©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The N-terminal Domain of a Glycolipid-anchored Prion Protein Is Essential for Its Endocytosis via Clathrin-coated Pits (*)

Show-Ling Shyng (§) , Krista L. Moulder , Alex Lesko , David A. Harris (¶)

From the (1)Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cellular prion protein (PrP) is a glycolipid-anchored protein that is involved in the pathogenesis of fatal spongiform encephalopathies. We have shown previously that, in contrast to several other glycolipid-anchored proteins, chPrP, the chicken homologue of mammalian PrP, is endocytosed via clathrin-coated pits in cultured neuroblastoma cells, as well as in embryonic neurons and glia (Shyng, S.-L., Heuser, J. E., and Harris, D. A.(1994) J. Cell Biol. 125, 1239-1250). In this study, we have determined that the N-terminal half of the chPrP polypeptide chain is essential for its endocytosis. Deletions within this region reduce the amount of chPrP internalized, as measured by surface iodination or biotinylation, and decrease its concentration in clathrin-coated pits, as determined by quantitative electron microscopic immunogold labeling. Mouse PrP, as well as two mouse PrP/chPrP chimeras, are internalized as efficiently as chPrP, suggesting that conserved features of secondary and tertiary structure are involved in interaction with the endocytic machinery. Our results indicate that the ectodomain of a protein can contain endocytic targeting information, and they strongly support a model in which the polypeptide chain of PrP binds to the extracellular domain of a transmembrane protein that contains a coated pit localization signal in its cytoplasmic tail.


INTRODUCTION

A number of important transmembrane receptors undergo endocytosis in clathrin-coated pits. Recruitment of receptors into coated pits depends on specific amino acid motifs in receptor cytoplasmic tails that bind clathrin-associated adaptor molecules on the inner surface of the plasma membrane (reviewed by Trowbridge et al.(1993)). These short amino acid motifs often include a tyrosine residue and are thought to adopt a -turn conformation.

A diverse group of proteins is attached to the plasma membrane by a phosphatidylinositol-containing glycolipid (GPI)() anchor (reviewed by Englund(1993)). This group includes lymphocyte and trypanosome surface antigens, adhesion molecules, exofacial enzymes, and receptors. Endocytic targeting of these proteins has been a subject of considerable debate, since they lack cytoplasmic domains that could interact directly with intracellular adaptors and clathrin. Some GPI-anchored proteins are not endocytosed at all, and some are internalized via pathways that do not involve clathrin-coated pits (Bretscher et al., 1980; Lemansky et al., 1990; Keller et al., 1992). The vitamin folic acid, for example, is internalized by binding to a glycolipid-anchored receptor that is concentrated in non-clathrin-coated invaginations of the plasmalemma called caveolae (Rothberg et al., 1990; Anderson et al., 1992).

We have been investigating the cellular trafficking of PrP, a glycolipid-anchored surface protein that is expressed by neurons, glia, and several peripheral cell types. Although the physiological function of PrP is unclear, the protein is known to be intimately involved in the pathogenesis of an unusual group of transmissible neurodegenerative diseases called spongiform encephalopathies or prion diseases (reviewed by Prusiner and DeArmond (1994)). This group includes Creutzfeldt-Jakob disease, kuru, Gerstmann-Sträussler syndrome, and fatal familial insomnia in humans, and scrapie in animals. The infectious agent responsible for these fatal disorders has been called a prion, and it appears to consist principally of PrP, a posttranslationally modified isoform of PrP. Conversion of PrP to PrP during prion replication appears to take place, at least in part, along an endocytic pathway in scrapie-infected neuroblastoma cells (Caughey et al., 1991; Borchelt et al., 1992).

We found previously that chPrP, the chicken homologue of mammalian PrP, is endocytosed via clathrin-coated pits in neuroblastoma cells, and in embryonic neurons and glia (Shyng et al., 1993, 1994). This surprising result raises the question of what molecular mechanism accounts for the localization of PrP in coated pits. To define the structural features of the PrP molecule that target it to coated pits, we have expressed deleted and chimeric forms of the protein in neuroblastoma cells. Our results indicate that the N-terminal half of the PrP polypeptide chain is essential for efficient endocytosis assayed biochemically, and for localization to coated pits analyzed by EM immunogold labeling. These data are consistent with a model whereby the polypeptide chain of PrP binds to the extracellular domain of a transmembrane protein that itself contains a coated pit localization signal (Harris et al., 1993a).


MATERIALS AND METHODS

Antibodies and Reagents

A rabbit antiserum raised against a bacterial fusion protein encompassing amino acids 35-96 of chPrP (Harris et al., 1993b) was used to recognize wild-type chPrP, 25-41, 25-65, 42-65, 66-91, 117-135, and moPrP(1-39/chPrP(42-267). A rabbit antiserum raised against a bacterial fusion protein encompassing amino acids 144-220 of chPrP (Harris et al., 1993b) was used to recognize 25-91 and moPrP(1-88)/chPrP(92-267). A new rabbit antiserum raised against a synthetic peptide encompassing amino acids 45-66 of moPrP was used to recognize wild-type moPrP. A rabbit anti-human decay-accelerating factor (DAF) serum (used for immunoprecipitations), and a mouse anti-human DAF monoclonal antibody (1H4) (used for immunogold labeling) were kindly provided by Dr. Doug Lublin (Coyne et al., 1992).

Cell culture reagents were from the Tissue Culture Support Center at Washington University. Gold-conjugated secondary antibodies were from Jackson ImmunoResearch. Osmium tetroxide, uranyl acetate, and Polybed 812 were from Polysciences, Inc. (Niles, IL).

Phosphatidylinositol-specific phospholipase C (PI-PLC) was prepared by a modification of the procedure of Low(1992). Supernatants (500 ml) from cultures of Bacillus subtilis carrying a plasmid encoding PI-PLC from Bacillus thuringiensis were concentrated 20-fold in an Amicon device and dialyzed against 50 mM Tris acetate (pH 7.4). The dialysate was then subjected to gel filtration in the same buffer using two columns (1.5 cm 120 cm) of Sephadex G-75 connected in series, and fractions containing PI-PLC activity were pooled and concentrated. The resulting preparations had a specific activity of 150 units/mg and had no detectable protease activity. Cells were treated at 4 °C with PI-PLC at a concentration of 1 unit/ml in Opti-MEM (Life Technologies).

DNA Expression Constructs

All mutant and chimeric constructs were created by recombinant PCR (Higuchi, 1989) using Vent® polymerase (New England Biolabs) and were sequenced in their entirety. The templates for primary PCR encoded chPrP (Harris et al., 1991), moPrP (Prn-p allele; Westaway et al., 1987), and human DAF (Lublin and Atkinson, 1989). Primers for secondary PCR contained a HindIII site at the 5` end and a BamHI site at the 3` end, each immediately adjacent to the coding region. Constructs were cloned into the expression vector pBC12/CMV (Cullen et al., 1986) after cleavage with HindIII and BamHI.

Transfected Cell Lines

N2a mouse neuroblastoma cells (ATCC CCL131) were grown in minimal essential medium (MEM) containing 10% fetal calf serum, nonessential amino acids, and penicillin/streptomycin in an atmosphere of 5% CO, 95% air. Cells were transfected with a 10:1 mixture of the pBC12/CMV expression plasmid and pRSVneo (Ulrich and Ley, 1990), using Lipofectin (Life Technologies, Inc.) according to the manufacturer's directions. Antibiotic-resistant clones were selected in 700 µg/ml Geneticin (G418), expanded, and then maintained in 300 µg/ml Geneticin. The line expressing wild-type chPrP is the clone designated A26 that we have described previously (Harris et al., 1993b). Even though N2a cells express endogenous moPrP, stably transfected lines were prepared that expressed increased levels of the wild-type protein.

Metabolic Labeling

Confluent cultures of N2a cells were labeled for 5 h with [S]methionine (ICN TranS-label, 250 µCi/ml, 1,000 Ci/mmol) in serum-free MEM lacking methionine. Cell lysates were treated with 0.01 units/ml N-glycosidase F (Boehringer Mannheim) at 37 °C overnight to remove N-linked oligosaccharides, and PrP immunoprecipitated as described previously (Harris et al., 1993b).

PrP Internalization Assays

Internalization of cell-surface PrP was measured by surface iodination as described previously (Shyng et al., 1993). Briefly, cells were incubated on ice with phosphate-buffered saline containing glucose, lactoperoxidase, glucose oxidase, and NaI for 20 min, and the reaction quenched with 1 mM tyrosine and 10 mM sodium metabisulfite. After warming to 37 °C for different times to allow internalization, cells were treated with PI-PLC for 2 h at 4 °C prior to lysis. PrP in the PI-PLC incubation media (surface) and cell lysates (internal) was immunoprecipitated (Harris et al., 1993b), and quantitated by imaging of SDS-PAGE gels using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Internalization was expressed as the percentage of the total amount of PrP from each dish that was present in the cell lysate and was corrected for PI-PLC digestion efficiency (>80% for plates held at 0 °C).

Internalization was also quantitated by surface biotinylation. Cells were incubated on ice for 10 min in 250 µg/ml sulfo-biotin-X-NHS (Calbiochem) in 20 mM HEPES, 150 mM NaCl (pH 7.2). The reaction was quenched by addition of 20 mM glycine in MEM, and the cells rinsed three times in phosphate-buffered saline. After warming to 37 °C in Opti-MEM for various times, cells were treated with PI-PLC, and PrP immunoprecipitated from PI-PLC incubation media and cell lysates, as described above. Immunoprecipitates were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membrane. Blots were then developed with horseradish peroxidase-streptavidin and visualized using enhanced chemiluminescence (Amersham Corp.). Films were digitized using an HP ScanJet IIp scanner, and images analyzed using SigmaScan/Image (Jandel Scientific).

To test the effects of hypertonic treatment, cells were preincubated for 30 min at 37 °C in Opti-MEM containing 0.45 M sucrose, and 0.45 M sucrose was also included during the biotinylation and warm-up.

EM Immunogold Labeling

Labeling and morphometric analysis were carried out exactly as described by Shyng et al. (1994).


RESULTS

We have shown previously that chPrP constitutively cycles between the cell surface and an endosomal compartment in N2a cells, and that endocytosis of this glycolipid-anchored protein is mediated by clathrin-coated pits (Shyng et al., 1993, 1994). During each cycle through the cell, a portion of the molecules are proteolytically cleaved within a highly conserved segment of 24 amino acids near the center of the polypeptide chain (Harris et al., 1993b). Intact molecules, as well as membrane-anchored C-terminal fragments, are then returned to the cell surface. We noticed that, at steady state, the C-terminal fragment is present almost exclusively on the cell surface, in contrast to the full-length protein, about half of which is intracellular. This observation suggested that the C-terminal fragment might be internalized less efficiently than the full-length protein, and that sequences in the N-terminal half of the protein might be important for endocytic targeting.

To test this idea, and to further define the regions of chPrP that are essential for efficient endocytosis, we constructed a series of mutant proteins containing N-terminal deletions, as well as two chimeric proteins consisting of N-terminal segments of mouse PrP (moPrP) fused to C-terminal portions of chPrP (Fig. 1). These proteins were expressed in stably transfected clones of N2a mouse neuroblastoma cells. Metabolic labeling of each transfected line revealed a protein of the appropriate molecular weight that was specifically immunoprecipitated by an anti-PrP antibody (Fig. 2). Each of the proteins was attached to the plasma membrane by a GPI anchor, as demonstrated by the fact that >80% of the surface-labeled protein could be released from cells maintained at 0 °C by incubation with bacterial PI-PLC (see 0-min time points in Figs. 3, 4, 6, and 7). Endocytosis was quantitated by iodinating or biotinylating cells at 0 °C, warming them to 37 °C to initiate internalization, and then using PI-PLC to separate molecules on the cell surface from those that were intracellular.


Figure 1: PrP constructs used in this study. The wild-type and six deletion () mutants are derived from chPrP. In the two moPrP/chPrP chimeras, the narrow segments of the schematic represent moPrP and the wide segments chPrP. Numbers refer to amino acid residues. Note that chPrP contains eight hexapeptide repeats, and moPrP five octapeptide repeats rich in proline and glycine. In the mature proteins, the GPI anchor is attached at the position indicated by the arrowhead in the schematic.




Figure 2: Stably transfected lines of N2a cells express mutant PrPs. Cells were metabolically labeled with [S]methionine for 5 h, after which PrP was immunoprecipitated from cell lysates, using chPrP-specific antibodies (see ``Materials and Methods''), and subjected to SDS-PAGE and fluorography. The specific PrP bands are indicated by arrows and brackets next to each lane; immunoprecipitation of these bands is blocked by preincubation of antibodies with immunogen (not shown). Each of the samples was enzymatically deglycosylated prior to immunoprecipitation to facilitate molecular weight comparisons. chPrP sometimes migrates as a doublet under these conditions (lanes with brackets), due either to incomplete deglycosylation, or to the presence of modifications other than N-linked oligosaccharides (Shyng et al., 1993). Molecular size markers are given in kilodaltons. Wild-type chPrP has a molecular mass of 34 kDa, and the deletion mutants have correspondingly lower masses: 25-41, 32.5 kDa; 25-65, 29 kDa; 25-91, 27.5 kDa; 42-65, 31.5 kDa; 66-91, 31.5 kDa; 117-135, 32.5 kDa. The two moPrP/chPrP chimeras migrate at 32.5 kDa.



N-terminal Deletions of chPrP Reduce Internalization

The C-terminal fragment of chPrP generated by posttranslational cleavage begins near Lys (Harris et al., 1993b), suggesting that the 92 amino acids between this site and the end of the signal peptide may be important in mediating internalization. To analyze this region in more detail, we constructed three deletion mutants that were missing 17, 41, or 67 amino acids following the signal peptide. These deletions include 0, 4, or all 8 of the proline/glycine-rich hexapeptide repeats. As shown previously (Shyng et al., 1993), approximately 40-50% of wild-type chPrP is internalized at 30 min following surface iodination (Fig. 3). The three deletion mutants were each internalized less efficiently than the wild-type protein, with longer deletions producing a greater effect: the amount of iodinated protein endocytosed was 25.9%, 12.4%, and 6.3%, respectively, for 25-41, 25-65, and 25-91. Since 5% of the C-terminal fragment generated posttranslationally is internalized following surface iodination (data not shown), removal of additional amino acids between positions 92 and 116 has little further effect on endocytosis.


Figure 3: N-terminal deletions progressively reduce the amount of chPrP internalized by N2a cells; DAF is not internalized. Panel A, N2a cells expressing each protein were surface-iodinated at 0 °C, and then warmed to 37 °C for 0 min or 30 min. Cells were then treated with PI-PLC for 2 h at 0 °C prior to lysis. PrP or DAF in the PI-PLC incubation media (lanes marked E, for external) and cell lysates (lanes marked I, for internal) was immunoprecipitated and subjected to SDS-PAGE. The portions of the autoradiograms containing the chPrP and DAF bands are shown. In this and subsequent figures, PI-PLC digestion efficiency (the amount of protein released by the enzyme from cells maintained at 0 °C) was >80%. The slight decrease from 0 to 30 min in the total amount of DAF was not consistent and probably resulted from a small difference in the efficiency of iodination between the two dishes. Panel B, the amount of protein internalized at 30 min was quantitated by PhosphorImager analysis of the gels. Each bar shows the average and S.E. of values from 4 dishes.



As a negative control, we also analyzed N2a cells that had been transfected to express human DAF, a GPI-anchored protein that is unrelated in sequence to chPrP. We found that <1% of DAF was internalized at 30 min (Fig. 3), arguing that the glycolipid anchor itself is not sufficient to mediate efficient endocytic uptake, and that distinctive features of the chPrP polypeptide chain are critical for this process.

We chose a 30-min time point to assess internalization because we had previously shown that the distribution of wild-type chPrP after surface iodination approaches a steady state with a t of approximately 10 min (Shyng et al., 1993). The existence of a steady state results from recycling of internalized molecules back to the cell surface. To see if the mutant forms of chPrP behaved similarly, we analyzed the time course of their internalization (Fig. 4). We found that the 25-41 and 25-91 proteins attained a steady state distribution, which was maintained for the duration of each experiment (>90 min). By fitting the kinetic data to an exponential curve, we calculated that the steady state value for the amount of internalized protein was 25% for 25-41 and 7% for 25-91, compared to 50% for the wild-type protein (Shyng et al., 1993). Surprisingly, although the steady state amounts of internalized PrP were lower for the two mutants than for the wild-type protein, the t for approach to the steady state was similar for all forms of the protein (6.6 and 11.5 min for the two mutants, and 10 min for the wild-type protein). These results indicate that the deletions alter the final extent of internalization, without significantly affecting the rate at which a stable distribution is achieved.


Figure 4: Internalization of the 25-41 and 25-91 mutants approaches a steady state. Upper panels, N2a cells expressing each protein were surface-iodinated at 0 °C and then warmed to 37 °C for the indicated periods of time. Cells were then treated with PI-PLC for 2 h at 0 °C prior to lysis. chPrP in the PI-PLC incubation media (Surface) and cell lysates (Internal) was immunoprecipitated and subjected to SDS-PAGE and autoradiography. The autoradiogram for the 25-91 mutant is overexposed to better visualize the small amount of internalized protein. Lower panels, the amounts of surface and internal chPrP at each time point were quantitated by PhosphorImager analysis of the gels. Each point represents the value from a single dish. Curves were fit by least squares analysis to the following equations: 25-41 internal, 0.25(1 - e); 25-41 surface, 0.75 + 0.25e; 25-91 internal, 0.07(1 - e); 25-91 surface, 0.93 + 0.07e. These equations assume steady state values of 0.25 and 0.07 for the fraction of internalized 25-41 and 25-91, respectively. The calculated t values for approach to the steady state are 6.6 and 11.5 min for the two mutants. These values do not differ markedly from the t of 10 min calculated for wild-type chPrP (Shyng et al., 1993).



N-terminal Deletions Reduce the Concentration of chPrP in Coated Pits

We carried out quantitative EM immunogold labeling to analyze the localization of the deleted proteins at the ultrastructural level. As reported previously (Shyng et al., 1994), wild-type chPrP was approximately 4 times more concentrated in coated pits than over other areas of the plasma membrane (). In contrast, 25-41 was only 1.7 times more concentrated in coated pits, and 25-91, as well as DAF, were barely concentrated in these structures at all. Fig. 5shows representative micrographs illustrating the localization of wild-type and 25-91 chPrP, as well as DAF. These results demonstrate a correlation between the efficiency of internalization measured biochemically and the degree of concentration in clathrin-coated pits determined morphologically.


Figure 5: 25-91 chPrP and DAF are less concentrated in clathrin-coated pits than wild-type chPrP. N2a cells expressing wild-type chPrP (A), 25-91 chPrP (B), or DAF (C) were fixed with paraformaldehyde and incubated with either rabbit anti-chPrP or mouse anti-DAF antibodies. Cells were then processed for EM immunogold detection using goat anti-rabbit antibodies conjugated to 12 nm gold particles (A and B) or goat anti-mouse antibodies coupled to 6 nm gold particles (C). For wild-type chPrP, gold particles (arrowheads) are clustered around clathrin-coated pits (arrows), as well as over undifferentiated areas of plasma membrane. In contrast, for 25-91 chPrP and DAF, gold particles are almost never found near coated pits. Scale bar = 0.1 µm. Morphometric quantitation of these data is presented in Table I.



Hexapeptide Repeats and the Conserved Region Are Essential for Internalization

Our results indicated that sequences within the N-terminal half of the chPrP polypeptide chain are essential for normal endocytic targeting. To identify which motifs within this region are important, we constructed three additional deletions. We first focused on the eight hexapeptide repeats (consensus HNPGYP) found between residues 42 and 89. Mutants lacking either the first four (42-65) or last four (66-91) of these repeats were internalized with less than half the efficiency of wild-type chPrP (Fig. 6), indicating that both halves of the repeat region are essential, and suggesting that normal endocytosis is likely to require all eight repeats. This conclusion is consistent with the fact that internalization of the 25-91 mutant, which is missing all eight repeats, is more severely impaired than that of the 25-65 mutant, which is missing the first four repeats (Fig. 3).


Figure 6: Deletions of the hexapeptide repeats or the conserved region reduce internalization of chPrP. PanelA, N2a cells expressing each protein were surface-iodinated at 0 °C and then warmed to 37 °C for 0 or 30 min. Cells were then treated with PI-PLC for 2 h at 0 °C prior to lysis. chPrP in the PI-PLC incubation media (lanes marked E, for external) and cell lysates (lanes marked I, for internal) was immunoprecipitated and subjected to SDS-PAGE. The portions of the autoradiograms containing the chPrP bands are shown. Panel B, the amount of protein internalized at 30 min was quantitated by PhosphorImager analysis of the gels. Each bar shows the average and S.E. of values from 4 dishes.



Farther along the chPrP polypeptide chain is a segment of 24 largely hydrophobic amino acids (residues 112-135), which is identical in chPrP and mouse PrP (Prn-p allele) and is highly conserved in all known PrP species. We found that this conserved region was also essential for normal internalization, since a mutant protein (117-135) in which most of the region was deleted exhibited significantly impaired endocytosis (Fig. 6).

It was not possible to analyze the effect on internalization of a deletion (161-180) in the C-terminal half of the chPrP molecule because this protein was not expressed on the cell surface, but was instead retained intracellularly, as judged by lack of surface immunofluorescence staining and insensitivity to release by PI-PLC (data not shown). It is possible that C-terminal deletions interfere with N-glycosylation or disulfide bond formation, which involve residues in this half of the molecule.

MoPrP and moPrP/chPrP Chimeric Proteins Are Efficiently Internalized

Our data indicated that endocytosis of chPrP depends on distinctive features of its polypeptide chain which are not present in unrelated glycolipid-anchored proteins such as DAF. To further investigate the sequence specificity of these targeting signals, we analyzed internalization of a homologous mammalian PrP from mouse. Although moPrP and chPrP have an overall amino acid sequence identity of only 33%, they share common structural motifs, including proline- and glycine-rich peptide repeats, as well as the conserved central segment. They are therefore likely to adopt similar secondary and tertiary structures (Huang et al., 1994).

We found that moPrP, like chPrP, is internalized after surface iodination, and that the distribution of the protein reaches a steady state, with approximately half on the cell surface and half intracellular (data not shown); the rate for approach to this steady state is similar for the mouse and chicken proteins (t 10 min).

We have also analyzed internalization of both moPrP and chPrP using an assay that involves biotinylation of intact cells with a membrane impermeant reagent (Fig. 7). Inefficient internalization of deleted forms of chPrP is observed in the biotinylation assay (data not shown), as it is in the iodination assay, although the absolute amount of chPrP endocytosed is approximately 50% greater after biotinylation than after iodination, an effect resulting from slower recycling of biotinylated PrP to the cell surface (compare wild-type chPrP in Fig. 3and Fig. 7). Fig. 7shows that internalization of moPrP, like chPrP (Shyng et al., 1994), is inhibited by incubation of cells in hypertonic sucrose, a treatment that disrupts clathrin lattices, suggesting that endocytosis of the murine protein is also mediated by clathrin-coated pits. We have also found that, following endocytic uptake, moPrP is proteolytically cleaved within the conserved central region (Lehmann et al., 1994), a result similar to that obtained for chPrP.


Figure 7: MoPrP and moPrP/chPrP chimeric proteins are efficiently internalized. PanelA, N2a cells expressing each protein were surface-biotinylated at 0 °C and then warmed to 37 °C for 0 or 30 min. Cells were then treated with PI-PLC for 2 h at 0 °C prior to lysis. chPrP in the PI-PLC incubation media (lanes marked E, for external) and cell lysates (lanes marked I, for internal) was immunoprecipitated. Immunoprecipitates were separated by SDS-PAGE, blotted, and visualized with horseradish peroxidase-streptavidin and enhanced chemiluminescence. The portions of the films containing the PrP bands are shown. In one set of experiments, cells expressing wild-type moPrP were preincubated, biotinylated, and warmed in the presence of 0.45 M sucrose, to test the involvement of clathrin-coated pits in endocytosis. Panel B, the amount of protein internalized at 30 min was quantitated by densitometric scanning of films. Each bar shows the average and S.E. of values from 4 dishes.



Two chimeric proteins, consisting of N-terminal segments of moPrP fused to C-terminal segments of chPrP, were as efficiently internalized as the wild-type chicken and mouse proteins (Fig. 7). The moPrP(1-39)/chPrP(42-267) construct includes 17 amino acids of moPrP between the signal peptide and the octapeptide repeat region, a segment that is 59% identical to the corresponding region of chPrP. The moPrP(1-88)/chPrP(92-267) construct includes 66 amino acids of moPrP beyond the signal peptide, a segment that contains all five of the octapeptide repeats, and that is only 27% identical to the corresponding segment of chPrP. These results indicate that, although the N-terminal primary amino acid sequences of moPrP and chPrP are quite divergent, the features responsible for efficient internalization are conserved.


DISCUSSION

We have reported previously that chPrP, in contrast to several other GPI-anchored proteins, is endocytosed via clathrin-coated pits in neuroblastoma cells, as well as in glia and neurons from chicken brain (Shyng et al., 1994). In the present study, we have investigated the structural features of the chPrP molecule that target it for coated pit-mediated endocytosis. Our results indicate that sequences in the N-terminal half of the chPrP polypeptide chain are essential for this process. Deletions within the first 111 amino acids following the signal sequence reduce internalization of chPrP as measured by surface iodination and biotinylation. They also decrease the concentration of the protein in coated pits, as determined by quantitative EM immunogold labeling. The correlation we observed between the internalization of the mutant proteins and their concentration in coated pits further strengthens the conclusion that coated pits represent the primary pathway for endocytosis of chPrP. Since the chPrP polypeptide chain is entirely extracellular, our results sharply distinguish chPrP from transmembrane receptors such as those for transferrin and low density lipoprotein, whose cytoplasmic domains contain coated pit targeting information (Trowbridge et al., 1993). In this way, chPrP, along with other PrPs, define a novel mechanism for endocytic uptake.

Endocytosis of chPrP does not depend on a single, short segment of amino acids, as is the case for internalization of transmembrane receptors, which display tyrosine- or dileucine-based motifs encompassing 4-5 residues (Trowbridge et al., 1993). Progressive N-terminal deletions of chPrP, ranging from 17 to 67 amino acids, cause graded reductions of internalization and concentration in coated pits, with no indication that any single region plays a dominant role. In addition, two deletions within the hexapeptide repeat region, and one within the conserved central segment are all about equally effective in reducing endocytosis.

One possible explanation for the apparent absence of a dominant targeting signal is that each of the deletions alters the overall folding pattern of the chPrP polypeptide chain, and thereby disrupts structural features that are important for endocytosis. Gross alterations in protein folding seem unlikely, since each of the mutants was efficiently transported to the cell surface, in contrast to aberrantly folded proteins, which often accumulate in the endoplasmic reticulum and are degraded after synthesis (Bonifacino and Lippincott-Schwartz, 1991). An alternative explanation is that multiple regions within the N-terminal half of the molecule are involved in interactions with the endocytic machinery. This might be the case, for example, if chPrP bound to several different components of coated pits, or if one of these components bound to several different sites on the chPrP molecule (see below).

Whatever features of the polypeptide chain are important for endocytosis, they are likely to be specific for PrP. DAF, an unrelated GPI-anchored protein, is not internalized when expressed in N2a cells and is not concentrated in coated pits. Several other glycolipid-anchored proteins in other cell types are also not clustered in coated pits (Bretscher et al., 1980; Rothberg et al., 1990; Keller et al., 1992).

The fact that moPrP, as well as two moPrP/chPrP chimeras, are internalized as efficiently as chPrP in N2a cells suggests that secondary and/or tertiary structural features are more important than primary sequence in endocytic targeting. Although the avian and mammalian proteins are only 33% identical in amino acid sequence, recent spectroscopic and computer modeling studies predict that they are likely to adopt the same conformation, including four -helices in the C-terminal half that may be arranged in the form of an X-bundle (Gasset et al., 1992; Pan et al., 1993; Huang et al., 1994). In the N-terminal half of the protein, the octapeptide repeat unit in the murine protein (consensus PHGG(G/S)WGQ) is chemically similar to the hexapeptide unit in the chicken protein (consensus HNPGYP), since both are rich in proline and glycine and both contain an aromatic amino acid. Although conformational studies of this part of the PrP molecule have not been carried out, standard secondary structural algorithms predict that the peptide repeat region will form a series of -turns in both proteins (Bazan et al., 1987), and these could play a role in endocytic targeting.

It is interesting that the tyrosine-containing motifs essential for coated pit-mediated endocytosis of transmembrane receptors have also been found to adopt a -turn configuration (Collawn et al., 1990; Bansal and Gierasch, 1991; Eberle et al., 1991). Although the PrP repeats are extracellular, and the receptor tyrosine-containing motifs are cytoplasmic, the -turn might represent a common structural element involved in binding to other membrane-associated molecules.

Although our results highlight an important role for the polypeptide chain of PrP in endocytic targeting, they do not rule out the possibility that the GPI anchor might also be involved. All of our mutant PrP constructs are attached to the cell surface by a GPI anchor, indicating that the anchor itself is not sufficient for normal internalization. It is conceivable, however, that the anchor might assist in endocytic uptake, for example by increasing the lateral mobility of PrP and so facilitating its delivery to clathrin-coated pits. Investigation of the role of the GPI anchor will require analysis of transmembrane versions of PrP.

To explain the localization of chPrP in coated pits, we have previously proposed that its polypeptide chain binds, either directly or indirectly, to the extracellular domain of a transmembrane protein that contains a conventional coated pit targeting signal in its cytoplasmic domain (Shyng et al., 1994). The results reported here strongly support this model, since deletions in the polypeptide chain of chPrP would be expected to decrease its affinity for the transmembrane protein, and thereby reduce the efficiency of endocytosis. Since the mutations we have analyzed have all been in the N-terminal half of the protein, we would predict this region to be involved in contacting the transmembrane protein, although a role for more C-terminal regions cannot be ruled out. We are currently employing a variety of biochemical techniques to directly demonstrate the existence of a PrP binding protein in coated pits.

We observed that although wild-type and mutant chPrPs were internalized to different extents, the rate at which a steady state distribution was reached was approximately 10 min for all forms of the protein (Fig. 4). This effect might be understood in terms of our model if it is assumed that the rate of internalization of chPrP depends only on the rate at which the transmembrane binding protein is internalized, while the steady state distribution depends on the affinity of chPrP for the binding protein. If mutated chPrP molecules had reduced affinity, then a smaller proportion would be bound to the transmembrane protein, although those that were bound would be internalized at the normal rate.

The model outlined here is related to one that has been proposed to explain the function of the glycolipid-anchored receptor for urokinase-type plasminogen activator (uPA) (Nykjaer et al., 1992; Blasi et al., 1994). This receptor facilitates coated pit-mediated internalization of uPA bound to its inhibitor (PAI-1) by ``presenting'' the uPA-PAI-1 complex to the low density lipoprotein receptor-related protein, a large transmembrane protein with tyrosine-based localization signals in its cytoplasmic domain. Whether the uPA receptor itself binds to lipoprotein receptor-related protein, and whether it is internalized along with the ligand, is unclear. If so, then this mechanism would be analogous to the one we have proposed here for endocytosis of PrP. Ligand-induced internalization of the glycolipid-anchored receptor for ciliary neurotrophic factor might involve a similar process (Curtis and DiStefano, 1994). This receptor forms a signal-transducing complex with two transmembrane proteins LIF and gp130, the second of which has been shown to contain a di-leucine motif in its cytoplasmic domain that can mediate endocytic uptake (Dittrich et al., 1994). Consideration of these two examples prompts the speculation that binding of an extracellular ligand could play a role in the internalization of PrP.

The results reported here have implications for understanding the physiological as well as the pathological properties of PrP. The normal function of PrP is unknown, although recent electrophysiological studies of mice in which the PrP gene has been deleted suggest a role in synaptic transmission within the central nervous system (Collinge et al., 1994). The identification of molecules in coated pits with which PrP interacts is likely to provide additional clues to the cellular function of this isoform. These putative binding molecules may also serve as cell surface receptors for PrP or play some other role in the conversion of PrP to PrP, a process that has been found to take place in part along an endocytic pathway (Caughey et al., 1991; Borchelt et al., 1992). It is noteworthy that expansion of the octapeptide repeat region of human PrP is associated with familial forms of Creutzfeldt-Jakob disease (Poulter et al., 1992). Since we have found that deletions within this region interfere with endocytosis, it is possible that the expanded human PrPs are also internalized abnormally and that this feature plays a role in the pathogenic process. Finally, our observation that the PrP polypeptide chain is essential for efficient endocytosis may allow the design of peptide-based ligands that competitively block internalization of PrP and thereby inhibit prion replication.

  
Table: Quantitative analysis of immunogold labeling experiments in neuroblastoma cells transfected with various proteins

Neuroblastoma cells transfected with various proteins were incubated with primary antibodies and gold-conjugated secondary antibodies (see ``Materials and Methods'') after fixation in 4% paraformaldehyde and were processed for electron microscopy.



FOOTNOTES

*
This work was supported in part by grants (to D. A. H.) from the National Institutes of Health (Grant NS30137), the Alzheimer's Association (Richard F. Bristor Investigator-Initiated Research Grant), the March of Dimes Birth Defects Foundation (Basic Research Grant 0571), and the McDonnell Center for Cellular and Molecular Neurobiology at Washington University. Portions of this paper have been presented in abstract form (Harris et al., 1993a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of fellowship support from the McDonnell Center for Cellular and Molecular Neurobiology, and from National Institutes of Health Postdoctoral Training Grant T32 NS07071.

To whom all correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-4690; Fax: 314-362-7463.

The abbreviations used are: GPI, glycosyl phosphatidylinositol; PrP, cellular isoform of the prion protein; PrP, scrapie isoform of the prion protein; chPrP, chicken prion protein; moPrP, mouse prion protein; PI-PLC, phosphatidylinositol-specific phospholipase C; DAF, decay-accelerating factor; PCR, polymerase chain reaction; MEM, minimal essential medium; PAGE, polyacrylamide gel electrophoresis; uPA, urokinase-type plasminogen activator.


ACKNOWLEDGEMENTS

We thank Marilyn Levy and Lori LaRose for technical assistance with immunogold labeling, Doug Lublin for informative discussions, as well as for DAF antibodies and cDNAs, and Alex Gorodinsky and Sylvain Lehmann for comments on the manuscript and help with computer graphics.


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