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Originally published In Press as doi:10.1074/jbc.M702891200 on June 11, 2007

J. Biol. Chem., Vol. 282, Issue 32, 23362-23371, August 10, 2007
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Trafficking of a Secretory Granule Membrane Protein Is Sensitive to Copper*Formula

Mithu De{ddagger}, Giuseppe D. Ciccotosto§, Richard E. Mains{ddagger}, and Betty A. Eipper{ddagger}1

From the {ddagger}University of Connecticut Health Center, Farmington, Connecticut 06030-3401 and the §University of Melbourne, Victoria 3010, Australia

Received for publication, April 5, 2007 , and in revised form, May 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine {alpha}-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl2. Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Copper plays an essential role in cellular respiration, free-radical defense, connective tissue biosynthesis, iron metabolism, peptide amidation, and the synthesis of catecholamines (1). An adequate supply of copper must be distributed to copper-requiring enzymes in various subcellular compartments without allowing free cuprous ions to react with hydrogen peroxide and generate hydroxyl radical (2). Cuproproteins can be divided into two groups, those that are involved in the uptake and delivery of copper and those that require copper for catalytic activity. Plasma membrane copper transporters like Ctr1 and copper-transporting P type ATPases like ATP7A and ATP7B, along with various copper chaperones, fall into the first group (3, 4). Enzymes like tyrosinase, cytochrome c oxidase, Cu/Zn superoxide dismutase (SOD),2 and peptidylglycine {alpha}-amidating monooxygenase (PAM) fall into the second group.

Many proteins playing important roles in copper homoeostasis have been identified. Copper enters the cell via the high affinity uptake protein, CTR1 (5). Specific copper chaperones are involved in the distribution of copper to cytosolic SOD, mitochondrial cytochrome c oxidase and the secretory pathway (6–8); sequestration of excess copper by metallothioneins prevents cellular damage. ATP7A and ATP7B deliver copper to the lumen of the secretory pathway, a transport step that is essential both for metallation of secretory pathway enzymes and for removal of excess copper from the cell (9). As might be expected, expression and localization of the proteins essential for copper uptake and delivery are sensitive to copper levels (3, 10, 11).

The stability of several cuproenzymes is sensitive to copper levels. Both ceruloplasmin and hephaestin are normally loaded with copper in or immediately after they pass through the trans-Golgi network (12, 13). Ceruloplasmin synthesized in the absence of copper is secreted normally, but is rapidly degraded in plasma (14). When synthesized in the absence of copper, hephaestin, an integral membrane protein is rapidly degraded by the proteasome pathway (13). SOD3, tyrosinase, and lysyl oxidase also receive copper in the secretory pathway (15–17). ATP7A binds directly to SOD3, presumably facilitating the incorporation of copper (15). Tyrosinase requires the incorporation of two copper ions into the apoenzyme during synthesis (16).

Two cuproenzymes, PAM and dopamine beta-monooxygenase, are localized to the secretory granules that store bioactive peptides (18, 19). Little is known about the effects of copper on their stability. PAM catalyzes the C-terminal amidation of glycine-extended peptide precursors, a modification essential for the bioactivity of numerous neuropeptides (20). The amidation reaction is catalyzed by two enzymes: peptidylglycine-{alpha}-hydroxylating monooxygenase (PHM; EC 1.14.17.3 [EC] ) and peptidyl-{alpha}-hydroxyglycine-{alpha}-amidating lyase (PAL; EC 4.3.2.5 [EC] ). PHM, an ascorbate-dependent monooxygenase, contains two essential copper ions that cycle between Cu+ and Cu2+ during each reaction cycle (20). Both copper atoms are easily removed with chelators and purified PHM must be loaded with exogenous copper to have full activity (20).

PAM occurs naturally in both integral membrane and soluble forms, providing a unique opportunity to compare the effects of copper availability on membrane and soluble forms of the same enzyme. En route through the secretory pathway, inactive propeptides are activated through the sequential actions of several peptide processing enzymes and PAM (21, 22). PAM and these other processing enzymes are targeted together with their prohormone substrates to the regulated secretory pathway (23, 24). PAM is active in the TGN and immature secretory granules (25). The biologically active peptides produced are stored in secretory granules, ready to undergo regulated release in response to external stimuli. Upon exocytosis, soluble PAM is secreted along with the bioactive peptides and membrane PAM appears on the plasma membrane and is retrieved by endocytosis (26). The signals essential for the entry of membrane PAM into secretory granules and for its endocytic trafficking are contained within its cytosolic domain and include phosphorylation and dephosphorylation at multiple sites.

The small amount of PAM on the plasma membrane at steady state undergoes rapid internalization and may be packaged into new granules or undergo lysosomal degradation (27, 28). We used corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM to assess the effects of copper availability on PAM biosynthesis and trafficking. While the trafficking of soluble PHM was unaffected by copper availability, depleting cellular copper facilitated the return of internalized membrane PAM to secretory granules. Elevated copper levels increased the degradation of internalized PAM. The cytosolic domain of PAM is essential to its ability to respond to copper availability.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—PAM proteins are numbered according to GenBankTM NM_013000. Wild-type AtT-20 cells and stable lines expressing PAM-1 (PAM-(1–976)) (18) or PHM(stop) (PAM-(1–382)) (29) or PAM-1/899 (28) were grown in Dulbecco's modified Eagle's medium/F-12 supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 25 mM HEPES, 10% NuSerum, 10% fetal bovine serum, 0.5 mg/ml G-418. Chinese hamster ovary cells stably expressing PHMcc (PAM-(1–25) fused to (42–356)) (30) were grown in {alpha}MEM supplemented with penicillin/streptomycin, 25 mM HEPES, 1.25 µg/ml fungizone, 2 mM L-glutamine, and 10% dialyzed fetal bovine serum. Cells were loaded with copper by preincubation for 2 h in growth medium containing 10 or 20 µM CuCl2. Cells were depleted of copper by overnight (16 h) incubation in growth medium containing 50 µM bathocuproine disulfonic acid (BCS). Subsequent biosynthetic, biotinylation, and antibody internalization experiments continued the initial copper depletion or copper loading treatments. All cell culture materials were from Mediatech Inc. (Herndon, VA). Sera were from Fisher, Hampton, NH.

PAM Enzymatic Assays—Peptidylglycine {alpha}-hydroxylating monooxygenase assays were performed using acetyl-Tyr-Val-Gly (31). Recombinant PHMcc was purified as described (30).

Western Blot—Samples fractionated by SDS-polyacrylamide gel electrophoresis were electroblotted to polyvinylidene difluoride membranes. After blocking with 5% nonfat dried milk in Tris-Tween Buffered Saline (TTBS: 50 mM TrisHCl, 0.05% Tween-20, 150 mM NaCl, pH 7.5), primary antibody was applied in TTBS for 2 h at room temperature. Exon A was visualized using rabbit polyclonal antibody JH629 (rPAM-1-(394–498), 1:1000) (32). PHM was visualized using rabbit polyclonal antibody JH1761 (rPAM-1-(37–382), 1:1000)) (31). TGN-38 was visualized using a rabbit polyclonal antibody to the luminal domain (1:1000) (33). Bound antibody was visualized using horseradish peroxidase-conjugated secondary antibody and Super Signal West Pico chemiluminescent substrate (Pierce). When quantification was performed, non-saturated signals were acquired with a GeneGnome work station using Gene-Tools software (Syngene, Frederick, MD). The effect of copper status on phosphorylation of PAM-1/Ser937 and PAM-1/Ser949 was evaluated using phosphospecific antisera to analyze cell extracts prepared using SDS lysis buffer (27, 40); no significant differences were detected when levels of phosphorylation were normalized to amount of PAM-1, which was quantified using a monoclonal antibody to the cytosolic domain of PAM-1.

Localization by Immunofluorescence—PAM-1 AtT-20 cells were fixed with 4% paraformaldehyde in PBS (50 mM sodium phosphate, 150 mM NaCl, pH 7.4) for 30 min, permeabilized with 0.075% Triton X-100 in PBS for 30 min, and blocked with 2 mg/ml BSA in PBS for 1 h at room temperature (18). Fixed cells were incubated with primary antibodies diluted in blocking buffer for 2 h at room temperature or overnight at 4 °C. Following extensive rinsing with PBS, cells were incubated in the appropriate FITC- or Cy3-conjugated secondary antibody for 1 h at room temperature in the dark. After rinsing, slides were mounted with glass coverslips using Permafluor mounting medium (Immunotech, Marseilles, France). Cells were observed using a Nikon TE300 fluorescence microscope and images were acquired with a Hamamatsu digital CCD camera C4742-95-2ERG connected to an Orca-ER camera controller using OpenLab software (Improvision, Lexington, MA).

Measurement of Immunoactive ACTH and JP-amide—For radioimmunoassays, cells were extracted in 5 N acetic acid with protease inhibitors and then diluted 3-fold with water. Samples were divided into two aliquots: BSA (2 mg/ml) was added to one aliquot, and all samples were lyophilized. Samples were dissolved in radioimmunoassay buffer (50 mM sodium phosphate, pH 7.4, 0.1% Triton X-100) with protease inhibitors. Samples lacking BSA were assayed for protein content using the bicinchoninic acid assay (Pierce). Samples containing BSA were used for radioimmunoassay. ACTH radioimmunoassay used antibody Kathy (1:15,000), 125I-labeled ACTH-(1–39) (NEN Life Science Products) and synthetic ACTH-(1–39) (Peninsula) as the standard; this antibody only recognizes POMC products in which the C-terminal end of ACTH-(1–39) is exposed (31). Amidated Joining Peptide radioimmunoassay used antibody Jamie (1:5000), 125I-labeled Tyr-Joining Peptide(12–18)NH2 and amidated Joining Peptide(12–18)NH2 (PNPSPAN-NH2) as the standard (Vega Biotechnologies); this antibody does not cross-react significantly with Joining Peptide-Gly, ACTH biosynthetic intermediate or POMC but does recognizes N-terminally extended peptides (34). Rabbit polyclonal antibodies Jamie and JH93, which is specific for the N-terminal region of ACTH (35) were used for immunostaining.

Endocytic Trafficking of PAM-1—To assess endocytic trafficking, PAM-1 AtT-20 cells were incubated in complete serum-free medium (CSFM) (with 1 mg/ml BSA added) containing a 1:50 dilution of rabbit polyclonal antiserum JH629 for 10 min at 37 °C and then rinsed and either fixed immediately (Pulse; a measure of antibody uptake) or incubated in antibody-free medium for 30 min at 37 °C (Chase; a measure of antibody retention) before fixation. Internalized antibody was visualized using Cy3-tagged second antibody. Total PAM protein was visualized using a monoclonal antibody to the cytosolic domain of PAM and FITC-tagged second antibody. Non-saturated images were acquired under identical conditions and analyzed using Simple PCI software (Compix, Cranberry, PA). As shown previously (26), monovalent PAM antibody is internalized in a similar manner.

A quantitative assessment of endocytic trafficking was made using peroxidase-tagged antibody. Affinity-purified Exon A antibody (0.3 mg) was linked to EZlink Activated HRP (1 mg) (31496; Pierce) and purified by binding to protein A-agarose (Repligen Corp., Waltham, MA). Based on a solid phase ELISA using recombinant Exon A (50 ng/well), recovery of purified HRP conjugate was 99%. PAM-1 AtT-20 cells plated into a 96-well plate were incubated with HRP-Exon A antibody (8 µg/ml) for 10 min, rinsed, and either harvested immediately (Pulse; a measure of antibody uptake) or chased for 5, 15, 30, or 60 min in complete serum free medium before harvest; duplicate samples were analyzed. Chase media were centrifuged to remove debris and transferred to empty wells of the 96-well plate. Cells were washed three times and extracted into TMT (20 mM NaTES, 10 mM mannitol, 1% Triton X-100, pH 7.4) by incubation at 4 °C for 30 min followed by freeze/thawing. Peroxidase levels were assessed by adding 100 µl of TMB substrate solution (Pierce) to each well. Pure HRP (0.001–2.5 ng) (Pierce) was assayed at the same time, providing a standard curve. The reaction was stopped after 30 min by adding 2 M H2SO4 (100 µl) and A450 was recorded using a Wallac Spectrophotometer (450-nm absorbance filter, 20-nm bandwidth).

Transferrin Internalization—PAM-1 AtT-20 cells were incubated in CSFM (lacking transferrin) containing 1 mg/ml BSA and 0.1 mg/ml AlexaFluor546 Transferrin (Invitrogen, Carlsbad, CA) for 10 min at 37 °C, rinsed and either fixed immediately or chased in CSFM for 30 min at 37 °C before fixation. PAM was visualized using antibody to Exon A and FITC-tagged second antibody; AlexaFluor546 Transferrin was visualized directly.

Biosynthetic Labeling—Cell lines plated into 4-well dishes were allowed to reach 70–80% confluency. Following a 5-min incubation in medium lacking Met, cells were incubated in Met-minus CSFM-air containing [35S]Met (1 mCi/ml; PerkinElmer, Waltham, MA or GE Healthcare, Piscataway, NJ) for 20 min and harvested immediately or chased for different times in CSFM-air. Cells were harvested in TMT; chase medium was centrifuged to remove cell debris. PAM was immunoprecipitated using antibody to PHM (JH1761) or Exon A (JH629). Immunoprecipitates were resolved by SDS-PAGE and newly synthesized proteins were visualized by fluorography following impregnation of the gel with Enhance.

Surface Biotinylation—PAM-1 AtT-20 cells were plated in triplicate in 12-well dishes. On the day of the experiment, cells were equilibrated in CSFM-air supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, insulin/transferrin/selenium for 20 min at 37 °C. Before addition of the biotin derivative, cells were rinsed three times with pre-warmed 15 mM HEPES-KOH, pH 7.5, 120 mM NaCl, 2 mM CaCl2, 4 mM KCl, 25 mM glucose. Sulfo-NHS-LC-biotin (1.25 mM) (Pierce) dissolved in the same buffer was allowed to react for 10 min at 37 °C. The reaction was quenched by incubating the cells with CSFM-air supplemented with 2 mg/ml BSA. Cells were either extracted immediately (Pulse) or incubated for 30 min (Chase). Following extraction into TMT supplemented with protease inhibitors (30 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 10 µg/ml {alpha}2-macroglobulin, and 16 µg/ml benzamidine), clarified lysates and media were incubated with neutravidin beads (40 µl slurry) (Pierce) for 60 min at 4 °C. Beads were rinsed twice with TMT containing protease inhibitors and bound proteins were eluted into Laemmli sample buffer by heating for 5 min at 95 °C.

Co-immunoprecipitation—Membranes prepared by centrifugation (430,00 x g for 15 min) of PAM-1 AtT-20 cells extracted into 10 mM NaTES, 20 mM mannitol, pH 7.4 were solubilized by tumbling in TMT for 30 min at 4 °C. Solubilized membranes were incubated with antibody to ATP7A (5 µl CT77) (36) or PAM cytosolic domain (PAM-(898–976)) (5 µl 6E6) (25) for 2 h at 4 °C. Antibody/antigen complexes were isolated using protein A/G-agarose beads (Pierce) (1 h, 4 °C). Beads were rinsed twice with TMT; proteins eluted into Laemmli sample buffer (5 min, 95 °C) were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Efficient immunoprecipitation of ATP7A and PAM-1 was verified by comparison to input samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Copper Slows the Processing of PAM and Secretion of PHM—Overnight incubation of cells with bathocuproinedisulfonate is frequently used to reduce the availability of copper (12, 13, 37). The ability of BCS to limit copper availability in AtT-20 cells was verified by monitoring peptide amidation, a reaction that is dependent upon the binding of copper to PAM (36). Proopiomelanocortin normally yields two amidated peptides, Joining Peptide and {alpha}MSH (34). Levels of Joining Peptide amide were reduced at least 5-fold following overnight BCS treatment while levels of ACTH were unaltered (supplemental Fig. S1). The medium used to maintain AtT-20 cells nominally contains 5 nM CuCl2; to load the system with copper, 10 or 20 µM CuCl2 was added to the medium for 2 h. Although higher copper levels are frequently utilized, concentrations above 20 µM had an inhibitory effect on protein synthesis in AtT-20 cells.

To determine whether copper levels affect the synthesis and secretion of PAM, AtT-20 cells stably expressing PAM-1 were either depleted of copper by overnight incubation with BCS, kept in normal medium (Control) or exposed to medium containing elevated levels of CuCl2 (Copper). Newly synthesized proteins were labeled by incubating cells in [35S]Met/Cys-containing medium of varying copper content and either harvested immediately (Pulse, P) or chased for 3 h before harvest (Chase, C) (Fig. 1). Based on precipitation of total protein with trichloroacetic acid, the copper content of the medium did not affect total protein synthesis (data not shown). PAM was immunoprecipitated from cell extracts and spent media, and newly synthesized PAM was visualized by fluorography (Fig. 1A). During the 3-h chase, the newly synthesized 120-kDa PAM protein present after the pulse incubation moves through the Golgi complex, enters immature secretory granules and begins to undergo cleavage (28). Endoproteolytic cleavage within Exon A yields soluble PHM (45 kDa) and membrane PAL (70 kDa, PALm). Soluble PHM can be stored or secreted, while intact PAM-1 and PALm undergo endocytosis following fusion of granules with the plasma membrane (28). Analysis of short chase times (30 and 60 min) failed to reveal any effect of BCS treatment on the time at which newly synthesized PAM first undergoes endoproteolytic cleavage (supplemental Fig. S2).


Figure 1
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  		FIGURE 1.
Metabolic labeling of membrane PAM. A, AtT-20 cells stably expressing PAM-1 were depleted of copper by overnight incubation with 50 µM BCS, kept in normal medium (Control) or exposed to medium containing 10 or 20 µM CuCl2 for 2 h. Duplicate wells of pretreated cells were incubated in medium containing [35S]Met/Cys for 20 min and harvested immediately (Pulse, P) or chased for 3 h before preparation of cell extract (C) and spent medium (M). Cell extracts and spent media were immunoprecipitated with antibody to Exon A and newly synthesized PAM was visualized by fluorography. Recoveries were calculated taking the 120-kDa PAM present after the pulse as 100%, taking into account the number of methionine residues in each molecule. B, for chase samples, % Processed = 100 x total PHM/total. C, for chase samples, % Secreted = 100 x medium PHM/total. The films shown are representative of the three experiments used for quantification.

 
Because the stability of many cuproenzymes is decreased when copper is not available (13, 16, 38), we quantified the recovery of PAM proteins after the chase. Setting the amount of labeled 120-kDa PAM recovered after the pulse to 100%, it is clear that neither lack of copper nor excess copper significantly alters the recovery of PAM proteins (Fig. 1A; p for BCS versus Con or BCS versus Cu > 0.05).

A significant amount of the newly synthesized PAM-1 and PALm reaches the plasma membrane and undergoes endocytosis during the 3-h chase period. Determinants in its cytosolic domain govern whether endocytosed PAM re-enters secretory granules, where it is again subject to endoproteolytic cleavage to 45-kDa PHM plus 70-kDa PALm, or undergoes degradation. We next asked whether cleavage of 120-kDa PAM to 45-kDa PHM were altered by copper levels. BCS pretreatment increased the amount of 120 kDa PAM cleaved to produce 45 kDa PHM (Fig. 1B). Soluble PHM can be stored or secreted. After the 3-h chase, almost 55% of the total radiolabeled PAM protein recovered is soluble PHM in the medium (Fig. 1C). The presence of 20 µM CuCl2 decreased secretion of 45-kDa PHM. Whereas lack of copper produces catalytically inactive enzyme (39), PAM turnover is not increased when copper levels are low. High levels of copper diminish PAM processing and secretion. The effect of CuCl2 on secretion is not due to blockage of Ca+2 entry, which is required for secretion, because similar levels of other divalent metals had no effect on secretion (Zn2+, Ni2+, Cd2+, Mn2+, Mg2+).

Copper Decreases the Rate at Which Antibody Internalized by PAM-1 Leaves the Endocytic Pathway—Exocytosis of secretory granules containing membrane PAM and soluble PHM along with stored peptide hormone leads to the appearance of PAM-1 on the plasma membrane and to the secretion of PHM. To assess the effect of copper levels on the endocytic trafficking of PAM, copper-loaded, control, and copper-depleted AtT-20 PAM-1 cells were incubated in medium containing peroxidase-tagged antibody to Exon A for 10 min. The peroxidase-tagged antibody was removed, and cells were either harvested immediately (Pulse; antibody uptake) or chased before harvesting. The peroxidase-antibody content of the cell extracts (Fig. 2) and chase media (data not shown) was determined using TMB as a substrate; uptake by wild type AtT-20 cells defined the assay background (supplemental Fig. S3). Copper loading did not affect antibody uptake (Fig. 2A). Under all conditions, 85–90% of the internalized peroxidase-antibody was released from the cells during the first 5-min chase. Cells depleted of copper lost internalized antibody more quickly than control cells (Fig. 2B). Copper-loaded cells retained internalized antibody for a longer time than control cells (Fig. 2B). The effect was most dramatic after the 30-min chase.

At the steady state, most of the PAM protein in AtT-20 cells is localized to endocytic compartments concentrated in the perinuclear region (18). Staining for total PAM was not detectably altered by copper levels (supplemental Fig. S4A). To visualize the effect of copper on the endocytic trafficking of PAM, AtT-20 PAM-1 cells loaded with copper or depleted of copper were allowed to internalize PAM antibody and then chased in antibody-free medium before fixation and permeabilization. As for the peroxidase-tagged antibody, copper loading resulted in the retention of internalized antibody while copper depletion resulted in the more rapid clearance of internalized antibody (supplemental Fig. S4, A and B); initial uptake of antibody was not affected by copper status. Copper levels had no effect on the retention of internalized transferrin (supplemental Fig. S4C), confirming the specificity of the copper effect.


Figure 2
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  		FIGURE 2.
Endocytic trafficking of PAM: peroxidase-conjugated antibody. AtT-20 cells stably expressing membrane PAM were depleted of copper (BCS), loaded with copper (10 or 20 µM), or kept under control conditions as described in Fig. 1. Cells were plated into a 96-well plate, and triplicates were analyzed for each condition and time point. Cells were incubated in medium containing peroxidase tagged antibody to Exon A for 10 min and harvested immediately (pulse, A) or chased for 5, 15, 30, or 60 min in CSFM (B). Chase media were centrifuged to remove any non-adherent cells. The peroxidase-antibody content of cell extracts and chase media (not shown) was determined using TMB as a substrate. A, peroxidase-antibody uptake after the 10-min pulse was indistinguishable (p > 0.05). B, values for chase cell extracts are plotted as percentage of the 10-min pulse; error bars are S.D. of triplicates. This experiment was replicated three times with similar results.

 
Copper Decreases the Ability of Internalized PAM to Return to Secretory Granules—The endocytic trafficking of membrane PAM is regulated by multiple determinants in its cytosolic domain (27, 28, 40, 41). Antibody uptake is only an indirect means of following the internalization of membrane PAM. Surface biotinylation provides a direct means of evaluating the effect of copper on the endocytic trafficking of PAM. AtT-20 PAM-1 cells depleted of copper by pretreatment with BCS, loaded with copper (20 µM) or kept as controls were exposed to sulfo-NHS-LC-biotin for 10 min at 37 °C and either extracted immediately (Pulse) or chased for 30 min before harvesting spent medium and extracting cells (Chase). The biotinylated proteins in cell lysates and spent media were isolated using Neutravidin-beads and biotinylated PAM proteins were visualized using antibody to Exon A (Fig. 3A). Input samples contain intact 120-kDa PAM-1, 70-kDa PALm, PHM, and small amounts of soluble PAL (sPAL), and 100-kDa PAM (Fig. 3B). While PHM and sPAL are generated in secretory granules by prohormone convertase cleavage at pairs of basic amino acids (18), the cleavage that generates sPAM does not occur in secretory granules.

Only a small percentage of the total 120-kDa PAM and 70-kDa PALm in the cell is on the plasma membrane and accessible to cell impermeant-activated biotin. In control cells, 1.7% of the PAM-1 and 1.3% of the PALm is biotinylated during a 10-min period at 37 °C (Fig. 3C). In copper-depleted cells, the fraction of PAM-1 accessible to biotin is decreased. Elevated levels of copper increase the fraction of the PAM-1 that is biotinylated during a 10-min period. The amount of PALm accessible to biotin is not affected by copper levels. A decrease in the amount of PAM-1 biotinylated in a 10-min period could reflect decreased delivery of PAM-1 to the plasma membrane or more rapid removal of PAM-1 from the plasma membrane.

To distinguish between these possibilities, cell extracts and spent media from the chase samples were analyzed (Fig. 3, A and D). During the chase, PAM-1 that was biotinylated while it was on the plasma membrane can be converted into sPAM or into PHM plus PALm. The endoproteolytic cleavage required to produce PALm plus PHM occurs only in secretory granules. Normalized to the amount of biotinylated 120-kDa PAM present after the pulse, more 120-kDa PAM-1 remains in copper-depleted cells than in copper-loaded cells. The amount of biotinylated PALm present in the cells after the chase is not altered by copper availability.


Figure 3
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  		FIGURE 3.
Endocytic trafficking of PAM: surface biotinylation. A, PAM-1 AtT-20 cells were plated into 12-well dishes and depleted of copper (BCS), loaded with copper (20 µM), or kept as controls as described above. Cells were then exposed to 1.25 mM sulfo-NHS-LC-biotin at 37 °C for 10 min, quenched with BSA, and extracted immediately (pulse) or chased for 30 min before separating chase medium from chase cells; triplicate samples were analyzed. Neutravidin-bound PAM proteins in cell extracts (25% of total) and spent media (100% of total) were compared with total PAM proteins in the cell lysate (input is 0.1%) using antibody to Exon A. * indicates major background band bound to streptavidin beads. B, the proteins produced by cleavage of PAM-1 are shown; arrows mark cleavages that occur in secretory granules. The specificity of the exon A antibody is shown. C, films were densitized and biotinylated PAM-1 (120-kDa PAM) and PALm present after the pulse were expressed as a percentage of the total PAM-1 in the corresponding pulse cell lysates; p values were calculated using the Student's t test. D, the total amount of biotinylated PAM-1, PALm, sPAM (cell + medium), and PHM (cell + medium) remaining after the chase was normalized to biotinylated PAM-1 present after the pulse for each treatment. E, secretion was somewhat variable between experiments, so the effects of BCS and copper were compared with the control; for each sample, the ratio of biotinylated PHM in the chase medium to biotinylated PHM in the chase cell extract was calculated. The average ratio from control cells was normalized to 1.0 and the effects of BCS and copper were adjusted accordingly. For the calculations shown in C–E, data from two experiments were combined.

 
Biotinylated 100-kDa PAM and 45-kDa PHM generated from PAM-1 that was on the plasma membrane can remain in the cell or be secreted. Looking first at the total amount of biotinylated 45-kDa PHM generated from biotinylated PAM-1, it is clear that more is generated in copper-depleted cells (Fig. 3D). Total production of biotinylated sPAM is not sensitive to copper levels. Because the endoproteolytic cleavage that generates PHM occurs only in secretory granules, we looked at the effect of copper levels on secretion of biotinylated PHM (Fig. 3E). To average data from three experiments, the rate at which 45-kDa PHM was secreted by control cells was normalized to 1.0; copper depletion results in a 2-fold increase in secretion of biotinylated PHM while excess copper decreases release of biotinylated PHM. Copper depletion results in an increase in the cleavage of newly synthesized PAM-1 and in an increase in the secretion of 45-kDa PHM derived from newly synthesized PAM-1. Consistent with this observation, copper depletion both stabilizes PAM-1 that has been internalized from the cell surface and increases its entry into secretory granules and conversion into 45-kDa PHM that is available for basal secretion. An increase in copper levels decreases the stability of PAM-1 biotinylated on the plasma membrane; less is converted into 45-kDa PHM in secretory granules and less is secreted.

The Synthesis and Secretion of Soluble PHM Is Not Sensitive to Copper Availability—The effect of copper on the trafficking of PAM-1 could be through its binding to PHM or could be an indirect effect, mediated through trafficking determinants in the cytosolic domain of PAM-1. To test for direct effects of copper binding to the active site of PHM, cells stably expressing soluble PHM were depleted of copper by overnight incubation with BCS, kept in control medium or exposed to medium containing 20 µM CuCl2 for 2 h. Cells were then incubated in medium containing [35S]Met/Cys for 20 min and either harvested immediately (Pulse) or chased for 3 h, with sequential hourly collections of medium. Cell extracts and chase media were analyzed by quantitative immunoprecipitation of PHM (Fig. 4). Recoveries were calculated by comparing the amount of PHM recovered from chase samples (cells plus media) to the amount of PHM present after pulse. In both AtT-20 cells and CHO cells, recovery of PHM was unaffected by copper levels.

The PHM(stop) protein expressed in the AtT-20 cells includes a short pro-region that is cleaved by the endoproteases present in this neuroendocrine cell line (29), leading to storage of a smaller PHM protein. Newly synthesized PHM(stop) secreted early during the chase period is largely uncleaved. Neither the timing nor the extent of this cleavage is affected by copper levels (Fig. 4A). The PHMcc protein expressed in the CHO cells does not include the pro-region, a region known to facilitate transit of PHM through the regulated secretory pathway (20, 33). Unlike AtT-20 cells, CHO cells do not produce secretory granules that store bioactive peptides. Newly synthesized PHMcc accumulates in the medium, with roughly 50% of the total remaining in the cells after a 3 h chase. The secretion pattern is unaltered by copper availability (Fig. 4B).

PHM Mutants That Cannot Bind Copper Are Efficiently Secreted—To ensure elimination of normal copper binding to the active site of PHM, catalytically inactive mutants lacking a functional CuA site were examined (Fig. 5). CHO cells stably expressing wild-type PHMcc or PHMcc with one (H107A) or two (HH107–108AA) mutations at the CuA site were incubated with [35S]Met/Cys as described above and analyzed by quantitative immunoprecipitation. Recovery of newly synthesized PHM was not diminished by the presence of these mutations. Neither the time course for secretion nor the amount of protein remaining in the cells after the chase was altered (Fig. 5). We conclude from these experiments that soluble PHM traverses the secretory pathway in much the same manner whether copper is bound to it or not.


Figure 4
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  		FIGURE 4.
Synthesis and secretion of soluble PHM. AtT-20 cells stably expressing PHM(stop) (A) and CHO cells stably expressing PHMcc (B) were depleted of copper or loaded with copper as described above. The structure of each protein is shown; residues 1–25 constitute the cleaved signal sequence. Duplicates wells were incubated in medium containing [35S]Met/Cys for 20 min and either harvested immediately (pulse, P) or chased for 3 h (C). During the chase, medium was collected every hour (M1, M2, and M3). Following immunoprecipitation with antibody to PHM and SDS-PAGE, newly synthesized PHM was visualized by fluorography. Non-saturated films were densitized, and recoveries for each treatment were calculated by setting the amount of PHM present after the pulse to 100%. The films shown are representative of three independent experiments; data were averaged, and S.D. are shown.

 


Figure 5
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  		FIGURE 5.
Synthesis and secretion of mutant PHM. CHO cells stably expressing wild-type PHMcc (WT) or PHM mutants lacking a functional CuA site (H107A; HH107–108AA) were incubated with [35S]Met/Cys and analyzed as described in Fig. 4. The films shown are representative of three independent experiments; data were averaged and S.D. are shown. Based on synthesis of trichloroacetic acid precipitable protein, similar amounts of recombinant PHM were expressed in each cell line.

 


Figure 6
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  		FIGURE 6.
Metabolic labeling of PAM-1/899. A, AtT-20 cells stably expressing PAM-1/899 were depleted of copper by overnight incubation with BCS, kept in normal medium or exposed to medium containing 20 µM CuCl2 for 2 h. Duplicate wells of pretreated cells were incubated in medium containing [35S]Met/Cys for 20 min and harvested immediately (pulse, P) or chased for 3 h before preparation of cell extract (C) and spent medium (M). Cell extracts and spent media were immunoprecipitated with antibody to Exon A and newly synthesized PAM was visualized by fluorography. Recoveries were calculated as in Fig. 1. For the chase samples, % Processed to PHM = 100 x total PHM (M + C)/Total (B) and % Secreted as PHM = 100 x PHM(M)/Total (C). The results shown are the average of two experiments; error bars show range.

 
The Cytosolic Domain of PAM Plays a Key Role in Its Sensitivity to Copper—The trafficking of membrane PAM is regulated by phosphorylation of Ser and Thr residues in its cytosolic domain (40). A PAM-1 protein truncated immediately after the transmembrane domain, PAM-1/899, fails to remain in secretory granules and does not undergo endocytosis, remaining on the plasma membrane (28); PAM-1/899 is catalytically active. AtT-20 cells expressing PAM-1/899 were depleted of copper with BCS, kept in normal medium or exposed to medium containing 20 µM CuCl2 (Fig. 6) Metabolic labeling with [35S]Met/Cys, carried out as in Fig. 1, again yielded recoveries of ~100%, independent of copper levels. Because PAM-1/899 does not accumulate in secretory granules, a significant fraction of the newly synthesized protein is cleaved at a site between PAL and the transmembrane domain, yielding a doublet of 105–107-kDa proteins that do not accumulate in the cell (28). The processing of PAM-1/899 to 105–107-kDa PAM, PALm and PHM is not altered by copper availability (Fig. 6, A and B). The secretion of PHM during the chase is not altered by copper availability (Fig. 6C). The effect of copper on PAM-1 trafficking requires the presence of its cytosolic domain.

In AtT-20 PAM-1 cells, endogenous ATP7A and transfected PAM-1 are both localized to the perinuclear region (18, 36). To determine whether ATP7A and PAM-1 interact directly, we tried to co-immunoprecipitate the proteins. Membrane proteins were solubilized with 1% Triton X-100. Using a polyclonal antibody to ATP7A or a monoclonal antibody to the cytosolic domain of PAM, we could efficiently capture the target antigen, but no co-immunoprecipitation was detected (data not shown). While many protein/protein interactions cannot be captured by immunoprecipitation, this observation raises the possibility that some other copper-sensitive machinery affects the trafficking of PAM-1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
ApoPAM and ApoPHM Are Stable—Although limiting copper availability by pretreatment with BCS eliminates the catalytic activity of PAM and PHM, the newly synthesized apoenzymes are stable. After a 3-h chase, essentially all of the newly synthesized PAM and newly synthesized PHM(stop) could be accounted for, whether copper levels were normal, artificially low (BCS) or elevated (Fig. 1A). This behavior is distinct from that of other secretory pathway cuproenzymes. Apohephaestin, a type I membrane protein, undergoes rapid, proteasome-mediated degradation (13). In the absence of copper, apoceruloplasmin is secreted but rapidly degraded in plasma (14). Rapid elimination of catalytically inactive apoenzyme appears to be an appropriate response to limited copper availability. The fact that PAM does not exhibit this type of response suggests that it may perform a non-catalytic function or be involved in some way in countering a deficit in copper. For example, the high levels of PAM found in atrial myocytes do not correlate with high levels of peptidylglycine substrate (20). When copper homeostasis is evaluated in mice heterozygous for PAM, alterations may become apparent.

The Cytosolic Domain of PAM Plays an Essential Role in the Effects of Copper on Trafficking—Copper binds to two sites in PHM: the CuA site (His107, His108, His172) and the CuB site (His242, His244, Met314) (42). Mutation of any one of these conserved His residues eliminates the catalytic activity of PHM (30). A detailed time course study of the HH107–108AA mutant, which cannot bind copper at the CuA site confirmed earlier data demonstrating efficient secretion of inactive PHM (30). When produced as a monofunctional, soluble protein, the stability and secretion of PHM are not sensitive to copper levels. While the binding of copper to the CuA and CuB sites of PHM must affect its structure, these changes are not sufficient to alter its trafficking. The fact that secretion of monofunctional PHM generated from membrane PAM is sensitive to copper levels (Fig. 2C) focuses our attention on an indirect effect, reflecting alterations in the trafficking of membrane PAM.

The ability of newly synthesized PAM-1 to enter secretory granules as it exits the TGN is not affected by copper levels. Nevertheless, when examined after a 3-h chase, both the production of PHM from newly synthesized PAM-1 and the secretion of newly synthesized PHM were increased when copper levels were reduced (Fig. 2, B and C). Basal secretion of soluble secretory granule proteins/peptides from AtT-20 cells occurs at a rate of ~12% of cell content per hour (43). Thus a significant fraction of the newly synthesized PAM-1 will have appeared on the plasma membrane and undergone endocytosis during a 3-h chase period. PAM-1/899 has a transmembrane domain but lacks the cytosolic determinants that govern secretory granule entry, recycling of PAM from immature granules and the endocytosis of PAM (28). While less than 5% of the newly synthesized PAM-1 reaches the plasma membrane within 3 h of synthesis, 50–70% of the newly synthesized PAM-1/899 reaches the plasma membrane in this time (26). Cleavage of newly synthesized PAM-1 to produce 45-kDa PHM proceeds essentially to completion while only about a third of newly synthesized PAM-1/899 is cleaved at this site (26). Production of PHM from PAM-1/899 and secretion of the PHM generated from PAM-1/899 were not affected by copper availability (Fig. 6). If basal secretion were sensitive to copper levels, we would expect to see an effect on PHM secretion from PAM-1/899 cells. The fact that we do not see an effect on PHM secretion leads us to conclude that copper availability affects primarily the endocytic trafficking of PAM-1.

Copper Affects the Endocytic Trafficking of PAM-1—Antibody internalization experiments provided our first indication that the endocytic trafficking of PAM-1 was sensitive to copper (Fig. 2 and supplemental Fig. S4). Antibody uptake was not affected by copper status. However, when copper levels were limiting, antibody internalized by PAM-1 was lost from cells more quickly. In copper loaded cells, internalized PAM antibody accumulated in the perinuclear region. Antibody internalization experiments provide an indirect measure of protein localization and surface biotinylation experiments were the key to understanding the effect of copper on PAM trafficking (Fig. 3). We examined the fate of PAM proteins biotinylated over a 10 min period at 37 °C (Pulse) and after a 30-min chase. The stability of PAM-1 biotinylated on the surface of AtT-20 cells is increased when copper levels are low and decreased when copper levels are high (Fig. 3D). While either antibody binding or biotinylation could alter the behavior of PAM-1, the fact that both approaches yield similar results largely alleviates this concern. Even after this short chase time, it is clear that low copper conditions facilitate the formation of biotinylated 45-kDa PHM from PAM-1 that was previously on the cell surface. Because 45-kDa PHM is produced only in regulated secretory granules, we have to conclude that the re-entry of internalized PAM-1 into secretory granules is facilitated when copper levels are low. Cleavage of PAM-1 to produce PHM increases its specific activity severalfold (29); because peptide amidation is often the rate-limiting step in the pathway leading to production of bioactive product peptides, increased cleavage of PAM-1 is a compensatory response. In contrast, the stability of PALm biotinylated on the surface of AtT-20 cells is unaffected by copper levels (Fig. 3D). Only full-length integral membrane PAM is responsive to copper levels.


Figure 7
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  		FIGURE 7.
Trafficking of membrane PAM. The major steps in the biosynthetic and endocytic trafficking of PAM are shown. PAM is cleaved to yield PHM plus PALm. ISG, immature secretory granule; SG, secretory granule; EE, early endosomes; MVB/LE, multivesicular body/late endosomes; Lyso, lysosomes. The degradation of newly synthesized and endocytosed PAM is increased by high copper. The cleavage of both newly synthesized and recycled PAM-1 is increased when copper levels are low; cleavage of PAM-1 increases the activity of PHM severalfold (29). Trafficking through the endocytic pathway is accelerated when copper levels are low and inhibited when copper levels are high.

 
Data from the metabolic labeling and surface biotinylation experiments are summarized in Fig. 7, providing a simple working model. All of the effects observed are compatible with a copper-sensitive trafficking step that allows endocytosed PAM to return to the regulated secretory pathway or to undergo degradation. When copper levels are low, PAM returns to secretory granules; it can undergo cleavage to PHM, increasing its specific activity and yielding the observed increase in basal secretion. When copper levels are high, internalized PAM does not return to granules, limiting the generation of PHM, and decreasing basal release; instead, internalized PAM is degraded. Neither the entry of newly synthesized PAM into immature secretory granules, the maturation of secretory granules nor the basal release of granules need be affected. The decreased level of intact PAM-1 on the surface of copper-depleted cells can simply reflect the increased cleavage of PAM-1 in secretory granules. More detailed studies will be needed to identify the endocytic trafficking events that are sensitive to copper. Because PAM-1 proteins mutated at individual phosphorylation sites traverse the early parts of the endocytic pathway normally and show deficits in their ability to exit late endosomes/multivesicular bodies (27) the copper-sensitive steps are tentatively assigned to this step.

Copper Affects the Endocytic Trafficking of Other Cuproproteins—In what appears to be a homeostatic response, plasma membrane levels of Ctr1 are reduced when copper levels are high; elevated levels of copper stimulate the clathrin-mediated endocytosis of human Ctr1 (44). In contrast, surface levels of PAM-1 are increased slightly when copper levels are high. A similar response was observed for ATP7A; an increase in copper levels yielded more ATP7A on the plasma membrane and in non-Golgi vesicles (45, 46). For ATP7B, an increase in copper levels resulted in rapid movement of ATP7B into a cytoplasmic vesicular compartment (47, 48). The copper-induced trafficking of ATP7A and ATP7B is associated with their ability to form a phosphorylated intermediate (49). Whether the TGN retention sequence in transmembrane domain-3 (50), the di-Leu motif in the cytosolic domain (51) or cytosolic copper binding motifs 5 and 6 (52) play a role in the copper-dependent trafficking of ATP7A is not yet clear.

Copper could affect one of the many interactions mediated by the cytosolic domain of PAM. In addition to a Tyr internalization motif, the cytosolic domain of PAM contains multiple phosphorylation sites (32) as well as sites for interacting with Kalirin and Trio, Rho GDP/GTP exchange factors that affect cytoskeletal organization (53, 54). Protein kinase A, protein kinase C, casein kinase II, and P-CIP2 each phosphorylate sites in the cytosolic domain of PAM (32, 55). Inability to phosphorylate Ser937, a protein kinase C site, limits the secretion of soluble PHM and increases the turnover rate of newly synthesized PAM-1 (27). Phosphorylation and dephosphorylation of the casein kinase II/P-CIP2 sites in the cytosolic domain is essential for normal endocytic trafficking of PAM (40). PAM cytosolic domain mutations that eliminate its ability to interact with P-CIP2 eliminate the ability of PAM to affect cytoskeletal organization (41). However, using antisera specific for PAM phosphorylated on Ser937 or Ser949 to analyze whole cell lysates, we were unable to detect copper-dependent differences in phosphorylation; a more detailed analysis of specific subcellular organelles may reveal a role for copper-dependent phosphorylation at these or other sites. Further study will be required to identify the copper sensitive steps in the endocytic trafficking of PAM-1 and the essential sites in the cytosolic domain.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK-32949 (to B. A. E. and R. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 To whom correspondence should be addressed: 263 Farmington Ave., Farmington, CT 06030-3401. E-mail: eipper{at}uchc.edu.

2 The abbreviations used are: SOD, superoxide dismutase; BCS, bathocuproine disulfonic acid; PHM, peptidylglycine {alpha}-hydroxylating monooxygenase; PAL, peptidyl-{alpha}-hydroxyglycine {alpha}-amidating monooxygenase; TGN, trans-Golgi network; CSFM, complete serum-free medium; JP, joining peptide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; PAM, peptidylglycine {alpha}-amidating monooxygenase; PALm, membrane PAL. Back


   ACKNOWLEDGMENTS
 
We thank Darlene D'Amato for making these experiments possible, Chitra Rajagopal and Dr. Francesco Ferraro for sharing their newly developed surface biotinylation protocols, and Jaqueline Sobota for introducing us to Simple PCI.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Madsen, E., and Gitlin, J. D. (2007) Curr. Opin. Gastroenterol. 23, 187-192[Medline] [Order article via Infotrieve]
  2. Linder, M. C., and Hazegh-Azam, M. (1996) Am. J. Clin. Nutr. 63, 797S-811S
  3. Camakaris, J., Voskoboinik, I., and Mercer, J. F. (1999) Biochem. Biophys. Res. Commun. 261, 225-232[CrossRef][Medline] [Order article via Infotrieve]
  4. Harris, E. D. (2000) Annu. Rev. Nutr. 20, 291-310[CrossRef][Medline] [Order article via Infotrieve]
  5. Zhou, B., and Gitschier, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7481-7486[Abstract/Free Full Text]
  6. Culotta, V. C., Klomp, L. W., Strain, J., Casareno, R. L., Krems, B., and Gitlin, J. D. (1997) J. Biol. Chem. 272, 23469-23472[Abstract/Free Full Text]
  7. Amaravadi, R., Glerum, D. M., and Tzagoloff, A. (1997) Hum. Genet. 99, 329-333[CrossRef][Medline] [Order article via Infotrieve]
  8. Klomp, L. W., Lin, S. J., Yuan, D. S., Klausner, R. D., Culotta, V. C., and Gitlin, J. D. (1997) J. Biol. Chem. 272, 9221-9226[Abstract/Free Full Text]
  9. Voskoboinik, I., Camakaris, J., and Mercer, J. F. (2002) Adv. Protein Chem. 60, 123-150[Medline] [Order article via Infotrieve]
  10. Puig, S., and Thiele, D. J. (2002) Curr. Opin. Chem. Biol. 6, 171-180[CrossRef][Medline] [Order article via Infotrieve]
  11. Rees, E. M., and Thiele, D. J. (2004) Curr. Opin. Microbiol. 7, 175-184[CrossRef][Medline] [Order article via Infotrieve]
  12. Hellman, N. E., Kono, S., Manchini, G. M., Hoogeboom, A. J., De Jong, G. J., and Gitlin, J. D. (2002) J. Biol. Chem. 277, 46632-46638[Abstract/Free Full Text]
  13. Nittis, T., and Gitlin, J. D. (2004) J. Biol. Chem. 279, 25696-25702[Abstract/Free Full Text]
  14. Sato, M., and Gitlin, J. D. (1991) J. Biol. Chem. 266, 5128-5134[Abstract/Free Full Text]
  15. Qin, Z., Itoh, S., Jeney, V., Ushio-Fukai, M., and Fukai, T. (2005) FASEB J. 20, 334-336[Medline] [Order article via Infotrieve]
  16. Wang, N., and Herbert, D. N. (2006) Pigment Cell Res. 19, 3-18[CrossRef][Medline] [Order article via Infotrieve]
  17. Li, S., and Kagan, H. M. (1997) Matrix Biol. 16, 387-398[CrossRef]
  18. Milgram, S. L., Johnson, R. C., and Mains, R. E. (1992) J. Cell Biol. 117, 717-728[Abstract/Free Full Text]
  19. Klinman, J. P. (2006) J. Biol. Chem. 281, 3013-3016[Free Full Text]
  20. Prigge, S. T., Mains, R. E., Eipper, B. A., and Amzel, L. M. (2000) Cell Mol. Life Sci. 57, 1236-1259[CrossRef][Medline] [Order article via Infotrieve]
  21. Steiner, D. F. (1998) Curr. Opin. Chem. Biol. 2, 31-39[CrossRef][Medline] [Order article via Infotrieve]
  22. Seidah, N. G., and Chretien, M. (1999) Brain Res. 848, 45-62[CrossRef][Medline] [Order article via Infotrieve]
  23. Hutton, J. C. (1990) Curr. Opin. Cell Biol. 2, 1131-1142[CrossRef][Medline] [Order article via Infotrieve]
  24. Halban, P. A., and Irminger, J. C. (1994) Biochem. J. 299, 1-18[Medline] [Order article via Infotrieve]
  25. Milgram, S. L., Kho, S. T., Martin, G. V., Mains, R. E., and Eipper, B. A. (1997) J. Cell Sci. 110, 695-706[Abstract]
  26. Milgram, S. L., Eipper, B. A., and Mains, R. E. (1994) J. Cell Biol. 124, 33-41[Abstract/Free Full Text]
  27. Steveson, T. C., Keutmann, H. T., Mains, R. E., and Eipper, B. A. (1999) J. Biol. Chem. 274, 21128-21138[Abstract/Free Full Text]
  28. Milgram, S. L., Mains, R. E., and Eipper, B. A. (1993) J. Cell Biol. 121, 23-36[Abstract/Free Full Text]
  29. Husten, E. J., Tausk, F. A., Keutmann, H. T., and Eipper, B. A. (1993) J. Biol. Chem. 268, 9709-9717[Abstract/Free Full Text]
  30. Kolhekar, A. S., Keutmann, H. T., Mains, R. E., Quon, A. S. W., and Eipper, B. A. (1997) Biochemistry 36, 10901-10909[CrossRef][Medline] [Order article via Infotrieve]
  31. Ciccotosto, G. D., Schiller, M. R., Eipper, B. A., and Mains, R. E. (1999) J. Cell Biol. 144, 459-471[Abstract/Free Full Text]
  32. Yun, H.-Y., Milgram, S. L., Keutmann, H. T., and Eipper, B. A. (1995) J. Biol. Chem. 270, 30075-30083[Abstract/Free Full Text]
  33. Milgram, S. L., Mains, R. E., and Eipper, B. A. (1996) J. Biol. Chem. 271, 17526-17535[Abstract/Free Full Text]
  34. Eipper, B. A., Park, L., Keutmann, H. T., and Mains, R. E. (1986) J. Biol. Chem. 261, 8686-8694[Abstract/Free Full Text]
  35. Zhou, A., Bloomquist, B. T., and Mains, R. E. (1993) J. Biol. Chem. 268, 1763-1769[Abstract/Free Full Text]
  36. Steveson, T. C., Ciccotosto, G. D., Ma, X. M., Mueller, G. P., Mains, R. E., and Eipper, B. A. (2003) Endocrinology 144, 188-200[Abstract/Free Full Text]
  37. Gordge, M. P., Hothersall, J. S., Neild, G. H., and Dutra, A. A. (1996) Br. J. Pharmacol. 119, 533-588
  38. Sato, S. M., and Mains, R. E. (1985) Endocrinology 117, 773-786[Abstract/Free Full Text]
  39. Eipper, B. A., Stoffers, D. A., and Mains, R. E. (1992) Annu. Rev. Neurosci. 15, 57-85[CrossRef][Medline] [Order article via Infotrieve]
  40. Steveson, T. C., Zhao, G. C., Keutmann, H. T., Mains, R. E., and Eipper, B. A. (2001) J. Biol. Chem. 276, 40326-40337[Abstract/Free Full Text]
  41. Alam, M. R., Steveson, T. C., Johnson, R. C., Back, N., Abraham, B., Mains, R. E., and Eipper, B. A. (2001) Mol. Biol. Cell 12, 629-644[Abstract/Free Full Text]
  42. Prigge, S. T., Kolhekar, A. S., Eipper, B. A., Mains, R. E., and Amzel, L. M. (1999) Nat. Struct. Biol. 6, 976-983[CrossRef][Medline] [Order article via Infotrieve]
  43. Mains, R. E., and Eipper, B. A. (1981) J. Cell Biol. 89, 21-28[Abstract/Free Full Text]
  44. Petris, M. J., Smith, K., Lee, J., and Thiele, D. J. (2003) J. Biol. Chem. 278, 9639-9646[Abstract/Free Full Text]
  45. Petris, M. J., Mercer, J. F., Culvenor, J. G., Lockhart, P., Gleeson, P. A., and Camakaris, J. (1996) EMBO J. 15, 6084-6095[Medline] [Order article via Infotrieve]
  46. Petris, M. J., Camakaris, J., Greenough, M. L., LaFontanie, S., and Mercer, J. F. (1998) Hum. Mol. Genet. 7, 2063-2071[Abstract/Free Full Text]
  47. Schaefer, M., Hopkins, R. G., Failla, M. L., and Gitlin, J. D. (1999) Am. J. Physiol. 276, G639-G646[Medline] [Order article via Infotrieve]
  48. Cater, M., La Fontanie, S., and Mercer, J. F. (2007) Biochem. J. 401, 143-153[CrossRef][Medline] [Order article via Infotrieve]
  49. Petris, M. J., Voskoboinik, I., Cater, M., Smith, K., Kim, B., Llanos, R. M., Strausak, D., Camakaris, J., and Mercer, J. F. (2002) J. Biol. Chem. 277, 46736-46742[Abstract/Free Full Text]
  50. Francis, M. J., Jones, E. E., levy, E. R., Ponnambalam, S., Chelly, J., and Monaco, A. P. (1998) Hum. Mol. Genet. 7, 1245-1252[Abstract/Free Full Text]
  51. Francis, M. J., Jones, E. E., levy, E. R., Martin, R. L., Ponnambalam, S., and Monaco, A. P. (1999) J. Cell Sci. 112, 1721-1732[Abstract]
  52. Goodyer, I. D., Jones, E. E., Monaco, A. P., and Francis, M. J. (1999) Hum. Mol. Genet. 8, 1473-1478[Abstract/Free Full Text]
  53. Hansel, D. E., Quinones, M. E., Ronnett, G. V., and Eipper, B. A. (2001) J. Histochem. Cytochem. 49, 833-844[Abstract/Free Full Text]
  54. Ma, X. M., Johnson, R. C., Mains, R. E., and Eipper, B. A. (2001) J. Comp. Neurol. 429, 388-402[CrossRef][Medline] [Order article via Infotrieve]
  55. Caldwell, B. D., Darlington, D. N., Penzes, P., Johnson, R. C., Eipper, B. A., and Mains, R. E. (1999) J. Biol. Chem. 274, 34646-34656[Abstract/Free Full Text]

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D. Bousquet-Moore, X. M. Ma, E. A. Nillni, T. A. Czyzyk, J. E. Pintar, B. A. Eipper, and R. E. Mains
Reversal of Physiological Deficits Caused by Diminished Levels of Peptidylglycine {alpha}-Amidating Monooxygenase by Dietary Copper
Endocrinology, April 1, 2009; 150(4): 1739 - 1747.
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