Search

Many interaction partners of β-arrestins intervene in the control of mRNA translation. However, how β-arrestins regulate this cellular process has been poorly explored. In this study, we show that β-arrestins constitutively assemble a p70S6K/ribosomal protein S6 (rpS6) complex in HEK293 cells and in primary Sertoli cells of the testis. We demonstrate that this interaction is direct, and experimentally validate the interaction interface between β-arrestin 1 and p70S6K predicted by our docking algorithm. Like most GPCRs, the biological function of follicle-stimulating hormone receptor (FSHR) is transduced by G proteins and β-arrestins. Upon follicle-stimulating hormone (FSH) stimulation, activation of G protein-dependent signaling enhances p70S6K activity within the β-arrestin/p70S6K/rpS6 preassembled complex, which is not recruited to the FSHR. In agreement, FSH-induced rpS6 phosphorylation within the β-arrestin scaffold was decreased in cells depleted of Gα s . Integration of the cooperative action of β-arrestin and G proteins led to the translation of 5' oligopyrimidine track mRNA with high efficacy within minutes of FSH input. Hence, this work highlights new relationships between G proteins and β-arrestins when acting cooperatively on a common signaling pathway, contrasting with their previously shown parallel action on the ERK MAP kinase pathway. In addition, this study provides insights into how GPCR can exert trophic effects in the cell.-Tréfier, A., Musnier, A., Landomiel, F., Bourquard, T., Boulo, T., Ayoub, M. A., León, K., Bruneau, G., Chevalier, M., Durand, G., Blache, M.-C., Inoue, A., Fontaine, J., Gauthier, C., Tesseraud, S., Reiter, E., Poupon, A., Crépieux, P. G protein-dependent signaling triggers a β-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

With the advent of next-generation sequencing technologies, identifying the translatome, which includes genome-wide ribosome-associated mRNAs, provides new opportunities to define faithfully the protein repertoire of a cell, as opposed to transcriptomic approaches. In addition, the role that extracellular signals such as hormonal modulations could play on the translatome remains to be deciphered. In particular, the regulation of the translatome by G protein-coupled receptors (GPCR) is still poorly described, albeit the trophic role that many receptors of this family play in their target cells. Here, we provide an overview of the current methods that are used to study the translatome, applied to the GPCR receptor family., (Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.)

In addition to their role in desensitization and internalization of G protein-coupled receptors (GPCRs), β-arrestins are essential scaffolds linking GPCRs to Erk1/2 signaling. However, their role in GPCR-operated Erk1/2 activation differs between GPCRs and the underlying mechanism remains poorly characterized. Here, we show that activation of serotonin 5-HT 2C receptors, which engage Erk1/2 pathway via a β-arrestin-dependent mechanism, promotes MEK-dependent β-arrestin2 phosphorylation at Thr 383 , a necessary step for Erk recruitment to the receptor/β-arrestin complex and Erk activation. Likewise, Thr 383 phosphorylation is involved in β-arrestin-dependent Erk1/2 stimulation elicited by other GPCRs such as β 2 -adrenergic, FSH and CXCR4 receptors, but does not affect the β-arrestin-independent Erk1/2 activation by 5-HT 4 receptor. Collectively, these data show that β-arrestin2 phosphorylation at Thr 383 underlies β-arrestin-dependent Erk1/2 activation by GPCRs.

The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β 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 p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β 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 p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β 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 p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β 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 p110β and AR have previously been reported to functionally interact.

Follicle-stimulating hormone (FSH) is a central regulator of male and female reproductive function. Over the last decade, there has been a growing perception of the complexity associated with FSH-induced cellular signaling. It is now clear that the canonical Gs/cAMP/PKA pathway is not the sole mechanism that must be considered in FSH biological actions. In parallel, consistent with the emerging concept of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. In this context, it is important to gain an integrative view of the signaling pathways induced by FSH and how they interconnect to form a network. In this review, we propose a first attempt at building topological maps of various pathways known to be involved in the FSH-induced signaling network. We discuss the multiple facets of FSH-induced signaling and how they converge to the hormone integrated biological response. Despite of their incompleteness, these maps of the FSH-induced signaling network represent a first step toward gaining a system-level comprehension of this hormone's actions, which may ultimately facilitate the discovery of novel regulatory processes and therapeutic strategies for infertility and non-steroidal contraception.

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 is the main endocrine control of mammalian reproduction. FSH triggers somatic cells of the gonads which support germ cells metabolically, i.e. Sertoli cells of the seminiferous tubules, and granulosa cells harboring the oocyte, within the ovarian follicle. FSH leads to similar biological responses in both cell types since it stimulates proliferation and differentiation, according to the developmental stage. However, FSH receptor knock-out female mice are infertile, unlike male mice. Hence, FSH is not equally important in both sexes. Nevertheless, does FSH induce distinct signalling mechanisms in its target cells ? Here, we compare the signalling mechanisms induced by FSH in ovarian and testicular physiology.

Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation.

The testis is devoted to two important tasks: haploid cell production and sexual steroid synthesis. A number of highly sophisticated and unique strategies operate during spermatogenesis, a process crucial for reproduction, heredity and evolution. It is particularly important to decipher the underlying molecular mechanisms whose function can be perverted in pathological situations, such as infertility and testicular cancers, which represent an increasing biomedical issue today. This review summarises the currently available data concerning some key molecular components that are altered or potentially involved in male infertility and testicular tumors, with the aim of defining some common "hot spots". We particularly focused on genetically engineered in vivo models in which testicular functions are altered and we pinpointed to the potential involvement of the targeted genes in testicular pathologies. Those molecular mechanisms peculiar to the male gonad can be envisioned as a basis for the design of novel drugs potentially dedicated to testicular dysfunction.