During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seventransmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1͞CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1␤͞ CCL4, and RANTES͞CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization.