonist, isoproterenol. DDT exposure decreased the level of FSH binding sites. Direct adenylyl cyclase activation by Forskolin was unaltered by DDT, while the activation of Galphas by cholera toxin was decreased by DDT. The DDT inhibitory effect on the FSH response was also observed in Ser W3 cells, a Sertoli cell-derived immortalized cell line. All these effects were reproduced by the lipophilic aromatic bisphenol A but not by structurally unrelated CisPlatin. In conclusion, these results are a first step in understanding the molecular basis of DDT deleterious effects in spermatogenesis.