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Etude des mécanismes d'activation des récepteurs chimiotactiques couplés aux protéines G : Biais de signalisation et rôle d'une proline dans le deuxième domaine transmembranaire

Abstract : G protein-coupled receptors (GPCRs) display a form of conservation in term of structure and signaling mode but also show diversification, allowing specialization. An important specialization during evolution is undoubtedly the pro-chemoattractant capability that enables the development of organs and vascularization, or immune responses. These chemotactic behaviors may involve G protein couplings but also interaction with intracellular proteins including β-arrestins. However, still reside the questions on i) their ability to detect low concentrations / molecular gradients, ii) transducing the extracellular binding domain signal to the intracellular domain of the receptor, and iii) the modes of interaction of β- arrestins to the cytosolic GPCR domains. Recent data provided by the high-resolution structures indicate that GPCRs are dynamic entities that adopt multiple conformations, i.e. active able to link different types of ligands to activate in a biased manner receptor coupling pathways, leading to functional selectivity. Based on previous works of the team, we hypothesized that urotensin II (UII)-derived peptides displayed unexpected physiological effects because of such biased signaling on the GPCR UT receptor. We determined the coupling to G proteins and β-arrestins of the UIIactivated UT receptor expressed in HEK293 using bioluminescence resonance energy transfer (BRET) biosensors, as well as the production of IP1-3 and cAMP using homogenous timeresolved Forster resonance energy transfer (FRET) (HTRF)-based assays. We showed that activated UT coupled to Gi1, GoA, Gq, and G13, excluding Gs, and recruited β-arrestins 1 and 2. Integration of these pathways led to a 2-phase kinetic phosphorylation of ERK1/2 kinases. The tested peptides induced three different profiles: UII, urotensin-related peptide (URP), and UII4–11 displayed the full profile; [Orn8]UII and [Orn5]URP activated G proteins, although with pEC50s 5–10× higher, and did not or barely recruited β-arrestin; urantide also failed to recruit β-arrestin but displayed a reversed rank order of pEC50s for Gi and Gq vs. Go and was a partial agonist of all G-protein pathways. Interestingly, the peptides differently modulated cell survival but similarly induced cell migration and adhesion. Thus, we demonstrate biased signaling between β-arrestin and G proteins, and between G-protein subtypes, which dictates the receptor's cellular responses and may open therapeutic new opportunities. We thus addressed whether the chemotactic properties of UT, quite unexpected since the urotensinergic system was previously characterized as a potent vasoactive system, may evolve from a structural signature acquired early during evolution. Based on previous published data, it is proposed that an evolutionary ancestral deletion in TM2, leading to a repositioning of a Abstract proline in 2.58, of some Rhodopsine-like GPCRs belonging to the PEP (peptide) receptor family, allowed expansion of the SO (somatostatinergic), CHEM (chemokine) and PUR (purinergic) receptors phylogenetically linked. Thus, by studying the PEP receptor prototype Kiss1R, exhibiting a proline in 2.59, and the UT and CXCR4 receptors, as respective prototypes of SO and CHEM receptor groups, we aimed to gain information on the role of this P2.58 in the acquisition of chemotactic behaviour. Thus, we have proceeded by engineering the P2.59 prototype Kiss1R and the two P2.58 UT and CXCR4 receptors by single mutation of Pro to Ala or insertion/deletion to reposition the Pro. We showed that wild-type and mutant Kiss1R, UT and CXCR4 expressed in HEK293 cells conserved expression levels and for most, binding capacities. The P2.58 UT and CXCR4 receptors activated by UII and SDF-1α, exhibited Gq/Ca2+/IP1 (UT) and/or Gi/o and β-Arrestin 2 couplings, as well ERK1/2 activation. They stimulated chemotactic migration via dynamic wave of assembly/disassembly of focal adhesions (FAs) associated with the selection of one prominent lamellipodia. In contrast, activation of the P2.59 Kiss1R receptor by Kisspeptin (KP-10) evoked Gq/Ca2+/IP1 couplings, and random migration through uncontrolled disassembly of FAs and production of many lamellipodia per cells. Single mutation or insertion/deletion mainly led to loss of Gq and/or Gi1 and Goa coupling capacities, establishing the key role of the proline in TM2 of Kiss1R, UT and CXCR4 in receptor activation. In addition, the P2.59 to P2.58 repositioning likely played a role in receptor re-sensitization, determinant for regulation of FAs assembly/disassembly and reduction of the number of lamellipodia. Thus, we propose that P2.58 receptors expanding during evolution, acquired a key competence to favor cell chemotactic migration under external ligand signals. This would be associated with a dynamic of receptor desensitization/resensitization and localized adhesionde- adhesion waves, selecting protrusions necessary for cell directed migration. This specific competence of P2.58 in general and the UT receptor in particular, likely involves a key functional selectivity towards β-arrestins.
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Jane-Eileen Joubert. Etude des mécanismes d'activation des récepteurs chimiotactiques couplés aux protéines G : Biais de signalisation et rôle d'une proline dans le deuxième domaine transmembranaire. Biologie cellulaire. Université Rouen Normandie, 2016. Français. ⟨tel-02415576⟩

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