Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment

Das, Sandhya; Rajagopalan, Lavanya; Guerrero-Plata, Antonieta; Sai, Jiqing; Richmond, Ann; Garofalo, Roberto P.; Rajarathnam, Krishna

doi:10.1371/journal.pone.0011754

Cited by 107 publications

(127 citation statements)

References 47 publications

(53 reference statements)

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“…8,43,44 Contrary to this, at low concentrations a trapped CXCL8 monomer was shown to be more active than the wild-type CXCL8 dimer in neutrophil migration. 18 Our biophysical studies indicate that a mutational variant of CXCL8 designed to remain monomeric (CXCL8M) binds only slightly more tightly to hCXCR1pep (Fig. 2).…”

Section: Discussionmentioning

confidence: 83%

“…In contrast with these results, CXCL8 dimerization has been shown to be critical for neutrophil recruitment, one of the major CXCL8 roles in regulating inflammatory responses. 18 These discrepancies may arise from the fact that most biophysical studies to date have been conducted with a peptide corresponding to the residues within the Nterminal region of a rabbit CXCR1 homolog, as opposed to its human counterpart, while biological studies reflect the interaction with the human CXCR1 (hCXCR1) sequence.…”

Section: Introductionmentioning

confidence: 99%

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The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

et al. 2014

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Interleukin-8 (CXCL8, IL-8) is a proinflammatory chemokine important for the regulation of inflammatory and immune responses via its interaction with G-protein coupled receptors, including CXC receptor 1 (CXCR1). CXCL8 exists as both a monomer and as a dimer at physiological concentrations, yet the molecular basis of CXCL8 interaction with its receptor as well as the importance of CXCL8 dimer formation remain poorly characterized. Although several biological studies have indicated that both the CXCL8 monomer and dimer are active, biophysical studies have reported conflicting results regarding the binding of CXCL8 to CXCR1. To clarify this problem, we expressed and purified a peptide (hCXCR1pep) corresponding to the N-terminal region of human CXCR1 (hCXCR1) and utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the binding of wild-type CXCL8 and a previously reported mutant (CXCL8M) that stabilizes the monomeric form. Our data reveal that the CXCL8 monomer engages hCXCR1pep with a slightly higher affinity than the CXCL8 dimer, but that the CXCL8 dimer does not dissociate upon binding hCXCR1pep. These investigations also showed that CXCL8 is dynamic on multiple timescales, which may help explain the versatility in this interleukin for engaging its target receptors.

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Section: Discussionmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

et al. 2014

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“…Under light anesthesia, mice were inoculated intranasally with 10 g of WT CXCL8 and CC-CXCL8 variant in Dulbecco's phosphate-buffered saline (D-PBS). Mice were sacrificed, and neutrophil levels in the broncheo-alveolar lavage fluid (BALF) were determined as described previously (30).…”

Section: Neutrophil Recruitment In a Mousementioning

confidence: 99%

Probing the Role of CXC Motif in Chemokine CXCL8 for High Affinity Binding and Activation of CXCR1 and CXCR2 Receptors*

Joseph¹,

Sarmiento²,

Mishra³

et al. 2010

Journal of Biological Chemistry

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All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/ transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC 3 CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K i > 1 M). Further, CC-CXCL8 failed to mobilize Ca 2؉ in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca 2؉ in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ϳ5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.Chemokines mediate a wide variety of biological functions from recruiting leukocytes to the site of injury and infection to organ development, wound healing, and angiogenesis (1-4). Chemokines are characterized by four conserved cysteines that form disulfide bonds, and are classified into CXC and CC families based on cysteine pattern near the N terminus. CXC ligands bind and activate only CXC receptors and CC ligands bind and activate only CC receptors, indicating functional divergence could be as old as the molecules themselves. Structure-function studies consistently show that binding and receptor activation for both classes involves two interactions: between the ligand N-loop and receptor N-domain residues (Site-I) and between the ligand N-terminal and receptor extracellular/transmembrane residues (Site-II) (5-9).Structures of several CXC and CC chemokines have been solved by NMR and x-ray crystallography, and reveal a common structural fold (known as chemokine fold) at the monomer level (10 -15). The CXC/CC motif connects the functionally important N-loop and N-terminal residues, and also plays a structural role by forming disulfide bonds that tether the N-terminal and N-loop residues to the protein core (Fig. 1). In the CXC chemokine CXCL8 (also known as interleukin-8, IL-8), the residue corresponding to X (Gln) in the CXC motif has been mutated with no effec...

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“…IL-8 has been shown to exist and function both as a monomer and dimer (10)(11)(12). The monomer binds to the N-terminal domain of CXCR1 with higher affinity than the dimer (13)(14)(15), and is believed to be the biologically active form of the protein.…”

Section: Introductionmentioning

confidence: 99%

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

Park

Berkamp

Radoicic

et al. 2017

Biophysical Journal

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The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G proteincoupled receptor, located in the cell membrane of neutrophils. Here, we describe 1 H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn 2þ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

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Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment

Cited by 107 publications

References 47 publications

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

Probing the Role of CXC Motif in Chemokine CXCL8 for High Affinity Binding and Activation of CXCR1 and CXCR2 Receptors*

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

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