Distinct but Overlapping Epitopes for the Interaction of a CC-Chemokine with CCR1, CCR3, and CCR5

Abstract

Chemokines play an important role in inflammation. The mechanism via which they bind to more than one receptor and activate them is not well understood. The chemokines are thought to interact with their receptors via two distinct sites, one necessary for binding and the other for activation of signal transduction. In this study we have used alanine scanning mutagenesis to identify residues on RANTES that specifically interact with its receptors CCR1, CCR3, and CCR5 for binding and activation. Residues within a potential receptor binding site known as the N-loop (residues 12-20) and near the N-terminus of RANTES were individually mutated to alanine. The results of this study show that, within the N-loop, the side chain of R17 is necessary for RANTES binding to CCR1, F12 for binding to CCR3, and F12 and I15 for binding to CCR5, thus forming distinct but overlapping binding epitopes. In addition, our finding that P2 is necessary for binding to CCR5 is the first to show that a residue near the N-terminus of a CC-chemokine is involved in binding to a receptor. We have also found that P2, D6, and T7 near the N-terminus are involved in activating signal transduction via CCR1, P2 and Y3 via CCR3, and Y3 and D6 via CCR5. These results indicate that RANTES interacts with each of its receptors in a distinct and specific manner and provide further evidence to support the two-site model of interaction between chemokines and their receptors.

Control of feeding behavior inC. elegansby human G protein-coupled receptors permits screening for agonist-expressing bacteria

“…5). The hydrophobic core includes F12, whose aromatic side chain has a pivotal role in CCR5 binding (6,(12)(13)(14)(15)(16)(17)(18). Indeed, our findings show a dramatic disruption in the ability to bind CCR5, when this residue is changed to alanine, validating the importance of the aromatic side chain for its binding activity.…”

“…The corresponding threonine residue in MIP-1␤ is important in dimer formation given its location at the hydrophobic dimer interface, where mutation to a charged residue will lead to a monomeric form (12). Interestingly, T8A mutation has no effect on the activity of RANTES with CCR1 (15). We propose that A9 could contribute to a hydrophobic core based around F12, which has been shown to have a role in CCR5 binding (Fig.…”

Molecular determinants for CC-chemokine recognition by a poxvirus CC-chemokine inhibitor

“…Chemokines are divided into four families based on the pattern of cysteine residues (C, CC, CXC, and CX 3 C), and they exert their effects by binding and signaling through seven transmembrane G protein-coupled receptors (GPCRs) on the surface of leukocytes. Although the molecular details remain unclear, mutagenesis and NMR studies have revealed some of the structural requirements for receptor binding and activation (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Collectively, these studies support a hypothesis whereby chemokines activate their receptor through a putative two-step mechanism.…”

“…One important feature of the interaction involves epitopes scattered on the face of chemokine opposite the C-terminal helix, and contacts between this face and the N terminus of the receptor (6,7,11,(14)(15)(16)(17)(18). A second interaction is mediated by the disordered N-terminal region of chemokines preceding the first disulfide motif, which has been shown to be necessary for receptor activation (5,6,8,9,12,14,15,(19)(20)(21)(22).…”

A novel role of hematopoietic CCL5 in promoting triple-negative mammary tumor progression by regulating generation of myeloid-derived suppressor cells

“…It is expressed inT lymphocytes, platelets, macrophages, tubular epithelium, synovial fibroblasts, endothelial cells and selected tumor cells [1,2]. CCL5 interacts with CCR1, CCR3, and CCR5 in various cell types [3].…”