Synthesis and Characterization of Fluorescent and Photoactivatable MIP-1α Ligands and Interactions with Chemokine Receptors CCR1 and CCR5

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Abstract
Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflammatory protein-1α (MIP-1α) were designed, synthesized, characterized, and applied to probe MIP-1α interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1α ligand, BP-MIP-1α, and the fluorescent ligand, Flu-MIP-1α were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1α. Both ligands BP-MIP-1α and Flu-MIP-1α 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 [125I]BP-MIP-1α 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-1α 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-1α was quenched by collision with iodide indicating that the N-terminal end of MIP-1α is accessible to the solvent. These data strongly suggest that the N-terminal end of MIP-1α interacts with domains of CCR1 or CCR5 receptors located at the extracellular surface. The photoactivatable BP-MIP-1α described here should prove valuable for the identification of contact sites on receptors by photoaffinity labeling experiments.