Análisis de los residuos de los receptores de quimioquinas implicados en su dimerización y en la unión a Janus quinasas: nuevas dianas terapéuticas para alterar la funcionalidad de las quimioquinas

Abstract

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-12-2005The chemokines are a large family of proteins that regulate leukocyte recruitment to inflammation sites and coordinate immune cell trafticking throughout the body. Chemokines mediate leukocyte function by binding to and activating specific G protein-coupled receptors (GPCR) expressed by these cell populations. 1t has been described that chemokine receptor dimerization plays an important role in signaling alter ligand binding. Chemokines trigger cell responses by stabilizing receptor dimerization and association of Janus kinases (JAK) to the receptor. JAK are then activated by transphosphorylation and phosphorylate the receptor, allowing G i activation and induction of the events that determine the final cell response. Bioinformatic analyses predicted that 11e52 in the CCR5 chemokine receptor transmembrane region-1 (TM1) and Va1150 in TM4 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated non-functional receptors that did not dimerize or trigger signaling, In vitro and in vivo studies in human cell fines and primary T cells showed that synthetic peptides containing these residues blocked the responses induced by the CCR5 ligand. CCL5. Fluorescence resonance energy transfer (FRET) techniques showed the presence of preforrned, ligand-stabilized chemokine receptor oligomers. This is the first description of residues involved in chemokine receptor dimerization, and indicates a potential target for modification of chemokine responses. JAK proteins are constitutively associated with many cytokine receptors. but this association takes place in chemokine receptors only alter ligand binding. Although the cytokine region responsible for JAK binding has been described, the chemokine receptor domain involved in JAK binding remained unknown. Using hCCR2 as a model, we identified the domains involved in JAK2 binding to this receptor. We found that Leu 140 and Ala 141 in the CCR2 second intracellular loop were essential for JAK association. By mutating these residues, we generated CCR2 variants that showed no alterations in ligand binding affinity, but did not bind JAK2. These mutants were unable to signa', indicating that JAK association is crucial for chemokine function. Through a combination of in silico analysis, imaging techniques, biochemistry, and in vitro and in vivo functional analysis, we identified amino acid residues that are potential targets in the design of drugs that that affect chemokine responses specifically. 1