Blanc SVSE 6 - Sciences de la vie, de la santé et des écosystèmes : Génomique, génomique fonctionnelle, bioinformatique, biologie systémique

Deciphering beta-arrestin-dependent signalling network engaged by G protein-coupled receptors. – GPCRnet

Deciphering the cell information system: a step towards new drugs.

Plasma membrane receptors are targeted by the majority of therapeutic drugs. To understand how, once activated, they can pilot cell fate is a major issue which requires multi-disciplinary approaches combining high throughput biological methods and mathematical modeling.

To clarify beta-arrestins’ roles to discover new drugs

G protein-coupled receptors (GPCR) are involved in most physiological and pathological processes. Moreover, they are targeted by almost 50% of currently marketed drugs. The goal of GPCRnet is to gain a better understanding of the mechanisms which transmit the information flux from activated GPCRs towards intracellular compartments. It has long been known that G proteins play a crucial role in these processes. However, it is now recognized that most GPCRs also transduce signals inside the cell through a beta-arrestin-dependent mechanism which is independent of G proteins. We want to dissect the respective contributions of beta-arrestins and G proteins in GPCR-mediated biological responses. The current project will allow, from instance, to determine whether beta-arrestins induce a unique signaling network or whether it changes as a function of the receptor to which they are associated. More generally, GPCRnet should provide a conceptual framework allowing us to understand and perhaps to anticipate how an extracellular signal that activates a GPCR triggers a given physiological or pathological response. So far, only G protein-dependent mechanisms have been taken into account in drug discovery. Adding béta-arrestins to the picture could lead to the discovery of new and potentially more specific drugs.

Three model GPCRs are compared in a unique cellular context: follicle stimulating hormone (FSHR) and type 2C or 4 serotonin (5HT2CR and 5HT4R) receptors. The effects of selective beta-arrestin depletion on signaling pathways and gene regulation are investigated using high throughput methods. Intracellular signaling pathways are known to involve protein phosphorylation cascades. We carry out quantitative and high throughput approaches for analyzing protein phosphorylation in order to identify beta-arrestin-dependent phosphoproteome. Results are then validated and expended using a protein microarray method, which allows comparing thousands of biological samples in a single experiment. In addition, beta-arrestin phosphorylation itself is extensively analyzed. In parallel, DNA chips are used to exhaustively study gene regulation and to identify beta-arrestin-regulated genes. The data are then used to build and to calibrate computational models. In depth study of these models through simulation should lead to original predictions which in turn will be experimentally challenged. The ultimate validations will be achieved in “authentic” cells such as neurons in the case of serotonin receptors or gonadal cells in the case of follicle stimulating hormone.

High throughput analyses for phosphoproteins and genes are coming along nicely and will fuel the computational modeling efforts.
Some interesting results have already been obtained:
• New phosphopeptides have been identified on beta-arrestins. One of them is induced upon activation of the 3 receptors. The functional impact of this ligand-induced phosphorylation is currently investigated. A paper is expected.
• DNA chips experiments demonstrate that beta-arrestin 1 and 2 differentially regulate gene expression. A paper is currently being prepared.
• Thanks to a protein docking algorithm developed by partner 1, a molecular model of the MAP kinase module scaffolded on beta-arrestins has been achieved. Validation experiments are currently ongoing. A paper is expected.
• A first dynamical mathematical model of the activation of MAP kinase ERK1,2 through G protein and beta-arrestin-dependent pathways has been built thanks to an original and powerful strategy for parameter optimization. This work has been published in Mol Syst Biol (Heitzler, Durand et al., Mol Syst Biol, 8:590 2012) and will serve as a reference for constructing other dynamical models as the project moves forward.

Data that will be generated in the coming months should put us in a position to propose dynamical models predicting the intracellular consequences of GPCR activation. To understand how these complex intracellular mechanisms are leading to a given biological outcome would certainly have an impact on drug discovery.

Biological samples for protein and gene analyses have been prepared and the quality of each experiment has been controlled. Analyses are ongoing.
Some interesting results have already been obtained:
New phosphopeptides have been identified on beta-arrestins. One of them is induced upon activation of 5HT2CR and 5HT4R. Analyses are ongoing in order to determine whether FSHR can also regulate this phosphorylation site as well as to determine the functional importance of this particular phosphorylation.
A first dynamical mathematical model of the activation of MAP kinase ERK1,2 through G protein and beta-arrestin-dependent pathways has been built thanks to an original and powerful strategy for parameter optimization. This work has been published in Mol Syst Biol (Heitzler, Durand et al., Mol Syst Biol, 8:590 2012) and will serve as a reference for constructing other dynamical models as the project moves forward.

The aim of GPCRnet is to gain an unprecedented understanding of the beta-arrestin-dependent mechanisms relaying the information flux from activated G protein-coupled receptors (GPCRs) to intracellular compartments. This question is of primary importance since GPCRs represent the largest class of membrane receptors, are capable of binding to a wide diversity of molecules that regulate nearly every physiological process and because they are targets for about 50% of currently marketed drugs. It is increasingly recognized that the majority of GPCRs can transduce signals via at least one generic G protein-independent mechanism involving beta-arrestins and their associated protein networks. The role of beta-arrestins in switching from G protein-dependent to independent signalling, places them in a pivotal position to dictate the downstream effects of ligand binding. A number of important biological functions such as dopaminergic neurotransmission, atherosclerosis and neointimal hyperplasia have been shown to depend upon beta-arrestin-dependent signalling. Moreover, several GPCR ligands have already been reported to selectively activate or inhibit beta-arrestin signalling. Accordingly, understanding the functioning of these molecules in a major challenge to discover and optimize new GPCR drugs with more selective actions and hence less pronounced side effects.
GPCRnet is an integrated project aimed at dissecting the respective contributions of beta-arrestin 1 & 2 to the biological responses triggered by three model GPCRs namely the follicle stimulating hormone (FSH-R), the serotonin type 2C and type 4 (5HT2C-R and 5HT4-R) receptors.These three GPCRs are involved in important biological processes and pathologies. FSH is centrally involved in both male and female reproductive functions. Moreover, defects in the FSH-induced signalling cascades are known to result in high incidence health problems such as male and female infertility and possibly ovarian epithelial cancer. 5HT4-Rs are important modulators of learning and memory. 5HT2C-Rs play an essential role in the regulation of mood and alteration of their functional status has been involved in the etiology of anxio-depressive states. Comparing beta-arrestin-dependent signalling initiated upon activation of these GPCRs in a unique cellular background should indicate whether beta-arrestins induce a generic signalling signature or whether beta-arrestin-associated is dependent of the nature of their associated GPCR.
To achieve that goal, we propose to combine large-scale determination of beta-arrestin-dependent signalling pathways and gene expression in transfected HEK293 cells followed by their validation in authentic cell contexts expressing native receptors, with state of the art bioinformatics approaches.

Project coordinator

Monsieur Eric Reiter (INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE - CENTRE DE RECHERCHE DE TOURS) – Eric.Reiter@tours.inra.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE LANGUEDOC-ROUSSILLON
INRA INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE - CENTRE DE RECHERCHE DE TOURS

Help of the ANR 599,970 euros
Beginning and duration of the scientific project: - 48 Months

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