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J Biol Chem, Vol. 273, Issue 50, 33107-33110, December 11, 1998
andFrom the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Prion diseases result from conformational
alteration of PrPC, a cell surface glycoprotein
expressed in brain, spinal cord, and several peripheral tissues, into
PrPSc, a protease-resistant isoform that is the principal
component of infectious prion particles. Although a great deal is known about the pathogenic role of PrPSc, the physiological
function of PrPC has remained a mystery. Several lines of
evidence have recently suggested the possibility that PrPC
may play a role in the metabolism of copper. To further investigate the
interaction of PrPC and copper, we have analyzed the effect
of this metal ion on the endocytic trafficking of PrPC in
cultured neuroblastoma cells. We report here that copper rapidly and
reversibly stimulates endocytosis of PrPC from the cell
surface. This effect may be physiologically relevant and suggests the
hypothesis that PrPC could serve as a recycling receptor
for uptake of copper ions from the extracellular milieu.
Prion diseases are neurodegenerative disorders that result from
conformational conversion of a normal cell surface glycoprotein (PrPC)1 into a
pathogenic isoform (PrPSc) that appears to be infectious in
the absence of nucleic acid (1, 2). Although a wealth of information is
now available about the role of PrPSc in the disease
process, relatively little is known about the normal, physiological
function of PrPC. PrPC is expressed in neurons
and glia of the brain and spinal cord and at lower levels in several
peripheral tissues and in leukocytes (3-7). Its localization on the
cell surface would be consistent with roles in cell adhesion and
recognition, ligand uptake, or transmembrane signaling. Mice in which
the endogenous PrP gene has been ablated do not display any gross
anatomical or developmental defects (8) but are reported in some
studies to have electrophysiological and structural abnormalities in
the hippocampus (9-12), loss of cerebellar Purkinje cells (13), or
alterations in circadian rhythm and sleep pattern (14). How these
abnormal phenotypes, when present, relate to the normal function of
PrPC is unclear. Identifying the function of
PrPC may be important in understanding the pathogenesis of
prion diseases, because loss of this function as a result of conversion
to PrPSc may explain some features of the disorders.
Several new pieces of data suggest that PrPC may play a
role in the metabolism of copper. First, the N-terminal half of
PrPC contains a series of histidine- and glycine-containing
peptide repeats that are capable of binding copper ions. Synthetic
peptides or recombinant PrP fragments
encompassing the repeats bind copper with a Kd of
5-10 µM, and binding of the metal induces conformational
alterations in the polypeptide chain (15-19). Copper also facilitates
restoration of protease resistance and infectivity during refolding of
guanidine-denatured PrPSc (20). Second, the total content
of copper but not of several other metals is only 20% of normal in
crude membranes, synaptosomes, and endosomes derived from the brains of
Prn-p0/0 mice that contain a disrupted PrP gene
(18). This result suggests that PrPC may be a major
copper-binding protein in brain. Third, neuronal Cu-Zn superoxide
dismutase (SOD) from Prn-p0/0 mice is less
enzymatically active and incorporates less radioactive copper than the
enzyme from normal mice; the opposite is true for SOD from transgenic
mice that over-express PrPC (21, 22). Neurons cultured from
Prn-p0/0 mice are also more sensitive to oxidative
insult, perhaps because of reduced SOD activity (21). These
observations raise the possibility that PrPC could be
involved in delivery of copper ions to SOD and perhaps other
cuproenzymes. Finally, PC12 cells selected for resistance to copper
toxicity have increased expression of PrPC, suggesting a
role for the protein in removal or detoxification of copper (23).
Exactly how copper and PrPC are functionally related is
unclear at present. One possibility is that bound copper serves as an
essential cofactor for an as yet undetermined enzymatic activity of
PrPC, as it does in cuproproteins such as SOD and
cytochrome c oxidase (24). By virtue of its presence on the
cell surface, PrPC could also function as a sink for
chelation of extracellular copper ions or as a carrier protein for
uptake and delivery of copper ions to intracellular targets. An
important piece of information that may shed light on the functional
interaction of PrPC and copper is how the metal affects the
cellular localization and trafficking of PrPC. We have
shown previously that PrPC is constitutively endocytosed
from the plasma membrane via clathrin-coated pits and transits an early
endosomal compartment before being recycled back to the cell surface
(25-28). We report here that copper has a dramatic effect on this cycle.
Reagents and Antibodies--
Cell culture reagents were from the
Tissue Culture Support Center at Washington University.
Phosphatidylinositol-specific phospholipase C (PIPLC) was prepared as
described previously (27). 125NaI was purchased from
Amersham Pharmacia Biotech. All other reagents were from Sigma.
Wild-type, Cell Lines--
N2a mouse neuroblastoma cells stably transfected
to express wild-type moPrP, wild-type chPrP, or N-terminally deleted
forms of chPrP ( Quantitation of Total Cell Surface PrP--
Cells were incubated
in Opti-MEM (Life Technologies, Inc.) for 90 min at 37 °C in the
presence or absence of CuSO4 and were then labeled for 20 min at 4 °C using 250 µg/ml sulfo-biotin-X-NHS (Calbiochem) in 20 mM HEPES, 150 mM NaCl (pH 7.2). Cell lysates were then subjected to SDS-PAGE, and blots of the gels were probed with
horseradish peroxidase-conjugated streptavidin and developed using ECL
(Amersham Pharmacia Biotech).
Endocytosis Assays--
Internalization of PrPC was
measured using surface iodination or biotinylation, as described
previously (25, 27). In the iodination assay, cells were incubated for
20 min on ice with 1 ml of phosphate-buffered saline containing 400 µg of glucose, 40 µg of lactoperoxidase, 20 µg of glucose
oxidase, and 100 µCi of 125NaI. After the reaction was
quenched with phosphate-buffered saline containing 1 mM
tyrosine and 10 mM sodium metabisulfite, cells were
incubated in Opti-MEM at 37 °C in the presence or absence of
CuSO4 and were then treated with PIPLC (1 unit/ml in
Opti-MEM) at 4 °C. PrP was immunoprecipitated from PIPLC incubation
media (surface) and cell lysates (internal), analyzed by SDS-PAGE, and quantitated using a PhosphorImager (Molecular Dynamics).
In the biotinylation assay, cells were labeled for 20 min at 4 °C
using 250 µg/ml sulfo-biotin-X-NHS in 20 mM HEPES, 150 mM NaCl (pH 7.2). After the reaction was quenched with 60 mM glycine in Opti-MEM, the cells were washed extensively
with phosphate-buffered saline and were then incubated in Opti-MEM at
37 °C in the presence or absence of CuSO4 for various
times. Cells were then treated with PIPLC (1 unit/ml in Opti-MEM) for
2 h at 4 °C, and PrP was immunoprecipitated from the PIPLC
incubation media and cell lysates. Immunoprecipitates were analyzed by
SDS-PAGE, and blots of the gels were probed with horseradish
peroxidase-conjugated streptavidin and developed using ECL. Films were
digitized using an HP ScanJet II scanner, and the bands were
quantitated using SigmaScan/Image (Jandel Scientific).
Reversibility and Recycling Assays--
One assay (see Fig. 3,
A and B) was designed to measure readjustment of
the steady-state distribution of PrP between the internal and external
pools after removal of copper. Surface-iodinated PrP was first allowed
to internalize for 20 min at 37 °C in the presence of 500 µM CuSO4. Cells were then washed and
incubated a second time at 37 °C in Opti-MEM with or without 500 µM CuSO4 for various lengths of time. Surface
and internal PrP was scored by PIPLC treatment as described above.
A second assay (see Fig. 3, C and D) was designed
to monitor the recycling of internalized PrP molecules back to the cell surface after removal of copper. Surface-iodinated PrP was allowed to
internalize for 20 min in the presence of 500 µM
CuSO4 as in the first assay. Surface PrP was then stripped
with PIPLC (2 h at 4 °C) prior to a second 37 °C incubation in
Opti-MEM containing PIPLC with or without 500 µM
CuSO4. Cells were then transferred to 4 °C and were
incubated for a further 2 h to ensure complete removal of surface
PrP. PrP was then immunoprecipitated from the medium and cell lysates
as above.
We have previously characterized the cellular trafficking of
PrPC using lines of N2a mouse neuroblastoma cells that have
been stably transfected to express either moPrP or chPrP (29, 25-27).
We therefore used these cells to examine the effects of copper on the
cellular distribution of PrPC. To determine whether copper
altered the total amount of PrPC on the cell surface, we
treated cells for 90 min with CuSO4 and then biotinylated
them with a membrane-impermeable reagent. The amount of biotinylated
PrP was then determined by immunoprecipitating PrP from cell lysates,
running the immunoprecipitates on SDS-PAGE, and developing blots of
the gel with horseradish peroxidase-streptavidin and ECL. We found that
at concentrations of 200 µM and above, CuSO4
dramatically reduced the amounts of both moPrP and chPrP on the cell
surface (Fig. 1). Similar results were
obtained when the amount of surface PrP was assayed by sensitivity to
externally applied trypsin (data not shown).
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
42-65, and 66-91 chicken PrP (chPrP) were recognized
with antiserum F35-96 (29); 25-91 chPrP was recognized with
antiserum F144-220 (29); and mouse PrP (moPrP) was recognized with
antiserum P45-66 (30).
25-91, 42-65, or 66-91) have been described
previously (27). Cells were maintained in minimal essential media
containing 10% fetal calf serum, nonessential amino acids,
penicillin/streptomycin, and 300 µg/ml of geneticin in an atmosphere
of 5% CO2/95% air.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (34K):
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Fig. 1.
Copper reduces the amount of PrP on the cell
surface. Transfected N2a cells expressing either chPrP or moPrP
were incubated in Opti-MEM containing the indicated amounts of added
CuSO4 for 90 min at 37 °C. The amount of surface PrP was
determined by biotinylation of the cells, immunoprecipitation of PrP,
and visualization of gel blots with horseradish peroxidase-streptavidin
and ECL.
Because PrPC in N2a cells is constitutively endocytosed from the plasma membrane (25, 26), we tested whether the effect of copper on the total amount of surface PrPC could be explained by a stimulatory effect on endocytosis. To measure endocytosis, we surface-iodinated cells at 4 °C, warmed them to 37 °C to initiate internalization, and then scored the amount of iodinated PrP that became inaccessible to externally applied phosphatidylinositol-specific phospholipase C (PIPLC), a bacterial enzyme that cleaves the glycolipid anchor responsible for attaching PrP to the membrane. Using this assay, we found that 500 µM CuSO4 rapidly stimulated internalization of both chPrP (Fig. 2, A and B) and moPrP (not shown). An increase in internalized PrP could be seen after as little as 5 min of incubation, and after 30 min 65% of the protein was internalized, compared with 15% in untreated cells. Stimulation of endocytosis could also be demonstrated using biotinylation rather than iodination to label surface PrP molecules. Using this procedure, the minimum concentration of CuSO4 required to produce an observable increase in internalization was ~100 µM (Fig. 2, C and D). At this concentration, ZnSO4 also stimulated endocytosis of chPrP, but no effect was seen with CoCl2, MnCl2, or FeSO4 at 500 µM (not shown). The effect of copper on PrP was not due to a generalized increased in endocytosis of surface proteins, because 500 µM CuSO4 did not significantly alter internalization of [125I]-transferrin (not shown).
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We found that the effect of copper was rapidly reversible. Within 15 min of removal of the metal, PrP began to redistribute from the inside to the outside of the cell, as assayed by accessibility to PIPLC (Fig. 3, A and B). By 60 min, a new steady-state was reached at which about 40% of the protein was internal, about the same percentage that is reached after endocytosis in the absence of copper (25). This readjustment of the steady-state distribution is presumably due to the previously documented ability of PrPC molecules to recycle back to surface of N2a cells after they are internalized (25). To directly measure this recycling process, we first incubated surface-iodinated cells for 20 min at 37 °C in the presence of 500 µM CuSO4 to stimulate internalization of PrP and then stripped surface PrP by treatment with PIPLC at 4 °C and finally re-warmed the cells in the presence of PIPLC to release recycled molecules into the medium. Using this procedure (Fig. 3, C and D), we found that when copper was absent from the re-warming medium, almost all of the previously internalized PrP recycled to the surface with a t1/2 of 10 min, consistent with the results of Fig. 3B. Interestingly, when copper was present in the re-warming medium, PrP also returned to the surface, although somewhat more slowly than in the absence of metal (t1/2 = 20 min). This result implies that although copper markedly stimulates the rate of PrP internalization, it has much less effect on the rate of recycling.
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Previous studies have implicated the N-terminal peptide repeats of both
mammalian and chicken PrP in copper binding (15, 16, 18, 19). We have
also shown that this region of the polypeptide chain is essential for
efficient endocytosis of PrP (27). We therefore tested the effect of
copper on endocytosis of N-terminally deleted forms of chPrP using a
surface biotinylation assay (Fig. 4).
chPrP normally contains eight hexapeptide repeats (31).
Deletion of the N-terminal one-third of the molecule (25-91), including all eight hexapeptide repeats, almost completely abolished the effect of copper, although the protein underwent some degradation during the course of the experiment (not shown). Deletion of the first
four repeats (42-65) or the last four repeats (66-91) attenuated but did not eliminate copper-induced internalization of PrP,
suggesting that the remaining repeats retain residual copper
coordinating activity.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Our previous studies have shown that PrPC is constitutively endocytosed from the cell surface via clathrin-coated pits and passes through an early endocytic compartment before being returned to the plasma membrane (29, 25-27). We demonstrate here that copper ions rapidly and reversibly stimulate the endocytic arm of this cycle.
It is attractive to speculate that this effect is attributable to binding of copper ions directly to PrPC, because purified, recombinant forms of PrP have been shown to bind copper ions, most likely via the peptide repeats (15, 16, 18, 19). Zinc also binds to PrPC, although less tightly than does copper (32). Consistent with a direct effect of copper on the internalization of PrPC, we find that N-terminally deleted forms of chPrP that are missing some or all of the peptide repeats are less efficiently endocytosed than the full-length protein in response to extracellular copper. One hypothesis to explain how binding of copper could stimulate endocytosis of PrPC is that the metal, by altering the conformation of PrPC, increases the affinity of the protein for a putative endocytic receptor that localizes PrPC in clathrin-coated pits. Because PrPC is attached to the cell surface by a glycosyl-phosphatidylinositol anchor, it lacks a cytoplasmic domain that could bind directly to the intracellular components of coated pits. We have therefore postulated the existence of a transmembrane receptor whose extracellular domain binds PrPC via its N-terminal region and whose cytoplasmic domain contains signals for interacting with adapter molecules and clathrin (27, 28).
Enhanced internalization of PrPC requires copper concentrations in the range of 100-500 µM, which is 10-50-fold greater than the estimated Kd for binding of copper to synthetic PrP peptides and recombinant PrP synthesized in bacteria (15, 16, 18, 19). This discrepancy may be due, in part, to binding of copper ions to other molecules such as amino acids that are present in the Opti-MEM medium used for our experiments, a phenomenon that would lower the concentration of free copper. It is also possible that the effect of copper on endocytosis occurs by binding to sites on cell-expressed PrPC that are distinct from the ones characterized on bacterially derived or synthetic PrP or to sites on other molecules involved in PrPC internalization. In any case, the average copper concentration in brain has been estimated to be ~100 µM and may be even higher in specific brain regions (19, 33). Therefore, the endocytic response we have described here is potentially relevant from a physiological standpoint.
One attractive hypothesis is that PrPC serves as a receptor
for uptake of copper ions from the extracellular milieu.
PrPC might bind copper ions on the cell surface and then
deliver them to an endocytic compartment within which the bound ions
dissociate from PrPC and are transferred to other
copper-carrier proteins that move the ions into the cytosol.
PrPC would then return to the cell surface to begin another
cycle. This proposed function for PrPC is analogous to that
of the transferrin receptor in uptake of iron (34), with the exception
that the metal ions bind directly to the receptor in the case of
PrPC rather than to a protein carrier comparable with
transferrin. Although plasma membrane copper transporters and
cytoplasmic copper chaperones have been identified in yeast and humans
(35), the molecular mechanism of copper uptake by mammalian cells is
still poorly understood (36). Further exploration of the interaction between PrPC and copper ions at the cellular level may shed
important light on this subject.
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ACKNOWLEDGEMENTS |
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We thank Jonathan Gitlin for helpful discussions of copper metabolism and members of the Harris laboratory for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Grants AG12925 and AG16462 from the National Institutes of Health (to D. A. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by Postdoctoral Training Grant 5T32NS07071 and NRSA
Award 1F32NS10467, both from the National Institutes of Health, and by
a fellowship from the McDonnell Center for Cellular and Molecular
Neurobiology at Washington University.
§ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-4690; Fax: 314-362-7463; E-mail: dharris{at}cellbio.wustl.edu.
The abbreviations used are: PrPC, cellular isoform of the prion protein; PrPSc, scrapie isoform of the prion protein; PrP, prion protein; chPrP, chicken prion protein; moPrP, mouse prion protein; PAGE, polyacrylamide gel electrophoresis; PIPLC, phosphatidylinositol-specific phospholipase C; SOD, superoxide dismutase.
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REFERENCES |
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References
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lanes) of
500 µM CuSO4. At the end of the chase period,
cells were treated with PIPLC, and chPrP was immunoprecipitated from
PIPLC incubation media (surface, S lanes) and cell lysates
(internal, I lanes), followed by analysis by SDS-PAGE and
autoradiography. B, the amount of internal PrP was expressed
as a percentage of the total amount of PrP (surface + internal) at each
chase time. C, the experiment was the same as that described
for panel A, except that surface PrP was stripped with PIPLC
after the initial 20-min incubation with CuSO4, and PIPLC
was included in the chase medium during the second 37 °C incubation
to recover internalized molecules of PrP that returned to the cell
surface. D, the amount of internal PrP was expressed as a
percentage of the total amount of PrP (surface + internal) at each
chase time. The t1/2 for return of internalized PrP
to the cell surface is probably overestimated, particularly in the
presence of copper, because PIPLC may not have sufficient time to
cleave recycled molecules before they are re-internalized. The
experiments shown in this figure are representative of at least three
similar ones.
