Structural Basis of the Interaction between Chemokine Stromal Cell-derived Factor-1/CXCL12 and Its G-protein-coupled Receptor CXCR4

Kofuku, Yutaka; Yoshiura, Chie; Ueda, Takumi; Terasawa, Hiroaki; Hirai, Toshihide; Tominaga, Sae; Hirose, Masanobu; Maeda, Yoshitake; Takahashi, Hideo; Terashima, Yuya; Matsushima, Kouji; Shimada, Ichio

doi:10.1074/jbc.m109.024851

Cited by 136 publications

(168 citation statements)

References 67 publications

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“…3 To whom correspondence should be addressed: Dept. experimental data (10). Both CXCL11 and CXCL12 interactions with their canonical receptors conform this model, with key roles for the chemokine N-loop in receptor recognition, and deletion of the chemokine N terminus reportedly affecting signaling (11)(12)(13).…”

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confidence: 79%

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

Benredjem

Girard

Rhainds

et al. 2017

Journal of Biological Chemistry

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Edited by Henrik G. DohlmanAtypical 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. Atypical chemokine receptors (ACKRs)4 are chemokine receptors that do not activate G protein-dependent signaling cascades upon agonist binding; they also do not mediate chemotaxis (1). Rather, chemokine binding to ACKRs leads to rapid chemokine internalization and degradation. Therefore, ACKRs act as chemokine scavengers and play a role in chemokine gradient reshaping, maintenance, and resolution.ACKR3, also known as CXCR7, is an atypical chemokine receptor for the constitutive chemokine CXCL12 (SDF-1), which also binds CXCR4 as a cognate canonical chemokine receptor (2). The second ACKR3 ligand is the highly regulated inflammatory chemokine CXCL11 (I-TAC), which also binds the canonical receptor CXCR3. Being unable to raise G protein responses in most cell types, agonist binding to ACKR3 nevertheless induces arrestin recruitment as a signaling response (3) and rapid degradation of both CXCL12 and CXCL11 (4). ACKR3 has been identified as a potentially promising drug target (2). Given the highly divergent role and regulation of its two ligands, selective interference with their ACKR3 interaction and scavenging may be of interest, warranting better understanding of ligand engagement and scavenging.The structures of chemokine receptors are beginning to be unraveled by crystallography, and the results are in line with those reported earlier from receptor mutagenesis-based approaches (5-7). The reported chemokine receptor crystals required co-crystallization in complex with receptor antagonists. CXCR4 was the first receptor crystallized in complex with an antagonistic chemokine ligand, the virally encoded vMIP-II, which has also permitted refined modeling of the putative CXCR4-CXCL12 complex (8). Overall, these structures refine an early two-step binding model that provides a conceptual framework for the interactions of chemokines with canonical chemokine receptors (for a recent critical review of this model see Kleist et al.; Ref. 9). Following this model, initial chemokine interaction with the receptor critically depends on the receptor N terminus, and some extracellular loop residues. This is then followed by secondary interaction mainly involving the chemokine N terminus with receptor binding pocket residues; this second interaction is required for receptor activation. Of note, for CXCR4 the two-step interaction model is supported by 4 The abbreviations used are: ACKR, atypical chemokine receptor; BRET, bioluminescence resonance energy transfer; CRS1, chemokine recognition...

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confidence: 79%

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

Benredjem

Girard

Rhainds

et al. 2017

Journal of Biological Chemistry

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“…CXCR4 residues involved in chemokine engagement. The interaction of CXCL12 with CXCR4 is known to be mediated by two distinct epitopes (1,8,12,(16)(17)(18)(19)(20)(21)(22). In the first [chemokine recognition site 1 (CRS1)], the unstructured N terminus of CXCR4 interacts with the globular core of the chemokine, specifically with the N loop and the 40s loop of CXCL12.…”

Section: Significancementioning

confidence: 99%

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

Wescott

Kufareva

Paes

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

103

183

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The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G proteincoupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling.T he CXC chemokine receptor 4 (CXCR4) belongs to the G protein-coupled receptor (GPCR) superfamily of proteins, the largest class of integral membrane proteins encoded in the human genome, comprising greater than 30% of current drug targets. Deregulation of CXCR4 expression in multiple human cancers, its role in hematopoietic stem cell migration, and the utilization of CXCR4 by HIV-1 for T-cell entry, make this receptor an increasingly important therapeutic target (1). One FDA-approved drug against CXCR4 is currently on the market (Mozobil, for hematopoietic stem cell mobilization), and multiple additional drugs against this target are in development for oncology and other indications (2).The crystal structures of class A GPCR superfamily members in their active and inactive conformations (reviewed in refs. 3 and 4) provide unprecedented insight into the structural basis of ligand binding, G protein coupling, and activation of GPCRs via rearrangements of transmembrane (TM) helices. GPCR helices V and VI in particular, and in some cases III and VII, are known to undergo significant conformational changes upon activation (5-7). However, static images alone have not been able to explain the residue-level mechanisms underlying the dynamic helical shifts that mediate GPCR signal transduction. Additionally, only inactive state structures have been solved for CXCR4 and most other GPCRs (8,9). Over the last two decades, extensive mutagenesis studies of GPCRs in general [collectively describing >8,000 mutations (gpcrdb.org)] and of CX...

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“…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)(6)(7)(8)(9).…”

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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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Structural Basis of the Interaction between Chemokine Stromal Cell-derived Factor-1/CXCL12 and Its G-protein-coupled Receptor CXCR4

Cited by 136 publications

References 67 publications

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

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

Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices

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

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