Molecular modulation of the copper and cisplatin transport function of CTR1 and its interaction with IRS-4.

The copper influx transporter CTR1 is also a major influx transporter for cisplatin (cDDP) in tumor cells. It influences the cytotoxicity of cDDP both in vivo and in vitro. Whereas Cu triggers internalization of CTR1 from the plasma membrane, cDDP does not. To investigate the mechanisms of these effects, myc-tagged forms of wild type hCTR1 and variants in which Y103 was converted to alanine, C189 was converted to serine, or the K178/K179 dilysine motif was converted to alanines were re-expressed in mouse embryo cells in which both alleles of CTR1 had been knocked out and also in HEK293T cells. The Y103A mutation and to a lesser extent the C189S mutation reduced internalization of CTR1 induced by Cu while the K178A/K179A had little effect. Both Y103 and C189 were required for Cu and cDDP transport whereas the K178/K179 motif was not. While Y103 lies in an YXXM motif that, when phosphorylated, is a potential docking site for phosphatidylinositol 3-kinase and other proteins involved in endocytosis, Western blot analysis of immunoprecipitated myc-CTR1, and proteomic analysis of peptides derived from CTR1, failed to identify any basal or Cu-induced phosphorylation. However, proteomic analysis did identify an interaction of CTR1 with IRS-4 and this was confirmed by co-immunoprecipitation from HEK cells expressing either FLAG-CTR1 or myc-CTR1. The interaction was greater in the Y103A-expressing cells. We conclude that Y103 is required for the internalization of hCTR1 in response to Cu, that this occurs by a mechanism other than phosphorylation and that mutation of Y103 modulates the interaction with IRS-4.