G protein-dependent signaling triggers a ß-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

Many interaction partners of ß-arrestins intervene in the control of mRNA translation. However, how ß-arrestins regulate this cellular process has been poorly explored. In this study, we show that ß-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 ß-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 ß-arrestins. Upon follicle-stimulating hormone (FSH) stimulation, activation of G protein-dependent signaling enhances p70S6K activity within the ß-arrestin/p70S6K/rpS6 preassembled complex, which is not recruited to the FSHR. In agreement, FSH-induced rpS6 phosphorylation within the ß-arrestin scaffold was decreased in cells depleted of Gαs. Integration of the cooperative action of ß-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 ß-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 ß-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.

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.

[Investigation methods to explore G protein-coupled receptor-regulated translatome]. / Méthodes d'étude du traductome régulé par les récepteurs couplés aux protéines G.

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.

G Protein-Coupled Receptors As Regulators of Localized Translation: The Forgotten Pathway?

G protein-coupled receptors (GPCRs) exert their physiological function by transducing a complex signaling network that coordinates gene expression and dictates the phenotype of highly differentiated cells. Much is known about the gene networks they transcriptionally regulate upon ligand exposure in a process that takes hours before a new protein is synthesized. However, far less is known about GPCR impact on the translational machinery and subsequent mRNA translation, although this gene regulation level alters the cell phenotype in a strikingly different timescale. In fact, mRNA translation is an early response kinetically connected to signaling events, hence it leads to the synthesis of a new protein within minutes following receptor activation. By these means, mRNA translation is responsive to subtle variations of the extracellular environment. In addition, when restricted to cell subcellular compartments, local mRNA translation contributes to cell micro-specialization, as observed in synaptic plasticity or in cell migration. The mechanisms that control where in the cell an mRNA is translated are starting to be deciphered. But how an extracellular signal triggers such local translation still deserves extensive investigations. With the advent of high-throughput data acquisition, it now becomes possible to review the current knowledge on the translatome that some GPCRs regulate, and how this information can be used to explore GPCR-controlled local translation of mRNAs.

Follicle-Stimulating Hormone Receptor: Advances and Remaining Challenges.

Follicle-stimulating hormone (FSH) is produced in the pituitary and is essential for reproduction. It specifically binds to a membrane receptor (FSHR) expressed in somatic cells of the gonads. The FSH/FSHR system presents many peculiarities compared to classical G protein-coupled receptors (GPCRs). FSH is a large naturally heterogeneous heterodimeric glycoprotein. The FSHR is characterized by a very large NH2-terminal extracellular domain, which binds FSH and participates to the activation/inactivation switch of the receptor. Once activated, the FSHR couples to Gαs and, in some instances, to other Gα-subunits. GPCR kinases and ß-arrestins are also recruited to the FSHR and account for its desensitization, the control of its trafficking and its intracellular signaling. Of note, the FSHR internalization and recycling are very fast and involve very early endosomes (EE) instead of EE. All the transduction mechanisms triggered upon FSH stimulation lead to the activation of a complex signaling network that controls gene expression by acting at multiple levels. The integration of these mechanisms not only leads to context-adapted responses from the target gonadal cells but also indirectly affects the fate of germ cells. Depending on the physiological/developmental stage, FSH elicits proliferation, differentiation, or apoptosis in order to maintain the homeostasis of the reproductive system. Pharmacological tools targeting FSHR recently came to the fore and open promising prospects both for basic research and therapeutic applications. This chapter provides an updated review of the most salient aspects and peculiarities of FSHR biology and pharmacology.

ß-arrestin signalling and bias in hormone-responsive GPCRs.

G protein-coupled receptors (GPCRs) play crucial roles in the ability of target organs to respond to hormonal cues. GPCRs' activation mechanisms have long been considered as a two-state process connecting the agonist-bound receptor to heterotrimeric G proteins. This view is now challenged as mounting evidence point to GPCRs being connected to large arrays of transduction mechanisms involving heterotrimeric G proteins as well as other players. Amongst the G protein-independent transduction mechanisms, those elicited by ß-arrestins upon their recruitment to the active receptors are by far the best characterized and apply to most GPCRs. These concepts, in conjunction with remarkable advances made in the field of GPCR structural biology and biophysics, have supported the notion of ligand-selective signalling also known as pharmacological bias. Interestingly, recent reports have opened intriguing prospects to the way ß-arrestins control GPCR-mediated signalling in space and time within the cells. In the present paper, we review the existing evidence linking endocrine-related GPCRs to ß-arrestin recruitement, signalling, pathophysiological implications and selective activation by biased ligands and/or receptor modifications. Emerging concepts surrounding ß-arrestin-mediated transduction are discussed in the light of the peculiarities of endocrine systems.