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The mammalian copper transporters CTR1 and CTR2 and their roles in development and disease
The International Journal of Biochemistry & Cell Biology 45 (2013) 960–963
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
The mammalian copper transporters CTR1 and CTR2 and their roles in development and disease Natalie K.Y. Wee a , Daniel C. Weinstein b,c , Stuart T. Fraser a,d,e,1 , Stephen J. Assinder a,d,e,∗,1 a Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney, Medical Foundation Building, Parramatta Road, Camperdown, NSW 2020, Australia b Biology Department, Queens College, City University of New York, Flushing, NY 11367, USA c The Graduate School and University Center, City University of New York, Flushing, NY 11367, USA d Discipline of Anatomy and Histology, Sydney Medical School, University of Sydney, Camperdown, NSW 2020, Australia e Bosch Institute, University of Sydney, Camperdown, NSW 2020, Australia
a r t i c l e
i n f o
Article history: Received 24 August 2012 Received in revised form 14 January 2013 Accepted 25 January 2013 Available online 4 February 2013 Keywords: Copper transporter Development Cancer Chemotherapy Immunity
a b s t r a c t Copper is vital to cell function. The influx of reduced copper ions is controlled by two functionally homologous transmembrane solute carrier transporters CTR1 (encoded by SLC31A1) and CTR2 (encoded by SLC31A2). These copper transporters vary in their expression profiles and intracellular localisation patterns. CTR1 plays roles in the developing embryo as well as regulating homeostasis in the adult mammal. In contrast, the regulation, expression and function of CTR2 is poorly defined. Both are capable of transporting other divalent metal ions and are the primary transporters for platinum-based chemotherapeutic drugs such as cisplatin. This review summarises our current understanding of these two copper transporters and highlights their roles in cellular processes, embryonic development, differentiation, cancer, immunity and disease. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Copper is crucial to cell function, as reflected by the many cuproenzymes described in both prokaryotes and eukaryotes. Copper metabolism is a complex field and is currently the focus of numerous research groups (for reviews of mechanisms of copper homeostasis, see Kaplan and Lutsenko, 2009; Festa and Thiele, 2011; Nevitt et al., 2012). Here, we will focus on the two mammalian transporters responsible for the influx of reduced copper ions across cell membranes, namely CTR1 and CTR2. 2. Structure The yeast Sacchromyces has served as a crucial model for identifying molecules regulating copper influx, highlighting the
Abbreviations: copper tra, nsporter; SLC, solute carrier. ∗ Corresponding author at: Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney, Medical Foundation Building, Parramatta Road, Camperdown, NSW 2020, Australia. Tel.: +61 2 9036 3614; fax: +61 2 9036 3316. E-mail address: [email protected] (S.J. Assinder). 1 Joint senior authors. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.01.018
conserved requirement for copper in cell function. The human CTR1 cDNA was isolated according to its capacity to rescue copper transporter in defective yeast cells (Zhou and Gitschier, 1997). In the human, CTR1 and CTR2 are encoded by the genes SLC31A1 and SLC31A2 respectively (Fig. 1a). These genes are located within close proximity to each other on human chromosome arm 9q32. Paradoxically, the mRNA encoding CTR2 was originally isolated according to homology to CTR1 (Zhou and Gitschier, 1997). However, BLAST analysis reveals 0% homology between the human CTR1 and CTR2 mRNAs, and also between the mouse Ctr1 and 2 mRNAs. SLC31A1 consists of 5 exons and encodes a transcript of 4744 bps. Translation of this transcript results in a 190 amino acid protein. SLC31A2 consists of 4 exons with a protein-coding transcript of 1785 bps and is translated to a 143 amino acid peptide. BLAST analysis of CTR1 and CTR2 demonstrates that these proteins share 33% protein identity with greatest homology in the second of the three membrane-spanning domains (Fig. 1b). Unlike CTR2, CTR1 has a long extracellular N-terminus that contains two methionine rich clusters at residues 7–12 and 40–45, and two histidine clusters at residues 3–6 and 22–24 (Fig. 1b: Larson et al., 2010). Histidine cluster 3–6, at least, is required for copper uptake (Haas et al., 2011).
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2008). Analyses under copper depleted and copper replete conditions identified a consensus sequence for specificity protein 1 (SP1), a transcription factor that responds to changes in copper levels, in SLC31A1 (Fig. 1a: Liang et al., 2012). SLC31A1 is a bone fide target gene of the transcriptional regulator c-Myc (Li et al., 2003). CTR1 and 2 differ markedly in their intracellular localisation. CTR1 is predominantly found at the plasma membrane (reviewed in Kaplan and Lutsenko, 2009; Nevitt et al., 2012). During normal cell function, CTR1 may alternate its location between the plasma membrane and intracellular vesicles (van den Berghe and Klomp, 2010). At high extracellular copper concentration, CTR1 is rapidly internalised and degraded, a process mediated by the N-terminus methionine clusters (Guo et al., 2004). Additional factors that increase CTR1 and modulate localisation include low extracellular pH and hypoxia, whilst degradation is induced by cisplatin entry and increases in glutathione levels (reviewed in Abada and Howell, 2010). At the quartenary level, hormonal stimuli (oestrogen, progesterone and insulin) induce CTR1 multimerisation (Hardman et al., 2006). In contrast to CTR1, CTR2 appears to be localised more in the membranes of intracellular organelles such as vacuoles, vesicles, endosomes and lysosomes in mammalian cells (van der Berghe et al., 2007). Hence, CTR2 may have a role in recycling copper from intracellular stores (Bertinato et al., 2008). However, this localisation varies according to cell type and local copper levels. CTR2 may also function at the plasma membrane. Over-expression of CTR2 in copper-depleted COS-7 cells resulted in the hyper-accumulation of copper when subsequently exposed to copper-enriched media (Bertinato et al., 2008). Post-translational modification regulates CTR1 protein function. Removal of the N-linked glycosylation at residue 15 did not alter localisation of CTR1 but significantly reduced copper transport activity (Maryon et al., 2007). CTR1 can also be O-linked glycosylated at Thr27 (Maryon et al., 2007). This protects against N-terminus cleavage and subsequent reduction in protein function (Maryon et al., 2007). Post-translational regulation of CTR2 function has not been reported.
4. Biological function
Fig. 1. (a) Genomic location of SLC31A1 and SLC31A2 with putative/known transcription factor binding sites. (b) Primary structure of CTR1 and CTR2. The transmembrane domains (TM1-3) shaded yellow with homologous TM2 highlighted. Known sites of N-linked and O-linked glycosylation are coloured orange. Copper binding domains of CTR1 are labelled: H1 – blue, M1 – purple, H2 – green and M2 – red. (c) Representation of the trimerisation of CTR1 to form a pore to facilitate copper uptake. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
3. Expression, activation and turnover While CTR1 is known to be broadly, though not ubiquitously expressed, our knowledge of the expression profile of CTR2 in the mammalian body is extremely limited. The highest levels of Ctr1 have been found in the liver, small intestine, heart and kidneys of mice (Lee et al., 2001; Kuo et al., 2006). Increased levels of Ctr1 are observed in mouse mammary tissue during pregnancy and lactation (Kuo et al., 2006). The sole report examining the histological expression pattern of CTR2 showed high levels of Ctr2 in the rat heart and placenta (Bertinato et al., 2008). At the genomic level, SLC31A1 and SLC31A2 respond to changes in microenvironmental copper levels and cellular copper requirements (Bertinato et al.,
Copper is essential for a range of cellular processes however due to its redox reactivity free copper ions are extremely cytotoxic. Intracellular copper levels must therefore be exquisitely regulated. The biological role of copper includes the incorporation into cuproenzymes for: oxidative phosphorylation (cytochromec oxidase); cellular antioxidant activity (superoxide dismutase); connective tissue formation (lysyl oxidase); neurological function (dopamine -hydroxylase); and iron metabolism (ceruloplasmin) (reviewed in Festa and Thiele, 2011). Intracellular copper is bound to chaperones and other metal-binding proteins such as glutathione to prevent the formation of hydroxyl radicals and facilitate the safe movement of copper. CTR1 is characterised as a high affinity copper uptake transporter. CTR1 monomers assemble into trimers to form a structure containing a pore for Cu2+ and other metals to pass through (Fig. 1c). CTR1 is responsible for the uptake of at least 80% of copper and other metals into cells (Larson et al., 2010). CTR2 has been proposed to facilitate the movement of copper from vesicular organelles to the cytoplasm. This is based on the structural homology of CTR2 to the second transmembrane domain of CTR1 which is essential for Cu2+ transport (Fig. 1b: Zhou and Gitschier, 1997). Comparisons of CTR2 to homologues in other organisms suggest that CTR2 may be associated with the vacuolar membrane and may be responsible for the mobilisation of copper from intracellular stores (Rees et al., 2004). No Ctr2-homozyous null mouse model has been reported. In contrast to the influx regulated by CTRs, copper
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embryonic fibroblasts. This may be due to a loss of Cu+ -dependent MEK activity (Turski et al., 2012). Intriguingly, Ctr1(−/−) embryonic stem cells are recalcitrant to differentiation cues in vitro (Haremaki et al., 2007). Whether this is due to a lack of cuproenyzme function or inhibition of FGF signalling is currently under investigation. There have been no reports of the expression profile or putative function of CTR2 in embryogenesis. 5.2. Cancer
Fig. 2. (a) The multiple roles of copper transporters in metal ion import and cell signalling. CTR1 and CTR2 transport copper and platinum ions into cells. CTR1 can transport copper ions to the mitochondrion, or interact with the FGFR adaptor protein Laloo and promote canonical MAPK/ERK1/2 signalling. CTR2 transported metal ions can be stored in vacuoles or enter the nucleus. (b) Roles of CTR1 and 2 in cancer. High levels of expression of CTR1 is linked with a good prognosis and cisplatin sensitivity. In contrast, CTR2 is associated with cisplatin insensitivity, with a greater relative levels indicating a poorer cancer patient outcome (Lee et al., 2011). The balance point might be targeted to improve prognosis and response to platinum based drug therapies.
export is modulated by the ATPases, ATP7A and ATP7B. Deficiencies in ATP7A and ATP7B function lead to Wilson’s and Menke’s disease respectively (as reviewed in Gupta and Lutsenko, 2009). 5. CTRs in development and disease 5.1. Embryogenesis CTR1 is crucial for embryonic development where it may function as an importer of reduced copper ions and/or by modulating critical intracellular signalling pathways. Ctr1(−/−) mouse embryos fail to gastrulate and die in utero (Lee et al., 2001; Kuo et al., 2001). Mice lacking both copies of the copper chaperone Cox17a demonstrate an identical embryonic lethality suggesting a significant portion of the copper transported by Ctr1 during embryogenesis is targeted to the mitochondria (Takahashi et al., 2002). In frog embryos, Xctr1 interacts with fibroblast growth factor receptors via the adaptor protein Laloo to induce mesodermal development via activation of the ERK pathway (Fig. 2; Haremaki et al., 2007). In support of a role for Ctr1 in inducing signalling pathways, neither FGF nor insulin could induce ERK phosphorylation in Ctr1(−/−) mouse
That CTR1 modulates activation of cell signalling pathways in embryogenesis may be pertinent to the development and progression of cancers where dysregulation of cell signalling cascades is a classical hallmark. CTR1 itself may therefore serve as a novel target for chemotherapies. Perhaps of greater importance, the CTRs are the principal transporters of platinum-based chemotherapeutic agents such as cisplatin (reviewed extensively in Abada and Howell, 2010). CTR expression levels correlate to responsiveness to these drugs with CTR1 the major control point of cisplatin uptake (Ishida et al., 2002). In addition, decreased CTR1 expression in non-smallcell lung cancer and ovarian cancer is associated with reduced survivability (Chen et al., 2012). High levels of expression of CTR1 are linked to a good prognosis possibly due to increased uptake of cisplatin (Ishida et al., 2002). In contrast, CTR2 expression is associated with cisplatin resistance. Knock-down of CTR2 leads to enhanced uptake of cisplatin (reviewed in Abada and Howell, 2010). The loss of SLC31A1 expression with concomitant up-regulation of SLC31A2 is an indicator of poor prognosis in ovarian and breast cancer (Fig. 2b). Recent attention has focused on treatment regimes that deplete microenvironmental copper levels, subsequently inducing surface expression of CTR1 and enhancing chemoresponsiveness of previously recalcitrant cells. This approach extends life span in mice xenotransplanted with cisplatin-resistant breast cancer cells (Ishida et al., 2010). However, in our hands this has failed to increase responsiveness of prostate cancer cell lines to cisplatin in vitro (unpublished observations NW, STF, SJA). Thus the modulation of CTR levels, by genomic manipulation or by copper chelation, could therefore serve to re-invigorate the use of platinum-based drugs in the treatment of previously resistant cancers (Fig. 2b). Enhancing the chemosensitivity of cancer cells could also allow for a reduction in the dose of platinum-based drugs given to the patient, possibly reducing side effects such as ototoxicity, nausea and kidney damage. 5.3. Role of CTRs in immunity The involvement of copper ions in the constant battle between the mammalian immune system and microbial pathogens has recently become a topic of considerable focus. In response to bacterial infection, macrophages up-regulate SLC31A1, exhibit increased surface CTR1, and accumulate copper within their phagosomes via ATP7A (reviewed in Hodgkinson and Petris, 2012). An up-regulation of Ctr1, Ctr2, and ATP7A, is observed in murine bone-derived macrophages infected with Salmonella typhimurium or treated with LPS (Achard et al., 2012). Copper transport also contributes to the pathogenicity of micro-organisms. The human pathogenic fungus Cryptococcus neoformans requires CTR2 to form a polysaccharide capsule that allows the fungal cell to evade engulfment and destruction by host macrophages (Chun and Madhani, 2010). 5.4. CTRs in neurodegenerative disease Consistent with a role for CTRs in macrophage function, microglia also upregulate SLC31A1 in response to
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interferon-gamma activation (Zheng et al., 2010). Chronic perturbations to copper homeostasis results in neuronal degeneration (reviewed in Nevitt et al., 2012). However, the role of copper transporters is only just being defined. Increased copper uptake and trafficking by microglia may have a neuroprotective role in Alzheimer’s disease (Zheng et al., 2010). Similarly, up-regulation of CTR1 in astrocytes has a protective effect in the brain against copper toxicity (reviewed in Nevitt et al., 2012). Mechanisms that regulate the influx of reduced copper ions into cells are clearly playing critical roles in cell function in normal and pathological settings. The roles of CTR1 and CTR2 are only now being elucidated and the similarities and differences between these functionally homologous proteins are becoming evident. Clearly these solute carriers and the molecules they transport warrant further investigation. Acknowledgements This review is a concise summary of a rapidly developing and complex field. We therefore apologise to any authors who could not be cited in the restricted number of references allowed. This work was supported by funding from the Sydney Medical School New Staff Grant and from the Disciplines of Physiology, Anatomy and Histology, School of Medical Sciences, University of Sydney (S.T.F.), a Novel Concept Award from the National Breast Cancer Foundation (S.T.F. & S.J.A) and a National Health and Medical Research Project Grant (#632778; S.J.A). References Abada P, Howell SB. Regulation of cisplatin cytotoxicity by Cu influx transporters. Metal-based Drugs 2010;2010, Article ID 317581, 9 pages, http://dx.doi.org/10.1155/2010/317581 Achard ME, Stafford SL, Bokil NJ, Chartres J, Bernhardt PV, Schembri MA, Sweet MJ, McEwan AG. Copper redistribution in murine macrophages in response to Salmonella infection. Biochemical Journal 2012;444:51–7. Bertinato J, Swist E, Plouffe LJ, Brooks SPJ, L’Abbè MR. Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells. Biochemical Journal 2008;409:731–40. Chen HH, Yan JJ, Chen WC, Kuo MT, Lai YH, Lai WW, Liu HS, Su WC. Predictive and prognostic value of human copper transporter 1 (hCtr1) in patients with stage III non-small-cell lung cancer receiving first-line platinum-based doublet chemotherapy. Lung Cancer 2012;75:228–34. Chun CD, Madhani HD. Ctr2 links copper homeostasis to polysaccharide capsule formation and phagocytosis inhibiton in the human fungal pathogen Cryptococcus neoformans. PLoS ONE 2010;5:e12503. Festa RA, Thiele DJ. Copper an essential metal in biology. Current Biology 2011;21, R877-R833. Guo Y, Smith K, Lee J, Thiele DJ, Petris MJ. Identification of methionine-rich clusters that regulate copper-stimulated endocytosis of the human Ctr1 copper transporter. Journal of Biological Chemistry 2004;279:17428–33. Gupta A, Lutsenko S. Human copper transporters: mechanism, role in human diseases and therapeutic potential. Future Medicinal Chemistry 2009;1:1125–42. Haas KL, Putterman AB, White DR, Thiele DJ, Franz KJ. Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1. Journal of the American Chemical Society 2011;133:4427–37. Hardman B, Manuelpillai U, Wallace EM, Monty JF, Kramer DR, Kuo YM, Mercer JF, Ackland ML. Expression, localisation and hormone regulation of the human
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copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells. Placenta 2006;27:968–77. Haremaki T, Fraser ST, Kuo YM, Baron MH, Weinstein DC. Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation. Proceedings of the National Academy of Sciences of the United States of America 2007;104:12029–34. Hodgkinson V, Petris MJ. Copper homeostasis at the host–pathogen interface. Journal of Biological Chemistry 2012;287:13549–55. Ishida S, Lee J, Thiele DJ, Herskowitz I. Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proceedings of the National Academy of Sciences of the United States of America 2002;99:14298–302. Ishida S, Mccormick F, Smith-Mccune K, Hanahan D. Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator. Cancer Cell 2010;17:574–83. Kaplan JH, Lutsenko S. Copper transport in mammalian cells: special care for a metal with special needs. Journal of Biological Chemistry 2009;284:25461–5. Kuo YM, Zhou B, Cosco D, Gitschier J. The copper transporter CTR1 provides an essential function in mammalian embryonic development. Proceedings of the National Academy of Sciences of the United States of America 2001;98: 6836–41. Kuo YM, Gybina AA, Pyatskowit JW, Gitschier J, Prohaska JR. Copper transport protein (Ctr1) levels in mice are tissue specific and dependent on copper status. Journal of Nutrition 2006;136:21–6. Larson CA, Adams PL, Jandial DD, Blair BG, Safaei R, Howell SB. The role of the Nterminus of mammalian copper transporter 1 in the cellular accumulation of cisplatin. Biochemical Pharmacology 2010;80:448–54. Lee J, Prohaska JR, Thiele DJ. Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proceedings of the National Academy of Sciences of the United States of America 2001;98:6842–7. Lee YY, Choi CH, Do IG, Song SY, Lee W, Park HS, Song TJ, Kim MK, Kim TJ, Lee JW, Bae DS, Kim BG. Prognostic value of the copper transporters, CTR1 and CTR2, in patients with ovarian carcinoma receiving platinum-based chemotherapy. Gynecologic Oncology 2011;122:361–5. Li Z, Van Calcar S, Qu Chunxu Cavenee WK, Zhang MQ, Ren B. A global transcriptional regulatory role for c-Myc in Burkitt’s lymphoma cells. Proceedings of the National Academy of Sciences of the United States of America 2003;100: 8164–9. Liang ZD, Tsai W-B, Lee M-Y, Savaraj N, Kuo MT. Specificity protein 1 (SP1) oscillation is involved in copper homeostasis maintenance by regulating human high-affinity copper transporter 1 expression. Molecular Pharmacology 2012;81:455–64. Maryon EB, Molloy SA, Kaplan JH. O-linked glycosylation at threonine 27 protects the copper transporter hCTR1 from proteolytic cleavage in mammalian cells. Journal of Biological Chemistry 2007;282:20376–87. Nevitt T, Öhrvik H, Thiele DJ. Charting the travels of copper in eukaryotes from yeast to mammals. Biochemistry and Biophysics Acta 2012;1823:1580–93. Rees EM, Lee J, Thiele DJ. Mobilization of intracellular copper stores by the Ctr2 vacuolar copper transporter. Journal of Biological Chemistry 2004;279:54221–9. Takahashi Y, Kako K, Kashiwabara S-C, Takehera A, Inada Y, Arai H, Nakada K, Kodama H, Hayashi J-I, Tadashi B, Munekata E. Mammalian copper chaperone cox17p has an essential role in activation of cytochrome C oxidase and embryonic development. Molecular and Cellular Biology 2002;22:7614–21. Turski ML, Brady DC, Kim HJ, Kim B-E, Nose Y, Counter CM, Winge DR, Thiele DJ. A novel role for copper in Ras/mitogen-activated protein kinase signaling. Molecular and Cellular Biology 2012;32:1284–95. van der Berghe PV, Folmer DE, Malingre HE, van Beurden E, Klomp AE, van de Sluis B, Merkx M, Berger M, Klomp LW. Human copper transporter 2 is localised in late endosomes and lysosomes and facilitates cellular copper uptake. The Biochemical Journal 2007;407:49–59. van den Berghe PE, Klomp LW. Posttranslational regulation of copper transporters. Journal of Biological Inorganic Chemistry 2010;15:37–46. Zheng Z, White C, Lee J, Peterson TS, Bush AI, Sun GY, Weisman GA, Petris MJ. Altered microglial copper homeostasis in a mouse model of Alzheimer’s disease. Journal of Neurochemistry 2010;114:1630–8. Zhou B, Gitschier J. hCTR1: a human gene for copper uptake identified by complementation in yeast. Proceedings of the National Academy of Sciences of the United States of America 1997;94:7481–748.
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
The mammalian copper transporters CTR1 and CTR2 and their roles in development and disease Natalie K.Y. Wee a , Daniel C. Weinstein b,c , Stuart T. Fraser a,d,e,1 , Stephen J. Assinder a,d,e,∗,1 a Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney, Medical Foundation Building, Parramatta Road, Camperdown, NSW 2020, Australia b Biology Department, Queens College, City University of New York, Flushing, NY 11367, USA c The Graduate School and University Center, City University of New York, Flushing, NY 11367, USA d Discipline of Anatomy and Histology, Sydney Medical School, University of Sydney, Camperdown, NSW 2020, Australia e Bosch Institute, University of Sydney, Camperdown, NSW 2020, Australia
a r t i c l e
i n f o
Article history: Received 24 August 2012 Received in revised form 14 January 2013 Accepted 25 January 2013 Available online 4 February 2013 Keywords: Copper transporter Development Cancer Chemotherapy Immunity
a b s t r a c t Copper is vital to cell function. The influx of reduced copper ions is controlled by two functionally homologous transmembrane solute carrier transporters CTR1 (encoded by SLC31A1) and CTR2 (encoded by SLC31A2). These copper transporters vary in their expression profiles and intracellular localisation patterns. CTR1 plays roles in the developing embryo as well as regulating homeostasis in the adult mammal. In contrast, the regulation, expression and function of CTR2 is poorly defined. Both are capable of transporting other divalent metal ions and are the primary transporters for platinum-based chemotherapeutic drugs such as cisplatin. This review summarises our current understanding of these two copper transporters and highlights their roles in cellular processes, embryonic development, differentiation, cancer, immunity and disease. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Copper is crucial to cell function, as reflected by the many cuproenzymes described in both prokaryotes and eukaryotes. Copper metabolism is a complex field and is currently the focus of numerous research groups (for reviews of mechanisms of copper homeostasis, see Kaplan and Lutsenko, 2009; Festa and Thiele, 2011; Nevitt et al., 2012). Here, we will focus on the two mammalian transporters responsible for the influx of reduced copper ions across cell membranes, namely CTR1 and CTR2. 2. Structure The yeast Sacchromyces has served as a crucial model for identifying molecules regulating copper influx, highlighting the
Abbreviations: copper tra, nsporter; SLC, solute carrier. ∗ Corresponding author at: Discipline of Physiology and Bosch Institute, Sydney Medical School, University of Sydney, Medical Foundation Building, Parramatta Road, Camperdown, NSW 2020, Australia. Tel.: +61 2 9036 3614; fax: +61 2 9036 3316. E-mail address: [email protected] (S.J. Assinder). 1 Joint senior authors. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.01.018
conserved requirement for copper in cell function. The human CTR1 cDNA was isolated according to its capacity to rescue copper transporter in defective yeast cells (Zhou and Gitschier, 1997). In the human, CTR1 and CTR2 are encoded by the genes SLC31A1 and SLC31A2 respectively (Fig. 1a). These genes are located within close proximity to each other on human chromosome arm 9q32. Paradoxically, the mRNA encoding CTR2 was originally isolated according to homology to CTR1 (Zhou and Gitschier, 1997). However, BLAST analysis reveals 0% homology between the human CTR1 and CTR2 mRNAs, and also between the mouse Ctr1 and 2 mRNAs. SLC31A1 consists of 5 exons and encodes a transcript of 4744 bps. Translation of this transcript results in a 190 amino acid protein. SLC31A2 consists of 4 exons with a protein-coding transcript of 1785 bps and is translated to a 143 amino acid peptide. BLAST analysis of CTR1 and CTR2 demonstrates that these proteins share 33% protein identity with greatest homology in the second of the three membrane-spanning domains (Fig. 1b). Unlike CTR2, CTR1 has a long extracellular N-terminus that contains two methionine rich clusters at residues 7–12 and 40–45, and two histidine clusters at residues 3–6 and 22–24 (Fig. 1b: Larson et al., 2010). Histidine cluster 3–6, at least, is required for copper uptake (Haas et al., 2011).
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2008). Analyses under copper depleted and copper replete conditions identified a consensus sequence for specificity protein 1 (SP1), a transcription factor that responds to changes in copper levels, in SLC31A1 (Fig. 1a: Liang et al., 2012). SLC31A1 is a bone fide target gene of the transcriptional regulator c-Myc (Li et al., 2003). CTR1 and 2 differ markedly in their intracellular localisation. CTR1 is predominantly found at the plasma membrane (reviewed in Kaplan and Lutsenko, 2009; Nevitt et al., 2012). During normal cell function, CTR1 may alternate its location between the plasma membrane and intracellular vesicles (van den Berghe and Klomp, 2010). At high extracellular copper concentration, CTR1 is rapidly internalised and degraded, a process mediated by the N-terminus methionine clusters (Guo et al., 2004). Additional factors that increase CTR1 and modulate localisation include low extracellular pH and hypoxia, whilst degradation is induced by cisplatin entry and increases in glutathione levels (reviewed in Abada and Howell, 2010). At the quartenary level, hormonal stimuli (oestrogen, progesterone and insulin) induce CTR1 multimerisation (Hardman et al., 2006). In contrast to CTR1, CTR2 appears to be localised more in the membranes of intracellular organelles such as vacuoles, vesicles, endosomes and lysosomes in mammalian cells (van der Berghe et al., 2007). Hence, CTR2 may have a role in recycling copper from intracellular stores (Bertinato et al., 2008). However, this localisation varies according to cell type and local copper levels. CTR2 may also function at the plasma membrane. Over-expression of CTR2 in copper-depleted COS-7 cells resulted in the hyper-accumulation of copper when subsequently exposed to copper-enriched media (Bertinato et al., 2008). Post-translational modification regulates CTR1 protein function. Removal of the N-linked glycosylation at residue 15 did not alter localisation of CTR1 but significantly reduced copper transport activity (Maryon et al., 2007). CTR1 can also be O-linked glycosylated at Thr27 (Maryon et al., 2007). This protects against N-terminus cleavage and subsequent reduction in protein function (Maryon et al., 2007). Post-translational regulation of CTR2 function has not been reported.
4. Biological function
Fig. 1. (a) Genomic location of SLC31A1 and SLC31A2 with putative/known transcription factor binding sites. (b) Primary structure of CTR1 and CTR2. The transmembrane domains (TM1-3) shaded yellow with homologous TM2 highlighted. Known sites of N-linked and O-linked glycosylation are coloured orange. Copper binding domains of CTR1 are labelled: H1 – blue, M1 – purple, H2 – green and M2 – red. (c) Representation of the trimerisation of CTR1 to form a pore to facilitate copper uptake. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
3. Expression, activation and turnover While CTR1 is known to be broadly, though not ubiquitously expressed, our knowledge of the expression profile of CTR2 in the mammalian body is extremely limited. The highest levels of Ctr1 have been found in the liver, small intestine, heart and kidneys of mice (Lee et al., 2001; Kuo et al., 2006). Increased levels of Ctr1 are observed in mouse mammary tissue during pregnancy and lactation (Kuo et al., 2006). The sole report examining the histological expression pattern of CTR2 showed high levels of Ctr2 in the rat heart and placenta (Bertinato et al., 2008). At the genomic level, SLC31A1 and SLC31A2 respond to changes in microenvironmental copper levels and cellular copper requirements (Bertinato et al.,
Copper is essential for a range of cellular processes however due to its redox reactivity free copper ions are extremely cytotoxic. Intracellular copper levels must therefore be exquisitely regulated. The biological role of copper includes the incorporation into cuproenzymes for: oxidative phosphorylation (cytochromec oxidase); cellular antioxidant activity (superoxide dismutase); connective tissue formation (lysyl oxidase); neurological function (dopamine -hydroxylase); and iron metabolism (ceruloplasmin) (reviewed in Festa and Thiele, 2011). Intracellular copper is bound to chaperones and other metal-binding proteins such as glutathione to prevent the formation of hydroxyl radicals and facilitate the safe movement of copper. CTR1 is characterised as a high affinity copper uptake transporter. CTR1 monomers assemble into trimers to form a structure containing a pore for Cu2+ and other metals to pass through (Fig. 1c). CTR1 is responsible for the uptake of at least 80% of copper and other metals into cells (Larson et al., 2010). CTR2 has been proposed to facilitate the movement of copper from vesicular organelles to the cytoplasm. This is based on the structural homology of CTR2 to the second transmembrane domain of CTR1 which is essential for Cu2+ transport (Fig. 1b: Zhou and Gitschier, 1997). Comparisons of CTR2 to homologues in other organisms suggest that CTR2 may be associated with the vacuolar membrane and may be responsible for the mobilisation of copper from intracellular stores (Rees et al., 2004). No Ctr2-homozyous null mouse model has been reported. In contrast to the influx regulated by CTRs, copper
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embryonic fibroblasts. This may be due to a loss of Cu+ -dependent MEK activity (Turski et al., 2012). Intriguingly, Ctr1(−/−) embryonic stem cells are recalcitrant to differentiation cues in vitro (Haremaki et al., 2007). Whether this is due to a lack of cuproenyzme function or inhibition of FGF signalling is currently under investigation. There have been no reports of the expression profile or putative function of CTR2 in embryogenesis. 5.2. Cancer
Fig. 2. (a) The multiple roles of copper transporters in metal ion import and cell signalling. CTR1 and CTR2 transport copper and platinum ions into cells. CTR1 can transport copper ions to the mitochondrion, or interact with the FGFR adaptor protein Laloo and promote canonical MAPK/ERK1/2 signalling. CTR2 transported metal ions can be stored in vacuoles or enter the nucleus. (b) Roles of CTR1 and 2 in cancer. High levels of expression of CTR1 is linked with a good prognosis and cisplatin sensitivity. In contrast, CTR2 is associated with cisplatin insensitivity, with a greater relative levels indicating a poorer cancer patient outcome (Lee et al., 2011). The balance point might be targeted to improve prognosis and response to platinum based drug therapies.
export is modulated by the ATPases, ATP7A and ATP7B. Deficiencies in ATP7A and ATP7B function lead to Wilson’s and Menke’s disease respectively (as reviewed in Gupta and Lutsenko, 2009). 5. CTRs in development and disease 5.1. Embryogenesis CTR1 is crucial for embryonic development where it may function as an importer of reduced copper ions and/or by modulating critical intracellular signalling pathways. Ctr1(−/−) mouse embryos fail to gastrulate and die in utero (Lee et al., 2001; Kuo et al., 2001). Mice lacking both copies of the copper chaperone Cox17a demonstrate an identical embryonic lethality suggesting a significant portion of the copper transported by Ctr1 during embryogenesis is targeted to the mitochondria (Takahashi et al., 2002). In frog embryos, Xctr1 interacts with fibroblast growth factor receptors via the adaptor protein Laloo to induce mesodermal development via activation of the ERK pathway (Fig. 2; Haremaki et al., 2007). In support of a role for Ctr1 in inducing signalling pathways, neither FGF nor insulin could induce ERK phosphorylation in Ctr1(−/−) mouse
That CTR1 modulates activation of cell signalling pathways in embryogenesis may be pertinent to the development and progression of cancers where dysregulation of cell signalling cascades is a classical hallmark. CTR1 itself may therefore serve as a novel target for chemotherapies. Perhaps of greater importance, the CTRs are the principal transporters of platinum-based chemotherapeutic agents such as cisplatin (reviewed extensively in Abada and Howell, 2010). CTR expression levels correlate to responsiveness to these drugs with CTR1 the major control point of cisplatin uptake (Ishida et al., 2002). In addition, decreased CTR1 expression in non-smallcell lung cancer and ovarian cancer is associated with reduced survivability (Chen et al., 2012). High levels of expression of CTR1 are linked to a good prognosis possibly due to increased uptake of cisplatin (Ishida et al., 2002). In contrast, CTR2 expression is associated with cisplatin resistance. Knock-down of CTR2 leads to enhanced uptake of cisplatin (reviewed in Abada and Howell, 2010). The loss of SLC31A1 expression with concomitant up-regulation of SLC31A2 is an indicator of poor prognosis in ovarian and breast cancer (Fig. 2b). Recent attention has focused on treatment regimes that deplete microenvironmental copper levels, subsequently inducing surface expression of CTR1 and enhancing chemoresponsiveness of previously recalcitrant cells. This approach extends life span in mice xenotransplanted with cisplatin-resistant breast cancer cells (Ishida et al., 2010). However, in our hands this has failed to increase responsiveness of prostate cancer cell lines to cisplatin in vitro (unpublished observations NW, STF, SJA). Thus the modulation of CTR levels, by genomic manipulation or by copper chelation, could therefore serve to re-invigorate the use of platinum-based drugs in the treatment of previously resistant cancers (Fig. 2b). Enhancing the chemosensitivity of cancer cells could also allow for a reduction in the dose of platinum-based drugs given to the patient, possibly reducing side effects such as ototoxicity, nausea and kidney damage. 5.3. Role of CTRs in immunity The involvement of copper ions in the constant battle between the mammalian immune system and microbial pathogens has recently become a topic of considerable focus. In response to bacterial infection, macrophages up-regulate SLC31A1, exhibit increased surface CTR1, and accumulate copper within their phagosomes via ATP7A (reviewed in Hodgkinson and Petris, 2012). An up-regulation of Ctr1, Ctr2, and ATP7A, is observed in murine bone-derived macrophages infected with Salmonella typhimurium or treated with LPS (Achard et al., 2012). Copper transport also contributes to the pathogenicity of micro-organisms. The human pathogenic fungus Cryptococcus neoformans requires CTR2 to form a polysaccharide capsule that allows the fungal cell to evade engulfment and destruction by host macrophages (Chun and Madhani, 2010). 5.4. CTRs in neurodegenerative disease Consistent with a role for CTRs in macrophage function, microglia also upregulate SLC31A1 in response to
N.K.Y. Wee et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 960–963
interferon-gamma activation (Zheng et al., 2010). Chronic perturbations to copper homeostasis results in neuronal degeneration (reviewed in Nevitt et al., 2012). However, the role of copper transporters is only just being defined. Increased copper uptake and trafficking by microglia may have a neuroprotective role in Alzheimer’s disease (Zheng et al., 2010). Similarly, up-regulation of CTR1 in astrocytes has a protective effect in the brain against copper toxicity (reviewed in Nevitt et al., 2012). Mechanisms that regulate the influx of reduced copper ions into cells are clearly playing critical roles in cell function in normal and pathological settings. The roles of CTR1 and CTR2 are only now being elucidated and the similarities and differences between these functionally homologous proteins are becoming evident. Clearly these solute carriers and the molecules they transport warrant further investigation. Acknowledgements This review is a concise summary of a rapidly developing and complex field. We therefore apologise to any authors who could not be cited in the restricted number of references allowed. This work was supported by funding from the Sydney Medical School New Staff Grant and from the Disciplines of Physiology, Anatomy and Histology, School of Medical Sciences, University of Sydney (S.T.F.), a Novel Concept Award from the National Breast Cancer Foundation (S.T.F. & S.J.A) and a National Health and Medical Research Project Grant (#632778; S.J.A). References Abada P, Howell SB. Regulation of cisplatin cytotoxicity by Cu influx transporters. Metal-based Drugs 2010;2010, Article ID 317581, 9 pages, http://dx.doi.org/10.1155/2010/317581 Achard ME, Stafford SL, Bokil NJ, Chartres J, Bernhardt PV, Schembri MA, Sweet MJ, McEwan AG. Copper redistribution in murine macrophages in response to Salmonella infection. Biochemical Journal 2012;444:51–7. Bertinato J, Swist E, Plouffe LJ, Brooks SPJ, L’Abbè MR. Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells. Biochemical Journal 2008;409:731–40. Chen HH, Yan JJ, Chen WC, Kuo MT, Lai YH, Lai WW, Liu HS, Su WC. Predictive and prognostic value of human copper transporter 1 (hCtr1) in patients with stage III non-small-cell lung cancer receiving first-line platinum-based doublet chemotherapy. Lung Cancer 2012;75:228–34. Chun CD, Madhani HD. Ctr2 links copper homeostasis to polysaccharide capsule formation and phagocytosis inhibiton in the human fungal pathogen Cryptococcus neoformans. PLoS ONE 2010;5:e12503. Festa RA, Thiele DJ. Copper an essential metal in biology. Current Biology 2011;21, R877-R833. Guo Y, Smith K, Lee J, Thiele DJ, Petris MJ. Identification of methionine-rich clusters that regulate copper-stimulated endocytosis of the human Ctr1 copper transporter. Journal of Biological Chemistry 2004;279:17428–33. Gupta A, Lutsenko S. Human copper transporters: mechanism, role in human diseases and therapeutic potential. Future Medicinal Chemistry 2009;1:1125–42. Haas KL, Putterman AB, White DR, Thiele DJ, Franz KJ. Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1. Journal of the American Chemical Society 2011;133:4427–37. Hardman B, Manuelpillai U, Wallace EM, Monty JF, Kramer DR, Kuo YM, Mercer JF, Ackland ML. Expression, localisation and hormone regulation of the human
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