Chimiokines adhésives: structure moléculaire, imagerie cellulaire et outils thérapeutiques – Adhékines

Rationale and background Among chemokines, which are secreted chemotactic cytokines, CX3CL1 and CXCL16 are exceptions: they are also expressed in transmembrane form. In this form, they surprisingly function (hence the acronym 'adhekines'), with their respective receptors CX3CR1 and CXCR6, as strong adhesive molecule pairs, leading to intercellular adhesion. CX3CR1 and CXCR6 are G-protein Coupled Receptors (GPCR) from the Rhodopsin sub-family, like any chemokine receptor. The interface ligand-receptor is usually small for such GPCR, while it is large in the case of adhesion molecules. In addition, the CXCL16/CXCR6 couple is endowed with only small adhesion potency: it does not resist to under flow adhesion. So two questions rise: What are the features that account for the fact that certain GPCR can function as adhesion molecules' How small difference (CX3CL1, CXCL16) could give rise to such variation in adhesive function' There are the central questions of our project. It is of importance since both molecules are involved in inflammatory and vascular diseases and cancer. So our project will identify new anti-inflammatory compound based on chemokines analogues. State of the art and originality of the project Our preliminary data using transfer fluorescence technique (BRET) showed that, as expected for any GPCR, CX3CR1 is aggregated. Surprisingly, we found that CX3CL1 is also oligomeric, while CXCL16 does not seem. As for integrins, a better oligomerization could give a larger adhesiveness [Hermand et al. J.Biol.Chem. 2008]. Besides, we showed that in some cases the adhesion via CX3CR1/CX3CL1 is dependent on intracellular signaling (Gi, RhoA) [Daoudi et al. J.Biol.Chem. 2004]. So our project consists in systematically comparing the adhesive capacities of CX3CL1/CX3CR1 and CXCL16/CXCR6 couples, visualizing the interacting molecules during intercellular adhesion, finding the protein domains (ligand-receptor interface, monomer-monomer interface, post-traductional modification) involved in adhesiveness either in adhesive chemokine or in their receptors. Our working program has three parts: 1- The fluorescence transfer techniques (BRET, HTRF in collaboration with CisBio International) will allow characterization of the clustering features of chemokines and receptors. By directed mutagenesis and selected deletion, we seek to identify the protein domains involved in agregation, ligand-receptor interaction and adhesion. To monitor the lateral diffusion rate, which gives access to the agregation degree, we will use the original FRAPP technique (bleaching with interference fringe pattern, collaboration with UMR CNRS 8550), which is more precise than standard FRAP. 2- Beyond standard methods to assay the intercellular adhesion (static, flow adhesion), we will quantify adhesion by the original dual pipette aspiration technique, which monitors the force required to dissociate a cell doublet (collaboration with UMR CNRS 8550). This system will be extended to imaging the dynamics of the molecules involved in the adhesion using confocal fluorescence microscopy. The same imaging will be used to follow up by homo-FRET and FRAPP the agregation state and the lateral diffusion of the molecules involved in the adhesion, i.e. chemokines and receptors, and the signaling proteins implicated in adhesion (Gi, RhoA,?). 3- By molecular modeling, we will analyze the key residues of the differences between CX3CR1 and CXCR6, as well as between natural CX3CR1 variants. The molecular determinants of the ligand/receptor interface will be studied using the 'docking' method. This will be done in collaboration with the INSERM U773 lab, which works on the structure/function relationships of another GPCR, the VIP receptor. This modeling task will help to design analogues of CXCL16 and of CX3CL1, endowed with antagonistic or super-agonistic properties. This could be the starting point of a new chemokine-based therapeutic. Expected results This interdisciplinary work gathers laboratories which are already collaborating. It should permit to investigate a still unknown field, say how some chemokines could work as adhesion molecules. It will identify the cardinal features for capture and distribution of leukocytes across endothelial barrier. It will determine the molecular domains differentially involved in adhesion and migration, allowing combating against inflammation. These would be the basis for a new pharmacology against cardiovascular diseases and cancer. Finally, it will set up an original imaging technique (confocal imaging and simultaneous monitoring of diffusion by FRAPP during adhesion process with micromanipulated cells) which would be useful in other cases of intercellular adhesion.