Synthesis and characterization of fluorescent and photoactivatable MIP-1α ligands and interactions with chemokine receptors CCR1 and CCR5

Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) were designed, synthesized, characterized, and applied to probe MIP-1 alpha interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1 alpha ligand, BP-MIP-1 alpha, and the fluorescent ligand, Flu-MIP-1 alpha were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1 alpha. Both ligands BP-MIP-1 alpha and Flu-MIP-1 alpha retained high binding affinity and agonist potency at CCR1 and CCR5. Photoaffinity labeling of CCR1 and CCR5 receptors stably expressed in CHO cells resulted in specific covalent attachment of [I-125]BP- MIP-1 alpha and production of protein complexes of 54 and 48 kDa, respectively, on SDS-PAGE. This represents the first photo-cross-linking between a chemokine and its receptor. Flu-MIP-1 alpha selectively labeled CCR1 or CCR5 receptors expressed in CHO cells and was used to characterize receptor binding domains. When bound to CCR1 or CCR5 receptors, the fluorescence signal of Flu-MIP-1 alpha was quenched by collision with iodide indicating that the N-terminal end of MIP-1 alpha is accessible to the solvent. These data strongly suggest that the N-terminal end of MIP-1 alpha interacts with domains of CCR1 or CCR5 receptors located at the extracellular surface. The photoactivatable BP-MIP-la described here should prove valuable for the identification of contact sites on receptors by photoaffinity labeling experiments.