CXCR4 is a G-coupled receptor for the stromal cellderived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH 2 -terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/ Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu 14 and/or Glu 15 and Tyr 21 . Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp 187 ), TMII (Asp 97 ), and TMVII (Glu 288 ). The first residues (2-9) of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.

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...

Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumorbearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited β-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and β-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.malignancy | functional selectivity | cellular idling | cancer therapeutics | chemokine oligomer C hemokines are chemoattractant cytokines that bind G-protein-coupled receptors and are mediators for many physiological processes including cell trafficking, angiogenesis, and embryogenesis (1-3). Chemokine receptor signaling is linked with cancer metastasis as well as infiltration of tumor-associated immune cells, neoangiogenesis, and proliferation (4, 5). Chemokines as primary mediators of metastasis were first identified by Muller and colleagues who implicated the chemokine receptor CXCR4 in tumor cell trafficking (6-8). At least 23 different cancers have been shown to express elevated levels of CXCR4, sensitizing these cancers to CXCL12 gradients in distant tissues (9). CXCL12 is constitutively expressed in the bone marrow, lungs, and liver, which are common tissues of metastatic growth. Efforts to block metastatic dissemination have mainly used small molecule antagonists of CXCR4 (10) to limit cancer malignancy (11, 12). However, this avenue has proven difficult to move into the clinic (5, 12), suggesting alternative strategies to interfere with CXCR4-guided metastatic homing (for example, by the use of agonists rather than antagonists) are required. Our previous data indicate that epigenetic silencing of the Cxcl12 promoter enhances metastasis of colonic and mammary carcinoma, implicating...