The bicyclam AMD3100 is known as a small synthetic inhibitor of the CXCL12-binding chemokine receptor CXCR4. Here, we show that AMD3100 also binds to the alternative CXCL12 receptor CXCR7. CXCL12 or AMD3100 alone activate ␤-arrestin recruitment to CXCR7, which we identify as a previously unreported signaling pathway of CXCR7. In addition, AMD3100 increases CXCL12 binding to CXCR7 and CXCL12-induced conformational rearrangements in the receptor dimer as measured by bioluminescence resonance energy transfer. Moreover, small but reproducible increases in the potency of CXCL12-induced arrestin recruitment to CXCR7 by AMD3100 are observed. Taken together, our data suggest that AMD3100 is an allosteric agonist of CXCR7. The finding that AMD3100 not only binds CXCR4, but also to CXCR7, with opposite effects on the two receptors, calls for caution in the use of the compound as a tool to dissect CXCL12 effects on the respective receptors in vitro and in vivo.Chemokine receptors belong to the G-protein coupled receptor (GPCR) family. GPCR binding by ligands is believed to alter receptor conformation in a way that is transmitted to the cytoplasmic face of the receptor and triggers the activation of the heterotrimeric G-proteins as well as that of other G-protein-independent effectors. Although ligand-induced conformational change of the receptor has for long been deduced from functional data, the advent of new biophysical methods has eventually permitted direct measurement of such changes in native receptors present in the plasma membrane of live cells. bioluminescence resonance energy transfer (BRET) is one of the resonance energy transfer techniques used to show constitutive dimerization of chemokine receptors and other GPCRs (Terrillon and Bouvier, 2004). Constitutively dimeric GPCR BRET couples can also be used to probe receptor conformation, because BRET depends on the distance between the luminescence donor [Renilla reniformis luciferase (RLuc)] and the acceptor [the yellow fluorescent protein (YFP)]. For instance, the use of CXCR4-RLuc/ CXCR4-YFP dimers as sensors permitted the detection of different conformations in a panel of CXCR4 mutants (Berchiche et al., 2007). Moreover, dimeric BRET sensors permit the measurement of ligand-induced conformational changes in the receptor dimer (Ayoub et al., 2004;Percherancier et al., 2005). These effects are not only observed for cognate agonists but also for small synthetic ligands that may be orthosteric or allosteric modulators.Allosteric modulation of receptor-ligand interactions results from binding of a second (allosteric) ligand to a distinct site on the receptor, in a way that does not directly compete with binding of the cognate (orthosteric) ligand. Binding of the allosteric ligand may decrease the affinity of the cognate ligand, resulting in negative allosteric modulation. Conversely, the presence of the allosteric modulator may increase binding of the cognate ligand, called positive allosteric modulation (May et al., 2007). Allosteric ligands may also have ...

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...

The G protein-coupled receptor (GPCR) C-X-C chemokine receptor 3 (CXCR3) is a potential drug target that mediates signaling involved in cancer metastasis and inflammatory diseases. The CXCR3 primary transcript has three potential alternative splice variants and cell-type specific expression results in receptor variants that are believed to have different functional characteristics. However, the molecular pharmacology of ligand binding to CXCR3 alternative splice variants and their downstream signaling pathways remain poorly explored. To better understand the role of the functional consequences of alternative splicing of CXCR3, we measured signaling in response to four different chemokine ligands (CXCL4, CXCL9, CXCL10, and CXCL11) with agonist activity at CXCR3. Both CXCL10 and CXCL11 activated splice variant CXCR3A. Whereas CXCL10 displayed full agonistic activity for Gαi activation and extracellular signal regulated kinase (ERK) 1/2 phosphorylation and partial agonist activity for β-arrestin recruitment, CXCL9 triggered only modest ERK1/2 phosphorylation. CXCL11 induced CXCR3B-mediated β-arrestin recruitment and little ERK phosphorylation. CXCR3Alt signaling was limited to modest ligand-induced receptor internalization and ERK1/2 phosphorylation in response to chemokines CXCL11, CXCL10, and CXCL9. These results show that CXCR3 splice variants activate different signaling pathways and that CXCR3 variant function is not redundant, suggesting a mechanism for tissue specific biased agonism. Our data show an additional layer of complexity for chemokine receptor signaling that might be exploited to target specific CXCR3 splice variants.

The Gag-Pol polyprotein of the human immunodeficiency virus type 1 (HIV-1) is the precursor of the virus enzymatic activities and is produced via a programmed -1 translational frameshift. In this study, we altered the frameshift efficiency by introducing mutations within the slippery sequence and the frameshift stimulatory signal, the two elements that control the frameshift. These mutations decreased the frameshift efficiency to different degrees, ranging from approximately 0.3% to 70% of the wild-type efficiency. These values were mirrored by a reduced incorporation of Gag-Pol into virus-like particles, as assessed by a decrease in the reverse transcriptase activity associated to these particles. Analysis of Gag processing in infectious mutant virions revealed processing defects to various extents, with no clear correlation with frameshift decrease. Nevertheless, the observed frameshift reductions translated into equivalently reduced viral infectivity and replication kinetics. Our results show that even moderate variations in frameshift efficiency, as obtained with mutations in the frameshift stimulatory signal, reduce viral replication. Therapeutic targeting of this structure may therefore result in the attenuation of virus replication and in clinical benefit.