Ligand-specific conformational transitions and intracellular transport are required for atypical chemokine receptor 3-mediated chemokine scavenging.

The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses. Although unable to trigger G protein activation, both ligands activate G protein-independent ACKR3 responses and prompt arrestin recruitment. This offers a model to specifically study ligand-specific receptor conformations leading to G protein-independent signaling and to functional parameters such as receptor transport and chemokine degradation. We here show chemokine specificity in arrestin recruitment, by different effects of single amino acid substitutions in ACKR3 on arrestin in response to CXCL12 or CXCL11. Chemokine specificity in receptor transport was also observed, as CXCL11 induced faster receptor internalization, slower recycling, and longer intracellular sojourn of ACKR3 than CXCL12. Internalization and recycling rates of the ACKR3 R1423.50A substitution in response to each chemokine were similar; however, ACKR3 R1423.50A degraded only CXCL12 and not CXCL11. This suggests that ligand-specific intracellular receptor transport is required for chemokine degradation. Remarkably, the failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine. This suggests the involvement of another, yet unidentified, ACKR3 effector in scavenging. In summary, our study correlates ACKR3 ligand-specific conformational transitions with chemokine-dependent receptor transport dynamics and points toward unexpected ligand specificity in the mechanisms of chemokine degradation.

Mutational Analysis of Atypical Chemokine Receptor 3 (ACKR3/CXCR7) Interaction with Its Chemokine Ligands CXCL11 and CXCL12.

Atypical chemokine receptors do not mediate chemotaxis or G protein signaling, but they recruit arrestin. They also efficiently scavenge their chemokine ligands, thereby contributing to gradient maintenance and termination. ACKR3, also known as CXCR7, binds and degrades the constitutive chemokine CXCL12, which also binds the canonical receptor CXCR4, and CXCL11, which also binds CXCR3. Here we report comprehensive mutational analysis of the ACKR3 interaction with its chemokine ligands, using 30 substitution mutants. Readouts are radioligand binding competition, arrestin recruitment, and chemokine scavenging. Our results suggest different binding modes for both chemokines. CXCL11 depends on the ACKR3 N terminus and some extracellular loop (ECL) positions for primary binding, ECL residues mediate secondary binding and arrestin recruitment potency. CXCL12 binding required key residues Asp-1794.60 and Asp-2756.58 (residue numbering follows the Ballesteros-Weinstein scheme), with no evident involvement of N-terminal residues, suggesting an uncommon mode of receptor engagement. Mutation of residues corresponding to CRS2 in CXCR4 (positions Ser-1032.63 and Gln-3017.39) increased CXCL11 binding, but reduced CXCL12 affinity. Mutant Q301E7.39 did not recruit arrestin. Mutant K118A3.26 in ECL1 showed moderate baseline arrestin recruitment with ablation of ligand-induced responses. Substitutions that affected CXCL11 binding also diminished scavenging. However, detection of reduced CXCL12 scavenging by mutants with impaired CXCL12 affinity required drastically reduced receptor expression levels, suggesting that scavenging pathways can be saturated and that CXCL12 binding exceeds scavenging at higher receptor expression levels. Arrestin recruitment did not correlate with scavenging; although Q301E7.39 degraded chemokines in the absence of arrestin, S103D2.63 had reduced CXCL11 scavenging despite intact arrestin responses.

Design, synthesis, and biological evaluation of CXCR4 ligands.

A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.

Mode of binding of the cyclic agonist peptide TC14012 to CXCR7: identification of receptor and compound determinants.

The chemokine receptor CXCR7 is an atypical CXCL12 receptor that, as opposed to the classical CXCL12 receptor CXCR4, signals preferentially via the ß-arrestin pathway and does not mediate chemotaxis. We previously reported that the cyclic peptide TC14012, a potent CXCR4 antagonist, also engaged CXCR7, albeit with lower potency. Surprisingly, the compound activated the CXCR7-arrestin pathway. The reason underlying the opposite effects of TC14012 on CXCR4 and CXCR7, and the mode of binding of TC14012 to CXCR7, remained unclear. The mode of binding of TC14012 to CXCR4 is known from cocrystallization of its analogue CVX15 with CXCR4. We here report the the mode of binding of TC14012 to CXCR7 by combining the use of compound analogues, receptor mutants, and molecular modeling. We find that the mode of binding of TC14012 to CXCR7 is indeed similar to that of CVX15 to CXCR4, with compound positions Arg2 and Arg14 engaging CXCR7 key residues D179(4.60) (on the tip of transmembrane domain 4) and D275(6.58) (on the tip of transmembrane domain 6), respectively. Interestingly, the TC14012 parent compound T140 is not a CXCR7 agonist, because of conformational constraints in its pharmacophore, which in TC14012 are relieved through C-terminal amidation. However, an engineered salt bridge between the CXCR7 ECL2 substitution R197D and compound residue Arg1 permitted T140 agonism by repositioning the compound in the binding pocket. In conclusion, our results show that the opposite effect of TC14012 on CXCR4 and CXCR7 is not explained by different binding modes. Rather, engagement of the interface between transmembrane domains and extracellular loops readily triggers CXCR7, but not CXCR4, activation.

The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and ßarrestin.

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

Structure-based ligand discovery for the protein-protein interface of chemokine receptor CXCR4.

G-protein-coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein-protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC(50) values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein-protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.

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 ß-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 ß-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.

Restoration of renal function by a novel prostaglandin EP4 receptor-derived peptide in models of acute renal failure.

Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or ß-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.

The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element.

Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5' untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle.

Different effects of the different natural CC chemokine receptor 2b ligands on beta-arrestin recruitment, Gαi signaling, and receptor internalization.

The chemokine receptor CCR2, which has been implicated in a variety of inflammatory, autoimmune, and cardiovascular conditions, binds several natural chemokine ligands. Here, we assessed the recruitment of ß-arrestin to CCR2 in response to these ligands using bioluminescence resonance energy transfer technology. Compared with CCL2, which was considered as a full agonist, other CCR2 ligands were partial agonists with reduced efficacy and potency. Agonist potencies were not a function of their affinity for CCR2. Efficacy of arrestin recruitment matched that of agonist-induced CCR2 internalization. Although the potency and efficacy rank orders of the ligands in arrestin recruitment were similar to those observed for Gα(i1) activation, arrestin recruitment was at least in part resistant to Gα(i/o)-inactivating pertussis toxin, suggesting partial independence from Gα(i/o). The degree of pertussis toxin resistance of arrestin recruitment was different between the chemokines. Moreover, qualitative differences between the arrestin responses to the different ligands were identified in the stability of the response: although CCL7-induced arrestin recruitment had a half-life of less than 15 min, CCL8 and CCL13 induced stable CCR2-arrestin interactions. Finally, the ligands stabilized different conformations of the CCR2 homodimer. Our results support the validity of models for receptor-ligand interactions in which different ligands stabilize different receptor conformations also for endogenous receptor ligands, with corresponding implications for drug development targeting CCR2.

The peptidomimetic CXCR4 antagonist TC14012 recruits beta-arrestin to CXCR7: roles of receptor domains.

CXCR7 is an atypical chemokine receptor that signals through ß-arrestin in response to agonists without detectable activation of heterotrimeric G-proteins. Its cognate chemokine ligand CXCL12 also binds CXCR4, a chemokine receptor of considerable clinical interest. Here we report that TC14012, a peptidomimetic inverse agonist of CXCR4, is an agonist on CXCR7. The potency of ß-arrestin recruitment to CXCR7 by TC14012 is much higher than that of the previously reported CXCR4 antagonist AMD3100 and differs only by one log from that of the natural ligand CXCL12 (EC(50) 350 nM for TC14012, as compared with 30 nM for CXCL12 and 140 µM for AMD3100). Moreover, like CXCL12, TC14012 leads to Erk 1/2 activation in U373 glioma cells that express only CXCR7, but not CXCR4. Given that with TC14012 and AMD3100 two structurally unrelated CXCR4 antagonists turn out to be agonists on CXCR7, this likely reflects differences in the activation mechanism of the arrestin pathway by both receptors. To identify the receptor domain responsible for these opposed effects, we investigated CXCR4 and CXCR7 C terminus-swapping chimeras. Using quantitative bioluminescence resonance energy transfer, we find that the CXCR7 receptor core formed by the seven-transmembrane domains and the connecting loops determines the agonistic activity of both TC14012 and AMD3100. Moreover, we find that the CXCR7 chimera bearing the CXCR4 C-terminal constitutively associates with arrestin in the absence of ligands. Our data suggest that the CXCR4 and CXCR7 cores share ligand-binding surfaces for the binding of the synthetic ligands, indicating that CXCR4 inhibitors should be tested also on CXCR7.

HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165.

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.

An engineered GM-CSF-CCL2 fusokine is a potent inhibitor of CCR2-driven inflammation as demonstrated in a murine model of inflammatory arthritis.

CCR2 is a chemokine receptor widely expressed by lymphomyeloid cells involved in maladaptive autoimmune ailments. Therefore CCR2 is of great interest as a biological target for immune suppression due to its direct implication in autoimmune diseases such as rheumatoid arthritis. We have generated a novel fusion protein using GM-CSF and an N-terminal truncated version of MCP-1/CCL2 (6-76, GMME1) and investigated its utility as a CCR2-specific immune suppressor. Using BRET studies, we found that distinct to CCL2, GMME1 binding to CCR2 led to altered conformational changes in the CCR2 homodimer and did not induce the recruitment of beta-arrestin 2 to the receptor. However, CCR2-dependent calcium mobilization, BAX induction and caspase-3 activation followed by cell death was observed. Using Th17 cells harvested from DBA/1 mice ill with bovine collagen-induced arthritis, we demonstrate that GMME1 is capable of blocking their production of IL-17 in vitro. Upon its delivery to mice symptomatic with inflammatory arthritis, a robust clinical recovery occurred with decreased paw thickness to normal levels and a significant reduction in anti-collagen Ab titer and rheumatoid factor titer, as well as reduction of proinflammatory cytokines levels both intraarticular and systemic. Our data demonstrate that GMME1 is a powerful synthetic suppressor cytokine that coopts CCR2-dependent cellular signaling and blunts the effects of CCR2-expressing lymphomyeloid cells causative of autoimmune arthritis.

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1.

The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.

The presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation.

HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5' and 3' end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal.

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 beta-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.

Functional selectivity of natural and synthetic prostaglandin EP4 receptor ligands.

Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.

The Distinct Roles of CXCR3 Variants and Their Ligands in the Tumor Microenvironment.

First thought to orchestrate exclusively leukocyte trafficking, chemokines are now acknowledged for their multiple roles in the regulation of cell proliferation, differentiation, and survival. Dysregulation of their normal functions contributes to various pathologies, including inflammatory diseases and cancer. The two chemokine receptor 3 variants CXCR3-A and CXCR3-B, together with their cognate chemokines (CXCL11, CXCL10, CXCL9, CXCL4, and CXCL4L1), are involved in the control but also in the development of many tumors. CXCR3-A drives the infiltration of leukocytes to the tumor bed to modulate tumor progression (paracrine axis). Conversely, tumor-driven changes in the expression of the CXCR3 variants and their ligands promote cancer progression (autocrine axis). This review summarizes the anti- and pro-tumoral activities of the CXCR3 variants and their associated chemokines with a focus on the understanding of their distinct biological roles in the tumor microenvironment.

Structure-Activity Relationship Study of Cyclic Pentapeptide Ligands for Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3)/CXC chemokine receptor 7 (CXCR7) recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and is involved in a number of physiological and pathological processes. Here, we investigated the SAR of the component amino acids in an ACKR3-selective ligand, FC313 [ cyclo(-d-Tyr-l-Arg-l-MeArg-l-Nal(2)-l-Pro-)], for the development of highly active ACKR3 ligands. Notably, modification at the l-Pro position with a bulky hydrophobic side chain led to improved bioactivity toward ACKR3.

Neuraminidase 1 activates insulin receptor and reverses insulin resistance in obese mice.

OBJECTIVES: Neuraminidase 1 (NEU1) cleaves terminal sialic acids of glycoconjugates during lysosomal catabolism. It also modulates the structure and activity of cellular surface receptors affecting diverse pathways. Previously we demonstrated that NEU1 activates the insulin receptor (IR) and that NEU1-deficient CathAS190A-Neo mice (hypomorph of the NEU1 activator protein, cathepsin A/CathA) on a high-fat diet (HFD) develop hyperglycaemia and insulin resistance faster than wild-type animals. The major objective of the current work was to reveal the molecular mechanism by which NEU1 desialylation activates the IR and to test if increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance. METHODS: To test if desialylation causes a conformational change in the IR dimer we measured interaction between the receptor subunits by Bioluminescence Resonance Energy Transfer in the HEK293T cells either overexpressing NEU1 or treated with the NEU1 inhibitor. The influence of NEU1 overexpression on insulin resistance was studied in vitro in palmitate-treated HepG2 cells transduced with NEU1-expressing lentivirus and in vivo in C57Bl6 mice treated with HFD and either pharmacological inducer of NEU1, Ambroxol or NEU1-expressing adenovirus. NEU1-deficient CathAS190A-Neo mice were used as a control. RESULTS: By desialylation of IR, NEU1 induced formation of its active dimer leading to insulin signaling. Overexpression of NEU1 in palmitate-treated HepG2 cells restored insulin signaling, suggesting that increased NEU1 levels may reverse insulin resistance. Five-day treatment of glycemic C57Bl6 mice receiving HFD with the activator of the lysosomal gene network, Ambroxol, increased NEU1 expression and activity in muscle tissue, normalized fasting glucose levels, and improved physiological and molecular responses to glucose and insulin. Ambroxol did not improve insulin sensitivity in obese insulin-resistant CathAS190A-Neo mice indicating that the Ambroxol effect is mediated through NEU1 induction. Sustained increase of liver NEU1 activity through adenovirus-based gene transfer failed to attenuate insulin resistance most probably due to negative feedback regulation of IR expression. CONCLUSION: Together our results demonstrate that increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance suggesting that a pharmacological modulation of NEU1 activity may be potentially explored for restoring insulin sensitivity and resolving hyperglycemia associated with T2DM.