(Received for publication, September 21, 1994; and in revised form, December 9, 1994)
From the
The analysis of potential sorting signals in amyloid precursor
protein (APP) by site-directed mutagenesis and the disturbance of
metabolic pathways by drugs is used here to define the parameters that
determine polarized secretion of APP in Madin-Darby canine kidney
cells. Endogenously produced APP751/770 and APP695 produced from
transfected constructs are secreted almost exclusively into the
basolateral compartment. The sorting mechanism is highly dependent on
intracellular pH as demonstrated by its sensitivity to primary amines
and inhibitors of the acidifying vacuolar proton ATPase. The role of
potential basolateral sorting signals in the cytoplasmic,
transmembrane, and A4 amyloid region of APP was investigated.
Neither deletion of the endocytosis and putative basolateral sorting
signal GY.NPTY nor complete deletion of the cytoplasmic domain causes
apical secretion of soluble APP. Further deletion of the transmembrane
domain and of the
A4 amyloid region confirmed that the major
basolateral sorting determinant resides in the extracellular domain of
APP. Increased
-secretase cleavage of APP after introduction of
the ``swedish'' double mutation causes apical missorting of
about 20% of
-secretase-cleaved APP. The data underline the
complexity of processing and sorting APP in polarized cells and suggest
a possible problem of protein sorting in Alzheimer's Disease.
Amyloid precursor protein (APP), an integral
membrane protein with unknown function, is the precursor of a series of
soluble proteins and peptides, including the
A4 amyloid peptide ((1, 2, 3, 4, 5) ; reviewed
in (6) ). This peptide is the main component of the amyloid
plaques, one of the neuropathological hallmarks of Alzheimer's
Disease (AD)(7, 8) . Although the direct relationship
between
A4 amyloid peptide deposition and the progressive neuronal
death observed in the brains of these patients remains to be clarified,
genetic data demonstrate the central role of APP in the pathogenesis of
AD, since point mutations in APP are linked to the familial form of
AD(9, 10, 11, 12, 13) .
Furthermore, a gene dosage effect of the APP gene on chromosome 21 is
implicated in AD associated with trisomy 21 (Down's
syndrome)(14) . Finally, the
A4 peptide is under certain
experimental conditions toxic for neuronal cells in
vitro(15, 16) .
Proteolytic processing of APP
occurs at three sites in the protein, either amino-terminal and
carboxyl-terminal of the A4 sequence, producing the
A4
peptide, or in the middle of the
A4 peptide to release the
ectodomain and thus precluding
amyloidogenesis(6, 17) . The
``amyloidogenic'' cleavages are mediated by hypothetical
- and
-secretases, whereas the ``nonamyloidogenic''
cleavage is mediated by
-secretase(s)(17, 18) .
Although none of these enzymes have been identified yet, available
evidence and conjecture points to some unusual characteristics. The
-secretase has to cleave in the APP transmembrane region, raising
conceptual problems regarding the mechanism. The
-secretase has an
extremely relaxed specificity regarding the primary amino acid sequence
cleaved but requires the presence of the transmembrane domain of
APP(19, 20, 21) . It was proposed that
several proteinases can perform
-secretase cleavage(18) ,
which in turn raises the question of how other integral membrane
proteins avoid this activity. An ``secretase compartment'' to
which APP is specifically targeted was proposed to be the trans-Golgi
network (TGN) or the transport vesicles en route toward the cell
surface(21, 22, 23, 24) . Since
deletion of the cytoplasmic domain carrying the putative sorting
signals GY and NPTY increased the secretion of APP, these signals were
suggested to target APP toward nonsecretory, intracellular pathways (21) . The different intracellular pathways that are open for
APP, characterized by specific processing and sorting steps in specific
subcellular compartments, have to be studied. Given the complexity of
the cellular protein sorting mechanisms, it is conceivable that the
different genetic and epigenetic factors believed to underlie the
pathogenesis of AD (25, 26, 27) interfere at
one or another level with the mechanisms governing the cellular
processing and sorting pathways followed by APP. In that regard, it
should be noted that the amyloid lesions of AD are associated with two
types of polarized cells, i.e. neurons and endothelial cells.
The best characterized model to study protein sorting is the Madin-Darby canine kidney (MDCK) cell line(28, 29, 30) . These cells form a well polarized, tight epithelial cell layer in vitro, separating apical and basolateral compartments. Moreover, an interesting parallel between MDCK cells and neuronal cells is the fact that proteins of the apical and basolateral membrane of MDCK cells are targeted to axonal and somatodendritic processes, respectively, in primary cultures of hippocampal neurons(31, 32, 33) .
To
extend our previous studies of APP processing and secretion in
unpolarized cells, we decided to approach the problem of polarized
sorting and metabolism of APP in the MDCK model. Previously we have
demonstrated that APP secretion is subject to modulation by metabolic
factors and structural determinants(21) . We now report that
APP is secreted in MDCK cells in a strictly polarized fashion,
confirming a recent report(34) . The correct sorting of wild
type, soluble APP is extremely sensitive to pH changes in intracellular
compartments as demonstrated with primary amines and bafilomycins,
indicating the involvement of an acid-sensitive sorting mechanism. We
further demonstrate that potential basolateral sorting signals in the
cytoplasmic domain of APP do not determine the polarized secretion of
APP and that the major basolateral sorting signal is located in the
extracellular domain. Furthermore, we find that a fraction of APP
terminating at either the - or the
-secretase cleavage site
is missorted to the apical compartment. We finally demonstrate that APP
containing the ``swedish'' mutation(12) , which is
linked to familial AD, not only causes overproduction of
A4
peptide in certain cell types (35, 36) but is also
partially missorted after
-secretase cleavage to the apical
compartment in polarized MDCK cells.
Rabbit antiserum B2/3 against
mouse APP was previously characterized (21, 22) ; goat
antiserum 207 against soluble APP (37) was generously provided
by Dr. B. Greenberg (Cephalon, West Chester, PA); rabbit antiserum
R1736 against residues 2-15 of the A4 peptide (4, 34) was kindly provided by D. Selkoe (Boston).
The cDNA for mouse APP has previously been characterized(38) . CDNA coding for human APP695 was kindly provided by Dr. R. Scott (Cephalon, West Chester, PA). CDNA coding for human APP695 containing the early onset familial Alzheimer's Disease (EOFAD) mutations APP695(H)617A:G (13) , APP695(H)618E:Q(9) , APP695(H)595K:N/596 M:L, swedish mutation(12) , and APP695(H)642V:I (11) were synthesized by site-directed mutagenesis (L. Hendriks and C. Van Broeckhoven, Antwerp, Belgium).
The tightness of cell
layers was tested by adding [H]inulin (Amersham
Corp.) to the apical compartment and measuring the amount of label that
leaked to the basolateral compartment after 1 h. This was always less
than 1%. The polarity of the cells in the culture conditions used was
ascertained by confirming the polarized secretion of gp80 (39) and the polarized uptake of
[
S]methionine. In addition, preliminary
experiments proved that the secretion of endogenous APP751/770 was
actually a very reliable basolateral marker, which functions in all
transfection experiments as an internal control (see
``Results'').
For transfection, 30 µg of linearized
pRC/RSV plasmid containing the cDNA of mouse APP695 or mutants thereof
or 30 µg of circular pSG5 plasmid containing the EOFAD APP695(H)
mutants with 1 µg of circular pRC/RSV plasmid was added to 2.5
10
cells suspended in 0.5 ml of phosphate-buffered
saline. Electroporation was done at 260 V, 960 microfarads
(Gene-Pulser; Bio-Rad). The time constant varied between 16 and 22 ms.
After transfection, cells were cultured for 24-48 h in medium
supplemented with penicillin/streptomycin. Selection of transfected
cells was performed in medium containing 700 µg/ml G418 sulfate
(Geneticin; Life Technologies, Inc.). After 2 weeks, cells were tested
for expression of the transfected constructs. Transfected cells were
used for a maximum of five passages. To demonstrate that a high level
of overexpressed, transfected APP per se is not sufficient to
target APP toward the apical compartment, high expression clones of
MDCK cells transfected with APP695(M) and APP695(M)Ala666*, were
isolated by limiting dilution.
Concentrated stocks of drugs were diluted into culture medium. For the alkalization experiments, cell layers were preincubated with the drugs for 30 min, and labeling was performed in the presence of the drug. Phorbol esters (PMA and Pdbu) and cholera toxin, pertussis toxin, and forskolin were present only during labeling. Control experiments ascertained that dimethyl sulfoxide used as a solvent for certain drugs at the dilutions used (1:1000) did not affect the cells.
cDNA coding for human APP695(H) was cloned as a HindIII fragment in the expression vector pSG5 (Stratagene). This vector allows transcription of the cloned cDNA from the SV40 early promoter. Mutants of mouse and human APP695 (see Fig. 1) were generated by site-directed mutagenesis in the pSG5 plasmid(40) . Codons coding for Tyr 653, Leu613, and Asp597, respectively, were mutated toward a TAG stop codon, yielding APP695(M)Y653*, APP695(M)L613*, and APP695(M)D597* (see Fig. 1). APP695(M)A626* was made by introducing a cassette in the BbeI restriction site of APP(M)695, changing codon 626 to a stop codon. The generation of APP695(M)A666*, previously named APP695TRUNC, has been described in detail elsewhere(21) .
Figure 1:
Deletion mutants of mouse APP695. The
domain structure for mouse APP (which is 97.6% identical to human APP) (38) is shown. SP, signal peptide; A4,
amyloid peptide; TM, transmembrane domain; CD,
cytoplasmic domain. The
-secretase cleavage site is indicated by a solid triangle. The displayed deletion mutants were obtained
by introducing stop codons at the indicated positions (see
``Experimental Procedures''). Below the structure of
APP695(M), the regions to which antibodies 207, B2/3, and R 1736 were
raised are indicated.
All constructs were analyzed by restriction analysis and by sequencing of the mutated sites.
In
double immune precipitation assays, 1 ml of basolateral medium or 0.6
ml of apical medium was used. Immune precipitation was performed with
20 µl of rabbit antiserum B2/3 against mouse APP, 3 µl of goat
antiserum 207 against soluble APP, 3 µl of R1736 prepared against
synthetic human A4 residues 2-15, 20 µl of rabbit
antiserum B7/7, or 20 µl of antiserum SGY2134 prepared against
synthetic
A4 peptide. All samples were brought to equal volumes
with phosphate-buffered saline and 10
double immune
precipitation buffer (Tris-buffered saline containing 10% Triton X-100,
10% sodium deoxycholate, and 1% SDS(41) ). Recovery of the
immune complexes was done with protein A-Sepharose CL4B (Pharmacia
Biotech Inc.) or protein G-agarose (Immunopure; Pierce). Processing of
immune complexes was essentially as described
previously(21, 41) . 6% polyacrylamide gels were
impregnated with dimethyl sulfoxide/2,5-diphenyloxazole, dried, and
exposed to preflashed Hyperfilm MP (Amersham Corp.)(21) .
Exposure varied from 1 day to 3 weeks. Quantitation was performed by
densitometric laser scanning(21) .
Figure 2:
Basolateral secretion of APP in polarized
MDCK cells. Panel a, endogenous APP was immune precipitated
with antibody 207 from apical (A) and basolateral culture
media (B) of untransfected MDCK cells after a 2-h metabolic
labeling with [S]methionine. Negative controls,
using only protein G-sepharose are shown on the right. Note
the presence of a protein of about 500 kDa in the apical compartment,
precipitated by protein G. Panel b, MDCK cells were
transfected with mouse APP695 cDNA. A high expressing clone was
obtained by limiting dilution. Cells were labeled for 2 h, and immune
precipitation was performed with antibody B2/3. Lanes on the right are negative controls using MDCK cells transfected with
pRC/RSV vector alone. Panel c, transfected mouse APP695 and
endogenous canine APP751/770 were immune precipitated with antibody
B2/3. Cells were pulse-labeled for 10 min and chased for the time
periods indicated. A, apical, B, basolateral
compartment. Soluble mouse APP695 as well as canine APP751/770 is
weakly visible after 20 min of chase in the basolateral compartment,
whereas no APP is precipitated from the apical compartment, even after
180 min of chase.
MDCK cells were transfected with recombinant DNA constructs coding for mouse or human APP695 or for human APP770. Transfected APP695 yielded proteins that migrated with a higher mobility in SDS-PAGE than endogenous APP (Fig. 2, b and c), whereas transfected APP770 co-migrated with the endogenous protein (result not shown), demonstrating that the endogenously produced canine APP contained the Kunitz type proteinase inhibitor domain. We could not determine whether either APP751 or APP770 or both were present, which is without further consequences for our experiments. Overexposure of immunoblots or immune precipitations indicated that the MDCK cells also produced very small amounts of endogenous APP695. The basolateral secretion of endogenous canine APP was used in subsequent transfection experiments as an internal control, demonstrating that the sorting machinery remained operational in the transfected cells (Fig. 2c and all other figures).
When transfected mouse APP695 was expressed at a level that was at least 10-fold higher than that of endogenous APP, secretion remained almost exclusively into the basolateral compartment (Fig. 2b), which indicated that the APP-sorting mechanism is characterized by a high efficiency and a high capacity. In addition, pulse-chase experiments demonstrated that endogenous and transfected APP were secreted with similar fast kinetics into the basolateral compartment (Fig. 2c). This demonstrated that endogenous and transfected APP were transported directly to the basolateral side and that secretion of APP does not involve transcytosis. After a 3-h chase, no APP was detected in the apical compartment, confirming the strongly polarized secretion of APP (Fig. 2c).
Figure 3: Effect of methylamine and pH on secretion of endogenous APP in MDCK cells. Panel a, untransfected MDCK cells were preincubated for 30 min and labeled for 120 min in culture medium buffered at the indicated pH, without (-MA) or with the addition of 20 mM methylamine (+MA). APP was immune precipitated with antibody 207. Panel b, quantitative analysis of three independent experiments performed as in panel a. The increased apical (A) and decreased basolateral (B) secretion of APP in the presence of methylamine (+MA), compared with the basolateral secretion of APP in the absence (-MA) of methylamine is demonstrated. Changing the pH of the medium alone does not influence basolateral APP secretion (B). The apical secretion in the absence of methylamine was always lower than 5% and is not displayed. Every point is the mean ± S.E. of three separate experiments in which the secretion in every compartment was compared with the total secretion of APP under control conditions.
The effect of methylamine was concentration-dependent (Fig. 4, a and b). Total metabolic incorporation of radiolabeled methionine in trichloroacetic acid-precipitable protein in the presence of 10 or 30 mM methylamine was 100.8 and 99.2%, respectively, of the control, indicating that cell viability and metabolism, as measured by overall protein synthesis, was not affected. Finally, the effect of methylamine was reversible after 1 h of incubation with normal medium (Fig. 4c).
Figure 4:
Effect of primary amines, bafilomycin A1,
and other drugs on polarized APP secretion. Panel a, MDCK
cells transfected with APP695(M) were preincubated (30 min) and
metabolically labeled (120 min) in medium containing the following
drugs: no additions (control), 0.2 mM monodansylcadaverine (MDCV), 20 mM 6-amino-1-hexanol (Am.Hex), 40 mM glycylglycine (Glygly), 30 mM methylamine (30 mM MA), 10 mM methylamine (10 mM MA),
30 mM ammonium chloride (30 mM NH),
10 mM ammonium chloride (10 mM NH
), 0.3 mM chloroquine (chlq).
Double immune precipitation was performed with antibody B2/3. Panel
b, Untransfected MDCK cells were treated as in panel a with no drug (control), 10 mM NH
Cl,
10 or 30 mM methylamine (MA), or 100 mM and
1 µM bafilomycin A1 (Baf A1). Bars represent the mean (with S.E.) apical (A) and basolateral (B) secretion of canine APP in three separate experiments. The
total secretion (apical + basolateral) is taken as 100%. Panel
c (upper part), untransfected MDCK cells were
preincubated and labeled in the presence of 20 mM methylamine (MA), 100 nM bafilomycin A1 (Baf), or 40
mM glycyl-glycine (Glygly). Cont, control,
with no drugs added. Apical (A) and basolateral (B)
medium was recovered and immune precipitated with antibody 207. Panel c (lower panel), cells were further incubated
with medium containing no drugs during 1 h. A second round of labeling
(30 min of preincubation, 2 h of labeling) was performed without any
addition of drugs. Immunoprecipitation of the medium was again
performed with antibody 207. Note the presence of a 65-kDa protein in
the apical and basolateral compartment appearing after washing out
bafilomycin A1. This represents a proteolytic fragment of APP, the
significance of which is unclear at this
moment.
Other primary amines such as ammonium chloride (10 or 30 mM), ammonium acetate (20 mM), and 6-amino-1-hexanol (10 or 30 mM) caused secretion of endogenous APP751/770 and of transfected APP695 in the apical compartment (Fig. 4a). Glycyl-glycine (20-40 mM), chloroquine (0.3 mM), and monodansylcadaverine (0.1 or 0.2 mM), in contrast, did not significantly affect the polarized secretion of APP toward the basolateral compartment of MDCK cells (Fig. 4a).
Other drugs such as forskolin (10 µM), cholera toxin (2 µg/ml), and pertussis toxin (2 µg/ml) did not significantly affect the polarized secretion of APP in the conditions used, whereas the phorbol esters PMA (1 µM) and Pdbu (1 µM) were confirmed to increase the basolateral secretion of APP (34) (results not shown).
The known alkalization by primary amines of intracellular
compartments, such as the basolateral endosome, the lysosomes, or the
trans-Golgi network, can be mimicked by drugs of the macrolide family
that inhibit specifically the vacuolar proton ATPase(44) .
Incubation of MDCK cells with bafilomycin A1 (10 nM, 100
nM, and 1 µM), bafilomycin B1 (10 nM and
100 nM), concanamycin A (10 nM, 100 nM, and
1 µM), and concanamycin C (100 nM) essentially
randomized the secretion of APP to the apical and the basolateral
compartment (Fig. 4b, and results not shown). The
effect of bafilomycin A1 was not reversed by incubation for 1 h without
the drug (Fig. 4c). A possible explanation is the high
affinity of bafilomycin A1 for the vacuolar H-ATPase,
although this was not further investigated. The concentrations used
here are in the same range as previously demonstrated by others to
inhibit specifically the vacuolar proton
pump(44, 45) .
Figure 5:
Missorting of deletion mutants of
APP695(M) in MDCK cells. Panels a and b, MDCK cells
were transfected with wild type APP695(M) (wild type),
APP695(M)A666* (A666*), APP695(M)Y653* (Y653*),
APP695(M)A626* (A626*), APP695(M)L613* (L613*), and
APP695(M)D597* (D597*), respectively; see Fig. 1for a
schematic representation of these mutants. Cells were labeled for 2 h,
and immune precipitation was performed with antibody 207. Apical (A) secretion of APP (10-18%) was observed after
transfection with mutant APP terminating at the -secretase
cleavage site (D597*), at the
-secretase cleavage site (L613*), or at the transmembrane domain (A626*). Panel c, a high expression MDCK clone transfected with
APP695(M)A666* was obtained by limiting dilution. Cells were labeled
for 2 h and APP in the apical and basolateral medium was immune
precipitated with antibody B2/3. MDCK cells overexpressing wild type
APP695 and transfected with pRC/RSV plasmid (control), containing no
insert (see Fig. 2b) are also shown. Whereas the
expression level of the transfected constructs in these cells is at
least 10-fold higher than that of endogenous APP, both transfected wild
type APP695(M) and APP695(M)A666* are secreted exclusively into the
basolateral compartment.
To investigate the role of the
amyloid domain in the polarized secretion of APP, stop codons were
introduced at positions Leu (APP695(M)L613*) and
Asp
(APP695(M)D597*), to produce soluble APP terminating
at the
- and at the
-secretase cleavage sites,
respectively(6) . Both soluble APP forms were still observed to
be mainly secreted into the basolateral compartment, although
10-15% of the total secreted APP was sorted toward the apical
compartment (Fig. 5b).
Figure 6:
Apical missorting after transfection with
the swedish EOFAD APP mutant. Panel a, MDCK cells were
transfected with APP695(H)617A:G (617A:G), APP695(H)618E:Q (618E:Q), APP695(H)595K:N/596 M:L (Swed, swedish
mutation), and APP695(H)642V:I (642V:I). These APP mutations
cause familial Alzheimer's Disease or hereditary cerebral
hemorrhage with amyloidosis-Dutch type. Labeling was performed
overnight, and double immune precipitations on apical (A) and
basolateral (B) compartments were performed with antibody 207.
Apical secretion (10-20%) is observed only by transfection with
the swedish mutant APP. Panel b, apically secreted APP
containing the swedish mutation is generated by -secretase. MDCK
cells transfected with APP695(H)595K:N/596 M:L (swedish mutation) were
labeled for 2 h. Apical (A) and basolateral (B)
medium were consecutively immune precipitated two times with antibody
R1736, which recognizes residues 2-15 of the
-amyloid
peptide and therefore precipitates only
-secretase-cleaved APP.
Antibody 207, which recognizes both
- and
-secretase-cleaved
APP, was used in one further round of immune precipitation to recover
-secretase-cleaved APP. Note that APP containing the swedish
mutation and secreted into the apical compartment is only precipitated
with antibody 207. Most
-cleaved APP, however, remains secreted
into the basolateral compartment. Note the slight difference in
mobility of
-secretase-cleaved APP in the apical compartment in panel a.
In
other transfection experiments, MDCK cells were obtained in which no
apical missorting of the swedish APP-mutant was observed (result not
shown). Further analysis demonstrated that in these cases also no
-secretase activity (Fig. 6) could be demonstrated.
Although further investigations are needed to explain this variation in
-secretase activity, the conclusion that only
-secretase-cleaved APP is missorted is supported by this
observation.
We investigated the secretion of APP in polarized MDCK cells. This cell line is by far the best studied model system for protein sorting (29, 30) . Moreover, certain aspects of the sorting mechanism operate similarly to that of hippocampal neurons(31, 32) . The physical separation between apical and basolateral compartments in MDCK cells cultured on polycarbonate filters permits the analysis and comparison of sorting of wild type and mutant APP as well as the study of drugs in a qualitative and quantitative fashion. This is currently not feasible in primary cultures of hippocampal neurons because of practical limitations.
The major observations are, first, that endogenously produced canine
APP is secreted in a strictly polarized fashion in MDCK
cells(34) , being sorted to the basolateral compartment by a
mechanism that is characterized by its high fidelity and capacity. More
than 10-fold overexpression of wild type APP695 did not result in any
apparent apical secretion. Advantage of this observation can be taken
by using the basolateral secretion of endogenous canine APP751/770 as
an internal control and marker for the basolateral compartment. This
result also demonstrates that APP (which is transported by fast axonal
transport in neurons) is an exception to the rule that basolateral
sorted proteins in MDCK cells are sorted somatodendritically in neurons (31, 32, 33) . Second, we demonstrate the
extreme sensitivity of the sorting mechanism to intracellular
alkalization. This indicates that the sorting mechanism resides in an
intracellular acid compartment, e.g. the trans-Golgi network
or the basolateral endosome, and that sorting per se is a
pH-sensitive process. Third, deletion of the cytoplasmic domain with
the NPTY endocytosis and potential basolateral sorting signal (46, 47) did not markedly affect the basolateral
secretion of APP. Deletion of nearly the entire cytoplasmic domain by
introducing a stop codon at Tyr affected the polarized
secretion of APP also only marginally (5-14%). Although the
putative sorting signals in the cytoplasmic domain could be important
for the targeting of uncleaved, membrane-bound APP, our results
demonstrate that the fraction of APP destined for secretion is sorted
by means of a determinant in the extracellular domain. This is
confirmed by the results obtained with soluble APP mutants terminating
at the transmembrane domain, at the
-cleavage site, or at the
-cleavage site. Fourth, all of the soluble mutants tested
displayed a small but significant leakage to the apical compartment,
amounting to 10-18% of the total pool of secreted APP. The direct
consequence is that about 5-20-fold more soluble APP is
accumulating in the apical compartment, compared with wild type APP.
Finally, we observed that a fraction of soluble,
-secretase-cleaved APP was targeted to the apical compartment
after introduction of the swedish point mutations (12) in the
APP protein.
In MDCK cells, integral membrane and soluble proteins
destined for the apical or the basolateral surface are sorted in the
trans-Golgi network (TGN)(28, 29, 30) . A
second sorting site is the basolateral endosome, from which endocytosed
basolateral proteins can recycle to the basolateral domain, travel to
the lysosomes, or transcytose to the apical domain (29, 30) . Similar basolateral sorting signals are
operative in the TGN and the basolateral endosome(47) . Since
APP is synthesized as an integral membrane protein to become processed
into a soluble form, it is unclear whether APP is sorted as an integral
membrane protein (in which case the cytoplasmic domain is expected to
contain the major basolateral sorting determinants) or as a soluble
protein (in which case the sorting determinants must reside in the
extracellular domain). The subcellular localization of the processing
-secretase is crucial in this regard: if cleavage of APP occurs
before the TGN is reached, APP would be sorted as a soluble protein.
Our results obtained with the cytoplasmic deletion mutants and with the
soluble APP mutants terminating at the
- and the
-secretase
cleavage site provide clear evidence for a major basolateral sorting
determinant in the extracellular domain of APP: all APP mutants,
soluble or not, remain essentially targeted to the basolateral
compartment. Moreover, addition of the broad spectrum proteinase
inhibitor
2-macroglobulin (48) in both apical and
basolateral compartments did not affect APP secretion, which is in
agreement with the idea that the bulk of
-secretase activity in
MDCK cells is located
intracellularly(21, 22, 23, 24) .
The soluble mutants (APP695(M)A626*, APP695(M)L613*, and
APP695(M)D597*) are all, however, less efficiently sorted than wild
type APP. The apical sorting of a fraction (10-18%) of soluble
APP is not the consequence of overexpression of the mutants, since low
expression of the APP695(M)L613 mutant yielded similar results, and a
more than 10-fold overexpression of wild type APP695 and
APP695(M)Ala666* did not cause apical secretion. Moreover, the measured
impermeability of the cell layer for [H]inuline
and the exclusive basolateral secretion of endogenous canine APP751/770
excluded leakage from the basolateral to the apical compartment in
these experiments. As stated above, this endogenous APP functions as a
nearly perfect marker for the basolateral secretion compartment of
MDCKII cells. Some apical missorting of soluble APP via apical
transport vesicles in the TGN, following the bulk of soluble proteins
that are secreted apically in MDCK cells, is therefore the most likely
explanation.
Methylamine and other primary amines disturb secretion
and sorting of APP in MDCK cells, resulting in the preferential
secretion of APP into the apical compartment. At the concentrations
used, the metabolic incorporation of, and labeling by
[S]methionine was not affected, and the cell
layer impermeability was not significantly altered. The drugs were
easily washed out and the basolateral secretion restored by incubating
the cells in normal medium for a relatively short time period. The same
effect is observed with related drugs such as ammonium chloride,
ammonium acetate, and 6-amino-1-hexanol. Glycyl-glycine, used as a
control, did not affect the polarity of APP secretion. The effect of
methylamine was accentuated by alkalization of the medium, which also
caused an unexpected substantial increase in the total amount of
(mainly apically) secreted APP. This increase in secretion is most
easily explained by blocking an as yet undefined but
degradation-oriented pathway and redirection of APP into the apical
secretory pathway. This is corroborated by data showing that treatment
of MDCK cells with primary amines results in randomized sorting of
lysosomal proteinases and secretion in the extracellular
medium(43) . In unpolarized cells an unknown fraction of APP is
targeted to the lysosomes(2) . Whereas the inhibition of
basolateral APP secretion in MDCK cells after methylamine treatment is
in line with the inhibition of APP secretion observed in unpolarized
cells(21) , the stimulation of APP secretion along the apical
pathway in MDCK cells suggests differences between the apical and the
basolateral secretory transport machinery, at least as far as APP is
concerned. It is clear that the effect of methylamine in MDCK cells is
partially mimicked by bafilomycins and concanamycins, drugs that
specifically block the vacuolar H
-ATPase(44) .
Therefore, the combined observations with primary amines and specific
H
-ATPase inhibitors in MDCK cells demonstrate that an
acid compartment is involved in the correct sorting of APP and that at
least part of the missorting of APP is explained by alkalization of
this compartment. It is likely that binding of the ectodomain of APP to
receptors or chaperones that are responsible for its specific
basolateral sorting are pH-sensitive. This phenomenon is essential for
recycling of endocytosis receptors (50) and was also postulated
for the basolateral sorting of soluble laminin and proteoglycan in MDCK
cells(43) . On the other hand, recent evidence suggests that
maintenance of the acid pH of microsomal vesicles is essential to bind
small GTP binding proteins and ADP ribosylation factor, which are
involved in the specific coupling of transport vesicles to their target
membranes(51, 52) . The possibility that vesicular
alkalization interferes with such incompletely understood targeting
mechanisms is therefore an alternative interpretation, which can be
approached experimentally.
Interestingly, -secretase cleavage
of the swedish mutant APP, as opposed to
-secretase cleavage of
normal APP, resulted in apical leakage of a fraction of soluble APP.
The results obtained with soluble APP mutants terminating at either the
-secretase cleavage site or the
-secretase cleavage site,
rule out the possibility that the basolateral sorting signal is in the
A4 amyloid sequence itself. The missorting of truncated soluble
APP mutants, and of
-cleaved APP with the swedish mutation
suggests, therefore, that merely the soluble character (defined as
absence of the cytoplasmic and integral membrane domain) is responsible
for the partial mistargeting of APP to the apical compartment, in line
with what we discussed above. The fact that the
-secretase-cleaved
swedish mutant APP was mistargeted to the apical compartment
(10-20%) to a similar degree as the synthetic soluble APP mutants
is compatible with the hypothesis that the swedish mutant is cleaved by
-secretase early in the biosynthetic pathway, before the TGN is
reached. Its further behavior is then identical to that of the soluble
transfectants.
In conclusion, our results demonstrate that small structural or metabolic persistent disturbances of the protein-sorting machinery of polarized cells could be involved in the abnormal processing of amyloid precursor protein. The possibility that missorting of APP plays a role in the pathogenesis of certain forms of Alzheimer's Disease requires further investigation.
Addendum-After this paper was submitted, we learned that the work of C. Haass et al.(53) confirms our findings completely.