Nikolaus Heveker

Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers

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.

AMD3100 Is a CXCR7 Ligand with Allosteric Agonist Properties

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 β-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.

Identification of Residues of CXCR4 Critical for Human Immunodeficiency Virus Coreceptor and Chemokine Receptor Activities

CXCR4 is a G-coupled receptor for the stromal cell-derived 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 NH2-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 Glu14 and/or Glu15 and Tyr21. Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp187), TMII (Asp97), and TMVII (Glu288). 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.

Antigenically Distinct Conformations of CXCR4

ABSTRACT The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1α and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways

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 tumor-bearing 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.

Neuropilin-1 is involved in human T-cell lymphotropic virus type 1 entry.

ABSTRACT Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted through a viral synapse and enters target cells via interaction with the glucose transporter GLUT1. Here, we show that Neuropilin-1 (NRP1), the receptor for semaphorin-3A and VEGF-A165 and a member of the immune synapse, is also a physical and functional partner of HTLV-1 envelope (Env) proteins. HTLV-1 Env and NRP1 complexes are formed in cotransfected cells, and endogenous NRP1 contributes to the binding of HTLV-1 Env to target cells. NRP1 overexpression increases HTLV-1 Env-dependent syncytium formation. Moreover, overexpression of NRP1 increases both HTLV-1 and HTLV-2 Env-dependent infection, whereas down-regulation of endogenous NRP1 has the opposite effect. Finally, overexpressed GLUT1, NRP1, and Env form ternary complexes in transfected cells, and endogenous NRP1 and GLUT1 colocalize in membrane junctions formed between uninfected and HTLV-1-infected T cells. These data show that NRP1 is involved in HTLV-1 and HTLV-2 entry, suggesting that the HTLV receptor has a multicomponent nature.

Dissociation of the signalling and antiviral properties of SDF-1-derived small peptides

BACKGROUND The chemokine receptor CXCR4 (a receptor for the Cys-X-Cys class of chemokines) is a CD4-associated coreceptor for T-cell-tropic strains of human immunodeficiency virus 1 (HIV-1) and represents a target for antiviral therapy. Infection by T-tropic HIV-1 can be blocked by stromal-cell-derived factor-1 (SDF-1), the natural ligand of CXCR4. The broad variety of cells expressing CXCR4 and the perturbations observed in mice deficient for SDF-1 suggest that antiviral compounds antagonizing the signalling activity of CXCR4 might have severe side effects in vivo. Compounds that interfere selectively with HIV entry and not with SDF-1 signalling would therefore be useful. RESULTS A series of peptides, each of 13 residues, spanning the whole SDF-1alpha sequence were tested for their ability to block HIV-1 infection. The antiviral and signalling properties of SDF-1 were retained by a peptide corresponding to its amino terminus. Removal of the first two residues resulted in an antiviral antagonist of the SDF-1-CXCR4 signalling pathway. We prepared 234 single-substitution analogues and identified one antiviral analogue that had drastically reduced agonistic or antagonistic properties. The antiviral peptides competed with the monoclonal antibody 12G5 for CXCR4 binding. Their antiviral activity seems to be due to receptor occupancy rather than induction of receptor endocytosis. CONCLUSIONS The amino terminus of the SDF-1 chemokine is sufficient for signal transduction via CXCR4 and for inhibition of HIV-1 entry, but these activities could be dissociated in a peptide analogue. This peptide represents a lead molecule for the design of low molecular weight antiviral drugs.

Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay

ABSTRACT We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.

BRET analysis of GPCR oligomerization: newer does not mean better

James et al.(1) proposed a “rigorous” treatment of bioluminescence resonance energy transfer (BRET) data to distinguish random (non-specific) from true oligomeric protein interactions. The question is not trivial and the intention laudable since BRET has become increasingly popular with more than 100 original articles published. However, the authors dismissed many studies that addressed similar issues and several points deserve comments. The authors preface their study by the statement: “Conventional BRET experiments are presently done at high levels of expression and at a single acceptor:donor ratio”. Although it is true of some studies, such general statement ignores a large body of work where these parameters were taken into account. Expression levels were monitored in many studies and found to be within physiological range (2–5) and at least 15 papers performed BRET titration assay where the ratio donor:acceptor was varied (ex: 2,4–6). Specificity of the BRET signals allowing to distinguish oligomerization from random collisions has also been verified by several authors using BRET competition assays where the occurrence of BRET between two partners expressed at a given donor:acceptor ratio can be inhibited by expression of the non-tagged partners but not of a non-tagged non-interacting protein (ex: 3,6–8). James et al. propose to differentiate random from true oligomeric interactions based on theoretical considerations summarized in a seminal article by Kenworthy and Edidin (9). The first approach consists in studying BRET efficiency (BRETeff) in experiments where the acceptor:donor (GFP/Luc) ratio is varied (type I assay). Random interactions are expected to be less sensitive to the acceptor:donor ratio if the surface density of the acceptor remains low. In multiple previous studies reported in the literature, the change of acceptor:donor ratio was obtained by maintaining the donor concentration fixed and progressively increasing the acceptor and true oligomeric interactions deduced from the hyperbolic progression of the BRET (ex: 2,4–6). In the experiments conducted by James et al., the total concentration of acceptor+donor was maintained constant by inversely changing the concentrations of both donor and acceptor and the difference between pseudo- and true- hyperbolic BRET curves used to define random collision. Such analysis is technically difficult and complicated by the fact that the efficiency of transfer for random collisions becomes independent of donor/acceptor ratio only if the acceptor concentration is kept constant (see table I in ref 9). In type I assays, James et al. also interpreted lower maximal BRET value as evidence for the lack of dimerization or equilibrium between dimers and monomers. Such interpretation is very hazardous given that the extent of RET signals vary with the distance between donors and acceptors within a dimer. Thus no direct conclusion can be drawn on the amount of dimers simply based on the maximal BRET signals observed. In type II assay, BRET for true oligomers should be independent of the concentrations of BRET partners at a fixed acceptor:donor ratio. This was previously shown to be the case for class A GPCRs (1, 11, 12) and in particular for the β2AR at receptor concentration below 15pmol/mg of protein (1). From their data, James et al. concluded otherwise. However, a close examination of Fig 4, reveals that the β2AR BRET curve has a slope that appears closer to that of the constitutive CTLA-4 dimer than that of the CD2 or CD86 monomers, consistent with the notion that the BRET between β2AR-luc and β2AR-GFP may reflect constitutive oligomerization. Also, the BRET signal observed for the constitutive dimer CD80 increase more readily with increasing expression levels than that of the β2AR, further complicating data interpretation. The fact that BRET falls below detection level at low donor/acceptor ratio may reflect lack of detector sensitivity for pairs yielding low BRET signals. Despite these interpretational difficulties, the authors concluded that the entire concept of GPCR oligomerization needs reappraisal. In their discussion concerning the specificity of the BRET signals observed in previous studies, James et al. argued that the GABAb type-2 receptor (GBR2) is a poor choice because it can itself dimerize. The reason to use this receptor as a negative control was precisely its demonstrated ability to dimerize thus offering a reliable selectivity test using a dimerization competent receptor. Also, contrary to what was implied by the authors, GBR2 is not the only negative control that was used in BRET studies. A number of other receptors were used as negative controls in BRET studies addressing class A GPCR oligomerization (ex: 3, 13–15). The observation that in some studies, ligand binding affects the maximal BRET signal between the proposed protomers of class A GPCR oligomers, is difficult to reconcile with the implicit conclusion of James et al. that the BRET signals observed for class A GPCRs most likely result from random collisions. In many of these studies, the ligand-promoted changes in BRET signal were interpreted as conformational changes within pre-existing dimers that changed the distances between the energy acceptor and donor. Finally, the notion that family A GPCRs may form constitutive oligomers is not only based on BRET studies. Many other biochemical and biophysical approaches support this notion. These include: co-immunoprecipitation, various types of FRET, atomic force microscopy, covalent cross-linking, gel filtration, neutron scattering experiments, functional complementation, cell biology studies demonstrating cross-internalization and co-processing of GPCRs as well as binding studies showing positive and negative cooperativity. These approaches, their relative strengths and caveats, including methodological considerations and potential functional outcomes have recently been reviewed (16, 17). It is therefore premature to dismiss the GPCR oligomer hypothesis based on the interpretations of a single BRET study. In conclusion, we believe that the results reported in the article by James et al. can be interpreted in different ways and that more controls would have been necessary to challenge the multidisciplinary work conducted on this topic by many groups over the past 10 years. Clearly, BRET is gaining in popularity to assess protein-protein interaction in living cells and additional quantitative approaches will certainly be forthcoming. Maybe more importantly, additional studies performed in native tissues are needed to establish the generality of GPCR dimerization in physiologically relevant systems.

Mutation of a Conserved Residue (D123) Required for Oligomerization of Human Immunodeficiency Virus Type 1 Nef Protein Abolishes Interaction with Human Thioesterase and Results in Impairment of Nef Biological Functions

ABSTRACT Nef is a myristoylated protein of 27 to 35 kDa that is conserved in primate lentiviruses. In vivo, Nef is required for high viral load and full pathological effects. In vitro, Nef has at least four activities: induction of CD4 and major histocompatibility complex (MHC) class I downregulation, enhancement of viral infectivity, and alteration of T-cell activation pathways. We previously reported that the Nef protein from human immunodeficiency virus type 1 interacts with a novel human thioesterase (hTE). In the present study, by mutational analysis, we identified a region of the Nef core, extending from the residues D108 to W124, that is involved both in Nef-hTE interaction and in Nef-induced CD4 downregulation. This region of Nef is located on the oligomer interface and is in close proximity to the putative CD4 binding site. One of the mutants carrying a mutation in this region, targeted to the conserved residue D123, was also found to be defective in two other functions of Nef, MHC class I downmodulation and enhancement of viral infectivity. Furthermore, mutation of this residue affected the ability of Nef to form dimers, suggesting that the oligomerization of Nef may be critical for its multiple functions.