The trace metal copper is an essential cofactor for a number of biological processes including mitochondrial oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal physiology. Little is known about the molecules and biochemical mechanisms responsible for copper uptake at the plasma membrane in mammals. Here, we demonstrate that human Ctr1 (hCtr1) is a component of the copper transport machinery at the plasma membrane. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated 64 Cu transport is an energy-independent process and is stimulated by extracellular acidic pH and high K ؉ concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. This is the first report on the biochemical characterization of the human copper transporter hCtr1, which is important for understanding mechanisms for mammalian copper transport at the plasma membrane.

The trace element copper (Cu) is a cofactor for biochemical functions ranging from energy generation to iron (Fe) acquisition, angiogenesis, and free radical detoxification. While Cu is essential for life, the molecules that mediate dietary Cu uptake have not been identified. Ctr1 is a homotrimeric protein, conserved from yeast to humans, that transports Cu across the plasma membrane with high affinity and specificity. Here we describe the generation of intestinal epithelial cell-specific Ctr1 knockout mice. These mice exhibit striking neonatal defects in Cu accumulation in peripheral tissues, hepatic Fe overload, cardiac hypertrophy, and severe growth and viability defects. Consistent with an intestinal Cu absorption block, the growth and viability defects can be partially rescued by a single postnatal Cu administration, indicative of a critical neonatal metabolic requirement for Cu that is provided by intestinal Ctr1. These studies identify Ctr1 as the major factor driving intestinal Cu absorption in mammals.

c Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. In vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer. Copper (Cu) is a metal ion that functions as a redox-active cofactor for a broad range of biochemical reactions, including mitochondrial oxidative phosphorylation, protection from reactive oxygen species, connective tissue maturation, iron absorption, neuropeptide biogenesis, and other processes (28, 43). Numerous studies point to the essentiality of Cu for normal growth and development, while aberrant Cu accumulation in tissues, as manifested in Wilson's disease patients, results in significant pathologies (33,35,42,47,60,61). However, the precise roles Cu plays and the mechanistic processes by which Cu drives cellular proliferation and growth are not well understood.The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is an evolutionarily conserved pathway involved in the control of many fundamental biological processes, including cell proliferation, apoptosis, survival, differentiation, motility, and metabolism (26,30). Aberrant Ras/MAPK signaling has significant consequences; loss of function of several components of the Ras/MAPK signaling cascade results in lethality, whereas gain-offunction mutations in many of the Ras/MAPK signaling components underlie cancer (2,12,26,55).Here we identify the Ctr1 high-affinity Cu ϩ transporter, conserved from yeast to humans, as being important for stimulation of the MAPK Erk in response to extracellular growth factor-mediated activation of the Ras signaling pathway. Moreover, genetic, physiological, and biochemical experiments point to a direct role for Cu in the ability of the MAPK kinase Mek1 to phosphorylate Erk in fruit flies, cultured cells, and mice. These studies suggest that the MAPK signaling pathway is a key cellular proliferation pathway that is stimulated by Cu and may be a direct target of potent cancer chemotherapeutics that function via Cu chelation. MATERIALS AND METHODS Drosophila melanogaster stocks and crosses. Phantom Gal4, UAS mCD8::GFP/TM6, Tb flies were from Michael O'Connor, University of Minnesota (44). The UAS-Ctr1ARNA...

Copper (Cu) is an essential cofactor for a variety of metabolic functions and the regulation of systemic Cu metabolism is critical to human health. While dietary Cu is absorbed through the intestine, stored in the liver and mobilized into the circulation, systemic Cu homeostasis is poorly understood. We generated mice with a cardiac specific knock out of the Ctr1 Cu transporter, resulting in cardiac Cu deficiency (Ctr1hrt/hrt) and severe cardiomyopathy. Unexpectedly, Ctr1hrt/hrt mice exhibited an increase in serum Cu levels and a concomitant decrease in hepatic Cu stores. Expression of the ATP7A Cu exporter, thought to function predominantly in intestinal Cu acquisition, was strongly increased in liver and intestine of Ctr1hrt/hrt mice. These studies identify ATP7A as a candidate for hepatic Cu mobilization in response to peripheral tissue demand and illuminate systemic regulation that signals the Cu status of the heart to Cu uptake and storage organs.

Significance Copper is essential for normal growth and development because it serves roles in catalysis, signaling, and structure. Cells acquire copper through the copper transporter 1 (Ctr1) protein, a copper transporter that localizes to the cell membrane and intracellular vesicles. Both copper and the anticancer drug cisplatin are imported by Ctr1 by virtue of an extracellular domain rich in metal-binding amino acids. In this report we demonstrate that a protein structurally related to Ctr1, called Ctr2, plays a role in the generation or stability of a truncated form of Ctr1 lacking a large portion of the extracellular domain. Retention of this domain in mice or cells lacking Ctr2 enhances copper and cisplatin uptake, thereby establishing Ctr2 as a regulator of Ctr1 function.

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu ؉ importer that is structurally and functionally

Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1 int/int ). DMT1 int/int mice exhibited a profound hypochromicmicrocytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1 int/int mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1 int/int mice compared with DMT1 ϩ/ϩ mice but no meaningful change in copper, manganese, or zinc. DMT1 int/int mice absorbed 64 Cu and 54 Mn from an intragastric dose to the same extent as did DMT1 ϩ/ϩ mice but the absorption of 59 Fe was virtually abolished in DMT1 int/int mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc. copper absorption; iron deficiency; iron-deficiency anemia; iron-refractive iron-deficiency anemia; manganese absorption; SLC11A2; zinc metabolism IRON DEFICIENCY is the most prevalent micronutrient deficiency worldwide (4, 50). Its deficiency leads to irondeficiency anemia, and to neurological and developmental disorders in children (3, 4). Since there exists no regulated mechanism for the excretion of iron, regulation of the whole body iron economy is achieved by tightly controlling absorption of the metal (21). Failure to regulate iron absorption in a manner appropriate to iron status is characteristic of several hereditary iron-overload disorders and iron-refractive iron-deficiency anemia (12,19,21).Divalent metal-ion transporter-1 (DMT1; reviewed in Ref. 52) is a widely expressed mammalian proton-coupled iron transporter (23,38). Mice in which the SLC11A2 gene coding for DMT1 was globally inactivated (i.e., SLC11A2 Ϫ/Ϫ ) exhibited a severe hypochromic-microcytic anemia and did not survive more than 7 days (22). A critical role for DMT1 in erythroid iron acquisition was confirmed by the following observations: 1) transfusion of red blood cells, but not parenteral iron injections, improved survival of SLC11A2 Ϫ/Ϫ mice; and 2) lethal dose-irradiated wild-type mice into which the investigators transplanted hematopoietic stem cells from SLC11A2 Ϫ/Ϫ mice exhibited defective erythropoiesis (22). The microcytic (mk) mouse and Belgrade (b) rat models, inbred strains that harbor a Gly185¡Arg mutation in DMT1 (17, 18), also exhibited an anemia phenotype. Parenteral iron injections partially improved the condition, and tissue or vesicle preparations from the m...

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