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Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon; and Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
ABSTRACT I. INTRODUCTION: THE ESSENTIAL ROLE OF COPPER-TRANSPORTING ATPases ATP7A AND ATP7B IN HUMAN PHYSIOLOGY II. COPPER DISTRIBUTION IN TISSUES A. Major Pathways of Copper Distribution 1. Copper uptake in intestine 2. ATP7B in hepatocytes is essential for homeostatic regulation of copper in the body 3. Copper distribution in nonhepatic tissues B. Function of Cu-ATPases in Copper Delivery to the Secretory Pathway 1. Copper transfer to tyrosinase can be used to measure Cu-ATPase activity in cells 2. Lysyl oxidase dependent processes are particularly sensitive to ATP7A inactivation 3. ATP7A-mediated copper delivery to peptidylglycine alpha-amidating monooxygenase does not require additional protein chaperones 4. Ceruloplasmin biosynthesis and ATP7B function are interdependent III. TISSUE-SPECIFIC FUNCTIONS OF Cu-ATPases A. Expression Patterns of Cu-ATPases in Tissues Correlate With Phenotypic Manifestations of Menkes Disease and Wilson Disease B. Both ATP7A and ATP7B Are Involved in Copper Transport to Milk C. Cu-ATPases Are Likely to Contribute to Tight Homeostatic Control of Copper in Kidneys D. Localization and Protein Levels of Cu-ATPases in Placenta Are Regulated by Hormones E. Multifaceted Contribution of Cu-ATPases to Central Nervous System Function F. Developmental Changes in Expression of Cu-ATPases IV. GENOMIC AND PROTEIN ORGANIZATION OF HUMAN Cu-ATPases A. Genomic Organization of ATP7A and ATP7B B. General Architecture of Cu-ATPases and the Functional Roles of Domains 1. The NH2-terminal copper-binding domain regulates Cu-ATPase activity 2. ATP-binding domain of Cu-ATPases has distinct nucleotide-coordination environment 3. Roles of the A-domain and the COOH terminus C. Alternative Splicing and Protein Variants of ATP7A and ATP7B 1. Splicing variants of ATP7B mRNA 2. PINA 3. Alternative splicing of ATP7A D. Promoters and Transcriptional Regulation of ATP7A and ATP7B V. TRANSPORT MECHANISM OF HUMAN Cu-ATPases A. Heterologous Expression of Cu-ATPases and Functional Assays B. ATP7A and ATP7B Have Distinct Enzymatic Characteristics C. Transport Activity of Cu-ATPases 1. Factors that may affect copper transport 2. Transport activity of Cu-ATPases in different cell locations D. Copper Delivery From Cytosol to the Intramembrane Sites of Cu-ATPase E. Atox1-Mediated Transfer of Copper to Cu-ATPases VI. REGULATION OF Cu-ATPase FUNCTION THROUGH INTRACELLULAR TRAFFICKING A. Copper-Dependent Trafficking of ATP7A 1. Physiological relevance of copper-dependent relocalization of Cu-ATPases 2. Does copper stimulate forward trafficking or inhibit endocytosis? B. Localization and Trafficking of ATP7B C. Molecular Mechanism of Copper-Dependent Trafficking of Cu-ATPases 1. Does copper binding to the NH2-terminal domain serve as a signal for trafficking? 2. The link between the catalytic cycle of Cu-ATPases and their trafficking D. Protein Machinery Involved in Cu-ATPase Trafficking 1. Does ATP7A use a clathrin-mediated pathway for trafficking? 2. Role of Rab GTPases, Cdc4, and kinase-mediated phosphorylation in Cu-ATPase trafficking 3. Regulation of Cu-ATPase trafficking by copper chaperone Atox1 4. Murr1 (COMMD1) and dynactin subunit p62 interact specifically with ATP7B VII. CONCLUSION NOTE ADDED IN PROOF GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION: THE ESSENTIAL ROLE OF COPPER-TRANSPORTING ATPases ATP7A AND ATP7B IN HUMAN PHYSIOLOGY |
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-hydroxylase, tyrosinase, lysyl oxidase, peptidylglycine-
-amidating monooxygenase, ceruloplasmin, and others. The physiological importance of Cu-ATPases in humans can be illustrated by the deleterious consequences of the Cu-ATPase inactivation on cell metabolism. Mutations or deletions in the gene encoding the Cu-ATPase ATP7A are associated with a fatal childhood disorder, Menkes disease (OMIM 309400). Patients with Menkes disease display dramatic developmental and neurological impairment due to disrupted delivery of copper to the brain (114). In addition, the patients have a variety of other symptoms that are caused by decreased function of copper-dependent enzymes and include connective tissue abnormalities, lack of pigmentation, and tortuosity of blood vessels (114, 130, 131, 266, 281, 297). The majority of Menkes disease patients die in early childhood.
Mutations in the gene encoding the Cu-ATPase ATP7B also result in a severe metabolic disorder, known as Wilson disease (OMIM 277900). The phenotypic manifestations of this disorder differ from those of Menkes disease. ATP7B inactivation is associated with copper accumulation in several tissues, particularly in the liver and the brain, and a spectrum of hepatic and neurological abnormalities. These may include liver dysfunction or failure, movement disorders, and psychiatric manifestations (65, 75, 90). Although copper chelation therapy using D-penicillamine is available for treatment of Wilson disease, this therapy is not always successful, especially if diagnosis is delayed, and may have severe side effects (28, 110, 201). In recent years, other therapies have been developed, including treatments with trientine, tetrathiomolobdate, or zinc; however, their efficacy and potential side effects remain to be fully evaluated (29, 182, 259).
The two diseases of copper metabolism illustrate the fundamental need for tight homeostatic control of intracellular copper as either copper deficiency (Menkes disease) or copper accumulation (Wilson disease) are extremely deleterious to cell function. Such precise control is mediated through the coordinated action of several proteins, including the high-affinity copper uptake protein Ctr1, a set of small cytosolic copper carriers called metallochaperones, which distribute copper to various cell destinations, and Cu-ATPases. Descriptions of cellular machinery involved in copper uptake and in the subsequent distribution of copper can be found in a number of excellent recent reviews (96, 178, 209, 220). In this review we focus primarily on the physiological role of Cu-ATPases in various tissues and the molecular mechanisms of their function and regulation. Clinical aspects of Menkes disease and Wilson disease as well as currently available animal models have been described in recent literature (66, 90, 130, 131, 175, 250, 280, 281) and are not discussed here.
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II. COPPER DISTRIBUTION IN TISSUES |
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Copper plays a critical role in human metabolism as a cofactor of key metabolic enzymes, which are involved in respiration, neurotransmitter biosynthesis, radical detoxification, iron metabolism, and many other physiological processes. The average daily intake of copper is between 1 and 3 mg, and this amount is adequate for body needs. The majority of copper absorption appears to take place in the duodenum; however, the molecular pathways through which dietary copper is absorbed by the intestinal epithelium are not well understood. The high-affinity copper transporter Ctr1 has been detected at the apical membrane of intestinal cells in agreement with a role in copper uptake from the lumen (139). However, this localization is seen only in suckling mice, while in adult animals Ctr1 has mostly an intracellular localization (139). This observation suggests a role for Ctr1 in regulated, rather than constitutive, copper transport and/or possible involvement of Ctr1 in the export of copper from intracellular stores for further utilization by the cell.
These possible roles of Ctr1 in the intestinal copper uptake were highlighted in the experiments on targeted deletion of Ctr1 in mouse intestine. Genetic inactivation of Ctr1 was shown to effectively block copper absorption into the blood and result in copper deficiency in other tissues (195). However, the deletion did not prevent copper entering and accumulating in the intestinal cells (195). This observation rules out the essential role for Ctr1 in apical uptake of copper, indicating that other pathways/transporters could be involved.
The presence of low-affinity copper transporter in Ctr1–/– embryonic cells has been demonstrated (149). One of the candidates for such a transporter is a relatively nonselective, divalent metal transporter DMT1 (12, 67), which mediates intestinal uptake of dietary iron (8). The siRNA-mediated knock-down of DMT1 in cultured cells significantly decreases both iron and copper uptake (12); whether DMT1 transports copper in tissue has not been directly examined. Another candidate is the ATP-driven copper transport system detected at the brush-border membrane (127); however, the molecular nature of this putative Cu-ATPase remains unknown. Lastly, such mechanisms of copper uptake as pinocytosis may also play a role in copper uptake (172), particularly during maturation of the gastrointestinal tract.
Copper is exported from the enterocytes into the blood by Cu-ATPase ATP7A (Fig. 1) in a process that involves trafficking of the transporter towards the basolateral membrane (184, 235). In Menkes disease, ATP7A is inactivated and copper export from the enterocytes is greatly impaired. As a result, copper accumulates in intestinal cells and less copper is delivered to the blood, resulting in restricted copper supply to other tissues (131). A close relationship appears to exist between iron and copper absorption in the intestine (160, 316). Under conditions of iron deficiency in rats, DMT1 expression is increased and could be responsible for the increase in absorption of both iron and copper at the brush border (44). The expression of ATP7A in duodenum is also markedly elevated in iron deficiency, most likely contributing to the overall increase of copper transport across intestinal epithelium (44, 235).
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2. ATP7B in hepatocytes is essential for homeostatic regulation of copper in the body
The majority of copper that emerges from the intestinal epithelium into the blood is delivered to the liver (23), and less to kidney and other tissues (159). The liver is the central organ of copper homeostasis and is primarily responsible for the export of excess copper out of the body (Fig. 1). The copper uptake into the liver does not appear to be highly regulated. In contrast, the export of copper from the liver is a regulated copper-dependent process, which is mediated by a copper-transporting ATPase ATP7B.
After entry into hepatocytes, copper is distributed to various intracellular destinations (Fig. 2). In the cytosol, copper is utilized by a radical-detoxifying enzyme copper, zinc-dependent superoxide dismutase (SOD1), which acquires copper with the help of a specific metallochaperone, copper chaperone of superoxide dismutase (CCS) (45). Copper also enters the mitochondria, where it is incorporated into cytochrome-c oxidase (COX). Several candidate proteins have been proposed to contribute to this latter process (101, 221, 223); however, the exact mechanism of copper delivery to the mitochondria is not yet understood.
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Excess cytosolic copper is excreted into the bile in a form that is less easily reabsorbed (159). In rats, biliary copper excretion can be detected as early as 15 min after gastrointestinal injection of the Cu isotope, although more time is necessary to reach saturation (23). ATP7B is required for copper excretion into the bile, and to perform this function, ATP7B relocalizes towards canalicular membrane of hepatocyte (for details on intracellular trafficking of Cu-ATPases, see sect. VI). ATP7B-dependent copper export into the bile and subsequently to feces represents the major route of copper excretion from the body. In Wilson disease patients, both copper transport to the secretory pathway and copper release into the bile are greatly impaired (Fig. 1), resulting in marked accumulation of copper in the liver, very low levels of copper-bound ceruloplasmin in the serum, and low biliary copper (146, 252, 267).
3. Copper distribution in nonhepatic tissues
Experiments in rats using radioactive copper revealed that newly absorbed copper appears in the bloodstream in two waves: an initial peak (after 2 h) corresponds to copper exiting the intestine, and the second peak (after 
6 h) represents copper incorporated into ceruloplasmin, which is secreted by the liver (159). All serum copper is thought to be bound to protein carriers (161) and, possibly, low-molecular-weight compounds. In vitro, copper can be presented to a cell in various forms (as a free ion, or in a protein-, peptide-, or amino acid-bound form), and in all these cases, copper uptake takes place. In which form copper is recognized by the uptake machinery in vivo is yet to be fully understood.
Copper, when delivered to various organs, is utilized to produce copper-dependent enzymes with general (cytochrome-c oxidase or superoxide dismutase) and tissue-specific functions [for example, dopamine--hydroxylase in the adrenals (74), peptidylglycine -amidating monooxygenase in the pituitary (262), or tyrosinase in melanocytes (213)]. The set of proteins regulating copper distribution within the cells is thought to be the same in all tissues (Fig. 2). Some organs, though, express only one Cu-ATPase [for example, ATP7A in adrenal gland (74) or ATP7B in hepatocytes (270)], and in these tissues, a single Cu-ATPase appears to perform both biosynthetic and copper export functions.
Many tissues, however, such as brain, developing kidney, placenta, mammary gland, eye, lung, and some others, express both Cu-ATPases [for details on expression profiles, see NCBI database UniGene Hs. 496414 (ATP7A) and UniGene Hs. 492280 (ATP7B)]. The cell specificity of Cu-ATPase expression as well as the presence of two Cu-ATPases in the same cells can be associated with different functional characteristics of ATP7A and ATP7B, their distinct developmental regulation, or different targeting and trafficking behavior in polarized epithelia. Recent studies (see sect. III) suggest that these differences between ATP7A and ATP7B could be linked to their distinct physiological roles in tissues. The conclusion that the roles of two human Cu-ATPases are not identical is most apparent when one considers intestine. In the intestine, both Cu-ATPases are expressed (20, 138, 165), and ATP7B does not compensate for the lack of ATP7A function, as evident from the Menkes disease phenotype. In contrast, in the cerebellum of Atp7b–/– mice, ATP7A appears to substitute for missing ATP7B (19), and in vitro, the ATP7A inactivity can be suppressed by heterologous expression of ATP7B (144, 164). Determining specific roles for each ATPase and the extent of functional complementation in vivo is important as it may help to better understand consequences of ATP7A and ATP7B inactivation in such tissues as lung, heart, and kidney.
B. Function of Cu-ATPases in Copper Delivery to the Secretory Pathway
The first evidence for ATPase-mediated copper transport to the secretory pathway was obtained in the yeast Saccharomyces cerevisiae that contains the ATP7A/ATP7B homolog Ccc2p (315). Ccc2p is located in the late Golgi compartment, where it transports copper from the cytosol to the Golgi lumen for biosynthetic incorporation into Fet3p (a copper-dependent plasma membrane metallo-oxidase involved in iron uptake). Genetic deletion of ccc2 abolishes copper incorporation into Fet3p and perturbs iron uptake, as illustrated by the inability of 
ccc2 cells to grow under iron-limiting conditions (315). Heterologous expression of either ATP7A or ATP7B restores the copper-dependent Fet3 activity and permits the growth of ccc2 mutants on an iron-deficient medium (61, 102, 104, 107). Complementation of the ccc2 phenotype serves as a convenient screening assay for evaluation of the ATP7A and ATP7B activity (for details, see sect. VA).
The initial results showing ATP7A/ATP7B-mediated transport of copper to the secretory pathway in yeast were further confirmed by more recent studies in mammalian cells, which yielded direct evidence for the role of Cu-ATPases in the biosynthesis of copper-dependent enzymes (see below). The biosynthetic events utilizing the Cu-ATPase function were shown to take place in specific compartments of Golgi, require prefolded acceptor protein, and be regulated by copper (26, 61, 134, 216, 248, 260, 262). The mechanism of coupling between copper transport and copper incorporation into target proteins is not understood, although it does not seem to involve additional proteins (61). In some cases, the Cu-ATPase and an acceptor protein can be coimmunoprecipitated, as was shown for ATP7A and superoxide dismutase 3 (SOD3) (230). This observation points to close spatial proximity of the Cu-ATPase and the copper-accepting enzymes (230) and also raises an interesting possibility that copper binding to the acceptor protein modulates its protein-protein interactions with the transporter. Much remains to be learned about the mechanism of metallation of copper-dependent enzymes and coordination of their biosynthetic rates with the transport activity of Cu-ATPases. Here we will briefly describe the copper-dependent enzymes for which dependence on Cu-ATPase function has been directly demonstrated and discuss how this information is being used for better understanding of the Cu-ATPase function.
1. Copper transfer to tyrosinase can be used to measure Cu-ATPase activity in cells
An elegant and convincing demonstration of the direct role of Cu-ATPases in the delivery of copper to the secretory pathway of mammalian cells was provided by Petris et al. (213) using tyrosinase as an example. Tyrosinase is a copper-dependent enzyme that participates in the formation of pigment melanin by catalyzing the hydroxylation of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPA-quinone. The incorporation of copper into two binding sites takes place during tyrosinase passage through the secretory pathway and is required for its activity. Menkes disease patients are known to have abnormal pigmentation, which can be partially corrected by subcutaneous copper injections (258). Therefore, it was proposed that ATP7A, which is not functional in Menkes disease patients, was responsible for delivering copper to tyrosinase (213).
This prediction was experimentally verified using fibroblasts derived from the skin of Menkes patients (Menkes fibroblasts, Me32, Me52). The heterologous expression of tyrosinase in these fibroblasts yields an inactive apo-tyrosinase; however, the biosynthesis of copper-bound tyrosinase and the generation of the pigment are restored by the transfection of the ATP7A-expression construct into these cells (213). Altogether, these functional data and the Menkes disease phenotype indicate that one of the physiological functions of ATP7A is to metallate apo-tyrosinase. ATP7B does not seem to play a role in copper delivery to tyrosinase in tissues; however, in an in vitro system, such as cultured hepatocytes, ATP7B can mediate copper delivery to heterologously expressed tyrosinase (85). Therefore, the measurements of tyrosinase activity can be used as a coupled assay to assess the ability of various ATP7A and ATP7B mutants to transport copper into the secretory pathway in mammalian cells.
2. Lysyl oxidase dependent processes are particularly sensitive to ATP7A inactivation
Copper-containing amine oxidases contain peptidyl-2,4,5-tri(oxo)phenylalanine (TOPA) at their active centers. TOPA is formed by copper-catalyzed oxidation of tyrosine, which takes place in Golgi or trans-Golgi (242). Mature lysyl oxidase catalyzes oxidative deamination of lysine and hydroxy-lysine residues in collagen and elastin; this step is required for subsequent protein cross-linking and polymer formation. Collagen and elastin fibers are important components of connective tissue and perform essential mechanical functions. The lack of functional ATP7A is associated with severe connective tissue defects in Menkes disease patients, likely due to disrupted delivery of copper to lysyl oxidase in the secretory pathway. The connective tissue defects including vascular tortuosity, loose skin, hyperextensible joints, and bone fragility (80, 136, 258) are commonly observed in Menkes disease patients.
The functional relationship between ATP7A and lysyl oxidase is further supported by the observation of a similar temporal expression of these two proteins during embryonic development in rats (272). It is notable that lysyl oxidase or lysyl oxidase-dependent processes are particularly sensitive to ATP7A inactivation. This conclusion was made based on studies of the occipital horn syndrome (OHS), a mild form of Menkes disease that primarily affects connective tissues (OMIM 304150). In one such study, Dagenais et al. (46) characterized a frameshift mutation of ATP7A in the OHS patient resulting in a COOH-terminal truncation of the protein. This mutation greatly diminished levels of ATP7A protein, but did not affect functional domains of the transporter. Unlike classical Menkes disease patients who show marked neurological impairment and die in early childhood, the OHS patient had relatively normal development and low-to-average cognitive abilities (46), suggesting that low level of ATP7A activity is sufficient to prevent severe neurological problems. At the same time, production of functional lysyl oxidase in the patient was diminished, and the patient displayed significant skeletal abnormalities including occipital horns, broad scapular necks, and radial bowing of the forearms (46). Similar phenotype has been observed in other OHS patients (84, 222, 225).
Studies in cultured cells offer a possible explanation for high sensitivity of connective tissue to ATP7A inactivation. It was shown that defect in ATP7A is associated not only with lysyl oxidase inactivation but also with dysregulation of mRNA transcription and/or turnover for such matrix proteins as elastin (72). Understanding the multifaceted role of ATP7A in connective tissues may have practical consequences. For example, precise control of intracellular copper levels has potential uses in tissue engineering to increase mechanical strength (47).
3. ATP7A-mediated copper delivery to peptidylglycine -amidating monooxygenase does not require additional protein chaperones
Peptidylglycine -amidating monooxygenase (PAM) is a copper-dependent enzyme primarily expressed in the pituitary gland, adrenal medulla, atrium of the heart, and the central nervous system. The enzyme has an important physiological role in catalyzing the conversion of over half of all neuropeptides into -amidated peptides (262). RT-PCR experiments demonstrate that tissues abundant in PAM, such as pituitary and adrenal gland, also express ATP7A (74, 262). ATP7A and PAM are both present in the TGN, suggesting that ATP7A provides copper to PAM in this compartment (262). Inactivation of ATP7A has important consequences for PAM function, as illustrated by experiments in the mottled-brindled mutant mouse that lack functional ATP7A (262). In these mice, protein levels of PAM are normal; however, the level of amidated peptides is markedly reduced consistent with diminished PAM activity due to the lack of copper cofactor. It was proposed that a deficiency in peptide amidation is an important contributing factor to the developmental problems associated with Menkes disease (262).
Studies of PAM have also examined whether copper transfer from the Cu-ATPase to the acceptor protein in the secretory pathway requires the presence of additional helper molecules or chaperones. It was shown that expressing the catalytic core of PAM (PHM) in yeast produced active holoenzyme. This process was dependent on the presence in cells of an active Cu-ATPase, Ccc2p, a functional homolog of ATP7A. Since yeast cells do not have PAM homologs, yet Ccc2p can supply copper to PHM, it was concluded that luminal Golgi chaperones may not be required for coupling copper release from the transporter and incorporation into the acceptor enzyme (61).
4. Ceruloplasmin biosynthesis and ATP7B function are interdependent
Ceruloplasmin (CP) is a multi-copper oxidase that couples the reduction of O2 to H2O with the oxidation of Fe(II) to Fe(III). Unlike tyrosinase or PAM, which receive their metal cofactor from ATP7A, CP acquires copper from ATP7B. Similarly to biosynthesis of other copper-dependent enzymes, copper incorporation into apo-CP takes place in the secretory pathway (273). The direct involvement of ATP7B in this process was confirmed in the experiments in which recombinant adenovirus was used to introduce ATP7B cDNA into LEC rats. These animals lack functional ATP7B and only produce the apo-form of CP. Infusion of the recombinant adenovirus restores copper incorporation into CP in the LEC livers, providing strong evidence for the role of ATP7B in ceruloplasmin biosynthesis (273).
Studies of CP biosynthesis also demonstrated that ceruloplasmin enters the late secretory pathway and incorporates copper in an "all-or-none" process (99). It was suggested that such cooperativity permits a more sensitive response to copper concentrations if copper supplies become limiting (99). Whether or not ATP7B directly interacts with CP and "recognizes" the properly folded intermediate is unknown. Precise fit of the transporter and acceptor protein is not essential for copper delivery, since mammalian Cu-ATPases can also deliver copper to the yeast CP homolog Fet3, although with a noticeably lower efficiency (102, 104, 204, 205).
It is interesting that genetic inactivation of CP has a significant effect on the maintenance of intracellular copper levels in hepatocytes, a process that is thought to be mediated by ATP7B. In CP–/– mice, hepatic copper content is increased while copper absorption and biliary copper excretion appear unaltered (179). These data suggest that in the absence of CP, the transport activity of ATP7B is insufficient to efficiently remove all copper from hepatocytes. This could be due to lower transport activity of ATP7B in the absence of CP. CP, when present, may facilitate copper transport by binding and/or oxidizing released copper and thus serving as a copper "sink." Alternatively, and perhaps more likely, trafficking of ATP7B towards apical membrane may represent a rate-limiting step in the biliary copper export. In this case, in the absence of CP, significantly more copper would need to be removed via this pathway. The copper will be sequestered by ATP7B into vesicles, but will remain mostly in cells due to slow vesicle-mediated exocytosis. This would result in copper accumulation in hepatocytes even in the presence of fully functional ATP7B. (For details on Cu-ATPase trafficking, see section VI.)
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III. TISSUE-SPECIFIC FUNCTIONS OF Cu-ATPases |
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Characterizing patterns of ATP7A and ATP7B expression is the first step towards dissecting their specific roles in tissues. The symptoms of Menkes disease and Wilson disease suggest some tissue-specific functions for the ATP7A and ATP7B, respectively. Expression of ATP7A in intestine and in choroid plexus (138, 184, 193, 194, 235) correlates well with the key role of ATP7A in the generalized delivery of dietary copper via intestine to the body and subsequently into the brain. Consistent with this role, Menkes disease patients present with severe neurological deterioration, seizures, and mental retardation. ATP7A is also abundantly expressed in vascular smooth muscle cells, vascular endothelial cells, and aorta (230) as well as in cerebrovascular endothelial (CVE) cells (228). Inactivation of ATP7A is associated with vascular abnormalities (78) and may drastically affect the integrity of pulmonary arterial system (80). The marked effect of ATP7A inactivation on vasculature is not limited to insufficient supply of copper to lysyl oxidase. Recent studies in brindled mice (a mouse model of Menkes disease) also revealed a 50% decrease in the activity of SOD3, a copper-dependent enzyme which modulates levels of extracellular superoxide ions in vasculature. It was hypothesized that ATP7A may contribute to regulation of superoxide in the vasculature by supplying copper to SOD3 (230).
Similarly to Menkes disease, the major phenotypic manifestations of Wilson disease, such as liver disease and neurological abnormalities, parallel the primary expression of the corresponding Cu-ATPase, ATP7B, in the liver and the brain. Hepatic ATP7B is involved in the biosynthesis of CP and export of excess copper into the bile. In the brain, the major functions of ATP7B are likely to be in the basal ganglia, midbrain, and pons since these regions are often affected in Wilson disease patients (243). Whether the function of ATP7B in these regions is biosynthetic and/or homeostatic is currently unknown.
Kayser Fleischer corneal pigment ring is a characteristic and diagnostic sign of Wilson disease observed in the vast majority of patients with neurological abnormalities. The appearance of this ring is thought to reflect deposition of copper. The copper deposits are likely caused by inactivation of ATP7B, which is expressed in the retinal pigment epithelium and in the ciliary body during retinal development (25, 135). It was proposed that in the retina ATP7B is required for production of holo-ceruloplasmin (135); whether this is the only role of ATP7B in the eye remains to be determined.
Northern blot analyses demonstrated coexpression of two Cu-ATPases in the brain and several other tissues, such as kidneys, lung, placenta, and mammary gland (31, 39, 177, 207, 215, 270, 297, 309), raising questions about specific role of these transporters in these tissues. The sections below describe current data on division of labor between ATP7A and ATP7B in cells and tissues where these two transporters are coexpressed.
B. Both ATP7A and ATP7B Are Involved in Copper Transport to Milk
The respective roles of ATP7A and ATP7B in mammary gland are better understood compared with other tissues and illustrate distinct but complementary functions of Cu-ATPases. Copper is essential for neonatal growth and therefore has to be exported into the milk. The importance of copper as a nutrient is evident from a 20-fold increase in the copper uptake into mammary tissue that occurs upon lactation (56). After entering the mammary gland, copper is rapidly transferred to milk. The entire process is initiated by copper being taken up from maternal circulation into mammary epithelial cells most likely by a high-affinity copper transporter Ctr1 (121). When in cells, copper is utilized to produce ceruloplasmin, which is synthesized in substantial amounts in the mammary gland (111) and then secreted into milk. Copper also appears to be excreted directly from epithelial cells into the lumen of alveoli (56).
The role of ATP7A in copper delivery to milk was addressed by localizing the transporter in control and lactating mammary gland. In luminal cells of nonlactating tissue, ATP7A is confined to a perinuclear compartment (3) consistent with the low need for copper export. In a lactating gland, ATP7A appears in a diffuse pattern in cells of the areola and ducts, suggesting trafficking of ATP7A towards the plasma membrane and involvement of ATP7A in the cellular efflux of copper into the milk (3). The trafficking of ATP7A from the intracellular compartment towards the plasma membrane was also demonstrated in vitro following prolactin treatment of HC11 cells (121). In rats, ATP7A was detected in both the luminal (apical) and serosal (basolateral) membranes (120) and was suggested to have a dual role: exporting copper into the milk and back to the maternal circulation.
The involvement of ATP7B in copper export from the mammary gland is evident from the phenotypes of toxic (tx) milk and Atp7b–/– mice. The tx mice have an inactivating mutation in ATP7B (143), and the tx dams produce milk with low copper content. The tx pups have reduced copper levels in the stomach and often die unless nursed by nonmutant mice (hence the term "toxic milk") (181). Similar copper perinatal deficiency is observed in Atp7b–/– knock-out mice (30). Characterization of copper distribution in control animals demonstrated that the copper content in milk is three- to fourfold higher than in mammary gland (30). In contrast, in Atp7b–/– knock-out mice lacking functional ATP7B, copper accumulates in the mammary gland, and the copper content of milk is only 30% of the norm. Interestingly, some copper is being delivered into milk even in the absence of functional ATP7B (most likely by ATP7A); however, ATP7A does not fully compensate for the lack of ATP7B function.
It was proposed that the role of ATP7A at the plasma membrane is to directly export copper into the milk (120), while ATP7B in the mammary gland could be necessary to export copper in a CP-bound form. Recent studies investigating milk copper composition during early and late periods of lactation have led to an interesting alternative model, in which ATP7B was suggested to play a constitutive role in milk copper secretion by delivering copper to ceruloplasmin and to secretory vesicles, whereas ATP7A serves to facilitate copper export in response to hormonal stimulation (121).
This model is consistent with changes in trafficking behavior of both Cu-ATPases in mammary cells. Similarly to ATP7A, during lactation the intracellular distribution of ATP7B changes and can be seen as a granular diffuse cytoplasmic pattern in contrast to the perinuclear staining in nonlactating animals (181). Regulated trafficking of the Cu-ATPases has been described in cultured cells and tissues in response to changes in copper concentration or, more recently, to Ca2+ signaling and hormonal stimulation (see sect. VI for details). It is currently unknown whether the relocalization of ATP7A and ATP7B in mammary gland is triggered by increased intracellular copper levels upon lactation, and/or is caused by induction of signaling pathways activated by hormones produced during lactation. A kinase-mediated phosphorylation of ATP7B that correlated with the intracellular localization of the transporter has been previously demonstrated in hepatic cells (284). Whether or not the regulatory phosphorylation contributes to the localization and function of Cu-ATPases in mammary gland remains to be examined.
C. Cu-ATPases Are Likely to Contribute to Tight Homeostatic Control of Copper in Kidneys
The kidneys have one of the highest copper concentrations among organs (7–12 mg/g; Ref. 159) and show tight homeostatic control of their copper content. Compared with other tissues, the renal copper content is less affected by systemic copper deficiency caused by limited absorption via the intestine (195). Similarly, dietary copper overload does not significantly alter copper concentrations in kidneys (5), pointing to efficient mechanisms that regulate copper levels in this tissue. It is thought that under normal conditions little copper is filtered in the glomerulus, since most serum copper is bound to proteins with molecular weights exceeding filtration limit. Most of the filtered copper is likely to be reabsorbed, because the copper content in urine is normally low. However, copper concentrations in the urine can increase dramatically under disease conditions, as observed in Wilson disease patients (27, 308).
Currently, little is known about renal copper transport and regulation. At least two copper-dependent enzymes, diamine oxidase, involved in the oxidative deamination of histamine and other diamines, and the ferroxidase CP, are produced in kidneys (62, 73). Therefore, renal cells that express these enzymes require Cu-ATPase function for biosynthetic purposes along with the general maintenance of intracellular copper. Which Cu-ATPase plays a key role in the biosynthesis of renal copper-dependent enzymes and how two ATPases contribute to the renal copper homeostasis remains to be characterized. Studies on in-tissue localization of ATP7A and ATP7B provide some insight.
In mice, high copper content was found in the proximal and distal tubules as well as the glomeruli (123, 129). Thus these regions seem to represent the major sites where copper concentration could be regulated through either uptake or export. Inactivation of Cu-ATPase in murine models for either Wilson disease or Menkes disease results in copper accumulation in kidneys (30, 122, 187, 274, 307), with most significant accumulation detected in proximal tubules of the cortex (123, 314). The simplest explanation of this result is that ATP7A and ATP7B function is required in proximal tubules.
This hypothesis has been tested in more recent immunolocalization studies. In kidneys of 10-day-old mice, ATP7A was detected in proximal and distal tubules with very little, if any, staining seen in the glomeruli (82). The staining of ATP7A in the proximal tubules was diffuse, indicative of an intracellular localization and hence, perhaps, a biosynthetic function. In distal tubules, ATP7A was observed at the basolateral membrane, suggesting the role of ATP7A in the transport of copper into circulation (82). Subsequently, the presence of ATP7A mRNA in the proximal tubules and the lack of glomerular staining was confirmed using in situ hybridization in 4-wk-old mice (190). In this latter work, no labeling of ATP7A was detected in the distal tubules, either due to difference in methods, in which either protein (82) or mRNA (190) was detected, or difference in the age of animals. In cultured renal cell lines, ATP7A is detected in both Madin-Darby canine kidney (MDCK) cells, which have a distal origin (81), and opossum kidney (OK) cells, which originate from proximal tubules (unpublished data). Consistent with the role of ATP7A in copper reabsorption, in polarized MDCK cells ATP7A was shown to traffic to the basolateral membrane in response to high copper (81).
ATP7B mRNA was localized to the glomeruli, and this result correlated with the immunohistochemical localization of the protein (185). Staining of both ATP7B mRNA and protein was also reported in the inner and outer zone of the medulla, which may be the loops of Henle. The inactivation of ATP7B in these regions of nephron may explain massive accumulation of copper-bound metallothionein observed in the outer strip of outer medulla of the ATP7B-deficient LEC rats (141, 200). Different patterns of expression of two Cu-ATPases along with copper accumulation in kidneys of Wilson disease patients (63) also indicate that the lack of ATP7B cannot be fully compensated by ATP7A, and therefore, the roles of ATP7A and ATP7B in renal copper distribution are distinct.
D. Localization and Protein Levels of Cu-ATPases in Placenta Are Regulated by Hormones
Insufficient copper transport to the fetus during pregnancy may produce various abnormalities including embryonic mortality, neonatal growth retardation, and pulmonary and cardiovascular defects. Such deficiency can be dietary, or be caused by inactivation of major components in copper distribution, such as Ctr1 (140, 150), copper chaperone Atox1 (86), or Cu-ATPases. The placenta of both Menkes disease and Wilson disease patients accumulate copper (100, 197). Therefore, it seems likely that ATP7A and ATP7B both have a role in the transfer of copper across the placenta during gestation. This conclusion is supported by studies on expression and localization of ATP7A and ATP7B in human placenta (94). ATP7A and ATP7B were found to be both expressed throughout gestation; however, their localization within the placenta differed.
In general, nutrient uptake from maternal circulation to embryo is mediated by syncytiotrophoblasts and by embryo-derived endothelium cells, which surround the maternal blood vessels within the placenta. ATP7A is localized in the syncytiotrophoblast, the cytotrophoblast, and in the endothelial cells, suggesting that ATP7A may transfer copper from the basolateral surface of the syncytiotrophoblast directly into fetal circulation (94). The ATP7B was also detected in the syncytiotrophoblast, and it was proposed that ATP7B might be present on the apical surface of the placenta and function to return copper from the placenta to the mother, thus preventing accumulation of excess copper in the fetus (94). This hypothesis was recently tested in a cultured cells model (95).
Polarized Jeg-3 cells derived from placental trophoblasts express both ATP7A and ATP7B. In these cells, both Cu-ATPases are localized in the perinuclear compartment and in vesicles, but show little colocalization when costained (95). Further difference between Cu-ATPases was detected following treatment of these cells with hormones or growth factors known to regulate nutrient transport in placenta. Treatment with insulin induced relocalization of ATP7A towards the basolateral membrane and was associated with higher copper transport across this membrane. Earlier, the correlation between levels of ATP7A and copper efflux was observed in another choriocarcinoma cell line, BeWo (226). Altogether, these results are consistent with the proposed role of ATP7A in copper transport from placenta to the fetus.
In contrast, the protein levels of ATP7B were reduced in response to treatment with insulin, and the intracellular localization of ATP7B remained perinuclear. Hormonal treatment also decreased copper efflux across the apical membrane. This result was interpreted as evidence for the role of ATP7B in transport of excess copper from fetus to maternal circulation (95). A more direct support for this hypothesis would be provided by an observation of copper-induced trafficking of ATP7B towards apical membrane, accompanied by increased copper transport across this membrane. Such experiments remain to be done. Additional functions for ATP7B, for example, delivery of copper to CP, which is expressed in placenta (311), should also be considered.
E. Multifaceted Contribution of Cu-ATPases to Central Nervous System Function
Cu-ATPases play an essential role in biochemistry and physiology of the central nervous system (CNS), as evidenced by marked neurological, developmental, and behavioral abnormalities observed in patients lacking either ATP7A or ATP7B. Brain magnetic resonance imaging (MRI) of patients displaying classical Menkes disease often show progressive cerebral atrophy, delayed myelination or even demyelination of white matter, and abnormalities of intracranial vessels (21, 283). Similarly, significant changes in MRI are commonly observed in the basal ganglia of Wilson disease patients. Destruction of white matter and degeneration of cortex vary, although necrosis, spongiform degeneration, and demyelination have been reported (108, 255, 269, 287, 288). These phenotypic manifestations are complex and do not provide obvious insight into specific functions of each Cu-ATPase. The studies attempting to decipher the localization, function, and regulation of Cu-ATPases in the brain are still in their infancy.
Measurements of copper efflux from murine microvascular cells suggested that ATP7A can be involved in copper transfer across the blood-brain barrier (228). Also, the ATP7A mRNA was found highly expressed in the ependymal cells of choroid plexus (109, 138), a structure that regulates the concentration of molecules in the cerebrospinal fluid. Therefore, ATP7A may facilitate copper efflux from the neuropil and/or function with the blood-brain barrier mediating copper entry into the brain. Consistent with this role of ATP7A, the copper levels in the brain and cerebrospinal fluid of Menkes patients are low (132, 162). ATP7A was also detected in numerous other cells within the CNS, including a subset of astrocytes, microglia, oligodendrocytes, tanycytes, endothelial cells, and neurons (193). The widespread expression of ATP7A indicates that this Cu-ATPase is the major contributor to the maintenance of Cu homeostasis in the CNS. In vitro, ATP7A was detected in rat C6 and PC12 cells, further confirming that both glial and neuronal cells require Cu-ATPase function (227).
Several copper-dependent enzymes play an important role in the CNS; those include PAM, dopamine -monooxygenase, and CP (98, 218, 268, 298). Expression of ATP7A activity in astrocytes and the olfactory system is consistent with its role in delivery of copper to PAM, which is expressed in these cells (89, 124, 237). ATP7A is also found in myelinating oligodendrocytes ensheathing optic nerve, and copper deficiency in CNS due to ATP7A inactivation is known to cause neuronal demyelination (162). How copper regulates myelination is unclear, although transcription or mRNA stability for myelin components is altered by copper misbalance (162).
Expression of ATP7A in the brain is developmentally regulated (19, 138, 193). It was found to be high in the early postnatal period, reaching maximum in murine neocortex and cerebellum at day 4 after birth (193). Subsequently, in most neuronal cells, the expression of ATP7A decreases, while in the CA2 hippocampal layer the levels of ATP7A are increased (193). Great variability in detecting ATP7A protein and mRNA in Purkinje neurons has been reported (19, 193). Some studies found ATP7A abundant in adult Purkinje cells (189, 193), while other observed it at low levels (109), or greatly downregulated in these cells during development, but present in surrounding Bergmann glia (19). The reason for such differences in ATP7A detection is not entirely clear, but could be related to variations in sample preparation, such as time and intensity of fixing, mouse strains used, and method of detection.
High levels of expression during early brain development are likely to reflect a critical role of ATP7A in CNS at these stages. This conclusion is supported by a striking observation that injection of copper in human Menkes patients or in brindled mice, a murine model of Menkes disease, can prolong survival and alleviate some of the neurological problems (114, 173, 310). Specifically, copper injections can reverse disease-caused frequent tonic seizures and ataxia and partially correct morphological abnormalities of Purkinje neurons (119, 310), although the arborization of the dendrites remains poor. It is interesting that Purkinje neurons also express ATP7B (19), and when copper is delivered (via injections) to the circulation and eventually reaches Purkinje cells, ATP7B appears to at least partially compensate for the lack of ATP7A function.
The localization and function of ATP7B in the brain is much less characterized compared with ATP7A. The ATP7B distribution was analyzed using in situ blotting of 4-wk-old rat brain following direct transfer of native proteins from sectioned tissue to a blotting membrane (244). The ATP7B was detected in neuronal cells of the CA1-CA4 layers of the hippocampus, and this localization was confirmed by in situ hybridization. ATP7B was also detected in the glomerular cell layer of the olfactory bulb, and in the granular cell layer of the cerebellum (244). More recent high-resolution studies on ATP7B in adult and developing mouse brain demonstrated that Purkinje neurons are the major site of ATP7B expression in the cerebellum (19). In Purkinje neurons, ATP7B may function in the biosynthetic pathway, delivering copper to ceruloplasmin, which is also expressed in these cells (19). Interestingly, in genetically engineered mice lacking ATP7B, copper delivery to ceruloplasmin is not disrupted (19), suggesting that ATP7A compensates for the lack of ATP7B.
Recent studies suggest that in addition to their roles in maintenance of cytosolic copper concentrations and cofactor delivery to copper-dependent enzymes, Cu-ATPases may have signaling and protective functions in the CNS. In cultured hippocampal, cortical, and olfactory bulb neurons copper acts as a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor (275, 289, 302). Reciprocally, activation of the NMDA receptor was shown to result in trafficking of ATP7A and an ATP7A-dependent vesicular release of copper (253). This novel and interesting link between neuronal activation and copper homeostasis supplied supportive evidence to earlier suggestions that copper may play a role in regulation of neuronal excitability (83, 116).
It was further demonstrated that copper may provide protection of primary hippocampal neurons against NMDA-mediated excitotoxic cell death (254) and that cells lacking ATP7A are more sensitive to NMDA receptor-mediated excitotoxicity. In the mouse models of Menkes disease, insufficient supply of copper to the CNS leads to abnormal structure and function of mitochondria (137, 241), decreased expression of the antiapoptotic protein Bcl-2, and elevated levels of cytochrome c released from mitochondria (241). It seems likely that abnormal mitochondria in mutant mice may have lower capacity to buffer NMDA receptor-gated calcium fluxes (256). Poor calcium buffering capacity and increased apoptosis may explain observed susceptibility of mutant mice to hypoxic/ischemic insults and poor resistance to neuronal injury (254).
Another important function of ATP7A in the CNS is suggested by the peak of ATP7A expression just before synaptogenesis in numerous neuronal subpopulations. In developing neurons, ATP7A is initially present in cell bodies but subsequently found in extending axons. These observations point to a possible role for ATP7A in synapse formation and plasticity (60). It seems particularly interesting that amyloid precursor protein (a causative agent of Alzheimer's disease) is a copper-binding protein, which is located in neuronal cells predominantly at the synapse (219). How amyloid precursor protein receives its copper is currently unknown, but it is tempting to speculate that ATP7A-mediated copper transport may play an important role in this process. A negative effect of ATP7A overexpression on amyloid precursor protein abundance in cell culture system has recently been reported (22), pointing to a potentially interesting connection between these two proteins.
Altogether, the results described in this section illustrate a high level of integration of copper homeostasis in CNS metabolism and function. The new findings also raise numerous questions about molecular mechanisms that govern ATP7A involvement in various physiological processes in the CNS. They also emphasize challenges of separating direct functional contributions of ATP7A to these processes from indirect consequences of systemic copper deficiency caused by ATP7A inactivation.
F. Developmental Changes in Expression of Cu-ATPases
The developmental changes in the expression of ATP7A in the brain have been discussed in the above section. The data for other tissues are still limited; however, it is clear that ATP7A and ATP7B are regulated in a distinct fashion. ATP7A is widely expressed in both embryonic and adult tissues, and embryonic liver is, so far, the only tissue apart from the brain for which significant developmental regulation of ATP7A was reported (138, 207).
Northern blot analysis demonstrated the presence of ATP7A in the mouse liver at days E17, E19, and P2; however, by P10 and P15, this expression was barely detectable (207). The decline in ATP7A expression shortly after birth is opposite to a change in ATP7B levels, which are increased at this time. It could be that the developmental changes in liver function require Cu-ATPases with somewhat different functional characteristics (for functional comparison of ATP7A and ATP7B, see sect. VB). In addition, ATP7A and ATP7B export copper through different membranes in polarized epithelia (basolateral and apical membranes, respectively). In the fetus, all wastes and presumably excess copper are removed through the blood (via basolateral membrane), while in the postnatal organism, excess copper is excreted via bile. Thus the switch from ATP7A to ATP7B upon liver maturation may reflect the tissue's need to export excess copper via canalicular (apical) membrane into the bile for eventual removal with feces. Another likely explanation for ATP7A downregulation is that the high level of ATP7A in fetal liver could be due to active fetal hemopoesis and presence of hemopoetic cells, which greatly decline upon birth (207).
The expression of ATP7B during development was found to be regulated in a number of tissues. Iwase et al. (109) demonstrated the presence of ATP7B in the heart and liver at E9.5 and subsequent increased expression in the heart, lung, intestine, nasal epithelia, and liver at E11.5 (109). From E15.5 to E18 expression of ATP7B was detected in the lung, thymus, liver, intestine, and lining of the respiratory tract (109). In the developing intestine, both Cu-ATPases are expressed in the villous epithelium; the role of ATP7B in the intestine is unclear.
Kuo et al. (138) directly compared the expression patterns of the murine Atp7a and Atp7b genes during embryonic development (138). In agreement with other studies, Atp7a mRNA was found throughout the embryo during gestation, whereas the Atp7b mRNA was present in a limited set of tissues, including the liver, heart, CNS, intestine, thymus, and respiratory epithelium (138). The authors proposed that ATP7A might be required for the maintenance of cellular copper homeostasis and the extracellular microenvironment of multiple cell types during development, while ATP7B may perform a more specialized function such as copper delivery to cuproenzymes. For example, both ATP7A and ATP7B were found in the embryonic lung; however, ATP7A was localized to the lung parenchyma, whereas ATP7B was highly expressed in the bronchial epithelium. Expression of ATP7B in fetal lung correlates with the expression of CP, which is also detected in bronchial epithelium (126). Coexpression and colocalization of these two proteins suggests that, as in many other tissues, ATP7B plays a key role in the biosynthetic delivery of copper to this enzyme.
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IV. GENOMIC AND PROTEIN ORGANIZATION OF HUMAN Cu-ATPases |
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The complete exon-intron structure of the Wilson disease gene ATP7B was determined in 1994 (215) and was quickly followed by characterization of the Menkes disease gene ATP7A (54, 282) and the mouse Atp7a gene (37). Presently, the cDNA sequence and genomic information are available for several orthologs of ATP7A and ATP7B as a result of sequencing and characterization of genome of various organisms. This information can be found using Ensemble Genome Browser at http://www.ensembl.org/index.html and the following links for ATP7A http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000165240 and ATP7B http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000123191, respectively.
Both ATP7A and ATP7B are fairly large genes. ATP7A spans 150 kb of genomic DNA and contains 23 exons with the ATG start codon located in the second exon; the size of the exons varies from 77 to 726 bp (54). The overall structure of ATP7B is similar to that of ATP7A. ATP7B has 21 exons varying from 77 to 1234 bp with the ATG codon for the initiating Met located within exon 1 (215). From exon 5 (exon 3 in ATP7B) to the end of the gene, all of the splice sites in ATP7A occur at exactly the same nucleotide positions as in ATP7B, except for the boundary between exons 17 and 18 (exons 15 and 16 in ATP7B) and a single codon difference at the boundary between exons 4 and 5 of ATP7A (exons 2 and 3 in ATP7B). The only significant difference in the gene structures is observed in the 5'-region encoding the NH2-terminal regulatory metal binding domains (for protein structure, see Figs. 3 and 4). In ATP7B, the first four of six metal binding domains (MBDs) are encoded in one large exon (exon 2), while in ATP7A, the sequence corresponding to the MBDs1–4 is spread over three exons. At the protein level, this region shows considerable sequence diversity between ATP7A and ATP7B and could be responsible for different functional characteristics of ATP7A and ATP7B and/or their trafficking behavior in polarized cells (see sects. V and VI for details).
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ATP7A and ATP7B encode large membrane proteins with significant primary sequence homology (50–60% identity). In the past several years considerable progress has been made in analyzing the biochemical properties of these two proteins (for recent reviews on structure and function of Cu-ATPases, see Refs. 176, 279, 291). The studies have established that ATP7A and ATP7B belong to the large family of P-type ATPases and have identified several functional domains in their structure (Fig. 4A). It has also become apparent that ATP7A, ATP7B, and their orthologs form a separate subgroup within the P-type ATPase family (P1B-ATPases), which has distinct structural and mechanistic characteristics.
Cu-ATPases transport copper from the cytosol across cellular membranes using the energy of ATP hydrolysis. This process involves specific recognition of copper, delivery of copper to the membrane portion of the transporter with subsequent release at the other side of the membrane, as well as binding and hydrolysis of ATP (Fig. 4B). The central step in the catalytic cycle is the transfer of 
-phosphate from ATP to the invariant Asp residue in the DKTG motif (Fig. 4, B and C) with formation of a transient phosphorylated intermediate. The prerequisite for this reaction is the binding of copper to the sites within the membrane portion of the enzyme, while the release of copper from these sites stimulates dephosphorylation (Fig. 4B). The structural organization of Cu-ATPases reflects the need to accommodate and couple these reactions.
The copper translocation pathway is located in the transmembrane portion of Cu-ATPases, which is composed of eight transmembrane segments (TMS) (Fig. 4). The highly conserved CPC sequence in TMS6 (Figs. 3 and 4) is one of the signature motifs characterizing Cu-ATPases and ATPases involved in transport of Zn, Cd, Ni, Ag, and Pb, which together form the P1B subfamily (9). Recently, four amino acid residues in transmembrane segments 7 and 8 of the bacterial Cu-ATPase, CopA, were identified as being required for copper binding (171). These residues are conserved in the primary structure of P1B-ATPases involved in the transport of Cu(I). Along with Cys residues of the CPC motif, they are likely to form copper binding site(s) within the membrane portion; in human Cu-ATPases, the corresponding residues are Y1365N1366, Met1393, and Ser1396 in ATP7A and Y1331N1332, Met1359, and Ser1362 in ATP7B (Fig. 3). All other functional domains of Cu-ATPases are cytosolic.
1. The NH2-terminal copper-binding domain regulates Cu-ATPase activity
The NH2-terminal portion of human Cu-ATPases is large (>600 residues) and contains six repetitive sequences, each harboring the sequence motif GMT/HCxxCxxxIE (Figs. 3 and 4). Each of these repeats forms a subdomain with a single metal-binding site, i.e., the total stoichiometry is six copper ions per NH2-terminal domain (43, 51, 112, 167, 305). Copper binds in the reduced Cu(I) form, and the two Cys residues in the metal-binding motif CxxC are the only copper-coordinating ligands (50, 231–233). In vitro, and perhaps in vivo, other metals such as zinc (52) or lead (229) can bind to the NH2-terminal domain; however, the functional consequences and physiological significance of zinc or lead binding remain to be determined.
Structural information on individual MBDs of ATP7A has be
