(Received for publication, September 27, 1994; and in revised form, November 14, 1994)
From the
The -amyloid precursor protein (APP) is proteolytically
processed to generate
-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.
Alzheimer's disease (AD) ()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,
-amyloid (A
), a 39-43
amino acid peptide derived from the
-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
A
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 A
(Citron et al., 1992; Cai et al. 1993).
The derivation of A from APP and the factors
regulating this process remain unclear (for review, see Selkoe(1994)).
Generation of A
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
-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
) (Esch et al., 1990; Sisodia, 1992).
Significantly, this proteolytic event occurs within the A
peptide,
thus precluding A
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 A.
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
-secretase to generate
APP
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
release as an indirect measure of endocytosis as
mutations abrogating internalization of APP would be expected to
increase APP
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
(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 release from mutant full-length APP molecules.
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 3-59 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.
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).
To generate a stable CHO cell line
expressing APP770 (CHO770
), CHO cells were cotransfected with
plasmid p770
and pSV2neo (Subramani et al., 1981), and
selected in 0.4 mg/ml G418. Resistant colonies were expanded and
assayed for expression of APP770
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).
Figure 6:
Release of APP 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 CHO770
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
from CHO770 (b) or CHO770
(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
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 CHO770 (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.
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).
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.
Figure 2:
Rapid degradation of APP-TR chimeras in a
post-Golgi endocytic compartment. Equivalent cell numbers of CEF
expressing wild-type (WT), TR (), or APP-TR (
)
chimera were pulse-labeled for 30 min with Trans
S-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 (NHCl)
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
Cl and then
pulse-labeled with Trans
S-label and chased in the presence
of same concentration of NH
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 (), AENPTA
(
), ATSI (
), 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.
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
(
) or acid-insoluble
I-labeled Tf (
) released
into medium, as well as surface-bound
I-labeled Tf
(
) 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.
To address the hypothesis that YENPTY influences APP release by mediating plasma membrane endocytosis of APP, we
compared levels of APP
derived from the surface-resident
population of APP molecules from CHO770 or CHO770
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 CHO770
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
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 CHO770
(compare Fig. 6, b, lane 1, and c, lane 1)
which is unexpected given the apparent higher efficiency cleavage of
mutant APP.
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) 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
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; 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 NHCl 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 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
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 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 A
can be generated from APP following
internalization from the cell surface and delivery to the endocytic
pathway (Koo and Squazzo, 1994).