Identification of a glycosaminoglycan-binding site in chemokine macrophage inflammatory protein-1alpha.
Chemokines bind to receptors of the seven-transmembrane type on target cells and also bind to glycosaminoglycans (GAGs), including heparin. In this study, we have sought to identify structural motifs mediating binding of the beta-chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) to GAGs. Alignment of beta-chemokine amino acid sequences revealed the presence of several highly conserved basic amino acids, and molecular modeling predicted that the side chains of three of the basic amino acids fold closely together in MIP-1alpha. Site-directed mutagenesis was used to change the conserved basic residues in MIP-1alpha to alanines, and both wild-type and mutant proteins were produced in a transient COS cell expression system. Wild-type MIP-1alpha bound to heparin-Sepharose, while three of the mutants, R18A, R46A, and R48A, failed to bind. Mutant K45A eluted from heparin-Sepharose at lower NaCl concentrations than wild type, while the binding of K61A, with a mutation in the C-terminal alpha-helix, was indistinguishable from that of the wild-type protein. To determine whether GAG-binding capacity is required for receptor binding and cell activation, we performed competition radioligand binding and calcium mobilization experiments using one of the non-heparin-binding mutants, R46A. R46A bound as efficiently as wild-type MIP-1alpha to CCR1 and was equally active in eliciting increases in intracellular free calcium concentrations. Our data define a GAG binding site in MIP-1alpha consisting of three noncontiguous basic amino acids and show that the capacity to bind to GAGs is not a prerequisite for receptor binding or signaling in vitro.
Koopmann
W
W
Department of Immunology, Box 3010 DUMC, Duke University Medical Center, Durham, North Carolina 27701, USA.
Krangel
M S
MS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D016415:Sequence Alignment; D015398:Signal Transduction
D000596:Amino Acids; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D006025:Glycosaminoglycans; D019402:Macrophage Inflammatory Proteins; D006493:Heparin