hCTR1 Protein Research Articles

Copper transporter 1 in human colorectal cancer cell lines: Effects of endogenous and modified expression on oxaliplatin cytotoxicity

Oxaliplatin-based chemotherapy is the mainstay for the treatment of advanced colorectal cancer. Copper transporter proteins have been implicated in the transport of platinum-based anticancer drugs, but their expression in human colorectal cancer cell lines and roles in controlling their sensitivity to oxaliplatin are not well studied or understood. The endogenous and modified expression of copper uptake transporter 1 (hCTR1) was studied in a panel of human colorectal cancer cell lines (DLD-1, SW620, HCT-15 and COLO205) with ~20-fold variation in oxaliplatin sensitivity. hCTR1 protein was expressed more abundantly than ATP7A and ATP7B proteins, but with broadly similar levels and patterns of expression across four colorectal cancer cell lines. In a colorectal cancer cell-line background (DLD-1), stable transfection of the hCtr1 gene enhanced hCTR1 protein expression and increased the sensitivity of the cells to the cytotoxicity of copper and oxaliplatin. Treatment with copper chelators (ammonium tetrathiomolybdate, bathocuproinedisulfonic acid and D-penicillamine) increased expression of hCTR1 protein in DLD-1 and SW620 cells, and potentiated the cytotoxicity of oxaliplatin in DLD-1 but not SW620 cells. Treatment with copper chloride altered neither the expression of copper transporters nor cytotoxicity of oxaliplatin in colorectal cancer lines. In conclusion, human colorectal cancer cell lines consistently express hCTR1 protein despite their variable sensitivity to oxaliplatin. Genetic or pharmacological modification of hCTR1 protein expression may potentiate oxaliplatin sensitivity in some but not all colorectal cancer cell lines.

Comparison between copper and cisplatin transport mediated by human copper transporter 1 (hCTR1)

Copper transporter 1 (CTR1) is a transmembrane protein that imports copper(i) into yeast and mammalian cells. Surprisingly, the protein also mediates the uptake of platinum anticancer drugs, e.g. cisplatin and carboplatin. To study the effects of several metal-binding residues/motifs of hCTR1 on the transport of both Cu(+) and cisplatin, we have constructed Hela cells that stably express a series of hCTR1 variant proteins including H22-24A, NHA, C189S, hCTR1ΔC, H139R and Y156A, and compared their abilities to regulate the accumulation and cytotoxicity of these metal compounds. Our results demonstrated that the cells expressing the hCTR1 mutants of histidine-rich motifs in the N-terminus (H22-24A, NHA) resulted in a higher basal copper level in the steady state compared to those expressing wild-type protein. However, the cellular accumulation of both copper and cisplatin in these variants was found at a similar level to that of wild type when incubated with an excess of metal compounds (100 μM). The cells expressing hCTR1 variants of H139R and Y156A exhibit lower capacities towards accumulation of copper but not cisplatin. Significantly, cells with the C189S variant partially retained the ability of the wild-type hCTR1 protein to accumulate both copper and cisplatin, while for cells expressing the C-terminus truncated variant of hCTR1 (hCTR1ΔC) this ability was absolutely abolished, suggesting that this motif is crucial for the function of the transporter.

Turnover Rate and Cell Copy Number of Over-Expressed and Endogenous hCTR1, the Human High Affinity Cu Transporter

Copper is an essential trace element required as a co-factor in many enzymatic reactions including: cytochrome oxidase, lysl oxidase, Cu/Zn superoxidase dismutase and ferroxidases. The human copper transporter, hCTR1, is comprised of 190 aa with 3 transmembrane domains and forms a homotrimeric complex with a central pore which transports monovalent Cu. Cu levels and fluxes are low in human cells and we were interested in determining the basis of this phenomenon. Using 64Cu isotopic uptake measurements, cell surface biotinylation and quantification of hCTR1 using purified recombinant hCTR1 protein as a standard we have determined the number of copper ions transported per minute by each hCTR1 trimer.

O-Linked Glycosylation at Threonine 27 Protects the Copper Transporter hCTR1 from Proteolytic Cleavage in Mammalian Cells

The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wild-type hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.

Expression, Localisation and Hormone Regulation of the Human Copper Transporter hCTR1 in Placenta and Choriocarcinoma Jeg-3 Cells

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Stable Plasma Membrane Levels of hCTR1 Mediate Cellular Copper Uptake

The human copper transporter 1 (hCtr1), when heterologously overexpressed in insect cells, mediates saturable Cu uptake. In mammalian expression systems, a rapid Cu-dependent internalization of hCtr1 has been reported in cells that overexpress epitope-tagged hCtr1 when exposed to Cu in the external medium. This finding led to the suggestion that such internalization may be a step in the hCtr1 transmembrane Cu transport mechanism. We have demonstrated that preincubation in Cu-containing media of sf9 cells stably expressing hCtr1 has no effect on the initial rate of Cu transport. Furthermore, Western blot analyses of fractionated sf9 cell membranes show no evidence of a regulatory Cu-dependent internalization from the plasma membrane. In similar studies on human embryonic kidney (HEK) 293 cells, we showed that incubation with Cu does not alter the initial rate of Cu uptake mediated by endogenous levels of hCtr1 compared with untreated cells. Confirmation that hCtr1 mediates this transport is provided by specific small interfering RNA-dependent decreases in hCtr1 protein levels and in Cu transport rates. Western blot analysis and confocal microscopy of human embryonic kidney 293 cells showed that the majority of hCtr1 protein is localized at the plasma membrane and no significant internalization is detected upon Cu treatment. We concluded that internalization of hCtr1 is not a required step in the transport pathway; we suggest that oligomeric hCtr1 acts as a conventional transporter providing a permeation pathway for Cu through the membrane and that internalization of endogenous hCtr1 in response to elevated extracellular Cu levels does not play a significant regulatory role in Cu homeostasis.

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.

Identification of Methionine-rich Clusters That Regulate Copper-stimulated Endocytosis of the Human Ctr1 Copper Transporter

Copper uptake and subsequent delivery to copper-dependent enzymes are essential for many cellular processes. However, the intracellular levels of this nutrient must be controlled because of its potential toxicity. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. Recent studies have shown that elevated copper stimulates the endocytosis and degradation of the hCtr1 protein, and this response is likely an important homeostatic mechanism that prevents the overaccumulation of copper. The domains of hCtr1 involved in copper-stimulated endocytosis and degradation are unknown. In this study we examined the importance of potential copper-binding sequences in the extracellular domain and a conserved transmembrane (150)MXXXM(154) motif for copper-stimulated endocytosis and degradation of hCtr1. The endocytic response of hCtr1 to low copper concentrations required an amino-terminal methionine cluster ((40)MMMMPM(45)) closest to the transmembrane region. However, this cluster was not required for the endocytic response to higher copper levels, suggesting this motif may function as a high affinity copper-sensing domain. Moreover, the transmembrane (150)MXXXM(154) motif was absolutely required for copper-stimulated endocytosis and degradation of hCtr1 even under high copper concentrations. Together with previous studies demonstrating a role for these motifs in high affinity copper transport activity, our findings suggest common biochemical mechanisms regulate both transport and trafficking functions of hCtr1.

The SLC31 (Ctr) copper transporter family.

Copper is essential for many copper-dependent processes, including mitochondrial oxidative phosphorylation, free-radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. The identification of proteins for high affinity copper uptake and export has greatly expanded our understanding of cellular copper homeostasis. Copper export in human cells is mediated by the ATP7A and ATP7B P-type ATPases, which are, respectively, affected in the genetic disorders of copper metabolism, Menkes disease and Wilson disease. A different class of transporter known as the SLC31 or Ctr family of proteins mediates cellular copper uptake. These high-affinity copper transporters exist in all eukaryotes and their discovery has provided new insights into how cells acquire and regulate this essential nutrient. The following is a brief overview of the SLC31 copper transporter family with a focus on the human hCtr1 protein.

Copper-stimulated Endocytosis and Degradation of the Human Copper Transporter, hCtr1

Copper uptake at the plasma membrane and subsequent delivery to copper-dependent enzymes is essential for many cellular processes, including mitochondrial oxidative phosphorylation, free radical detoxification, pigmentation, neurotransmitter synthesis, and iron metabolism. However, intracellular levels of this nutrient must be controlled because it is potentially toxic in excess concentrations. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. In this study, we demonstrate that levels of the hCtr1 protein at the plasma membrane of HEK293 cells were reduced when cells were exposed to elevated copper. This decrease in surface hCtr1 levels was associated with an increased rate of endocytosis, and low micromolar concentrations of copper were sufficient to stimulate this process. Inhibitors of clathrin-dependent endocytosis prevented the trafficking of hCtr1 from the plasma membrane, and newly internalized hCtr1 and transferrin were co-localized. Significantly, elevated copper concentrations also resulted in the degradation of the hCtr1 protein. Our findings suggest that hCtr1-mediated copper uptake into mammalian cells is regulated by a post-translational mechanism involving copper-stimulated endocytosis and degradation of the transporter.

Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1).

The human copper transporter 1 gene (hCTR1) was previously identified by functional complementation in ctr1-deficient yeast. Overexpression of hCTR1 in wild-type yeast leads to increased sensitivity to copper toxicity, and mice with a homozygous disruption at the Ctr1 locus die early during embryogenesis. It is proposed that hCTR1 is responsible for high-affinity copper uptake into human cells, but the underlying molecular mechanisms are unknown. To begin to investigate the biochemical characteristics of hCTR1, a polyclonal antiserum was raised against recombinant hCTR1-fusion peptides. Biosynthetic studies using this antiserum revealed that hCTR1 was synthesized as a precursor protein of 28 kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx. 35 kDa, which is ubiquitously expressed. Immunofluorescence studies showed that subcellular hCTR1 localization differed markedly between cell types. In some cell lines, hCTR1 was located predominantly in an intracellular vesicular perinuclear compartment, and in others hCTR1 was located predominantly at the plasma membrane. In contrast with the copper export P-type ATPases mutated in Wilson disease and Menkes disease, the localization of hCTR1 was not influenced by copper concentrations. Inhibition of endocytosis by methyl-beta-cyclodextrin caused a partial redistribution of hCTR1 to the cell surface of HeLa cells. Taken together, the results in this study suggest a cell-specific control of copper uptake, which involves subcellular localization of the hCTR1 protein.

Characterization of the h CTR1 gene: Genomic organization, functional expression, and identification of a highly homologous processed gene

The human h CTR1 gene was originally identified by its ability to complement a yeast mutant deficient in high-affinity copper uptake (Zhou, B., Gitschier, J., 1997. A human gene for copper uptake identified by complementation in yeast. Proc. Natl. Acad. Sci. USA 94, 7481–7486). Here, we have determined the DNA sequence of the exon–intron borders of the h CTR1 structural gene and report that the coding sequence is disrupted by three introns, all of which comply with the GT/AG rule. Furthermore, human fibroblasts, transfected with h CTR1 cDNA, were shown to have a dramatically increased capacity for 64Cu uptake, indicating that the hCtr1 protein is functional in copper uptake in human cells. In contrast, no evidence was found for involvement of the h CTR2 gene product in copper uptake. Finally, we have identified a highly homologous processed pseudogene, h CTR1ψ, which was localized to chromosome 3q25/26. The processed gene was found to be transcribed, but due to a frame shift mutation, it only had the potential to encode a truncated protein of 95 amino acid residues, and cells transfected with h CTR1ψ DNA showed no increase of 64Cu uptake.