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  • br Experimental Procedures br Acknowledgments We


    Experimental Procedures
    Acknowledgments We thank R. Brink for providing HEL2x, M. Tanaka for CD169DTR mice, R. Lathe for making Cyp7b1+/− mice available, and M. Barnes and A. Reboldi for comments on the manuscript. T.Y. is an Irvington Institute Postdoctoral Fellow at the Cancer Research Institute, and J.G.C. in an Investigator at the Howard Hughes Medical Institute. This work was supported by National Institutes of Health grants AI40098 and HL20948. A.W.S is a current employee of Novartis and holds stock and stock options in the Novartis company.
    Introduction G-protein-coupled receptors (GPCRs) are integral cell membrane receptors organized in seven transmembrane α-helices associated to heterotrimeric G-proteins. They are involved in a plethora of physiological processes through a very efficient, specific and selective control of cell functions. In several pathologies, a dysregulation of their Radotinib and/or activity has been described, and their pharmacological targeting is at the basis of the most up-to-date therapeutic strategies for many relevant human diseases. Indeed, most marketed drugs have been developed for their ability to target GPCRs, preferentially the class-A ones [1]. In the last decades, classical in vitro and in vivo studies suggested that each GPCR has a specific pharmacological profile and is operated by highly specific ligands. All the pharmacological efforts of the last years have been focused on the development of drugs selective for a single class of GPCR, or for a specific receptor, in order to achieve a successful therapeutic strategy without serious and limiting side effects. However, recent evidence challenges the currently accepted dogma that each receptor responds to a single endogenous ligand or a single family of related signaling molecules. In this respect, oxysterols are well-known as natural and specific ligands of nuclear liver X receptor (LXR) α and β belonging to the cytoplasmic family of steroid receptors, whose activation controls lipid and cholesterol homeostasis inducing several target gene products. Furthermore, activation of LXRs by oxysterols can favor tumor progression by dampening native immune response [2]via activation of specific target genes and trans-repression of pro-inflammatory genes. In 2011, Nature published two letters, in which it was demonstrated that EBI2, an orphan class-A GPCR involved in the immune response, can be operated by oxysterols [3], [4]. Very recently, our research group contributed to demonstrating the ability of oxysterols to also operate a completely different class-A GPCR, CXCR2, a chemokine receptor involved in the control of the immune system and of cancer development [5]. All together, these observations suggest that a second level of operability, less specific and more transversal, exists, at least for some class-A GPCRs and for some ligands. The different affinity of oxysterols with respect to CXCR2 endogenous ligands also suggests that this second activation mode can become of great interest under specific pathological conditions, e.g. when oxysterols are produced locally at very high concentrations. These results also raise the hypothesis that activation of some class-A GPCRs by oxysterols represents a new way to modulate some specific immune system function. At the moment, no molecular data about the activation mechanism of CXCR2 by oxysterols have been reported. In order to carefully analyze at an atomistic level this mechanism, and to evaluate if oxysterols can act as transversal ligands also for other structurally related class-A GPCRs, we modeled, through comparative in silico modeling, the 3D structures of human CXCR2 and of two phylogenetically and structurally related receptors, EBI2 and GPR17, a P2Y-like receptor involved in central nervous system function [6], [7]. We then investigated the mechanism of molecular recognition between these three receptors and oxysterols through in silico molecular docking and well-established in vitro experimental approaches. Results show that oxysterols bind to and activate transversally the three receptors, thus extending the number of GPCRs and tissues involved in this new type of signaling, with significant implications for the biology and pharmacology of these systems.