ANALYSIS OF BIASED SIGNALING IN THE CHEMOKINE SYSTEM
Creators: name: VACCHINI, ALESSANDRO
Publisher: name: Universit� degli Studi di Milano
Year: 2016

description: Chemokines constitute a family of almost 50 small secreted cytokines, recognized by about 20 different 7TM spanning G protein coupled receptors (GPCRs), that activating pertussis toxin sensitive G proteins induce cell migration. These receptor are abundantly expressed by leukocytes and, controlling cell migration, they dictate leukocyte positioning during homeostatic patrolling within peripheral tissues, their maintenance in bone marrow during maturation and in addition mediate their recruitment to inflamed tissues. Upon inflammation in fact a number of chemokines are produced or activated by inflammatory mediators and diffuse within the tissue, generating a chemical gradient along which leukocytes migrate to reach the center of inflammation to contain and remove the insulting factor. This system needs an extremely tight control, since its dysregulation has been demonstrated to be at the basis of different inflammatory diseases, auto-immunity and has also been linked to cancer development. In particular, in this thesis we focused our attention on two regulatory system: post-translational modifications of chemokines, mediated by enzymes specifically released upon inflammation also by immune cells, and on the activity of atypical chemokine receptors, a subfamily of chemokine receptors that despite high structural homology and similar binding properties compared to conventional chemokine receptors, are unable to drive chemotaxis but act instead as key regulators of the chemokine system activity. In detail we looked at the ability of these regulatory mechanisms to modulate chemokine signaling properties generating a biased signaling, an emerging feature of GPCR pharmacology that describes the ability of a given receptor to elicit different or even opposite functional activities depending on the ability of different agonists to stabilize different receptor?s active structural conformation, resulting in different phenotypes mediated by the same receptor. In the chemokine system biased signaling has already been described to occur on different receptors upon binding of their different ligands, therefore during our investigation on chemokine regulatory system signaling we maintained our focus on the ability of these systems to bias chemokine signaling properties in order to better understand how this regulation occurs. To this point we assessed the ability of differently post-translationally modified chemokines to elicit signaling activities on different receptors by measuring in HEK293 cells their potential in inhibiting adenylyl cyclase, a proximal downstream signal of G? inhibitory proteins activation, and in inducing ?-arrestin recruitment to the receptors in energy transfer-based assays. We also compared signaling properties of an atypical chemokine receptor to the ones elicited by a conventional receptor analyzing the phosphoproteome modifications occurring constitutively and after stimulation with the same agonist. Our results indicate that regulation of CXCL5 and CXCL8 chemokine activity by post-translational modifications is more prone to regulate chemokine activity modifying chemokine potency, rather than generating a bias in their signaling properties. Truncation of chemokine NH2-terminus increases both CXCL5 and CXCL8 activity, while citrullination of the most NH2-terminal arginine results in opposite effects on the two agonists since on CXCL8 increases chemokine potency, while it reduces CXCL5 activity. We investigated the properties of ACKR2 in recruiting ?-arrestins, demonstrating that this receptor is able to associate both ?-arrestin 1 and 2 in basal conditions while upon agonist stimulation preferentially increases its association with ?-arrestin 1, resulting in a completely different agonist-induced outcome of proteome phosphorylation, compared to CCR5, in terms of kinetics, protein phosphorylation modifications, biological function of the regulated proteins and signal mediators activated. Taken together, these results indicate that chemokine system regulation is based not only on chemokine post-translational modifications that modulate chemokine potency, but also on the activity of structurally biased atypical chemokine receptors, as in the case of ACKR2 that interacts with different effectors and kinetics to generate distinct functional outcomes, compared to the conventional chemokine receptor CCR5. In this thesis it has also been attempted to translate the investigation of chemokine signaling to a clinical intervention for inflammatory diseases. We assessed the modulation of CXCR1 signaling activity exerted by Reparixin, a leukocyte migration inhibitor that blocks cell recruitment to inflamed tissues, that binds to its target receptors in an allosteric binding site, without inhibiting chemokine binding to the receptor. In our assays performed on HEK293 cells we could not detect any inhibition of the signaling pathways assayed, possibly indicating that HEK293 cells are not the best model to assay the activity of this molecule, with the need to assess in this cellular model drug inhibitory activity on read-outs already evaluated in literature on other cell types. In conclusion, we can say that observations described in this thesis allow to better understand chemokine system regulation, that occurs by biased signaling activity in the case of atypical chemokine receptors that by selected interaction with signaling mediators induce opposite biological outcomes compared to conventional chemokine receptors, while post-translational modifications regulate chemokines activity modulating their potency, rather than biasing their signaling properties.