Distinct but overlapping epitopes for the interaction of a CC-chemokine with CCR1, CCR3 and CCR5.
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.
Pakianathan
D R
DR
Department of Immunology, Genentech Inc., South San Francisco, California 94080, USA.
Kuta
E G
EG
Artis
D R
DR
Skelton
N J
NJ
Hébert
C A
CA
D016475:3T3 Cells; D000818:Animals; D018946:Chemokine CCL5 / Q000235:genetics; D018925:Chemokines / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D054389:Receptors, CCR1; D054397:Receptors, CCR3; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000378:metabolism*; D015728:Receptors, HIV / Q000378:metabolism
C515300:CCR1 protein, human; C515350:CCR3 protein, human; C515301:Ccr1 protein, mouse; C515351:Ccr3 protein, mouse; D018946:Chemokine CCL5; D018925:Chemokines; D000939:Epitopes; D054389:Receptors, CCR1; D054397:Receptors, CCR3; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D015728:Receptors, HIV