Bioluminescence Resonance Energy Transfer Reveals Ligand-induced Conformational Changes in CXCR4 Homo- and Heterodimers

Abstract

Homo-and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo-and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo-and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.In recent years, the concept of GPCR 1 dimerization has raised questions about the molecular details and functional role of such oligomeric assembly (for a recent review, see Ref.1). Given the clinical interest in GPCRs, insights into the structural and functional organization of the receptor complexes have the potential to facilitate the design of new drug candidates with increased efficacy and selectivity. Resonance energy transfer (RET) techniques have emerged as methods of choice to study receptor dimerization in living cells. Although most RET studies indicate that many if not all GPCRs exist as dimers or higher oligomers under basal conditions, apparent contradictions exist concerning their potential dynamic regulation upon ligand binding. Although numerous authors did not find any effects of ligands on constitutive RET signals in their systems (2-8), others observed ligand-promoted increases or decreases that were interpreted as either the formation (9 -11) or the dissociation (12-15) of GPCR dimers in response to receptor activation. Conformational changes within pre-existing constitutive dimers have also been proposed as alternative explanations for agonist or antagonist-induced changes in .Chemokine receptors such as CCR2 and CXCR4 have been reported to form homo-and heterodimers (3, 4, 19 -24). In early co-immunoprecipitation studies, proposed that the dimerization of CXCR4 is induced upon activation by its chemokine ligand SDF-1. In contrast, data obtained with RET techniques revealed that CXCR4 homo-dimers form spontaneously in the absence of ligand (3,4,24). In one study, no significant effect of SDF-1 was observed on the constitutive energy transfer (4), whereas a small but reproducible increase was detected by others (24). As for CXCR4, agonist stimulation of CCR2 was found to promote the formation of dimers as revealed by chemical cross-linking followed by immunoprecipitati...

Ligand stabilization of CXCR4/δ‐opioid receptor heterodimers reveals a mechanism for immune response regulation

“…Recent evidence shows that chemokines interfere with MOR and KOR antinociceptive activity [49,50]. CXCR4 forms homo- [22,51] and heterodimers [38,52] at the cell surface. Our data are compatible with a model in which the receptor conformation stabilized depends on several factors including (i) receptor levels at the cell surface, (ii) ligand levels in the cell microenvironment, and (iii) the presence of other factors that alter receptor or ligand expression and function.…”

“…The results confirmed that DOR-CXCR4 complexes also form in primary cells in a ligand-independent manner. Many reports show that CXCR4 and DOR form homoand heterodimers [8,10,22,37,38]. To determine whether these conformations are fixed or in dynamic equilibrium, we cotransfected HEK293 cells with CXCR4-CFP, CXCR4-YFP and increasing amounts of DOR.…”

“…We thus hypothesized that DOR-complexed CXCR4 binds CXCL12 in an inactive conformation. Several CXCR4 mutants were described that bind CXCL12 but do not signal [45], and recent evidence suggests that ligand binding induces conformational changes in CXCR4 [38]. GPCR oligomerization may also be crucial for modulating signal transduction triggered by individual receptors [6].…”

Le baclofène est un modulateur allostérique du récepteur CXCR4

“…Ce résultat contredit nos observations initiales d'un blocage de CXCR4 par ces substances. À l'aide de la technique TIRF, nous avons montré que les récepteurs CXCR4 ne forment pas de dimères lorsqu'ils sont exprimés à la membrane des ovocytes de xénope, contrairement à ce qui est décrit dans les cellules de mammifères [14,15]. Nous avons alors utilisé un autre système cellulaire, les cellules Nous avons d'abord pensé que les récep-teurs GABA B et CXCR4 pouvaient interagir dans la membrane des cellules : en effet, on sait que CXCR4 peut former des hétérodimères avec d'autres récepteurs couplés aux protéines G [12,13].…”

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“…Toutefois, il exclut clairement les antagonistes de CXCR4 de l'arsenal antirétroviral, dans une perspective de thérapie permanente, à moins que l'on ne soit en mesure de [27]. Grâce à cette technique, notre équipe a démontré que CXCR4 forme un homodimère constitutif [28]. De plus, nous avons établi que l'application de différents ligands naturels et synthétiques sur le dimère de récepteur module le BRET qui en résulte, ce qui permet l'observation des changements conformationnels du récepteur induits par ces ligands.…”