Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

The CXCL12/CXCR4/ACKR3 Axis in the Tumor Microenvironment: Signaling, Crosstalk, and Therapeutic Targeting.

Elevated expression of the chemokine receptors CXCR4 and ACKR3 and of their cognate ligand CXCL12 is detected in a wide range of tumors and the tumor microenvironment (TME). Yet, the molecular mechanisms by which the CXCL12/CXCR4/ACKR3 axis contributes to the pathogenesis are complex and not fully understood. To dissect the role of this axis in cancer, we discuss its ability to impinge on canonical and less conventional signaling networks in different cancer cell types; its bidirectional crosstalk, notably with receptor tyrosine kinase (RTK) and other factors present in the TME; and the infiltration of immune cells that supporttumor progression. We discuss current and emerging avenues that target the CXCL12/CXCR4/ACKR3 axis. Coordinately targeting both RTKs and CXCR4/ACKR3 and/or CXCL12 is an attractive approach to consider in multitargeted cancer therapies. In addition, inhibiting infiltrating immune cells or reactivating the immune system along with modulating the CXCL12/CXCR4/ACKR3 axis in the TME has therapeutic promise.

Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.

Herpesviruses are ubiquitous pathogens that establish lifelong, latent infections in their host. Spontaneous reactivation of herpesviruses is often asymptomatic or clinically manageable in healthy individuals, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with multiple proliferative cardiovascular and post-transplant diseases. Herpesviruses encode viral G protein-coupled receptors (vGPCRs) that alter the host cell by hijacking cellular pathways and play important roles in the viral life cycle and these different disease settings. In this review, we discuss the pharmacological and signaling properties of these vGPCRs, their role in the viral life cycle, and their contribution in different diseases. Because of their prominent role, vGPCRs have emerged as promising drug targets, and the potential of vGPCR-targeting therapeutics is being explored. Overall, these vGPCRs can be considered as attractive targets moving forward in the development of antiviral, cancer, and/or cardiovascular disease treatments. SIGNIFICANCE STATEMENT: In the last decade, herpesvirus-encoded G protein-coupled receptors (GPCRs) have emerged as interesting drug targets with the growing understanding of their critical role in the viral life cycle and in different disease settings. This review presents the pharmacological properties of these viral receptors, their role in the viral life cycle and different diseases, and the emergence of therapeutics targeting viral GPCRs.

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists.

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions.

The development of protein-protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment-based drug screening approach using the regulatory hub-protein 14-3-3 as a platform for identifying PPI stabilizers. A homogenous time-resolved FRET assay was used to monitor stabilization of 14-3-3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in-house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14-3-3 PPIs in a cooperative fashion with Fusicoccin-A. Mechanistically, fragment 2 appears to enhance 14-3-3 dimerization leading to increased client-protein binding. Functionally, fragment 2 enhanced potency of 14-3-3 in a cell-free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14-3-3, which could be used as a tool compound for investigating 14-3-3 client protein interactions.

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.

Kinetic Analysis of the Early Signaling Steps of the Human Chemokine Receptor CXCR4.

G protein-coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer-based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development. SIGNIFICANCE STATEMENT: The CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor-G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

The constitutive activity of the viral-encoded G protein-coupled receptor US28 supports a complex signalling network contributing to cancer development.

US28 is a viral G protein-coupled receptor (GPCR) encoded by the human cytomegalovirus (HCMV). This receptor, expressed both during lytic replication and viral latency, is required for latency. US28 is binding to a wide variety of chemokines but also exhibits a particularly high constitutive activity robustly modulating a wide network of cellular pathways altering the host cell environment to benefit HCMV infection. Several studies suggest that US28-mediated signalling may contribute to cancer progression. In this review, we discuss the unique structural characteristics that US28 acquired through evolution that confer a robust constitutive activity to this viral receptor. We also describe the wide downstream signalling network activated by this constitutive activation of US28 and discuss how these signalling pathways may promote and support important cellular aspects of cancer.

Viral G protein-coupled receptors as modulators of cancer hallmarks.

Herpesviruses encode transmembrane G protein-coupled receptors (GPCRs), which share structural homology to human chemokine receptors. These viral GPCRs include KSHV-encoded ORF74, EBV-encoded BILF1, and HCMV-encoded US28, UL33, UL78 and US27. Viral GPCRs hijack various signaling pathways and cellular networks, including pathways involved in the so-called cancer hallmarks as defined by Hanahan and Weinberg. These hallmarks describe cellular characteristics crucial for transformation and tumor progression. The cancer hallmarks involve growth factor-independent proliferation, angiogenesis, avoidance of apoptosis, invasion and metastasis, metabolic reprogramming, genetic instability and immune evasion amongst others. The role of beta herpesviruses modulating these cancer hallmarks is clearly highlighted by the proliferative and pro-angiogenic phenotype associated with KSHV infection which is largely ascribed to the ORF74-mediated modulation of signaling networks in host cells. For HCMV and Epstein-Bar encoded GPCRs, oncomodulatory effects have been described which contribute to the cancer hallmarks, thereby enhancing oncogenic development. In this review, we describe the main signaling pathways controlling the hallmarks of cancer which are affected by the betaherpesvirus encoded GPCRs. Most prominent among these involve the JAK-STAT, PI(3)K-AKT, NFkB and MAPK signaling nodes. These insights are important to effectively target these viral GPCRs and their signaling networks in betaherpesvirus-associated malignancies.

From Insight to Modulation of CXCR4 and ACKR3 (CXCR7) Function.

Chemokine receptors CXCR4 and atypical chemokine receptor 3 (ACKR3/CXCR7) are highly expressed in a range of tumors. Yet, their role in cancer progression is not well understood. This minireview series encompasses seven comprehensive reviews focusing on modulators (small molecules, pepducins, antibodies), structural aspects, spatio-temporal signaling properties, and phosphorylation/interactome of CXCR4 and ACKR3. Moreover, different (patho)physiologic aspects and roles of these receptors in immunologic and oncogenic processes are discussed. SIGNIFICANCE STATEMENT: CXCR4 and atypical chemokine receptor 3 are two oncogenic G protein-coupled receptors that are highly upregulated in various tumors. Insight into the signalling properties of these receptors and the availability of modulators targeting these receptors are essential to assess their role in cancer.

The Role of ACKR3 in Breast, Lung, and Brain Cancer.

Recent reports regarding the significance of chemokine receptors in disease have put a spotlight on atypical chemokine receptor 3 (ACKR3). This atypical chemokine receptor is overexpressed in numerous cancer types and has been involved in the modulation of tumor cell proliferation and migration, tumor angiogenesis, or resistance to drugs, thus contributing to cancer progression and metastasis occurrence. Here, we focus on the clinical significance and potential mechanisms underlying the pathologic role of ACKR3 in breast, lung, and brain cancer and discuss its possible relevance as a prognostic factor and potential therapeutic target in these contexts.

CXCR4/ACKR3 Phosphorylation and Recruitment of Interacting Proteins: Key Mechanisms Regulating Their Functional Status.

The C-X-C motif chemokine receptor type 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3/CXCR7) are class A G protein-coupled receptors (GPCRs). Accumulating evidence indicates that GPCR subcellular localization, trafficking, transduction properties, and ultimately their pathophysiological functions are regulated by both interacting proteins and post-translational modifications. This has encouraged the development of novel techniques to characterize the GPCR interactome and to identify residues subjected to post-translational modifications, with a special focus on phosphorylation. This review first describes state-of-the-art methods for the identification of GPCR-interacting proteins and GPCR phosphorylated sites. In addition, we provide an overview of the current knowledge of CXCR4 and ACKR3 post-translational modifications and an exhaustive list of previously identified CXCR4- or ACKR3-interacting proteins. We then describe studies highlighting the importance of the reciprocal influence of CXCR4/ACKR3 interactomes and phosphorylation states. We also discuss their impact on the functional status of each receptor. These studies suggest that deeper knowledge of the CXCR4/ACKR3 interactomes along with their phosphorylation and ubiquitination status would shed new light on their regulation and pathophysiological functions.

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3.

Dysregulation of the chemokine system is implicated in a number of autoimmune and inflammatory diseases, as well as cancer. Modulation of chemokine receptor function is a very promising approach for therapeutic intervention. Despite interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAbs) and antibody-based molecules have been successfully applied in the clinical therapy of cancer and represent a potential new class of therapeutics targeting chemokine receptors belonging to the class of G protein-coupled receptors (GPCRs). Besides conventional mAbs, single-domain antibodies and antibody scaffolds are also gaining attention as promising therapeutics. In this review, we provide an extensive overview of mAbs, single-domain antibodies, and other antibody fragments targeting CXCR4 and ACKR3, formerly referred to as CXCR7. We discuss their unique properties and advantages over small-molecule compounds, and also refer to the molecules in preclinical and clinical development. We focus on single-domain antibodies and scaffolds and their utilization in GPCR research. Additionally, structural analysis of antibody binding to CXCR4 is discussed. SIGNIFICANCE STATEMENT: Modulating the function of GPCRs, and particularly chemokine receptors, draws high interest. A comprehensive review is provided for monoclonal antibodies, antibody fragments, and variants directed at CXCR4 and ACKR3. Their advantageous functional properties, versatile applications as research tools, and use in the clinic are discussed.

Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells.

Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology.

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control in situ the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H3 receptor (H3R). From 16 newly synthesized photoswitchable compounds, VUF14738 (28) and VUF14862 (33) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H3R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical trans-/ cis conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H3R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H3R signaling.

CXCR4-Specific Nanobodies as Potential Therapeutics for WHIM syndrome.

WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: warts, hypogammaglobulinemia, infections, and myelokathexis, which refers to abnormal accumulation of mature neutrophils in the bone marrow. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4, giving these CXCR4-WHIM mutants a gain of function in response to their ligand CXCL12. Considering the broad functions of CXCR4 in maintaining leukocyte homeostasis, patients are panleukopenic and display altered immune responses, likely as a consequence of impairment in the differentiation and trafficking of leukocytes. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability, and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small-molecule clinical candidate AMD3100. The CXCR4-targeting nanobodies displace CXCL12 binding and bind CXCR4-wild type and CXCR4-WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The nanobodies' epitope was mapped to extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent, nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of human papillomavirus-immortalized cell lines in response to CXCL12 was effectively inhibited by the nanobodies. Based on these in vitro results, we conclude that CXCR4 nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases such as WHIM syndrome.

Cutting edge: GPR35/CXCR8 is the receptor of the mucosal chemokine CXCL17.

Chemokines are chemotactic cytokines that direct the traffic of leukocytes and other cells in the body. Chemokines bind to G protein-coupled receptors expressed on target cells to initiate signaling cascades and induce chemotaxis. Although the cognate receptors of most chemokines have been identified, the receptor for the mucosal chemokine CXCL17 is undefined. In this article, we show that GPR35 is the receptor of CXCL17. GPR35 is expressed in mucosal tissues, in CXCL17-responsive monocytes, and in the THP-1 monocytoid cell line. Transfection of GPR35 into Ba/F3 cells rendered them responsive to CXCL17, as measured by calcium-mobilization assays. Furthermore, GPR35 expression is downregulated in the lungs of Cxcl17(-/-) mice, which exhibit defects in macrophage recruitment to the lungs. We conclude that GPR35 is a novel chemokine receptor and suggest that it should be named CXCR8.

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function.

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

Chemokine cooperativity is caused by competitive glycosaminoglycan binding.

Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems and has been dubbed chemokine cooperativity. In this study, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, in which multiple chemokines are secreted simultaneously.