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.

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.

Structural basis for chemokine recognition by a G protein-coupled receptor and implications for receptor activation.

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein-dependent and ß-arrestin-dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein-dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance-based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical "two-site model" for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

Development of novel CXC chemokine receptor 7 (CXCR7) ligands: selectivity switch from CXCR4 antagonists with a cyclic pentapeptide scaffold.

The CXC chemokine receptor 7 (CXCR7)/ACKR3 is a chemokine receptor that recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and interferon-inducible T-cell α chemoattractant (I-TAC)/CXCL11. Here, we report the development of novel CXCR7-selective ligands with a cyclic pentapeptide scaffold through an SAR study of CXC chemokine receptor 4 (CXCR4) selective antagonist FC131 [cyclo(-d-Tyr-l-Arg-l-Arg-l-Nal-Gly-), Nal = 3-(2-naphthyl)alanine]. Substitution of Gly with l-Pro switched the receptor preference of the peptides from CXCR4 to CXCR7. The SAR study led to the identification of a potent CXCR7 ligand, FC313 [cyclo(-d-Tyr-l-Arg-l-MeArg-l-Nal-l-Pro-)], which recruits ß-arrestin to CXCR7. Investigations via receptor mutagenesis and molecular modeling experiments suggest a possible binding mode of the cyclic pentapeptide CXCR7 agonist.

Characterization of currently marketed heparin products: adverse event relevant bioassays.

The polyanion oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in heparin products and was associated with severe hypotensive responses and other symptoms in patients receiving the drug. The OSCS associated adverse reactions were attributed to activation of the contact system via the plasma mediator, activated factor XII (FXIIa), which triggers kallikrein (KK) activity. Unlike heparin alone, OSCS, is able to activate FXII in plasma and stably bind to FXIIa enhancing plasma KK activity and the induction of vasoactive mediators such as bradykinin (BK), C3a and C5a. Similarly OSCS can interfere with heparin neutralization by the polycationic drug protamine. Here, we assess heparin (heparin sodium, dalteparin, tinzaparin or enoxaparin)-protamine complex formation and plasma based bioassays of KK, BK and C5a in a 96-well plate format. We establish the normal range of variation in the optimized bioassays across multiple lots from 9 manufacturers. In addition, because other oversulfated (OS) glycosaminoglycans (GAGs) besides OSCS could also serve as possible economically motivated adulterants (EMAs) to heparin, we characterize OS-dermatan sulfate (OSDS), OS-heparan sulfate (OSHS) and their native forms in the same assays. For the protamine test, OS-GAGs could be distinguished from heparin. For the KK assay, OSCS and OSDS were most potent followed by OSHS, and all had similar efficacies. Finally, OSDS had a greater efficacy in the C5a and BK assays followed by OSCS then OSHS. These data established the normal range of response of heparin products in these assays and the alteration in the responses in the presence of possible EMAs.

Altered cardiac bradykinin metabolism in experimental diabetes caused by the variations of angiotensin-converting enzyme and other peptidases.

The peptidases angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) mediate most of the kinin catabolism in normal cardiac tissue and are the molecular targets of inhibitory drugs that favorably influence diabetic complications. We studied the variations of those kininases in the myocardium of rats in experimental diabetes. ACE and NEP activities were significantly decreased in heart membranes 4-8weeks post-streptozotocin (STZ) injection. However, insulin-dependent diabetes did not modify significantly bradykinin (BK) half-life (t(1/2)) while the effect of both ACE (enalaprilat) and ACE and NEP (omapatrilat) inhibitors on BK degradation progressively decreased, which may be explained by the upregulation of other unidentified metallopeptidase(s). In vivo insulin treatment restored the activities of both ACE and NEP. ACE and NEP activities were significantly higher in hearts of young Zucker rats than in those of Sprague-Dawley rats. BK t(1/2) and the effects of peptidase inhibitors on t(1/2) varied accordingly. It is concluded that kininase activities are subjected to large and opposite variations in rat cardiac tissue in type I and II diabetes models. A number of tissue or molecular factors may determine these variations, such as remodeling of cardiac tissue, ectoenzyme shedding to the extracellular fluid and the pathologic regulation of peptidase gene expression.

Bradykinin forming capacity of oversulfated chondroitin sulfate contaminated heparin in vitro.

UNLABELLED: Oversulfated chondroitin sulfate (OSCS) contaminated heparin has been associated with severe anaphylactoid reaction (AR), mainly in dialysed patients. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. In this study, we investigated the kinin forming capacity of OSCS and OSCS contaminated heparin when incubated in vitro with a pool of human plasma. At 100 microg/mL, OSCS liberates BK in a profile similar but not identical to dextran sulfate, a well known activator of the plasma contact system. The results have highlighted that the quantity of BK accumulated during contact system activation depends not only on the concentration of OSCS but also on the plasma dilution and the presence of an angiotensin converting enzyme inhibitor. We demonstrate a highly significant correlation between the concentration of OSCS present in the contaminated heparin and BK released concentration. In conclusion, for the first time, we show that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated heparin. DISCLAIMER: The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.