Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.     

The CC chemokine MCP-1 stimulates surface expression of CX3CR1 and enhances the adhesion of monocytes to fractalkine/CX3CL1 via p38 MAPK

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.     

Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.     

Receptor-ligand binding in the cell-substrate contact zone: a quantitative analysis using CX3CR1 and CXCR1 chemokine receptors

Receptor-ligand binding analyses have generally used soluble components to measure thermodynamic binding constants. In their biological context, adhesion receptors bind to an immobile ligand and the binding reaction is confined to the cell-substrate contact zone. We have developed a new procedure based on the spinning disk technology to measure the number of receptor-ligand bonds in the contact zone. Application of this methodology to the CX3CR1-fractalkine and the CXCR1-IL-8 receptor-ligand systems demonstrated that the level of binding to an immobilized ligand is reduced by several orders of magnitude in comparison to solution binding. A comparison of the solution binding and contact zone binding constants shows that the effect of ligand immobilization was similar for each system. In contrast, although the CXCR1-IL-8 bond had the higher affinity, the average bond strength was only 10% of that for the CX3CR1 bond. Because fractalkine can be expressed as a cell surface-bound protein, CX3CR1 has been proposed to function as an adhesion receptor. The higher bond strength suggests that the bond architecture has also evolved to serve an adhesion function.     

Mutational analysis of the vaccinia virus E3 protein defines amino acid residues involved in E3 binding to double-stranded RNA.

Alanine-substitution mutations were targeted to 14 amino acid residues within the double-stranded (ds) RNA binding motif (dsRBM) of the vaccinia virus E3 protein. Substitutions at six positions--Glu-124, Phe-135, Phe-148, Lys-167, Arg-168, and Lys-171--caused significant reductions in dsRNA binding. These six residues are conserved in the two dsRBMs for which structural information is available (Escherichia coli RNase III and Drosophila melanogaster staufen) and in many other members of the dsRBM protein family. Residues we show to be important for dsRNA binding by vaccinia virus E3 map to the same face of the dsRBM structure and are thus likely to compose part of the RNA binding site.     

Unique role of the chemokine domain of fractalkine in cell capture. Kinetics of receptor dissociation correlate with cell adhesion

The chemokine fractalkine (FK) has two structural features that make it unique in the chemokine family: a CX(3)C motif and an extended carboxyl terminus that anchors it to the cell surface. This mucin-like stalk or an equivalent spacer is required for FK to mediate the adhesion of cells expressing its receptor, CX(3)CR1. To determine whether the ability of FK to act as a cell adhesion molecule is due to the unique presentation of a chemokine domain on a stalk or to properties of the chemokine domain itself, we created a series of chimeras in which other soluble chemokines (RANTES (regulated on activation normal T cell expressed), monocyte chemoattractant protein 1, macrophage inflammatory protein 1 beta, secondary lymphoid tissue chemokine, and interleukin 8) were fused to the mucin stalk. When tested in a static-cell adhesion assay, many of these chemokine chimeras demonstrated activity equivalent to that of FK. In flow assays, however, none of the chimeras captured cells as efficiently as FK. Interestingly, FK captured cells expressing either CX(3)CR1 or the viral receptor US28. Cells bound to FK without rolling or detaching, whereas the interleukin 8 and monocyte chemoattractant protein 1 chimeras induced primarily cell rolling and detaching, respectively. In binding studies, FK has a significantly slower off-rate from its receptors than any of the other chemokine chimeras had for their cognate receptors. We conclude that presentation of a chemokine atop a mucin-like stalk is not, in and of itself, sufficient to capture cells. The unique ability of FK to mediate adhesion under flow may be a function of its slow receptor off-rate.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H⁺ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H⁺-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H⁺ in the course of cellular amino acid uptake by PAT1.     

Basic amino acid residues in the beta-structure region contribute, but not critically, to presynaptic neurotoxicity of ammodytoxin A

The molecular mechanism of action of presynaptically toxic secreted phospholipases A2 (sPLA2s) isolated from snake venoms is not completely understood. It has been proposed that the positive charge in the beta-structure region is important for their toxic activity. To test this hypothesis, we characterised several mutants of ammodytoxin A (AtxA) possessing substitution of all five basic residues in this region. The mutations had relatively little influence on the catalytic activity of AtxA, either on charge-neutral or anionic phospholipid vesicles. An exception was R72 when replaced by a hydrophobic (higher activity) or an acidic (lower activity) residue. Lethal potencies of the eight single site mutants were up to four times lower than that of the wild-type, whereas the triple mutant (K74S/H76S/R77L) was 13-fold less toxic. The substitutions also lowered the affinity of the toxin, slightly to moderately, for the neuronal receptors R25 and R180. Interaction with calmodulin was only slightly affected by substitutions of K86, more by those of the K74/H76/R77 cluster and most by those of R72 (up to 11-fold lower binding affinity). The results clearly indicate that the basic amino acid residues in the beta-region of AtxA contribute to, but are not necessary for, its neurotoxic effect.     

Two alcohol binding residues interact across a domain interface of the L1 neural cell adhesion molecule and regulate cell adhesion

Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule. Azialcohols photolabel Glu-33 and Tyr-418, two residues that are predicted by homology modeling to lie within 2.8 Å of each other at the interface between the Ig1 and Ig4 domains of L1 (Arevalo, E., Shanmugasundararaj, S., Wilkemeyer, M. F., Dou, X., Chen, S., Charness, M. E., and Miller, K. W. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 371–375). Using transient transfection of NIH/3T3 cells with wild type (WT-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent β-mercaptoethanol (βME) reversibly decreased the adhesion of E33C/Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of βME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.     

Association of polymorphisms in the chemokine receptor CX3CR1 gene with coronary artery disease

Two chemokine receptor CX3CR1 gene variants, V249I and T280M, have been implicated in coronary artery diseases (CAD). Currently no consistent effect has been revealed and their role in cardiovascular disease is still conflicting. In the present study the association of CX3CR1 genotypes with CAD and myocardial infarction (MI) was investigated in the Ludwigshafen Risk and Cardiovascular Health (LURIC) cohort, including 3316 individuals in whom cardiovascular disease angiographically has been defined or ruled out. Similarly to previous studies, the alleles I249 and M280 were in strong linkage disequilibrium and formed an I₂₄₉M₂₈₀ haplotype. However, there was no relationship between CX3CR1 genotypes or corresponding haplotypes and the prevalence of CAD or MI. Adjusted for classical risk factors (age, sex, hypertension, dyslipidemia, diabetes mellitus and smoking), the odds ratio (OR) of V249I for CAD was 0.95 (95% confidence interval (CI) = 0.78–1.15, p = 0.61). The OR of T280M for CAD was 0.83 (95% CI = 0.66–1.04, p = 0.11). Furthermore, CX3CR1 variants were not associated with C-reactive protein levels, age at onset of CAD, severity of CAD and MI. In conclusion, present data of LURIC do not support the hypothesis that common variants of the CX3CR1 gene are associated with the presence of CAD or MI.     

Decreased expression of the fractalkine receptor CX3CR1 on circulating monocytes as new feature of sepsis-induced immunosuppression

Although it is known that septic shock rapidly induces immune dysfunctions, which contribute to the impaired clearance of microorganisms observed in patients, the mechanisms for this phenomenon remain incompletely understood. We recently observed, in a microarray study, an altered circulating leukocyte CX3CR1 mRNA expression associated with patients' mortality. As monocytes play a central role in septic shock pathophysiology and express high levels of CX3CR1, we therefore further investigated the alteration of CX3CR1 expression and of its ligand fractalkine (CX3CL1) on those cells in this clinical condition. We observed that CX3CR1 expression (both mRNA and protein) was severely down-regulated in monocytes and consequently associated with a lack of functionality upon fractalkine challenge. Importantly, nonsurvivors presented with significantly sustained lower expression in comparison with survivors. This down-regulation was reproduced by incubation of cells from healthy individuals with LPS, whole bacteria (Escherichia coli and Staphylococcus aureus), and, to a lower extent, with corticosteroids-in accordance with the concept of LPS-induced monocyte deactivation. In addition, CX3CL1 serum concentrations were elevated in patients supporting the hypothesis of increased cleavage of the membrane-anchored form expressed by endothelial cells. As CX3CR1/CX3CL1 interaction preferentially mediates arrest and migration of proinflammatory cells, the present observations may contribute to patients' inability to kill invading microorganisms. This could represent an important new feature of sepsis-induced immunosuppression.     

Mutational analysis of the KIX domain of CBP reveals residues critical for SREBP binding

Structure-based mutagenesis was used to probe the binding surface for the activation domain of sterol-responsive element binding protein (SREBP) in the KIX domain of CREB binding protein. A set of conserved residues scattering in the alpha2 helix and the extended C-terminal region of alpha 3 helix in the KIX domain including two arginines previously characterized as a hot spot for cofactor-mediated methylation was shown to be crucial for SREBP-KIX interaction, and was not essential for phosphorylated KID recognition. Therefore, our results suggest the existence of a SREBP binding site formed by positively charged residues in the C-terminal part of the extended alpha 3 helix of the KIX domain distinct from the previously identified phosphorylated KID binding site.     

IL-15 alters expression and function of the chemokine receptor CX3CR1 in human NK cells

The chemokine receptor CX3CR1 is thought to regulate inflammation in part by modulating NK cell adhesion, migration, and killing in response to its ligand CX3CL1 (fractalkine). Recent reports indicate that IL-15, which is essential for development and survival of NK cells, may negatively regulate CX3CR1 expression, however, the effects of the cytokine on human NK cell CX3CR1 expression and function have not been fully delineated. Here, we demonstrate that short term culture in IL-15 decreases surface expression of CX3CR1 on cultured CD56+ cells from human blood resulting in diminished chemotaxis and calcium flux in response to CX3CL1. Cells cultured long term in IL-15 (more than five days) completely lost surface expression as well as mRNA and protein for CX3CR1. The effect was specific since mRNA for CCR5 was increased and mRNA for CXCR4 was unchanged in these cells by IL-15. Thus, exogenous IL-15 is a negative regulator of CX3CR1 expression and function in human CD56+ NK cells. The data imply that the use of IL-15 alone to expand NK cells ex vivo for immunotherapy may produce cells impaired in their ability to traffic to sites of inflammation.     

Functional adhesiveness of the CX3CL1 chemokine requires its aggregation. Role of the transmembrane domain

In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.     

Effect of electroacupuncture on the cervicospinal P2X7 receptor/fractalkine/CX3CR1 signaling pathway in a rat neck-incision pain model

Increasing evidence supports that acupuncture intervention is an effective approach for intraoperative and postoperative pain. Neuron–microglia crosstalk, mediated by the purinergic P2X7 receptor (R)/fractalkine/CX3CR1 cascade in the spinal cord dorsal horn, plays a pivotal role in pain processing. However, its involvement in the analgesic effect of electroacupuncture (EA) remains unclear. In this study, a rat neck-incision pain model was established by making a longitudinal incision along the midline of the neck and subsequent repeated mechanical stimulation. EA stimulation was applied to bilateral LI18, LI4-PC6, or ST36-GB34. The thermal pain threshold, cervicospinal ATP concentration, expression levels of purinergic P2XR and P2YR subunits mRNAs, and fractalkine, CX3CR1 and p38 MAPK proteins, were detected separately. The neck incision induced strong thermal hyperalgesia and upregulation of spinal ATP within 48 h. No significant change was found in thermal hyperalgesia after a single session of EA intervention. However, a single session of EA dramatically enhanced the neck incision-induced upregulation of ATP and upregulated the expression of P2X7R, which was reversed by two sessions of EA. Two sessions of EA at bilateral LI18 or LI4-PC6 attenuated hyperalgesia significantly, accompanied with downregulation of P2X7R/fractalkine/ CX3CR1 signaling after three sessions of EA. EA stimulation of LI18 or LI4-PC6 alleviates thermal hyperalgesia in neck-incision pain rats, which may be associated with its effects in regulating the neck incision-induced increase of ATP and P2X7R and subsequently suppressing fractalkine/CX3CR1 signaling in the cervical spinal cord.     

Mutational analysis of the DNA binding domain A of chromosomal protein HMG1.

We have mutated several residues of the first of the two HMG-boxes of mammalian HMG1. Some mutants cannot be produced in Escherichia coli, suggesting that the peptide fold is grossly disrupted. A few others can be produced efficiently and have normal DNA binding affinity and specificity; however, they are more sensitive towards heating and chaotropic agents than the wild type polypeptide. Significantly, the mutation of the single most conserved residue in the rather diverged HMG-box family falls in this 'in vitro temperature-sensitive' category, rather than in the non-folded category. Finally, two other mutants have reduced DNA binding affinity but unchanged binding specificity. Overall, it appears that whenever the HMG-box can fold, it will interact specifically with kinked DNA.     

Protective roles of the fractalkine/CX3CL1-CX3CR1 interactions in alkali-induced corneal neovascularization through enhanced antiangiogenic factor expression

Macrophages accumulate during the course of corneal neovascularization, but its mechanisms and roles still remain elusive. To address these points, we herein examined corneal neovascularization after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is normally expressed by macrophages. After alkali injury, the mRNA expression of CX3CR1 was augmented along with accumulation of F4/80-positive macrophages and Gr-1-positive neutrophils in the corneas. Compared with wild-type mice, CX3CR1-deficient mice exhibited enhanced corneal neovascularization 2 wk after injury, as evidenced by enlarged CD31-positive areas. Concomitantly, the accumulation of F4/80-positive macrophages, but not Gr-1-positive neutrophils, was markedly attenuated in CX3CR1-deficient mice compared with wild-type mice. The intraocular mRNA expression of vascular endothelial growth factor (VEGF) was enhanced to similar extents in wild-type and CX3CR1-deifient mice after the injury. However, the mRNA expression of antiangiogenic factors, thrombospondin (TSP) 1, TSP-2, and a disintegrin and metalloprotease with thrombospondin (ADAMTS) 1, was enhanced to a greater extent in wild-type than CX3CR1-deificient mice. A double-color immunofluorescence analysis demonstrated that F4/80-positive cells also expressed CX3CR1 and ADAMTS-1 and that TSP-1 and ADAMTS-1 were detected in CX3CR1-positive cells. CX3CL1 enhanced TSP-1 and ADAMTS-1, but not VEGF, expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 wk, along with enhanced intraocular expression of TSP-1 and ADAMTS-1 but not VEGF. Thus, these observations indicate that accumulation of CX3CR1-positive macrophages intraocularly can dampen alkali-induced corneal neovascularization by producing antiangiogenic factors such as TSP-1 and ADAMTS-1 and suggest the potential therapeutic efficacy of using CX3CL1 against alkali-induced corneal neovascularization.