IDENTIFICATION OF A GLYCOSAMINOGLYCAN-BINDING SITE IN CHEMOKINE MACROPHAGE INFLAMMATORY PROTEIN-1-ALPHA

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-1 alpha (MIP-1 alpha) to GAGs. Alignment of beta-chemokine amino acid sequence s 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-1 alpha. Site-directed mutagenesis was used to change the conserved basic residues in MIP-1 alpha to alanines, and both wild-type and mutant proteins were produce d in a transient COS cell expression system. Wild type MIP-1 alpha bou nd to heparin-Sepharose, while three of the mutants, R18A R46A, and R4 8A, 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 th at of the wild-type protein. To determine whether GAG-binding capacity is required for receptor binding and cell activation, we performed co mpetition radioligand binding and calcium mobilization experiments usi ng one of the non-heparin-binding mutants, R46A. R46A bound as efficie ntly as wild-type MIP-1 alpha to CCR1 and was equally active in elicit ing increases in intracellular free calcium concentrations. Our data d efine a GAG binding site in MIP-1 alpha consisting of three noncontigu ous basic amino acids and show that the capacity to bind to GAGs is no t a prerequisite for receptor binding or signaling in vitro.