Chemokines are critical mediators of cell migration during routine immune surveillance, inflammation, and development. Chemokines bind to G protein-coupled receptors and cause conformational changes that trigger intracellular signaling pathways involved in cell movement and activation. Although chemokines evolved to benefit the host, inappropriate regulation or utilization of these proteins can contribute to or cause many diseases. Specific chemokine receptors provide the portals for HIV to get into cells, and others contribute to inflammatory diseases and cancer. Thus, there is significant interest in developing receptor antagonists. To this end, the structures of ligands coupled with mutagenesis studies have revealed mechanisms for antagonism based on modified proteins. Although little direct structural information is available on the receptors, binding of small molecules to mutant receptors has allowed the identification of key residues involved in the receptor-binding pockets. In this review, we discuss the current knowledge of chemokine:receptor structure and function, and its contribution to drug discovery.

Immune modulators such as cytokines and growth factors exert their biological activity through high-affinity interactions with cell-surface receptors, thereby activating specific signaling pathways. However, many of these molecules also participate in low-affinity interactions with another class of molecules, referred to as proteoglycans. Proteoglycans consist of a protein core to which glycosaminoglycan (GAG) chains are attached. The GAGs are long, linear, sulfated, and highly charged heterogeneous polysaccharides that are expressed throughout the body in different forms, depending on the developmental or pathological state of the organ/organism. They participate in many biological functions, including organogenesis and growth control, cell adhesion, signaling, inflammation, tumorigenesis, and interactions with pathogens. Recently, it was demonstrated that certain chemokines require interactions with GAGs for their in vivo function. The GAG interaction is thought to provide a mechanism for retaining chemokines on cell surfaces, facilitating the formation of chemokine gradients. These gradients serve as directional cues to guide the migration of the appropriate cells in the context of their inflammatory, developmental, and homeostatic functions. In this review, we discuss GAGs and their interaction with proteins, with a special emphasis on the chemokine system.

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2⅐CCL13, CCL2⅐CCL11 and CCL8⅐CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8⅐CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8⅐CCL11⅐Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.

Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2 (MCP-1/ CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/ CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that, in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.