Cisplatin stabilizes a multimeric complex of the human Ctr1 copper transporter: requirement for the extracellular methionine-rich clusters.

Cisplatin is a highly effective cancer chemotherapy agent. However, acquired resistance currently limits the clinical utility of this drug. The human high affinity copper importer, hCtr1, and its yeast and murine orthologues have been shown to mediate the uptake of cisplatin. This transporter is located at the plasma membrane under low copper conditions, and excess copper concentrations stimulate its endocytosis and degradation. In this study we further examined the link between cisplatin and hCtr1 by examining whether cisplatin can also stimulate the endocytosis and degradation of hCtr1. The steady-state location of hCtr1 and its endocytosis from the plasma membrane were not altered by cisplatin treatment. Unexpectedly, cisplatin treatment of a cell line expressing hCtr1 revealed the time- and concentration-dependent appearance of a stable hCtr1 multimeric complex, consistent with a homotrimer, which was not observed following copper treatment of these same cells. Mutagenesis studies identified two methionine-rich clusters in the extracellular amino-terminal region of hCtr1 that were required for stabilization of the hCtr1 multimer by cisplatin, suggesting that these sequences bind cisplatin and form crosslinks between hCtr1 polypeptides. Treatment with the metal chelator dimethyldithiocarbamate disassembled the hCtr1 multimer following cisplatin exposure, suggesting that platinum was an integral component of this complex. These studies provide the first evidence for a direct interaction between cisplatin and the hCtr1 protein and establish that cisplatin and copper have distinct biochemical consequences on this transporter.