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...