Dimer Dissociation Is Essential for Interleukin-8 (IL-8) Binding to CXCR1 Receptor

Abstract

Chemokines play a fundamental role in trafficking of immune cells and in host defense against infection. The role of chemokines in the recruitment process is highly regulated spatially and temporally and involves interactions with G protein-coupled receptors and cell surface glycosaminoglycans. The dynamic equilibrium between chemokine monomers and dimers, both free in solution and in cell surface-bound forms, regulates different components of recruitment such as chemotaxis and receptor signaling. The binding and activity of the chemokine interleukin-8 (IL-8) for its receptors, previously studied using "trapped" non-associating monomers and non-dissociating dimers, show that the monomer has a native-like function but support conflicting roles for the dimer. We have measured the binding of native IL-8 to the CXCR1 N-domain, using isothermal titration calorimetry and sedimentation equilibrium techniques. The N-domain constitutes a critical binding site, and IL-8 binding affinity to the receptor N-domain is in the same concentration range as the IL-8 monomerdimer equilibrium. We observed that only the IL-8 monomer, and not the dimer, is competent in binding the receptor N-domain. Based on our results, we propose that IL-8 dimerization functions as a negative regulator for the receptor function and as a positive regulator for binding to glycosaminoglycans and that both play a role in the neutrophil recruitment process.

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

“…Residues in the N-terminal domain (binding site-I) and in extracellular loops (binding site-II) of CXCR1 interact with IL-8 (13,14,16,(30)(31)(32)(33)35,66,68,69). Specific residues in the N-terminal domain of CXCR1 contribute to both the selectivity and the affinity of the receptor for IL-8.…”

“…Although IL-8 exists as a homodimer in solution (8), monomeric IL-8 has been shown to bind to CXCR1 with similar or higher affinity than the dimer (13,14). Truncation of the last six C-terminal residues results in a stable monomeric form of IL-8 (1-66) (34).…”

“…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. Modification, mutation, or truncation of residues at the dimer interface stabilize the monomeric form of IL-8 (12,16).…”

“…1 B. Although none of the structures represent complexes of a receptor with one of its native chemokine agonists, the structures of receptors bound to viral chemokines provide information that is complementary to the many mutagenesis and binding studies of their interactions. There have been many solution NMR and other biophysical studies of IL-8 interacting with polypeptides whose sequences correspond to N-terminal residues of CXCR1 (13)(14)(15)(16)(30)(31)(32)(33). The current model for chemokine-receptor interactions involves two binding sites on the receptor.…”

Molecular Evolution of CXCR1, a G Protein-Coupled Receptor Involved in Signal Transduction of Neutrophils

“…A previous study indicated that the protein sequence of CXCR1 is conserved in the transmembrane and intracellular domains but less conserved in the extracellular domains (Rajagopalan and Rajarathnam 2004). The binding of IL-8 involves close interactions between the CXCR1 N-domain and IL-8 N-loop residues and between CXCR1 exoloops and the transmembrane and IL-8 N-terminal residues (Fernando et al 2004;Rajagopalan and Rajarathnam 2004). Both the sliding window analysis and the codon-based neutrality test tend to favor positive selection acting on the N-terminal domain of human CXCR1, the critical region for ligand/receptor signaling for neutrophils.…”