Human copper transporter ATP7B (Wilson disease protein) forms stable dimers in vitro and in cells.

ATP7B is a copper-transporting P1B-type ATPase (Cu-ATPase) with an essential role in human physiology. Mutations in ATP7B cause the potentially fatal Wilson disease, and changes in ATP7B expression are observed in several cancers. Despite its physiologic importance, the biochemical information about ATP7B remains limited because of a complex multidomain organization of the protein. By analogy with the better characterized prokaryotic Cu-ATPases, ATP7B is assumed to be a single-chain monomer. We show that in eukaryotic cells, human ATP7B forms dimers that can be purified following solubilization. Deletion of the four N-terminal metal-binding domains, characteristic for human ATP7B, does not disrupt dimerization, i.e. the dimer interface is formed by the domains that are conserved among Cu-ATPases. Unlike the full-length ATP7B, which is targeted to the trans-Golgi network, 1-4ΔMBD-7B is targeted primarily to vesicles. This result and the analysis of differentially tagged ATP7B variants indicate that the dimeric structure is retained during ATP7B trafficking between the intracellular compartments. Purified dimeric species of 1-4ΔMBD-7B were characterized by a negative stain electron microscopy in the presence of ADP/MgCl2 Single-particle analysis yielded a low-resolution 3D model that provides the first insight into an overall architecture of a human Cu-ATPase, positions of the main domains, and a dimer interface.

Communication between the N and C termini is required for copper-stimulated Ser/Thr phosphorylation of Cu(I)-ATPase (ATP7B).

The Wilson disease protein ATP7B exhibits copper-dependent trafficking. In high copper, ATP7B exits the trans-Golgi network and moves to the apical domain of hepatocytes where it facilitates elimination of excess copper through the bile. Copper levels also affect ATP7B phosphorylation. ATP7B is basally phosphorylated in low copper and becomes more phosphorylated ("hyperphosphorylated") in elevated copper. The functional significance of hyperphosphorylation remains unclear. We showed that hyperphosphorylation occurs even when ATP7B is restricted to the trans-Golgi network. We performed comprehensive phosphoproteomics of ATP7B in low versus high copper, which revealed that 24 Ser/Thr residues in ATP7B could be phosphorylated, and only four of these were copper-responsive. Most of the phosphorylated sites were found in the N- and C-terminal cytoplasmic domains. Using truncation and mutagenesis, we showed that inactivation or elimination of all six N-terminal metal binding domains did not block copper-dependent, reversible, apical trafficking but did block hyperphosphorylation in hepatic cells. We showed that nine of 15 Ser/Thr residues in the C-terminal domain were phosphorylated. Inactivation of 13 C-terminal phosphorylation sites reduced basal phosphorylation and eliminated hyperphosphorylation, suggesting that copper binding at the N terminus propagates to the ATP7B C-terminal region. C-terminal mutants with either inactivating or phosphomimetic substitutions showed little effect upon copper-stimulated trafficking, indicating that trafficking does not depend on phosphorylation at these sites. Thus, our studies revealed that copper-dependent conformational changes in the N-terminal region lead to hyperphosphorylation at C-terminal sites, which seem not to affect trafficking and may instead fine-tune copper sequestration.

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B.

Wilson disease (WD) is a monogenic autosomal-recessive disorder of copper accumulation that leads to liver failure and/or neurological deficits. WD is caused by mutations in ATP7B, a transporter that loads Cu(I) onto newly synthesized cupro-enzymes in the trans-Golgi network (TGN) and exports excess copper out of cells by trafficking from the TGN to the plasma membrane. To date, most WD mutations have been shown to disrupt ATP7B activity and/or stability. Using a multidisciplinary approach, including clinical analysis of patients, cell-based assays, and computational studies, we characterized a patient mutation, ATP7B(S653Y), which is stable, does not disrupt Cu(I) transport, yet renders the protein unable to exit the TGN. Bulky or charged substitutions at position 653 mimic the phenotype of the patient mutation. Molecular modeling and dynamic simulation suggest that the S653Y mutation induces local distortions within the transmembrane (TM) domain 1 and alter TM1 interaction with TM2. S653Y abolishes the trafficking-stimulating effects of a secondary mutation in the N-terminal apical targeting domain. This result indicates a role for TM1/TM2 in regulating conformations of cytosolic domains involved in ATP7B trafficking. Taken together, our experiments revealed an unexpected role for TM1/TM2 in copper-regulated trafficking of ATP7B and defined a unique class of WD mutants that are transport-competent but trafficking-defective. Understanding the precise consequences of WD-causing mutations will facilitate the development of advanced mutation-specific therapies.

Vesicular distribution of Secretory Pathway Ca²+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells.

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn(2+) leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn(2+) detoxification by excretion into bile. Although many pathways of cellular Mn(2+) uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn(2+) detoxification by the Secretory Pathway Ca(2+), Mn(2+)-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn(2+) as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn(2+). Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn(2+) toxicity. Taken together, our findings suggest a role for SPCA1 in Mn(2+) detoxification in liver.

JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells.

Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in approximately 50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (huDeltaC-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, huDeltaC-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.

Scribble associates with two polarity proteins, Lgl2 and Vangl2, via distinct molecular domains.

Scribble (Scrib) is a large multi-domain cytoplasmic protein that was first identified through its requirement for the establishment of epithelial polarity. We tested the hypotheses that Scrib asssociates with the basolateral membrane via multiple domains, binds specific protein partners, and is part of a multimeric complex. We generated a series of EGFP-tagged Scrib fusion proteins and examined their membrane localizations in two types of polarized mammalian epithelial cells using biochemical and morphological approaches. We found that Scrib's Leucine-rich-repeat (LRR) and PDS-95/Discs Large/ZO-1 (PDZ) domains independently associate with the plasma membrane in both cell types. We identified multiple large Scrib complexes, demonstrated that Scrib and the cytoplasmic protein Lethal giant larvae2 (Lgl2) co-IP and that this association occurs via Scrib's LRR domain. Further, this report demonstrates that the membrane protein Vangl2 binds selectively to specific PDZ domains in Scrib. Our identification of Scrib's associations highlights its function in multiple biologic pathways and sets the stage for future identification of more proteins that must interact with Scrib's remaining domains. J. Cell. Biochem. 99: 647-664, 2006. (c) 2006 Wiley-Liss, Inc.