Chemokines exert their function by binding the GPCR class of receptors on leukocytes and cell surface GAGs in target tissues. Most chemokines reversibly exist as monomers and dimers, but very little is known regarding the molecular mechanisms by which the monomer-dimer equilibrium modulates in vivo function. For the chemokine CXCL8, we recently showed in a mouse lung model that monomers and dimers are active and that the monomer-dimer equilibrium of the WT plays a crucial role in regulating neutrophil recruitment. In this study, we show that monomers and dimers are also active in the mouse peritoneum but that the role of monomer-dimer equilibrium is distinctly different between these tissues and that mutations in GAG-binding residues render CXCL8 less active in the peritoneum but more active in the lung. We propose that tissue-specific differences in chemokine gradient formation, resulting from tissue-specific differences in GAG interactions, are responsible for the observed differences in neutrophil recruitment. Our observation of differential roles played by the CXCL8 monomer-dimer equilibrium and GAG interactions in different tissues is novel and reveals an additional level of complexity of how chemokine dimerization regulates in vivo recruitment.

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.

CXCL8 (interleukin-8) interacts with two receptors, CXCR1and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (ϳ90% versus ϳ10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca Chemokines are a family of structurally related peptides that regulate inflammation through cell-surface G protein-coupled receptors on leukocytes. These peptides mediate diverse biological and biochemical activities, including endothelial adhesion, directed migration, and activation of cytotoxic activities such as the respiratory burst and exocytosis (1, 2). Chemokines have been classified into four families (C, CC, CXC, and CX3C) based on the number and positions of the N-terminally conserved cysteine residues. Most chemokines activate more than one chemokine receptor, and many chemokine receptors are activated by multiple chemokines (3). CXCL8 activates two receptors, CXCR1 and CXCR2. CXCR1 is specific for CXCL8, whereas CXCR2 also interacts with CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 (4). Upon activation, both receptors couple to pertussis toxin-sensitive G protein to mediate phosphoinositide (PI) 2 hydrolysis, intracellular Ca 2ϩ mobilization, chemotaxis, and exocytosis. CXCR1 (but not CXCR2) activates phospholipase D and mediates respiratory burst, suggesting that the two receptors may play different physiological roles (5, 6). Like many G protein-coupled receptors, both receptors become phosphorylated, desensitized, and internalized upon exposure to CXCL8. Over 95% of CXCR2 internalizes in the first 2-5 min of activation compared with ϳ10% of CXCR1 (7-10). CXCR2 also recovers more slowly (ϳ35% after 90 min) to the cell surface than does CXCR1 (ϳ100% after 90 min) upon removal of CXCL8 (7,8,(11)(12)(13)(14). This difference in receptor trafficking appears to be an important factor in the distinct ability of CXCR1 and CXCR2 to mediate leukocyte activation and regulation in response to CXCL8 (9, 10).To date, the molecular basis for the differential regulation of the CXCL8 receptors remains unclear. CXCR1 and CXCR2 are highly homologous (77%) (15, 16). The most divergent regions are the N terminus, the fourth transmembrane domain (TMD), the second extracellular loop (ECL), and the C terminus (15-18). Although both receptors internalize via a phosphorylation/ arrestin/dynamin-dependent mechanism,