Synthesis and characterization of fluorescent and photoactivatable MIP-1alpha ligands and interactions with chemokine receptors CCR1 and CCR5.
Photoaffinity and fluorescent analogues of the 70-amino acid chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) were designed, synthesized, characterized, and applied to probe MIP-1alpha interactions with the chemokine receptors CCR1 and CCR5. The photoactivatable MIP-1alpha ligand, BP-MIP-1alpha, and the fluorescent ligand, Flu-MIP-1alpha were prepared by selective chemical coupling of p-benzoylphenylthiocarbamyl or fluoresceinthiocarbamyl, respectively, at the N-terminus of MIP-1alpha. Both ligands BP-MIP-1alpha and Flu-MIP-1alpha 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 [(125)I]BP-MIP-1alpha 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-1alpha 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-1alpha was quenched by collision with iodide indicating that the N-terminal end of MIP-1alpha is accessible to the solvent. These data strongly suggest that the N-terminal end of MIP-1alpha interacts with domains of CCR1 or CCR5 receptors located at the extracellular surface. The photoactivatable BP-MIP-1alpha described here should prove valuable for the identification of contact sites on receptors by photoaffinity labeling experiments.
Zoffmann
S
S
Serono Pharmaceutical Research Institute, 14 chemin des Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland.
Turcatti
G
G
Galzi
J
J
Dahl
M
M
Chollet
A
A
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D019402:Macrophage Inflammatory Proteins / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D011869:Radioligand Assay; D054389:Receptors, CCR1; D019713:Receptors, CCR5 / Q000819:agonists / Q000378:metabolism*; D019707:Receptors, Chemokine / Q000819:agonists / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014162:Transfection
C515300:CCR1 protein, human; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D005456:Fluorescent Dyes; D008024:Ligands; D019402:Macrophage Inflammatory Proteins; D019920:Photoaffinity Labels; D054389:Receptors, CCR1; D019713:Receptors, CCR5; D019707:Receptors, Chemokine; D002118:Calcium