Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines. To characterize the CCR1-mediated cellular signal transduction mechanism, we used the yeast two-hybrid system to identify a cellular ligand for CCR1. LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein. Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established. We found that LZIP binds to CCR1 based on results of a mammalian two-hybrid assay and immunoprecipitation experiments. The 21-260 residues of LZIP were essential for interaction with CCR1. Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn-1-induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP-1alpha, RANTES, or HCC-4, were not affected by LZIP overexpression. These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn-1-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1.

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Leukotactin-1 (Lkn-1)/CCL15 is a recently cloned CC-chemokine that binds to the CCR1 and CCR3. Although Lkn-1 has been known to function as a chemoattractant for neutrophils, monocytes and lymphocytes, its cellular mechanism remains unclear. To understand the mechanism of Lkn-1-induced chemotaxis signaling, we examined the chemotactic activities of human osteogenic sarcoma cells expressing CCR1 in response to Lkn-1 using inhibitors of signaling molecules. Inhibitors of G i /G o protein, phospholipase C (PLC) and protein kinase CN N (PKCN N) inhibited the chemotactic activity of Lkn-1 indicating that Lkn-1-induced chemotaxis signal is transduced through G i /G o protein, PLC and PKCN N. The activities of PLC and PKCN N were also enhanced by Lkn-1 stimulation. Chemotactic activity of Lkn-1 was inhibited by the treatment of cycloheximide and actinomycin D suggesting that newly synthesized proteins are needed for chemotaxis. Nuclear factor-U UB (NF-U UB) inhibitor reduced chemotactic activity of Lkn-1. DNA binding activity of NF-U UB was also enhanced by Lkn-1 stimulation. These results suggest that Lkn-1 transduces the signal through G i /G o protein, PLC, PKCN N, NF-U UB and newly synthesized proteins for chemotaxis. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.