The Functional study of Histidine motif that regulates multimerization and copper stimulated endocytosis of Human Ctr1 Copper Transporter

The human copper transporter 1 (hCtr1) is a high affinity copper transporter at the plasma membrane of human cells. It plays an essential role in copper homeostasis and embryonic development. It has been shown that elevated copper stimulates the endocytosis and degradation of hCtr1 protein. Previous studies indicated that the cystine in 188HCH190, a histidine motif located at the end of C‐terminal region in cytoplasm is required for multimerization of hCtr1. However, the function of the histidine motif and the regulation of hCtr1 multimerization are still unknown. In this study we used mutagenesis and coimmunoprecipitation methods to examine the function of two histidines and cystine in the 188HCH190 motif. We found that hCtr1 indeed forms a homodimer and both 188Histidine and 189Cystine are required for copper stimulated hCtr1 endocytosis and degradation. Unexpectedly, hCtr1 can still form multimer which does not require 189Cystine. Our studies demonstrate that the 188HCH190 motif might be an intracellular copper sensor and play a very important and complicated role in copper induced endocytosis and degradation of hCtr1. It also suggests that the regulation of multimerization of hCtr1 through both covalent and non‐covalent interactions might be another regulation mechanism of hCtr1 copper uptake and trafficking. This work was supported by Grants DK59893 and DK66333 (to M. J. P.) from the National Institutes of Health.