Characterization of a monoclonal antibody capable of reliably quantifying expression of human Copper Transporter 1 (hCTR1).
ABSTRACT: Human copper transporter 1 (hCTR1) is the high-affinity copper influx transporter in mammalian cells that also mediates the influx of cisplatin. Loss of hCTR1 expression has been implicated in the development of resistance to this cancer chemotherapeutic agent. It has turned out to be very difficult to develop antibodies to hCTR1 and polyclonal antibodies produced by different laboratories have yielded conflicting results. We have characterized a newly-available rabbit monoclonal antibody that reacts with an epitope on the N-terminal end of hCTR1 that now permits rigorous identification and quantification of hCTR1 using Western blot analysis. Postnuclear membrane (PNM) preparations made from cells engineered to express high levels of myc-tagged hCTR1, and cells in which the expression of hCTR1 was knocked down, were used to characterize the antibody. The identity of the bands detected was confirmed by immunoprecipitation, surface biotinylation and deglycosylation of myc-tagged hCTR1. Despite the specificity expected of a monoclonal antibody, the anti-hCTR1 detected a variety of bands in whole cell lysates (WCL), which made it difficult to quantify hCTR1. This problem was overcome by isolating post-nuclear membranes and using these for further analysis. Three bands were identified using this antibody in PNM preparations that migrated at 28, 33-35 and 62-64kDa. Multiple lines of evidence presented here suggest that the 33-35 and 62-64kDa bands are hCTR1 whereas the 28kDa band is a cross-reacting protein of unknown identify. The 33-35kDa band is consistent with the expected MW of the glycosylated hCTR1 monomer. This analysis now permits rigorous identification and quantification of hCTR1.
Project description: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.
Project description:Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using (64)Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu(+) first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.
Project description:Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 A. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.
Project description:The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.
Project description:We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper-binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter.
Project description:The ?V integrin is expressed in most cancer cells where it regulates a diverse array of cellular functions essential to the initiation, progression and metastasis of solid tumors. However, little is known about how ?V integrin modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. In this study, we found that down-regulation of ?V sensitized human M21 cells to cisplatin (cDDP) through up-regulation of the copper influx transporter CTR1. Cells selected for low ?V integrin expression (M21L) were more sensitive to cDDP, accompanied by increase in CTR1 mRNA and CTR1 protein levels, more intracellular cDDP accumulation and cDDP DNA adduct formation. Basal copper (Cu) content, Cu uptake, and Cu cytotoxicity were also increased. Transfection of a luciferase reporter construct containing the hCTR1 promoter sequence revealed an increase of the hCTR1 transcription activity in M21L cells. The basis for the increased hCTR1 transcription was related to an increase in the steady-state level of Sp1, a transcription factor known to drive hCTR1 expression. These results indicate that the ?V integrin modulates sensitivity of human cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1, and introduce the concept that ?V expression is linked to Cu homeostasis.
Project description:The human copper transporter hCTR1 is a homotrimer composed of a plasma membrane protein of 190 amino acids that contains three transmembrane segments. The extracellular 65-amino acid amino terminus of hCTR1 contains both N-linked (at Asn(15)) and O-linked (at Thr(27)) sites of glycosylation. If O-glycosylation at Thr(27) is prevented, hCTR1 is efficiently cleaved, removing approximately 30 amino acids from the amino terminus. We have now investigated (i) the site of this cleavage, determining which peptide bonds are cleaved, (ii) the mechanism by which glycosylation prevents cleavage, and (iii) where in the cell the proteolytic cleavage takes place. Cleavage occurs in the sequence Ala-Ser-His-Ser-His (residues 29-33), which does not contain previously recognized protease cleavage sites. Using a series of hCTR1 mutants, we show that cleavage occurs preferentially between residues Ala(29)-Ser(30)-His(31). We also show that the O-linked polysaccharide at Thr(27) blocks proteolysis due to its proximity to the cleavage site. Moving the cleavage site away from the Thr(27) polysaccharide by insertion of as few as 5 amino acids allows cleavage to occur in the presence of glycosylation. Imaging studies using immunofluorescence in fixed cells and a functional green fluorescent protein-tagged hCTR1 transporter in live cells showed that the cleaved peptide accumulates in punctate structures in the cytoplasm. These puncta overlap compartments were stained by Rab9, indicating that hCTR1 cleavage occurs in a late endosomal compartment prior to delivery of the transporter to the plasma membrane.
Project description:Copper is an essential metal nutrient, yet copper overload is toxic. Here, we report that human copper transporter (hCtr) 1 plays an important role in the maintenance of copper homeostasis by demonstrating that expression of hCtr1 mRNA was up-regulated under copper-depleted conditions and down-regulated under copper-replete conditions. Overexpression of full-length hCtr1 by transfection with a recombinant hCtr1 cDNA clone reduced endogenous hCtr1 mRNA levels, whereas overexpression of N terminus-deleted hCtr1 did not change endogenous hCtr1 mRNA levels, suggesting that increased functional hCtr1 transporter, which leads to increased intracellular copper content, down-regulates the endogenous hCtr1 mRNA. A luciferase assay using reporter constructs containing the hCtr1 promoter sequences revealed that three Sp1 binding sites are involved in the basal and copper concentration-dependent regulation of hCtr1 expression. Modulation of Sp1 levels affected the expression of hCtr1. We further demonstrated that the zinc-finger domain of Sp1 functions as a sensor of copper that regulates hCtr1 up and down in response to copper concentration variations. Our results demonstrate that mammalian copper homeostasis is maintained at the hCtr1 mRNA level, which is regulated by the Sp1 transcription factor.
Project description:Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased (64)Cu radioactivity were visualized previously by PET using (64)CuCl2 as a radiotracer ((64)CuCl2 PET). This study aimed to determine whether the increased tumor (64)Cu radioactivity was due to increased cellular uptake of (64)Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic (64)CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed.A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular (64)Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of (64)Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper.RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of (64)Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer (64)CuCl2, the (64)Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the (64)Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 %ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm(3) for Lenti-hCtr1-shRNA-PC-3 or 39 ± 22 mm(3) for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm(3) for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm(3) for Lenti-SCR-shRNA-DU-145, P < 0.01).Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using (64)CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.
Project description:The high-affinity copper transporter (Ctr1; SCLC31A1) plays an important role in regulating copper homeostasis because copper is an essential micronutrient and copper deficiency is detrimental to many important cellular functions, but excess copper is toxic. Recent research has revealed that human copper homeostasis is tightly controlled by interregulatory circuitry involving copper, Sp1, and human (hCtr1). This circuitry uses Sp1 transcription factor as a copper sensor in modulating hCtr1 expression, which in turn controls cellular copper and Sp1 levels in a 3-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by copper stresses has also been described in the literature. Because hCtr1 can also transport platinum drugs, this finding underscores the important role of hCtr1 in platinum-drug sensitivity in cancer chemotherapy. Consistent with this notion is the finding that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies showed that elevated hCtr1 expression can be induced by depleting cellular copper levels, resulting in enhanced cisplatin uptake and its cell-killing activity. A phase I clinical trial using a combination of trientine (a copper chelator) and carboplatin has been carried out with encouraging results. This review discusses new insights into the role of hCtr1 in regulating copper homeostasis and explains how modulating cellular copper availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation.