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

Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macr ophage inflammatory protein-1 alpha (MIP-1 alpha) were designed, synthesize d, characterized, and applied to probe MIP-1 alpha interactions with the ch emokine receptors CCR1 and CCR5. The photoactivatable MIP-1 alpha ligand, B P-MIP-1 alpha, and the fluorescent ligand, Flu-MIP-1 alpha were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceint hiocarbamyl, respectively, at the N-terminus of MIP-1 alpha. Both ligands B P-MIP-1 alpha and Flu-MIP-1 alpha retained high binding affinity and agonis t potency at CCR1 and CCR5. Photoaffinity labeling of CCR1 and CCR5 recepto rs stably expressed in CHO cells resulted in specific covalent attachment o f [I-125]BP- MIP-1 alpha and production of protein complexes of 54 and 48 k Da, respectively, on SDS-PAGE. This represents the first photo-cross-linkin g 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 fluore scence signal of Flu-MIP-1 alpha was quenched by collision with iodide indi cating 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 interac ts with domains of CCR1 or CCR5 receptors located at the extracellular surf ace. The photoactivatable BP-MIP-la described here should prove valuable fo r the identification of contact sites on receptors by photoaffinity labelin g experiments.