©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Sorting Signals in the -Amyloid Precursor Protein Cytoplasmic Domain (*)

(Received for publication, September 27, 1994; and in revised form, November 14, 1994)

Albert Lai (1) (2) Sangram S. Sisodia (3) Ian S. Trowbridge (1)(§)

From the  (1)Department of Cancer Biology, The Salk Institute, San Diego, California 92186-5800, the (2)Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California 92037, and the (3)Neuropathology Laboratory, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The beta-amyloid precursor protein (APP) is proteolytically processed to generate beta-amyloid protein, the principal protein component of neuropathological lesions characteristic of Alzheimer's disease. To investigate potential sorting signals in the cytoplasmic tail of APP, we transplanted APP cytoplasmic tail sequences into the cytoplasmic tail of the human transferrin receptor (TR) and showed that two sequence motifs from the APP cytoplasmic tail promote TR internalization. One sequence, GYENPTY, is related to the low density lipoprotein receptor internalization signal, FDNPVY, but also involves a critical glycine residue; the other, YTSI, conforms to the 4-residue tyrosine-based internalization signal consensus sequence. Furthermore, a chimeric molecule (APP-TR) consisting of the cytoplasmic domain of APP and the transmembrane and external domains of TR was rapidly internalized enabling the transport of iron into the cell at 50% the rate of wild-type TR. Alanine scanning mutations indicated that the two sequences identified in transplantation experiments were required for internalization of the chimera. Metabolic pulse-chase experiments showed that the APP-TR chimeras were degraded in a post-Golgi membrane compartment within 2-4 h following normal glycosylation. Degradation was partially dependent upon the two internalization signals and was inhibited by ammonium chloride. A fraction of APP-TR chimeras traffic to a degradative endocytic compartment after appearing on the cell surface. Comparison of soluble APP released from cells expressing either full-length human APP or mutant APP with the sequence YENPTY deleted indicated that this sequence is required for sorting of full-length APP along similar trafficking pathways as the APP-TR chimera.


INTRODUCTION

Alzheimer's disease (AD) (^1)is a progressive neurodegenerative disorder affecting 1-6% of people over the age of 65. A characteristic neuropathological feature of AD is the senile plaque, which contains, beta-amyloid (Abeta), a 39-43 amino acid peptide derived from the beta-amyloid precursor protein (APP) (for reviews, see Hardy and Allsop(1991) and Selkoe(1994)). Encoded by a single gene on chromosome 21, APP is a family of alternatively spliced Type I integral transmembrane glycoproteins, whose cellular function is unknown. Considerable effort has been directed at understanding the mechanism by which APP is converted to Abeta because abnormal APP processing may be involved in the pathogenesis of AD. Genetic studies have revealed mutations in the coding region of the APP genes isolated from affected individuals with early-onset familial AD, and it has been shown that one of these mutations, the Swedish double mutation, leads to secretion of elevated levels of Abeta (Citron et al., 1992; Cai et al. 1993).

The derivation of Abeta from APP and the factors regulating this process remain unclear (for review, see Selkoe(1994)). Generation of Abeta involves two proteolytic cleavages: one in the extracellular domain and another in the transmembrane region; however, the enzyme(s) involved and the cellular location(s) of these proteolytic events are not known. Alternatively, proteolytic cleavage by another unidentified enzyme (referred to as alpha-secretase) thought to reside on or near the plasma membrane leads to production of a large soluble fragment comprised of most of the APP extracellular domain (APP(s)) (Esch et al., 1990; Sisodia, 1992). Significantly, this proteolytic event occurs within the Abeta peptide, thus precluding Abeta formation.

In 1990, it was pointed out that the cytoplasmic domain of APP contains the tetrapeptide sequence, NPTY, which conforms to the consensus sequence, NPXY, required for rapid endocytosis of the low density lipoprotein receptor (LDLR) (Chen et al., 1990). This observation raised the possibility that APP may participate in receptor-mediated endocytosis and that membrane trafficking of APP might influence the generation of Abeta. Subsequently, it was shown that APP expressed on the cell surface was internalized and delivered to the prelysosomal/lysosomal branch of the endocytic pathway consistent with the hypothesis that APP is transported to the cell surface via the constitutive biosynthetic pathway where it can be either cleaved by alpha-secretase to generate APP(s) or internalized and eventually degraded in lysosomes to yield amyloidogenic peptide fragments (Haass et al., 1992). APP is found in clathrin-coated vesicles, implying that it is recognized by clathrin-based sorting machinery either at the plasma membrane or in the trans-Golgi (Norstedt et al., 1993). Analysis of APP sorting signals has been limited to the use of APP(s) release as an indirect measure of endocytosis as mutations abrogating internalization of APP would be expected to increase APP(s) release according to this model. Truncation of the APP cytoplasmic domain which deletes the NPTY sequence or alteration of the tyrosine residue in the tetrapeptide motif to alanine leads to increased secretion of APP(s) (Haass et al., 1993; De Strooper et al., 1993; Jacobsen et al., 1994). However, the NPTY sequence has not been shown directly to be either required or sufficient for high efficiency internalization of APP. Furthermore, the delivery of APP to a degradative endocytic compartment by a direct intracellular route has not been ruled out.

Internalization signals of constitutively recycling receptors are self-determined interchangeable structural motifs that can be transplanted from Type I to Type II membrane proteins and vice versa without loss of activity (Collawn et al., 1991; Jadot et al., 1992). One approach, therefore, to determine whether specific sequences can function as internalization signals is to transplant them into the cytoplasmic tail of a heterologous receptor and examine whether they can promote rapid endocytosis (Trowbridge et al., 1993). In this study, we have identified putative internalization signals in the cytoplasmic domain of APP by assaying their internalization activity after transplantation into the cytoplasmic tail of the human transferrin receptor (TR). We have also constructed chimeric molecules (APP-TR) in which the TR cytoplasmic tail has been replaced by the APP cytoplasmic tail. Furthermore, we have measured APP(s) release from mutant full-length APP molecules.


MATERIALS AND METHODS

Human TR Transplants and APP-TR Constructs

Mutant human TR constructs were prepared as described previously (Jing et al., 1990) by the method of Kunkel(1985). Briefly, mutants were screened and selected by restriction mapping and cloned into the expression vector, BH-RCAS (Hughes et al., 1990).

To generate the APP-TR chimera, we cloned a PCR fragment encoding the APP tail into a ``tail-less'' TR construct. To create the tail-less TR vector, unique restriction sites (NheI and AflII) were introduced by oligo site-directed mutagenesis into the Delta3-59 deletion TR mutant cloned in pBluescript (Jing et al., 1990) which contained only a 4-residue cytoplasmic domain. This required addition of 3 residues, in-frame, ASL, not derived from TR resulting in the tail-less construct with a 7-residue cytoplasmic domain (see Fig. 1b), which was also used as the tail-less negative control for internalization. Plasmid p770 consisting of the cDNA of APP770 isoform inserted into a pUC19-based vector (Sisodia et al., 1990), was used as the PCR template to create a PCR fragment encoding the entire cytoplasmic domain of APP with flanking NheI and AflII sites, using as a 5`-primer the sequence 5`-CAT-GCT-AGC-CTG-AAG-AAG-AAA-CAG-3` and the sequence 5`-CAT-CTT-AAG-GTT-CTG-CAT-CTG-CTC-3` as the 3`-primer. This fragment was digested with NheI and AflII and ligated into the tail-less TR-pBluescript vector. Alanine scanning mutations were created using either mutagenic PCR primers or by oligonucleotide site-directed mutagenesis with the APP-TR template. All mutations were verified by dideoxynucleotide sequencing of the entire cytoplasmic domain in BH-RCAS constructs (Sanger et al., 1977; Tabor and Richardson, 1987).


Figure 1: Cytoplasmic tail amino acid sequences of TR transplantation constructs and APP-TR chimeric constructs. a, amino acid sequences of wild-type TR cytoplasmic tail (underlined tetrapeptide is endogeneous TR internalization signal) and mutant TR transplantation constructs showing where APP sequences have been inserted. Constructs are referred to in text by corresponding name at left. For double transplant, GYENPTY/YTSI, YTSI replaces the TR sequence, GDNS, which was selected based on Collawn et al.(1993) (see ``Results''). Residues are numbered from amino terminus, position of transmembrane is represented by TM , and - represents unchanged residues. b, top, schematic diagram of APP-TR construct showing combination of Type I (APP) and Type II (TR) transmembrane proteins. Shaded areas represent regions of APP-TR derived from TR; unshaded areas represent domains derived from APP where: CYT, cytoplasmic domain; TR, transmembrane region; and EC, extracellular domain. represents carboxyl terminus of the APP cytoplasmic domain. Bottom, cytoplasmic tail amino acid sequences of wild-type APP695, APP-TR, and mutant APP-TR constructs showing positions of various alanine mutations in APP-TR (YTSI and GYENPTY sequences are underlined). Constructs are referred to in text by corresponding names at left. Numbering and symbols are the same as a.



Expression of Wild-type TR, TR Transplants, and APP-TR Chimeras in CEF

TR transplants and chimeras were expressed in chicken embryo fibroblasts (CEF) as described previously (Jing et al., 1990). Surface expression levels of the wild-type TR and chimeric APP-TR constructs were determined by measuring the binding of I-labeled human transferrin (Tf) at 4 °C (Jing et al., 1990).

Determination of the Apparent Internalization Efficiencies of TR Transplants and APP-TR Chimeras

Apparent internalization efficiencies of the wild-type TR, TR transplants, and chimeric APP-TR constructs were estimated from measurement of the steady-state distribution of receptors at 37 °C (Tanner and Lienhard, 1987). CEF were plated in triplicate wells as described for surface-binding studies. The cells were preincubated in serum-free DME for 1 h and then incubated with 4 µg/ml I-labeled Tf in BSA/PBS for 1 h at 37 °C. After removal of labeling medium, the cells were washed three times with 1 ml of ice-cold BSA/PBS, incubated twice for 3 min with 0.5 ml of 0.2 M acetic acid, 0.5 M NaCl (pH 2.4) to remove surface-bound I-labeled Tf (Hopkins and Trowbridge, 1983), and removed from the wells with 1 M NaOH. Radioactivity in the acid wash and in the cell lysate was counted. More prolonged incubation with the acid wash did not change the amount of I released. At steady state, the rate of internalization, k, of cell surface TfbulletTR complexes, [TR], equals the rate of externalization, k, of the internal pool of apoTfbulletTR complexes, [TR]; i.e.kbullet[TR] = kbullet[TR], assuming an insignificant rate of degradation of internalized receptors during the time required to achieve steady state. The values of [TR] and [TR] can be obtained from steady-state binding of TR under saturating conditions at 37 °C. As k of apoTfbulletTR complexes is independent of signals in the TR cytoplasmic domain (Jing et al., 1990), k values of mutant and wild-type receptors are identical so that their k values are proportional to their steady-state distribution, [TR]/[TR]; i.e. mutant internalization efficiency percent = [TR] (mutant)bullet[TR] (wild-type) times 100/[TR] (mutant) bullet[TR] (wild-type). Kinetic studies demonstrated that the steady-state distributions of wild-type TR were achieved within 20 min and did not change if cells were incubated up to 1 h (Jing et al., 1990).

The internalization efficiencies of wild-type TR and the APP-TR chimera were also determined by measuring their ability to mediate iron uptake as described previously (Jing et al., 1990).

Measurement of Tf Proteolysis after Internalization

CEF were plated in triplicate wells as described for the binding studies. Cells were preincubated in serum-free DME for 30 min at 37 °C, then incubated with I-labeled Tf (4 µg/ml) in BSA/PBS for 1 h at 37 °C. The labeling medium was removed, and the cells were washed three times with ice-cold BSA/PBS and incubated at 37 °C with prewarmed (37 °C) DME, containing 0.1% BSA and 50 µg/ml unlabeled Tf for 0, 10, 20, 40, 60, or 90 min. After incubation, the medium from each well was collected, and radioactivity was counted in a -counter. 10% Trichloroacetic acid was added to the medium to precipitate protein which was then removed by centrifugation, allowing the acid-soluble radioactivity remaining in medium to be counted in a -counter. Acid-insoluble radioactivity was calculated by subtracting acid-soluble radioactivity from total radioactivity of collected media. The surface-bound and internalized Tf in CEF were determined by the acid wash procedure described for the steady-state distribution assay.

Metabolic Labeling and Immunoprecipitation of CEF

One day prior to the experiment, approximately 2 times 10^6 cells were plated on 6-cm tissue culture dishes and grown overnight. Cells were washed twice with methionine-free DME, preincubated in methionine-free DME for 20 min, and incubated for 30 min in 1.5 ml of methionine-free DME containing 0.12 mCi/ml TransS-label (ICN Biomedicals, Irvine, CA) and 2% defined calf serum. Pulse-labeled cells were chased for 0, 2, 4, or 8 h in complete medium. In one experiment, CEF expressing APP-TR were preincubated for 1 h in 0, 10, 25, or 50 mM concentrations of NH(4)Cl prior to addition of TransS-label and chased in same concentrations of NH(4)Cl for 0, 2, 4, and 8 h. In another experiment, cells were preincubated for 4 h in medium containing 100 µg/ml leupeptin and then pulse-labeled and chased for 0, 2, 4, or 8 h in leupeptin-containing medium. At each time point, labeled cells were placed on ice and solubilized with 1% Nonidet P-40/PBS. The wild-type TR and chimeric APP-TR constructs were immunoprecipitated from postnuclear supernatants using the B3/25 monoclonal antibody directed against the human TR external domain (Omary and Trowbridge, 1981). Immunoprecipitates were analyzed on 7.5% polyacrylamide gels. Gels were treated with 2,5-diphenyloxazole (U. S. Biochemical Corp., Cleveland, OH)/dimethyl sulfoxide and dried for fluorography. Dried gels were exposed to preflashed X-AR film (Eastman Kodak, Rochester, NY). Quantitation of radioactivity was performed on a model 425 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Generation of Stable CHO Cell Lines

Construction of plasmid p770Delta, encoding APP770 deleted of the YENPTY sequence, was achieved using a PCR-based approach. Plasmid p770 was used as the template with the 5`-primer, 5`-CCG-AGA-TCT-CTG-AAG-TG-3`, and the 3`-primer, 5`-CCG-TCT-AGA-CTA-GTT-CTG-CAT-CTG-CTC-AAA-GAA-CTT-GCC-GTT-CTG-CTG-CAT-C-3`, to create a 300-base pair PCR fragment which was digested with BglII and XbaI, then ligated to the vector fragment from p770SP (Sisodia et al., 1990) that had been digested with BglII and XbaI.

To generate a stable CHO cell line expressing APP770Delta (CHO770Delta), CHO cells were cotransfected with plasmid p770Delta and pSV2neo (Subramani et al., 1981), and selected in 0.4 mg/ml G418. Resistant colonies were expanded and assayed for expression of APP770Delta by immunoblotting with the monoclonal antibody 22C11 (Weidemann et al., 1989). The stable CHO cell line expressing APP770 (CHO770) was described previously (Wang et al., 1991).

Metabolic Labeling, Biotinylation, and Quantitative Immunoprecipitation of APP and APP(s) from CHO Cells

In Fig. 6and Fig. 7, monolayers of approximately 3 times 10^6 of either CHO770 or CHO770Delta cells were preincubated in methionine-free DME supplemented with 1% dialyzed fetal bovine serum, then labeled for indicated times with 0.125 mCi of [S]methionine in methionine-free DME supplemented with 1% dialyzed fetal bovine serum. Immediately (Fig. 6) or after a 25-min chase (Fig. 7), cell surface biotinylation was performed by washing pulse-labeled CHO monolayers on ice with cold PBS containing both 1 mM MgCl(2) and CaCl(2) (PBS). Cells were incubated with 0.5 mg/ml NHS-SS-biotin (Pierce) in PBS for 45 min at 4 °C. Cells were then washed in PBS containing 50 mM NH(4)Cl to quench unreacted biotin and subsequently washed in PBS. Following either a 10-min chase at the indicated temperature (Fig. 6) or directly after biotinylation (Fig. 7), cells were lysed at 4 °C in immunoprecipitation buffer containing detergent and protease inhibitors (Sisodia et al., 1990), and APP was immunoprecipitated from aliquots containing identical volumes of each cell lysate using either the antisera, CT-15 (Sisodia et al., 1993), generated against a synthetic peptide encompassing the carboxyl-terminal 15 amino acids of APP, in which the principal epitope(s) lies in the last 7 amino acids of the peptide, or monoclonal antibody P2.1 (Van Nostrand et al., 1992) which recognizes an epitope in the APP ectodomain. To isolate biotinylated APP molecules, CT-15 or P2.1 immunoprecipitates were boiled in a solution consisting of 10 mM Tris (pH 7.4), 50 mM NaCl, and 1% SDS for 3 min, and four-fifths of the eluted material were incubated with 30 µl of streptavidin-agarose beads (Pierce) for 2 h at 4 °C. After incubation, beads were washed in detergent containing immunoprecipitation buffer, then boiled in Laemmli sample buffer, and analyzed on SDS-PAGE. The remaining one-fifth of the original immunoprecipitate was loaded directly on the gel to represent total labeled APP. Immunoprecipitates were analyzed on SDS-PAGE.


Figure 6: Release of APP(s) from cell-surface resident APP deleted of the YENPTY sequence. a, parallel sets of dishes containing equivalent numbers of CHO770 cells (lanes 1 and 2) or CHO770Delta cells (lanes 3 and 4) were labeled with [S]methionine for 2 h, cooled, then reacted with NHS-SS-biotin on ice for 45 min. One set of dishes was incubated at 4 °C for 10 min and lysed. The other set was incubated at 37 °C for 10 min, then lysed following collection of conditioned medium. Lanes 1 and 3 show one-fifth of the immunoprecipitate (using monoclonal P2.1) of APP from cell lysates (L) prepared from cells at 4 °C, and lanes 2 and 4 show the same except from cells incubated at 37 °C. b and c, to assess levels of cell-surface APP and released APP(s) from CHO770 (b) or CHO770Delta (c), the remaining four-fifths of the immunoprecipitate shown in panel a were subsequently reacted with immobilized streptavidin. Lanes 1 and 2 represent 135-kDa cell surface-resident APP (L) after incubation at 4 or 37 °C, respectively. The 110-kDa band likely represents nonspecific binding of immature APP to streptavidin. Lanes 3 and 4 represent immunoprecipitable APP(s) forms in the conditioned medium (CM) of cells incubated at 4 or 37 °C, respectively, that bound to streptavidin. The percentage of total APP (at 0 h) found in CM of cells incubated at 37 °C is indicated.




Figure 7: Levels of newly synthesized APP appearing on cell surface. CHO770 (lane 1) or CHO770Delta (lane 2) cells were pulse-labeled with [S]methionine for 10 min and chased for 25 min. Cells were cooled to 4 °C and reacted with NHS-SS-biotin for 45 min. Equivalent trichloroacetic acid-precipitable counts/min from detergent lysates from each cell line were subject to immunoprecipitation with antisera CT-15, and four-fifths of the immunoprecipitate were reacted with immobilized streptavidin (b) while the remaining one-fifth was left unreacted (a).



In Fig. 6, b and c, an immunoprecipitation protocol was used in which the levels of immunopurified cellular or secreted APP-related molecules were not biased by variation in sample protein concentration between conditioned media and cell lysates. For example (Fig. 6b), pulse-labeled CHO770 cells chased for 10 min were lysed in buffer containing detergent, protease inhibitors, and the 10-min chase medium of CHO770 cells incubated for 2 h in ``labeling'' medium lacking [S]methionine. In parallel, the [S]methionine radiolabeled medium obtained after 10 min chase was adjusted with detergents and protease inhibitors and used to lyse a monolayer of CHO770 cells which had been chased for 10 min after incubated for 2 h in ``labeling medium'' which lacked [S]methionine. Identical volumes of each mixture were subject to immunoprecipitation. Immunoprecipitates were analyzed on SDS-PAGE and quantitated on a PhosphorImager.


RESULTS

Two APP Cytoplasmic Tail Sequences Transplanted into the Human TR Are Active as Internalization Signals

To obtain direct evidence that the APP cytoplasmic tail contains sequence motifs that function as internalization signals, putative signals from the APP cytoplasmic tail were transplanted into the cytoplasmic domain of the human TR replacing the endogeneous TR internalization signal, YTRF. Mutant receptors containing the transplanted signals were then stably expressed in CEF using BH-RCAS, a replication-competent retroviral vector derived from Rous sarcoma virus (Jing et al., 1990; Hughes et al., 1990). Internalization efficiency of the mutant receptors was determined by measuring the steady-state distribution of internalized receptors (Jing et al., 1990; Collawn et al., 1990).

Previous studies suggested that the LDLR internalization signal is the 6-residue sequence FDNPVY since its activity is crucially dependent on the amino-terminal phenylalanine residue in both the context of the LDLR cytoplasmic tail and the TR cytoplasmic tail following transplantation (Chen et al., 1990; Collawn et al., 1991). For this reason, we thought it likely that the amino-terminal tyrosine in the 6-residue APP sequence would also be required for internalization activity. Therefore, we initially transplanted the 6-residue APP sequence, YENPTY, into the human TR cytoplasmic tail rather than NPTY (Fig. 1a). As shown in Table 1, however, YENPTY had little or no activity in the context of the TR cytoplasmic tail since the mutant receptor containing this transplanted sequence was internalized only slightly more efficiently than the tail-less TR (15 versus 9%, respectively).



The cytoplasmic tails of several lysosomal transmembrane glycoproteins contain the sequence pattern Y-X-X-aromatic/large hydrophobic residue, characteristic of 4-residue tyrosine-based internalization signals (Trowbridge et al. 1993), flanked on the amino-terminal side by a conserved glycine residue (reviewed by Fukuda(1991)). It has been shown that this glycine residue is important for the internalization of lysosomal acid phosphatase (Lehmann et al., 1992), and indirect evidence has been provided that the GY motif is important for intracellular sorting of newly synthesized Lgp-A (Harter and Mellman, 1992). These considerations led us to test whether the glycine residue adjacent to the amino-terminal tyrosine residue of the YENPTY sequence in the APP cytoplasmic tail might be required for internalization activity. We found that more than 50% of mutant TRs displaying the transplanted GYENPTY sequence were internalized at steady-state representing an internalization efficiency of 47% relative to wild-type TR (Table 1).

To confirm that the glycine residue was itself important for the activity of GYENPTY rather than the loss of the wild-type proline residue it had replaced, another mutant TR was constructed in which the alanine was substituted for the proline residue (Fig. 1a). This mutant receptor containing the sequence AYENPTY was poorly internalized clearly indicating that the glycine residue is critical for the activity of the LDLR-related APP signal at least in the context of the TR cytoplasmic tail (Table 1).

In addition to the LDLR-related signal, the APP cytoplasmic tail also contains the sequence YTSI, which conforms to the sequence pattern of 4-residue tyrosine-based internalization signals (Trowbridge et al., 1993). Upon substitution into the TR cytoplasmic tail in place of YTRF, this sequence was found to promote internalization as effectively as the LDLR-related signal (Table 1). Furthermore, a mutant TR (GYENPTY/YTSI) containing both APP signals with the YTSI sequence inserted at a second position known to be compatible with internalization activity (Collawn et al., 1993) was internalized with an efficiency of 80% relative to wild-type TR. The roughly additive level of internalization shown by this mutant implies that both sequence motifs could be simultaneously recognized by clathrin-based sorting machinery at the plasma membrane (Table 1).

APP Cytoplasmic Tail Promotes Internalization of the Human TR

To test whether the entire APP cytoplasmic tail could promote internalization of a heterologous protein, we generated a chimeric protein, APP-TR, by fusing the carboxyl-terminal end of the cytoplasmic tail of APP to the transmembrane and extracellular domains of the TR. Construction of the APP-TR chimera required reversing the polypeptide polarity of the APP cytoplasmic tail with respect to the cell membrane (see Fig. 1b). However, previous studies have shown that internalization signals are three-dimensional structural motifs whose activity is independent of the polypeptide chain's orientation with respect to the cell membrane (Collawn et al., 1991; Jadot et al., 1992). Thus, we expected that the construction of the APP-TR chimera would not alter the activity of any internalization signals displayed by the APP cytoplasmic tail. APP-TR chimeras were stably expressed in CEF using BH-RCAS, and surface expression was 83.5 ± 2.8% (mean ± S.E. of four determinations) of wild-type TR. As shown in Table 2, the internalization efficiency of the APP-TR chimera was 34% relative to wild-type TR, a value almost 4-fold higher than the 9% internalization efficiency for tail-less TR measured in parallel. The ability of the APP cytoplasmic tail to promote rapid endocytosis of the human TR was independently confirmed by iron uptake experiments. As shown in Table 3, the APP-TR chimera mediated iron uptake with an efficiency of almost 50% relative to wild-type TR, implying that the chimeric molecules were not only internalized but also efficiently recycled back to the cell surface.





To investigate whether the putative internalization signals functionally identified by the transplantation experiments were required for internalization activity of APP-TR chimera, we inactivated each signal independently by altering the tyrosine residues in each signal to alanine. Alteration of the 7-residue signal GYENPTY to GAENPTA partially reduced the internalization efficiency of the APP-TR chimera (Table 2). Additional substitution of alanine for the critical glycine residue in this signal resulting in the mutation, AAENPTA, further reduced the internalization efficiency of the APP-TR chimera to 50% the activity of the chimera containing an intact GYENPTY signal (Table 2). Similarly, substitution of the tyrosine residue in the YTSI signal also partially reduced the internalization efficiency of the APP-TR chimera. Importantly, when both signals were inactivated, the internalization efficiency of the APP-TR chimera was comparable to that of the tail-less mutant TR (Table 2). To rule out the possibility that alanine substitutions decreased internalization through nonspecific structural changes, analysis of mutant APP-TR molecules in which the residues HLS have been altered to alanines showed no appreciable change in internalization compared to APP-TR (Table 2). These results indicate that the APP cytoplasmic tail contains two internalization signals of similar strength that can independently facilitate internalization of human TR.

APP-TR Chimeras Are Degraded in a Post-Golgi Endocytic Compartment

It has been suggested that after internalization, APP is targeted to lysosomes where it is degraded (Haass et al., 1992). To determine whether the APP cytoplasmic tail was sufficient to target APP-TR to the prelysosomal/lysosomal branch of the endocytic pathway, metabolic pulse-chase experiments were performed to compare the rates of degradation of the APP-TR chimera and wild-type TR. CEF expressing either the APP-TR chimera or wild-type human TR were pulselabeled with TransS-label for 30 min and chased for various lengths of time; TR and APP-TR chimeric molecules were then isolated by immunoprecipitation and analyzed by SDS-PAGE. As shown in Fig. 2, the APP-TR chimera is degraded much more rapidly than wild-type TR; greater than 90% of the chimera was degraded (t = 3 h) during an 8-h chase, whereas only 15% of the wild-type TR was degraded during the same period, consistent with previous results indicating that the wild-type TR has a t of 24 h in CEF (Jing and Trowbridge, 1990; Odorizzi et al., 1994). After 2 h (Fig. 2) the M(r) of both TR and APP-TR increased to that of the mature glycoprotein (Omary and Trowbridge, 1981), indicating that both molecules traverse the Golgi compartment where oligosaccharide processing and synthesis is completed. Taken together, these data indicate that the APP cytoplasmic tail targets TR to a post-Golgi compartment where it is rapidly degraded.


Figure 2: Rapid degradation of APP-TR chimeras in a post-Golgi endocytic compartment. Equivalent cell numbers of CEF expressing wild-type (WT), TR (bullet), or APP-TR (box) chimera were pulse-labeled for 30 min with TransS-label and chased for various periods of time (h) as indicated. TR or APP-TR molecules were then immunoprecipitated from post-nuclear supernatants and analyzed on SDS-polyacrylamide gels as described under ``Materials and Methods.'' Dried gels were exposed to X-AR film overnight (Kodak). Immunoprecipitates were quantitated on a model 425 PhosphorImager (Molecular Dynamics). Relative amounts were calculated as a percentage of labeled immunoprecipitate at 0 h. Data represented is mean ± S.E. where n = 4 for TR and n = 7 for APP-TR.



Intracellular degradation of APP is partially inhibited by lysosomotropic agents (Golde et al., 1992; Caporaso et al., 1992; Haass et al., 1992). To determine whether the rapid degradation of APP-TR chimera was also inhibited by lysosomotropic agents, metabolic pulse-chase experiments were performed using cells treated with different concentrations of ammonium chloride. Degradation of the APP-TR chimera was markedly inhibited by the lowest concentration of ammonium chloride tested (10 mM), and inhibition increased in a dose-dependent manner. Even at the highest concentration of ammonium chloride used (50 mM), virtually all the APP-TR chimeric molecules were transported through the Golgi and converted to the mature form of the glycoprotein (Fig. 3). Addition of 100 µg/ml of the protease inhibitor, leupeptin, also decreased the degradation rate of APP-TR molecules, although less dramatically (data not shown). These results are consistent with the idea that APP-TR chimeras traffic either by a direct intracellular route or via the plasma membrane to an acidic endocytic compartment where they are degraded.


Figure 3: Ammonium chloride (NH(4)Cl) inhibits degradation of the APP-TR chimera. Equivalent cell numbers of CEF expressing APP-TR were preincubated for 1 h at 37 °C in 0, 10, 25, and 50 mM concentrations of NH(4)Cl and then pulse-labeled with TransS-label and chased in the presence of same concentration of NH(4)Cl. APP-TR was then immunoprecipitated and analyzed on SDS-polyacrylamide gels as described in legend to Fig. 2. Data shown are from one of two similar experiments.



To investigate whether the tyrosine-based sorting signals mediating internalization were required for the degradation of the APP-TR chimera, we determined the degradation rates of mutant chimeras in which one or both signals were inactivated through alteration of their tyrosine residues to alanine (Fig. 4). Inactivation of only one of the sorting signals had only a modest effect on the degradation rate of the APP-TR chimeras. However, when the tyrosine residues in both signals were concomitantly changed to alanine, the mutant chimera was degraded significantly slower than the APP-TR chimera itself. Nevertheless, this mutant chimera was still degraded more rapidly than wild-type TR, implying that other structural features of the APP cytoplasmic tail were involved in targeting APP-TR to this degradative compartment. Additional modification of the glycine residue in the LDLR-related signal did not, however, increase the half-life of the mutant chimera in which the tyrosines residues of each signal were altered to alanine (data not shown).


Figure 4: Degradation of APP-TR mutant chimeras. Equivalent cell numbers of CEF expressing APP-TR (box), AENPTA (), ATSI (bullet), or AENPTA/ATSI () chimeras were pulse-chased and immunoprecipitated as described in the legend to Fig. 2. Analysis of immunoprecipitates was performed by SDS-PAGE, visualized on X-AR film, and quantitated on a PhosphorImager. Data represents averages ± S.E. from five to seven independent experiments.



A Fraction of APP-TR Chimeras Traffic from the Plasma Membrane to the Degradative Endocytic Compartment

To determine whether APP-TR chimeras traffic via the plasma membrane to the endocytic compartment where degradation occurs, cells were incubated with I-labeled Tf at 37 °C for 1 h to load APP-TR chimeras residing on the cell surface and throughout the endocytic pathway with ligand. After the cells were washed rapidly, the reappearance of intact and degraded Tf in the medium was monitored by measuring trichloroacetic acid-insoluble and -soluble radioactivity (Odorizzi et al., 1994). As shown previously, wild-type TR are efficiently recycled back to the cell surface through the sorting and recycling endosomal compartments as only 3.1 ± 0.7% (mean ± S.E. of two independent experiments) of I-labeled Tf released into the medium was degraded (Fig. 5). In contrast, 14.1 ± 1.1% (mean ± S.E. of three independent experiments) of the I-labeled Tf originally bound to the APP-TR chimera and released into the medium was degraded. These results indicate that a minor fraction of the internalized APP-TR molecules are sorted to an endocytic compartment where they are degraded, while the majority of endocytosed chimeras are recycled back to the cell surface.


Figure 5: Degradation of transferrin (Tf) bound to APP-TR chimeras at cell surface. Equivalent cell numbers of CEF, plated in triplicate for each time point, expressing either WT TR or APP-TR were preincubated in serum-free DME for 30 min at 37 °C, followed by incubation with I-labeled Tf for 1 h at 37 °C. The cells were then washed and reincubated at 37 °C in DME containing 50 µg/ml unlabeled Tf for various times. Acid-soluble radioactivity (bullet) or acid-insoluble I-labeled Tf (Delta) released into medium, as well as surface-bound I-labeled Tf (box) and internalized I-labeled Tf () were determined as described under ``Materials and Methods'' and are expressed as a percent of total radioactivity recovered. Each data point represents average ± S.E. of two to three independent experiments. Error bars representing S.E. are not visible.



Deletion of YENPTY Enhances Release of Surface-expressed APP and Increases Level of APP Found on Cell Surface

Studies from several laboratories have shown that cells expressing tail-less APP or APP mutated at tyrosine 686 (NPTY in APP695) secrete higher levels of APP(s) than cells expressing wild-type APP (Haass et al., 1993; De Strooper et al., 1993; Jacobsen et al., 1994). Consistent with these results, examination of APP(s) release from CHO cell lines stably expressing at equivalent levels either wild-type human APP770 (CHO770) or APP770, in which the YENPTY sequence has been deleted (CHO770Delta), revealed that CHO770Delta cells released 65% of newly synthesized APP as APP(s) compared to 20% for CHO770 cells (data not shown).

To address the hypothesis that YENPTY influences APP(s) release by mediating plasma membrane endocytosis of APP, we compared levels of APP(s) derived from the surface-resident population of APP molecules from CHO770 or CHO770Delta cell lines. Duplicate dishes of cells were labeled with [S]methionine for 2 h at 37 °C. Cells were then cooled and reacted at 4 °C for 45 min with NHS-SS-biotin, a membrane-impermeant biotinylation reagent. One set of dishes was incubated at 4 °C for 10 min, while the other set was incubated at 37 °C for the same period of time. From each dish, the conditioned medium was collected, and a detergent lysate was prepared. Immunoprecipitation was performed using monoclonal antibody P2.1, and four-fifths of the immunoprecipitates were subsequently reacted with immobilized streptavidin. Fig. 6a, showing the unreacted one-fifth of the immunoprecipitate, demonstrates that accumulated levels of immature 115-kDa and mature 125-135-kDa APP from either cell line are identical (compare lanes 1 and 3), and as expected, levels remained essentially unaltered after incubation of cells at 37 °C (compare lanes 2 and 4). However, PhosphorImager analysis of immunoprecipitates obtained from the conditioned medium revealed that CHO770 cells release 20% of cell surface APP after 10 min (Fig. 6b, lane 4), while CHO770Delta cells release 33% of the surface pool after 10 min (Fig. 6c, lane 4). These results are consistent with the hypothesis that enhanced release of APP(s) derived from APP molecules lacking the YENPTY sequence is the result of impaired endocytosis of surface-resident molecules. Interestingly, comparison of steady-state levels of 135-kDa APP residing on the cell surface showed that significantly more APP is present on cell surface of CHO770Delta (compare Fig. 6, b, lane 1, and c, lane 1) which is unexpected given the apparent higher efficiency cleavage of mutant APP.

Removal of YENPTY Increases the Amount of Newly Synthesized APP Found on the Cell Surface

To determine whether levels of newly synthesized APP found on the surface of CHO770 or CHO770Delta cells were different, both cell lines were pulse-labeled for 10 min with [S]methionine followed by a 25-min chase to allow newly synthesized proteins to reach the cell surface. As in the previous experiment, cells were then cooled to 4 °C and reacted with NHS-SS-biotin. APP was immunoprecipitated from cell lysates with the CT-15 antibody. Four-fifths of collected immunoprecipitates were reacted with immobilized streptavidin in order to recover molecules which were on the plasma membrane at the end of the chase period. Fig. 7a, representing the remaining unreacted one-fifth of the original immunoprecipitates, shows that overall levels of immature 110 kDa and modified 125-135-kDa APP from either cell line were indistinguishable, and results of phosphorimaging analysis confirmed that nearly identical levels of higher molecular mass forms were recovered contributing roughly one-third of total immunoprecipitable APP from both CHO770 and CHO770Delta cells. This indicates that post-translational modification and maturation of APP770Delta occurred at the same rate as APP770. In contrast, Fig. 7b shows that 2.2-fold higher levels of streptavidin-precipitable APP were recovered from CHO770Delta cells than from CHO770 cells, suggesting that YENPTY may also affect release of APP(s) by sorting APP away from biosynthetic pathway prior to appearance on the plasma membrane.


DISCUSSION

Quantitative studies of APP trafficking are complicated by several reasons including the fact that a variable fraction of APP molecules are cleaved to yield a large secreted soluble fragment (APP(s)) consisting of virtually the entire external domain; the observation that in many cell types, a substantial fraction of APP molecules are not processed to the fully glycosylated mature form of the molecule, implying that their transport along the biosynthetic pathway is blocked (Kuentzel et al., 1993); and the lack of a known naturally occurring ligand that can be used to monitor internalization. To overcome these difficulties, we have characterized putative APP sorting signals by assaying their activity after transplantation into the cytoplasmic tail of the human TR. To further analyze the role of the APP cytoplasmic tail in membrane protein trafficking, we also constructed chimeric molecules consisting of the APP cytoplasmic tail and the TR transmembrane region and external domain. Both of these experimental strategies have previously been used to characterize sorting signals of other membrane proteins (Collawn et al., 1991; Jadot et al., 1992; Garippa et al., 1994; Odorizzi et al., 1994). Last, we studied the effect of removal of one of these signals on surface expression of full-length APP molecules and APP(s) secretion.

Our results extend previous observations by demonstrating that two tyrosine-containing sequence motifs within the APP cytoplasmic domain can promote rapid internalization of the human TR when transplanted into the TR cytoplasmic domain, in which the internalization efficiency of either signal is 50% that of the wild-type TR internalization signal. One transplantable signal, GYENPTY, conforms closely to the 6-residue LDLR consensus sequence, FXNPXY (Chen et al., 1990), but includes, in addition, an amino-terminal glycine which is critical for endocytosis. Although several lysosomal membrane proteins contain a conserved GY motif found flanking sequences that conform to the consensus sequence of 4-residue tyrosine-containing internalization signals (Fukuda, 1991), the glycine residue is apparently not always required for activity. Mutagenesis of the cytoplasmic tail of lysosomal acid phosphatase suggests that glycine is an important element of its internalization signal, PGYRHV (Lehmann et al., 1992). However, transplantation of the tetrapeptide sequence, YRHV, is sufficient to promote internalization of the TR and Man-6-PR (Trowbridge et al., 1993; Jadot et al., 1992). In the case of Lgp-A, the conserved glycine residue seems to be important for intracellular sorting but not for rapid endocytosis (Harter and Mellman, 1992).

A second APP cytoplasmic domain sequence, YTSI, also has the potential to function as an internalization signal based on its activity when transplanted into the TR cytoplasmic tail, and this sequence could promote internalization additively with GYENPTY in this context. However, its ability to act as an internalization signal in the native APP molecule may be compromised by its proximity to the cell membrane as it is separated by only 4 residues from the predicted transmembrane region (Fig. 1). By comparison, the TR internalization signal has been shown to require a spacer region of 7 residues separating it from the transmembrane region for full activity (Collawn et al., 1990), which likely reflects a structural requirement for the signal to be a minimum distance from the cell membrane in order to interact with the clathrin-based sorting machinery. However, the sequence GPLY, reported to be the most active internalization signal of the insulin receptor, is separated by only 5 residues from the transmembrane region (Backner et al., 1992; Rajagopalan et al., 1991), suggesting that the spacer region length may vary between proteins. If the YTSI signal is active in APP, it might be predicted that alteration of the tyrosine to alanine in this sequence would lead to increased secretion of APP(s); however, this effect was not observed (Jacobsen et al., 1994).

Steady-state and iron uptake assays indicated that the APP cytoplasmic tail was sufficient to promote internalization of the APP-TR chimera with an efficiency of roughly one-third to one-half that of wild-type TR. Rapid endocytosis of the chimera was dependent on both tyrosine-based signals identified in the transplantation experiments as internalization was abolished if both signals were inactivated by alanine mutations but only partially inhibited if either one were inactivated independently. However, these results do not exclude the possibility that the YTSI sequence is inactive in APP because it is separated by 40 residues from the transmembrane region in the APP-TR chimera (Fig. 1).

The results of metabolic pulse-chase experiments showed that the APP-TR chimera was rapidly degraded. Virtually all of the APP-TR chimeras were converted from a precursor to a mature fully glycosylated form during the chase period, indicating that the chimeric molecules transit through the Golgi and are subsequently degraded in a post-Golgi membrane compartment. Trafficking to this compartment appears to be partially dependent upon tyrosine-based sorting signals as a mutant APP-TR chimera in which both internalization signals were inactivated was degraded more slowly. Degradation of the APP-TR chimera was inhibited by concentrations of NH(4)Cl that did not block transport along the biosynthetic pathway, indicating that the chimera was degraded in an acidic intracellular compartment. Evidence that the APP-TR chimeras can traffic to this compartment via the plasma membrane was obtained by loading chimeras transiently expressed on the cell surface with exogenous Tf and following its subsequent fate. As only 15% of the bound Tf was degraded after internalization, it appears that the APP-TR chimeras, like lysosomal acid phosphatase (Braun et al., 1989) and the major histocompatibility complex class II-associated invariant chain (Ii) (Odorizzi et al., 1994), undergo several rounds of internalization and recycling before being degraded. Since wild-type TR, in contrast, can undergo many rounds of endocytosis before being degraded, degradation of the APP-TR chimera must occur in a compartment along the prelysosomal/lysosomal branch of the endocytic pathway distinct from the sorting and recycling endosomal compartments traversed by wild-type TR. As the surface expression of the APP-TR chimera is comparable to that of the wild-type receptor, it is likely that a substantial fraction, if not all, of the chimeric molecules traffic via the plasma membrane to the endocytic compartment where they are eventually degraded.

These results are supported by our studies of APP(s) release from cells expressing full-length APP where surface biotinylation experiments establish that some mature APP is directed to the plasma membrane. Furthermore, demonstration that this surface pool of APP is released at higher levels into the medium from cells expressing APP lacking the YENPTY sequence implies that this sequence mediates endocytosis of APP. Additional biotinylation experiments reveal that deletion of YENPTY also appears to sharply increase the amount newly synthesized APP reaching the cell surface, suggesting the involvement of YENPTY in intracellular targeting into endocytic pathway without cell surface appearance. Therefore, increased overall release of APP(s) from APP deleted of YENPTY likely results from diminished plasma membrane endocytosis and decreased intracellular sorting, and both effects may contribute to elevated levels of APP remaining at the cell surface for secretory cleavage. Although we directly establish an endocytic pathway for APP-TR involving the cell surface, our data from this molecule can neither confirm nor rule out the possibility of a second intracellular route. Along these lines, we have recently shown that the efficient delivery of an Ii-TR chimera containing both the Ii cytoplasmic and transmembrane domains to a degradative endocytic compartment by a direct intracellular route requires a sorting signal in the Ii transmembrane region (Odorizzi et al., 1994). Trafficking of APP-TR is remarkably similar to an Ii-TR chimera containing only the Ii cytoplasmic region, suggesting the possibility that structural determinants in regions of APP not contained in the APP-TR chimera may be required to specify efficient targeting along the intracellular route.

In conclusion, we provide direct evidence that the APP cytoplasmic tail can promote rapid endocytosis and degradation of APP-TR chimeras. We have also defined two tyrosine-containing sequences in the APP cytoplasmic tail that can function as internalization signals analogous to those found in constitutively recycling receptors. Additionally, studies of cell-surface expression and APP(s) release from full-length APP deleted of one of these signals support these findings and provide evidence for an additional intracellular pathway. Overall, our data are consistent with the model for APP trafficking proposed by Haass et al.(1992), and recent evidence that Abeta can be generated from APP following internalization from the cell surface and delivery to the endocytic pathway (Koo and Squazzo, 1994).


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM07198 and the Lucille P. Markey Charitable Trust (to A. L.), by National Institutes of Health Grants NSAG05146 and NS20471 and the American Health Assistance Foundation (to S. S. S.), and by National Institutes of Health Grant RO1-CA34787 (to I. S. T.). 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.

§
To whom correspondence should be sent. Tel.: 619-453-4100 (ext. 1241); Fax: 619-455-1349.

(^1)
The abbreviations used are: AD, Alzheimer's disease; APP, human beta-amyloid precursor protein; Abeta, beta-amyloid protein; APP(s), soluble APP fragment; TR, human transferrin receptor; Tf, human transferrin; LDLR, low density lipoprotein receptor; CEF, chick embryo fibroblasts; CHO, Chinese hamster ovary; DME, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.


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