Báo cáo khoa học: Shedding of the amyloid precursor protein-like protein APLP2 by disintegrin-metalloproteinases Retinoic acid-induced upregulation of substrate and proteinase ADAM10 during neuronal cell differentiation ppt

Shedding of the amyloid precursor protein-like protein
APLP2 by disintegrin-metalloproteinases
Retinoic acid-induced upregulation of substrate and proteinase
ADAM10 during neuronal cell differentiation
Kristina Endres1, Rolf Postina1, Anja Schroeder2, Ulrike Mueller3 and Falk Fahrenholz1
1 Institute of Biochemistry, Johannes Gutenberg-University Mainz, Germany
2 ZVTE, Johannes Gutenberg-University Mainz, Germany
3 Institute for Pharmacia and Molecular Biotechnology, University of Heidelberg, Germany

Keywords
ADAM10; Alzheimer’s disease; amyloid
precursor protein-like protein 2; retinoic acid;
tumor necrosis factor-a converting enzyme
Correspondence
F. Fahrenholz, Institute of Biochemistry,
Johannes Gutenberg-University, Becherweg
30, D-55128 Mainz, Germany
E-mail:
(Received 27 June 2005, revised 14
September 2005, accepted 16 September
2005)
doi:10.1111/j.1742-4658.2005.04976.x

Cleavage of the amyloid precursor protein (APP) within the amyloid-beta
(Ab) sequence by the a-secretase prevents the formation of toxic Ab peptides. It has been shown that the disintegrin-metalloproteinases ADAM10
and TACE (ADAM17) act as a-secretases and stimulate the generation of
a soluble neuroprotective fragment of APP, APPsa. Here we demonstrate
that the related APP-like protein 2 (APLP2), which has been shown to be
essential for development and survival of mice, is also a substrate for both
proteinases. Overexpression of either ADAM10 or TACE in HEK293
cells increased the release of neurotrophic soluble APLP2 severalfold. The

strongest inhibition of APLP2 shedding in neuroblastoma cells was
observed with an ADAM10-preferring inhibitor. Transgenic mice with neuron-specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C-terminal fragments. To elucidate a possible
regulatory mechanism of APLP2 shedding in the neuronal context, we
examined retinoic acid-induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of
soluble APLP2.

The amyloid precursor protein (APP) is a member of a
protein family in mammals that includes the APP-like
proteins APLP1 and APLP2 [1]. All APP ⁄ APLP family members are type I integral membrane proteins
with large extracellular ectodomains and short cytoplasmic tails. Compared with APP, both APLPs are
highly homologous in their amino acid sequence (e.g.
APLP2 ⁄ APP 52% identical, 71% similar) [2] and are
proteolytically processed in a similar way. The N-terminal ectodomains are released by a shedding enzyme
[2,3], whereas the C-termini remain in the membrane

[2,4,5] and can be further processed to release a cytoplasmic fragment with signaling properties [4,6,7].
Further elucidation of APLP2-processing is of relevance with regard to the outstanding function of this
protein, which was derived from knockout experiments.
Whereas a double knockout of APP and APLP1 did
not show severe phenotypic changes in mice, the combined knockout of APLP2 with both of the other APP
family members resulted in postnatal lethality [8,9].
This shows that APLP2 and ⁄ or one of its proteolytic
fragments are essential for normal development and

Abbreviations
ADAM, a disintegrin and metalloproteinase; ADAM10DN, catalytically inactive dominant negative mutant form of ADAM10; APLP1, APP-like
protein 1; APLP2, APP-like protein 2; APLP2s, cleaved soluble APLP2; APP, amyloid precursor protein; BACE, b-site APP-cleaving enzyme;
CS-GAG, chondroitin sulfate glycosaminoglycan; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PVDF, poly(vinylidene
difluoride); RA, retinoic acid; TACE, tumor necrosis factor-a converting enzyme.

5808

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS


K. Endres et al.

survival, and may compensate the lack of either APP or
APLP1. Whereas APP orthologs have been identified in
lower and higher vertebrates, a recent publication
revealed the existence of the first nonmammalian
APLP2 in Xenopus laevis and its overall high percentage of conserved amino acids implies an important role
for this member of the APP superfamily [10]. Although
BACE [11–13] and the c-secretase [6,22] have previously been identified as proteinases involved in the proteolytic processing of the APP relatives, it remains to
be shown whether APLPs are also subject to cleavage
by disintegrin-metalloproteinases (ADAMs) which act
as a-secretases for APP [14–16].
Shedding of APLP2 can be induced by activation
of protein kinase C (PKC) in human corneal epithelial
cells [17]. Moreover, a decline in the membraneanchored C-terminal fragments of APLP1 and APLP2
by the hydroxamic acid-based inhibitors batimastat
and TAPI-2 was shown recently [11]. Using deletion
mutants, metalloproteinase-dependent cleavage of
APLPs was shown to occur at a similar distance to the
membrane as is known for APP. Thus, an a-secretaselike activity seems to release the APLP2 ectodomain,
but the proteinases involved are not yet identified.
Three members of the ADAM family have been
shown to act as a-secretases [14,15,18]. We restricted
our investigations on APLP2 shedding to ADAM10
and tumor necrosis factor-a converting enzyme

(TACE, ADAM17), because purified ADAM9 failed
to cleave a synthetic APP peptide at the major a-secretase cleavage site [19], and ADAM9 knockout mice
exhibit unchanged APP processing [20]. ADAM10, in
contrast, was recently shown to process APP in vivo
and to prevent plaque formation in an Alzheimer’s disease mouse model [16].
ADAM10 and TACE, which cleave APP, have
been implicated in ectodomain shedding of other substrates such as cytokines [21], growth factors and
their receptors [22,23], and adhesion molecules [24].
If ADAMs have several cellular substrates, how are
physiologically relevant processing events coordinated? One possibility is a common up- or downregulation of substrate and sheddase during cell-fate
decisions. Differentiation of neuronal cell types
through retinoic acid (RA) leads to the upregulation
of both APP [25] and APLP2 [26,27]. Therefore, we
investigated the effect of RA on ADAM10 and
TACE expression in neuroblastoma cell lines. In this
study we provide evidence for a common upregulation of ADAM10 and its newly identified substrate
APLP2 by RA-induced neuronal cell differentiation
which resulted in an enhanced release of neurotrophic
secreted APLP2 [28].
FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS

APLP2-shedding by disintegrin-metalloproteinases

Results
Phorbol-12-myristate-13-acetate-induced APLP2
ectodomain shedding
To study the effect of the PKC activator phorbol-12myristate-13-acetate (PMA) on endogenous APLP2
shedding, we stimulated HEK293, SKNMC and
SH-SY5Y cells with 1 lm PMA and performed Western
blot analysis of proteins from cell supernatants. It has

been shown that a large fraction of APLP2 and its secreted soluble derivative is modified by the addition of
chondroitin sulfate glycosaminoglycan (CS-GAG) at a
single site (Ser614) in the extracellular domain. This
gives rise to the secretion of molecules with an apparent
molecular mass between 130 and 170 kDa (Fig. 1). Two
minor sharp bands between 95 and 120 kDa probably
represent, according to earlier studies, APLP2s and
truncated APLP2s without CS-GAG-modification (for
post-translational modification of APLP2 see Slunt
et al. [2] and Thinakaran and colleagues [29,30]). PMA
treatment of all tested cell lines resulted in a significant
increase in secreted endogenous APLP2 indicating that
shedding of APLP2, like that of APP, is stimulated by
PMA in neuronal and non-neuronal cell lines (Fig. 1).
Inhibition of APLP2 ectodomain shedding
by metalloproteinase inhibitors
It is known that the shedding of various transmembrane substrates is inhibited by hydroxamic acid-based
inhibitors [31,23,32]. GM6001, a broad-spectrum
hydroxamic acid-based inhibitor of matrixmetalloproteinases (MMPs) and ADAMs, decreased basal APPsa
and APLP2s secretion to 60 and 75%, respectively, of
untreated cells (Fig. 2A,B, lanes 1 and 3) and reduced
the PMA-stimulated amount of both shed ectodomains
to almost the level of control cells without inhibitor
HEK293

SKNMC

SH-SY5Y

148 kDa

98 kDa

PMA

-

+

-

+

-

+

Fig. 1. Enhancement of APLP2 secretion in HEK293, SKNnc and
SH-SY5Y cells by the PKC activator PMA. PMA was added at a final
concentration of 1 lM for 4.5 h, proteins in the cell supernatants
were then precipitated and analyzed by western blotting using the
antibody D2II. A representative example of three independent experiments is shown. Arrows indicate differentially modified APLP2s.

5809


APLP2-shedding by disintegrin-metalloproteinases

A

APLP2s
5
2 3 4

1

K. Endres et al.

B

6

APPsα
5 6
1 2 3 4

98 kDa

98 kDa

APLP2FI
98 kDa

C

APPsα secretion
in % of control

APLP2 secretion
in % of control

D
150
100
50

0
PMA

-

+

-

+

GM

-

-

+

+

200
150
100

50

-

+

0
PMA

-

+

-

+

GM

GI

-

-

+

+

APLP2 secretion in % of control

E
125
100
75
50
10
0
-8

-7

-6

-5

log c [M]

98 kDa
GI254023X

0

0,3 0,6 1,3 2,5

5 10 µM

(Fig. 2A,B, lanes 2 and 4). This suggested participation
of either ADAMs and ⁄ or MMPs in the processing of
APLP2. There was more pronounced inhibition of

constitutive shedding by the inhibitor GI254023X,
which has a 100-fold higher potency to inhibit recombinant ADAM10 than recombinant TACE [33,34].
When compared with solvent-treated control cells,
both APPsa and APLP2s were decreased to 30%
(Fig. 2A,B, lanes 5 and 6), showing that ADAM10 is
strongly involved in the shedding of APLP2.
With both inhibitors the amount of full-length
APLP2 was comparable with control cells (Fig. 2A)
and did not increase upon inhibited processing.
Because a-secretase cleavage of APP occurs at the
surface of neuronal cells [35], only a small fraction of
5810

GI

-

+

Fig. 2. Influence of metalloproteinase inhibitors on APPsa and APLP2s secretion of neuroblastoma cells. Detection of shed (A) APLP2
and (B) APPsa in SKNMC cells treated with
metalloproteinase inhibitors. GI254023X,
GM6001 and the inactive GM6001NK were
added at a final concentration of 10 lM for
overnight preincubation, proteins of the cell
supernatant were then collected for 4.5 h.
PMA (1 lM) was added directly during the
collection period. Secreted APLP2s and
APPsa were detected as described in Experimental procedures (lane 1, control; lane 2,
PMA; lane 3, GM6001; lane 4, PMA ⁄ G6001;

lane 5, control; lane 6, GI254023X). Fulllength APLP2 (APLP2Fl) was analyzed in cell
lysates (A, lower) to confirm that the inhibitors did not alter steady-state levels of the
protein. Representative blots are shown.
Quantitative analysis of (C) APLP2 and (D)
APPsa secretion. Quantification for APLP2
was carried out taking into account all three
APLP2 protein forms. Values are the
mean ± SD of three independent experiments. Control cells treated with the solvent
or the inactive compound GM6001NK (indicated as GM –) were set to 100% (One-way
ANOVA: *P < 0.05, **P < 0.01). (E) Detection of constitutive APLP2 shedding in SHSY5Y cells. Cells were pretreated with
increasing doses of GI254023X (0.3–10 lM)
for 30 min. After 4 h treatment with freshly
added inhibitor, the conditioned media were
harvested and the amount of secreted
APLP2 was determined. Data represent the
mean ± SD of three independent experiments performed in duplicate. The inhibitor
dose–response curve was generated using
the software GRAPHPAD PRISM 4.02 (GraphPad
Software Inc., San Diego, CA, USA).

the total cellular APP is cleaved, which generally does
not result in a decrease in the full-length protein
[36,37]. Therefore, reduction of APLP2 proteolysis
by hydroxamic acid-based inhibitors might also affect
only minor pools of the cellular protein resulting in an
unchanged steady-state level.
To compare the cell-based inhibitory effect of
GI254023X on APLP2 shedding with recently published data for shedding of other ADAM substrates
like the interleukin-6 receptor [38], we applied the
inhibitor in concentrations ranging from 0.3 to 10 lm

to SH-SY5Y cells (Fig. 2E). The IC50 value for inhibition of APLP2 shedding by GI254023X was in the
micromolar range (1.7 lm) showing a reduction of
potency in cellular assays as compared to its effect on
FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS


K. Endres et al.

APLP2-shedding by disintegrin-metalloproteinases

recombinant ADAM10 with IC50 values in the nanomolar range [33]. In comparison, inhibition of the
interleukin-6 receptor shedding in COS cells occurred
with a potency of 1.8 lm [38] and therefore was in the
same range as found for cellular APLP2 shedding.
Inhibition of APLP2 ectodomain shedding by a
specific b-secretase inhibitor
Another proteinase suggested to be an APLP2-cleaving
enzyme is BACE-1 [11,13]. To elucidate whether, in
cells of neuronal origin, APLP2 is processed by b-secretase, we tested the effect of the tripeptidic b-secretase
inhibitor
[(N-benzyloxycarbonyl-val-leu-leu-leucinal)
Z-VLL-CHO] on APLP2 shedding in the human astroglioma cells U373. These cells overexpress human
wild-type APP and therefore allow detection of
BACE-1-generated secreted APPsb, which is normally
found at very low concentrations in the cell supernatant
[41]. As shown in Fig. 3, both ectodomains were
reduced significantly by applying the b-secretase-specific
inhibitor. For APPsb we found a decreased shedding of
 50% of control cells. For APLP2s shedding was
A

APLP2s

APPsb

148 kDa

98 kDa

Enhancement of APLP2 secretion by overexpression of the a-secretases ADAM10 and TACE
To identify the proteinases that participate in APLP2
shedding, we examined cells overexpressing the a-secretase ADAM10 or TACE (Fig. 4A–C). Stable overexpression of either proteinase resulted in  2.5–3.5-fold more
soluble APLP2 in the culture supernatant than in control cells (Fig. 4A,B). Because expression levels of the
two proteinases differed (TACE being expressed at
higher levels, Fig. 4C), we are not able to determine
from the data which of the two enzymes preferentially
cleaves APLP2. In all cases, overexpression did not significantly alter the steady-state levels of cellular APLP2
(data not shown), therefore the observed effects are
not due to enhanced expression levels of APLP2.

98 kDa

β-secretaseInhibitor II

-

+

+

-

B
secretion in % of control

inhibited to a significant but lesser extent (reduction of
 30% compared with control cells).
Because the antibody available against the APLP2
extracellular region (D2II) recognizes both the BACE1- and a-like cleavage product of APLP2, APLP2s in
cell supernatants reflect the effect of both shedding processes. Probably therefore the effects on the a-like cleavage of APLP2 by metalloproteinase inhibitors (Fig. 2)
or on the b-like cleavage by a BACE-1 inhibitor (Fig. 3)
are probably not as strong as for the processing of APP,
which is monitored by specific antibodies (a-cleavage,
6E10, Fig. 2B; b-cleavage 192 Wt, Fig. 3A).

100

*
*

50

0
APPsb

APLP2s

control

β-secretaseInhibitor II

Fig. 3. Effect of the b-secretase inhibitor II on the ectodomain
shedding of APLP2 in astroglioma cells overexpressing APP. (A)
Western blots of secreted APPsb and APLP2s upon b-secretase
inhibitor treatment of U373hwtAPP cells. Following preincubation
for 18 h with 25 lM of the b-secretase inhibitor II, shedding of
APPsb and APLP2s was analyzed in western blots with the antibodies 192 Wt or D2II. (B) Quantitation of APPsb and APLP2s. The
amount of shed proteins was quantified in three independent
experiments. For secreted APLP2 all detectable protein bands
above the 98 kDa marker band were taken into account (unpaired
Student’s t-test: *P < 0.05).

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS

Effect of a dominant negative mutant of ADAM10
on APLP2 shedding
To verify the APLP2-shedding activity of endogenous
ADAM10, we used a cell line with stable overexpression of a dominant negative form of ADAM10
(Fig. 4F). This mutant protein carries the E384A point
mutation in the zinc-binding region of ADAM10,
which is known in Drosophila melanogaster [40] and in
HEK293 cells [14] to suppress endogenous ADAM10
activity. HEK ADAM10DN cells showed a decreased
APLP2 secretion of  60% compared with nontransfected HEK293 cells (Fig. 4D,E), whereas expression
of full-length APLP2 was not significantly affected
(data not shown). Thus, dominant negative ADAM10
inhibits the endogenous APLP2 sheddase activity.
Influence of overexpressed ADAM10 on the
proteolytical processing of APLP2 in transgenic
mice

Cleavage of APLP2 in vivo was demonstrated by western blots comparing brain homogenates from FVB ⁄ N
5811


APLP2-shedding by disintegrin-metalloproteinases

A

HEK AD10

D

T

K. Endres et al.

HEK AD10
DN

98 kDa

98 kDa

∗∗

secretion of APLP2s
in % of control

400

∗∗
300

200
100

0

E
secretion of APLP2s
in % of control

B

125
100

∗∗

75
50
25
0

HEK

HEK
HEK
ADAM10 TACE

HEK

C

HEK
ADAM10DN

F
98 kDa

98 kDa
64 kDa

64 kDa

control ADAM10 TACE

control ADAM10DN

Fig. 4. Influence of ADAM10, TACE and dominant negative
ADAM10, overexpressed in HEK293 cells, on APLP2 shedding. (A)
Immunoblot of secreted APLP2 with antibody D2II in ADAM10
and TACE overexpressing cells. (B) Quantification of APLP2s in
ADAM10 and TACE overexpressing cells (mean ± SD of three
experiments performed in duplicate, unpaired Student’s t-test:
**P < 0.01). As control, HEK cells transfected with the empty vector pcDNA3 were used and set to 100%. A representative example
is shown. (C) Immunoblot of overexpressed ADAM10 and TACE.
The overexpressed proteinases were detected in cell lysates by an
anti-HA serum. (D) Immunoblot of secreted APLP2 in ADAM10DN
overexpressing cells. A longer exposure time as in (A) was chosen

to demonstrate the reduction of basal secretion of APLP2 by
ADAM10DN. (E) Quantification of APLP2s in ADAM10DN overexpressing cells (mean ± SD of three experiments performed in duplicates, unpaired Student’s t-test: **P < 0.01). (F) Immunoblot of
overexpressed dominant negative ADAM10. The overexpressed
mutated form of ADAM10 was detected in cell lysates by an antibody against the fused Flag-epitope.

mice and APLP2 knockout mice (Fig. 5A). In FVB ⁄ N
mice (Wt) the antibody D2II against the N-terminal
part of APLP2 detected a double band (Fig. 5A, lane
1). The CS-GAG-modified protein species were almost
not detectable according to the low levels of this form
in the brain as described for rat neuronal tissue [41].
By using antibody CT12, two C-terminal processing
products of APLP2 were identified (C-stub I and II,
Fig. 5A, lane 1). These stubs were also detected in
HEK cells which had been treated for 20 h with the
c-secretase inhibitor DAPT before cell lysis (results not
shown).
5812

To examine the a-like cleavage of APLP2 by
ADAM10 in vivo, we investigated the influence of
overexpressed ADAM10 in a transgenic mouse line.
These mice overexpress bovine ADAM10 under the
control of a neuron-specific Thy1 promoter [16].
Expression of the HA-tagged ADAM10 protein in
brains of transgenic mice was verified by immunoblotting with the anti-HA serum Y-11. Both the immature
and the mature forms of ADAM10 were detectable
with a dominance of the catalytically active, mature
form (Fig. 5B).
To analyze APLP2 processing, soluble and membrane-bound proteins from brain homogenates were

subjected to immunoblotting using either the D2II or
the CT12 antibody. We detected an enhanced amount
of secreted APLP2 protein fragments (170%) by comparing ADAM10 transgenic mice with wild-type littermates (Fig. 5C,D). When we examined the amount
of C-terminal stubs, we noticed a roughly twofold
increase in both C-stubs in ADAM10 transgenic mice
(Fig. 5C,D). No fragment corresponding to an APLP2
Cb-stub could be detected by immunoblotting with
the CT12 antibody in mouse brain homogenates, and
therefore both identified C-stubs probably correspond
to a-secretase-like cleavage products.
To exclude the possibility that the observed effects
result from an altered expression intensity due to overexpression of the proteinase, we performed realtime RT-PCR experiments with mouse brain mRNA.
APLP2-mRNA levels in transgenic and in control mice
were not significantly different (P > 0.4; n ¼ 5, data
not shown).
Effect of RA on APP, APLP2 and ADAM10
expression in neuroblastoma cell lines
Because APP and APLP2 expression is enhanced
during neuronal differentiation [26,27], we wanted to
elucidate the effect of RA-induced differentiation of
neuroblastoma cell lines on ADAM10 and TACE
expression and on the release of secreted APLP2 and
APPsa. For neuronal (N)-type SH-SY5Y cells, differentiation by RA was accompanied by the generation
of long cellular outgrowths. Under the same conditions, the more Schwann-like SKNMC cells changed
their morphology only slightly but revealed strongly
decreased proliferative properties (Fig. 6A); for a characterization of both cell lines during differentiation see
Voigt and Zintl [42].
The effect of RA-induced differentiation on either
the substrate APLP2 or the proteinase ADAM10 was
analyzed using real-time RT-PCR for quantification of

mRNAs. At the mRNA level, APLP2 was increased
FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS


K. Endres et al.

APLP2-shedding by disintegrin-metalloproteinases

A

B
APLP2s

98 kDa

immature

98 kDa

mature

64 kDa

14 kDa

C-stub I
C-stub II

Wt

C

D
APLP2s

98 kDa

14 kDa

C-stubs

Wt

AD10

APLP2
KO
expression in % of control

Wt

250

**

200

**

**

150
100
50
0
APLP2s

C-stub I

C-stub II

AD10
Wt

ADAM10

Fig. 5. Analysis of APLP2 proteolysis in transgenic mice overexpressing ADAM10. (A) APLP2 processing products in mice. The high specificity of the antibodies D2II and CT12 (recognizing either an N-terminal epitope or an epitope at the very end of the C-terminus) is demonstrated by comparing brain homogenates of wild-type with APLP2 knockout mice in western blots. (B) Detection of overexpressed ADAM10 in
transgenic mice. ADAM10 transgenic mice were 10 weeks old. As controls we used nontransgenic littermates (Wt) of the same age. (C)
Detection of APLP2s and the membrane-bound C-stubs. The amounts of shed APLP2 and the C-terminal stubs were quantified by Western
blotting using membrane and soluble fractions derived from brain homogenates. (D) Quantitation of APLP2 processing products in transgenic
mice. The values of shed APLP2 (APLP2s) and both C-stubs (C-stub I and C-stub II) were quantified for eight animals of each group in at
least two independent western blot experiments and normalized to the full-length protein form (mean ± SD, unpaired Student’s t-test:
*P < 0.05, **P < 0.01).

significantly in both RA-differentiated cell lines SHSY5Y and SKNMC (Fig. 6B). Also, ADAM10 mRNA
was strongly increased as we have recently shown for
A

control

RA

SH-SY5Y cells [45]. Interestingly, both mRNA species
were induced more strongly in the N-type neuroblastoma cell line SH-SY5Y than in the more SchwannB

SH-SY5Y

SKNMC

mRNA in % of control

SKNMC

SH-SY5Y ∗∗

300

200

∗∗

∗

∗∗

∗∗
100

0
APLP2 ADAM10 APLP2 ADAM10 BACE

control

RA

Fig. 6. Morphological changes and mRNA levels in neuroblastoma cell lines upon RA-treatment. Cells were treated for 4 days with 1 lM RA.
(A) Microscopic image of RA-differentiated neuroblastoma cell lines. Morphological changes as cellular outgrowths and loss of adherence
were determined as markers of differentiation using light microscopy. (B) Real-time RT-PCR for mRNA quantitation. Changes in mRNA for
APLP2 and the proteinases ADAM10 and BACE-1 were investigated using real-time RT-PCR. Experiments were performed three times in
duplicate and amounts of mRNAs were normalized to GAPDH mRNA. Values are given as mean ± SD and results obtained with control cells
were set to 100% (unpaired Student’s t-test: *P < 0.05, **P < 0.01).

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS

5813


APLP2-shedding by disintegrin-metalloproteinases

K. Endres et al.

like SKNMC cells. As APLP2 is also known to be
processed by BACE (see above), we also quantified
the mRNA of BACE-1 in SH-SY5Y cells. Although
ADAM10 mRNA was induced to  250% compared
with undifferentiated cells, we found only a slight, but
significant increase in the amount of BACE-1 mRNA
(Fig. 6B and 147% of control). At the protein level,
the enhancement of APLP2 and ADAM10 was confirmed for both cell lines (Fig. 7A,B). Again in the
N-type SH-SY5Y the increase in both, the APLP2 and

the ADAM10 protein was stronger than in SKNMC
cells, where significant increase occurred only in the
immature form (Fig. 7B).
In contrast to ADAM10 expression, we could not
detect increased TACE protein levels upon RA treatment in our experiments (Fig. 8). Although TACE
remained unchanged in SH-SY5Y cells, the amount of
the pro- and the mature form of this proteinase even
decreased in SKNMC cells, revealing reduced stability
compared with ADAM10. Therefore, the concerted
upregulation of APLP2 and its sheddase during
RA-induced neuronal differentiation appears to be
specific for ADAM10.
In both neuroblastoma cell lines we found, upon
RA treatment, an increase of APLP2 shedding. Soluble
APLP2 in supernatants of differentiated cells was
enhanced to 150% for SH-SY5Y and 180% for
SKNMC compared with undifferentiated control cells
(Fig. 9A). Also, in SH-SY5Y cells the secretion of
APPsa was found to be enhanced significantly to

A

SH-SY5Y

> 200% of control cells due to increased expression of
the a-secretase ADAM10. This phenomenon was also
seen in SKNMC cells although to a lesser extent
(Fig. 9B).

Discussion

We report the cleavage of the mammalian APP-related
protein APLP2 by the disintegrin and metalloproteinases ADAM10 and TACE (ADAM17), and a common
upregulation of ADAM10 and its substrate by RA.
The main criteria for the involvement of ADAMs,
the enhancement of APLP2 shedding by phorbolesters
and decreasing amounts of APLP2s by hydroxamic
acid derivatives, were fulfilled. Overexpression of
ADAM10 as well as of TACE resulted in increased
secretion of APLP2s from cultured cells. Also, a dominant negative form of ADAM10 reduced the shedding
of APLP2.
Because
the
ADAM10-preferring
inhibitor
GI254023X displayed the most pronounced effect by
reducing APLP2s to  30% of control cells, we conclude that ADAM10, as shown for APP [14], plays an
important role in the secretion of the APLP2 ectodomain. We were also able to demonstrate the
influence of the a-secretase ADAM10 on APLP2
processing in vivo. Transgenic mice with neuronal overexpression of ADAM10 showed significantly increased
amounts of shed APLP2 as well as C-terminal processing products.

SKNMC

APLP2 expression
in % of control

98 kDa

RA

-

+

-

∗∗

200

+

∗

150

control

100

RA

50
0

SH-SY5Y

B

SKNMC

150

RA

-

∗∗

+

∗∗

100

5814

50
0

immature

64 kDa

mature

ADAM10 expression
in % of control

200

98 kDa

mature
ADAM10 expression
in % of control

immature

200

RA

-

∗

+
control

150

RA

100
50
0

immature

mature

Fig. 7. Expression of APLP2 (A) and
ADAM10 (B) in differentiated neuroblastoma
cell lines. Cell lysates of RA-differentiated
SH-SY5Y and SKNMC cells were subjected
to 7.5% SDS ⁄ PAGE, and the proteins were
detected by immunoblotting using primary
antibodies against the C-termini. Experiments were performed three times in duplicate, representative immunoblots are
shown. Values are given as mean ± SD and
results obtained with control cells were
set to 100% (unpaired Student’s t-test:
*P < 0.05, **P < 0.01).

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS


K. Endres et al.

APLP2-shedding by disintegrin-metalloproteinases

SH-SY5Y
Fig. 8. Expression of TACE in differentiated
neuroblastoma cell lines. SH-SY5Y and SKNMC cells were differentiated with RA for
4 days, and the mature and the immature
form of the proteinase were detected by
immunoblotting. Values are given as mean
± SD of three independent experiments,
and results obtained with control cells were
set to 100% (unpaired Student’s t-test:

*P < 0.05, **P < 0.01).

A

immature
mature

98 kDa

-

200

RA

-

+

150

100
50
0

B

control

100

150

immature

SKNMC

+

200

TACE expression
in % of control

TACE expression
in % of control

RA

SH-SY5Y

RA

50
0

immature

mature

SH-SY5Y

mature

SKNMC

98 kDa

98 kDa

APLP2s

APPsα

300

secretion
in % of control

250

secretion
in % of control

SKNMC

200

200

150
100

100

50

0

0

SH-SY5Y
control

SKNMC
RA

SH-SY5Y
control

SKNMC
RA

Fig. 9. Proteolytical processing of APP and APLP2 in RA-treated neuroblastoma cells. Western blots and quantification of (A) APLP2s and of
(B) APPsa in RA-differentiated neuroblastoma cell lines. Cells were treated as described in Experimental procedures. Precipitated proteins of
cell supernatants were subjected to 7.5% SDS ⁄ PAGE and immunoblotted. Detection was performed with the antibodies 6E10 and D2II.
Values of the quantitative analysis are mean ± SD and significances were determined using paired Student’s t-test (*P < 0.05, **P < 0.01).
Experiments were performed three times in duplicate, representative immunoblots are shown.

Because soluble APLP2 was shown to induce neurogenesis in the subventricular zone of adult mouse brain

[44] and enhances neurite outgrowth [28], the proteolytical processes that generate APLP2s may be important
for the generation and survival of neuronal cells. The
elevation of APP and APLP1 and APLP2 in differentiated SH-SY5Y [27] suggests an important function for
the expression and proteolysis of APP family members
especially in neuronal cell populations. In support of
this hypothesis, we found enhanced secretion of the
extracellular domains of APP and APLP2 upon RA
treatment, which might correspond to increased expression of ADAM10 in both SH-SY5Y and SKNMC cell
lines. We cannot completely rule out the possibility
that the increase in secretion of soluble APLP2 following treatment with RA may also be due to the increase
in the amount of APLP2 and not because of the
increase in ADAM10 expression. But because the
BACE-1 mRNA level was increased to a lesser extent,
a major role of the nonamyloidogenic pathway and
FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS

ADAM10 in differentiating neuronal cells may be supposed. Recent findings [43] demonstrate a conserved
binding site for retinoid receptors in the promoter
sequence of ADAM10 and an increase of promoter
activity by RA. These results suggest a RA-induced
regulation of this disintegrin-metalloproteinase by nuclear receptors. Because TACE was not positively affected by RA, but even degraded in SKNMC cells, we
demonstrate again a higher stability of ADAM10 compared with TACE, which was also selectively degraded
after PMA treatment of cultured cells [45].
In late-onset Alzheimer’s disease there is genetic,
metabolic and dietary evidence for defective retinoid
transport and function [46–48]. In accordance with these
findings, is the observation that the impairment of longterm potentiation induced by experimental vitamin A
deficiency in adult mice can be reversed by direct
application of RA to hippocampal slices [49]. Recently,
we demonstrated that overexpression of ADAM10

in APP[V717I] transgenic mice prevented plaque
5815


APLP2-shedding by disintegrin-metalloproteinases

formation and rescued the impairments of hippocampal
long-term potentiation, thus suggesting a beneficial role
of the a-secretase ADAM10 in memory and learning
[16]. Because ADAM10 together with its substrates is
upregulated via RA our results suggest that bioactive
retinoids in the hippocampus could lead to an increased
a-secretase activity and to an increased release of the
neurotrophic-soluble ectodomains of APP and APLP2.
Further studies are necessary to support this conclusion
in vivo and to delineate the regulatory mechanism of
RA-induced a-like cleavage of APLP2.

Experimental procedures
Materials
PMA and all-trans-RA were purchased from Sigma (St.
Louis, MO, USA), the broad-spectrum inhibitor GM6001
(Galardin) and the corresponding inactive control compound (GM6001NK), as well as the b-secretase inhibitor II,
were from Calbiochem (San Diego, CA, USA). Each was
dissolved as stock in dimethylsufoxide and kept at )20 °C.

K. Endres et al.

HA-tagged TACE (named HEK ADAM10, HEK
ADAM10DN and HEK TACE, respectively) were cultured

in Dulbecco’s modified Eagle’s medium (DMEM; containing
10% fetal calf serum, 2 mm glutamine, 100 mL)1 penicillin, 100 lgỈmL)1 streptomycin). SKNMC cells were cultured
in DMEM complete medium supplemented with 1% sodium
pyruvate, and SH-SY5Y cells were cultivated in Ham’s F12
medium [containing 10% (v ⁄ v) fetal bovine serum, 2 mm
glutamine, 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin]. For the astroglioma cell line U373 MEM supplemented with 10% (v ⁄ v) fetal bovine serum, 2 mm glutamine,
100 mL)1 penicillin, 100 lgỈmL)1 streptomycin, 1% (w ⁄ v)
sodium pyruvate and 1% (w ⁄ v) nonessential amino acids
was used.
Stable transfections of HEK293 cells were performed by
using the calcium phosphate precipitation method followed
by selection of transfected cells with G418 (1 mgỈmL)1).
For differentiation of the neuroblastoma cell lines, cells
were seeded on 10 cm culture plates after adjusting the cell
number (SH-SY5Y 2.5–5 · 105, SKNMC 0.5–1.0 · 105
cells) and grown for 72 h. The medium was replaced by
fresh phenol red-free medium containing 1 lm RA, the cells
were incubated for 4 days, and the RA-containing medium
was changed daily.

Primary antibodies
The following antibodies were used for western blot analysis: D2II, a rabbit polyclonal antibody against the N-terminus of APLP2; CT12, a rabbit polyclonal antibody against
the C-terminus of APLP2 (both kindly provided by
G. Thinakaran, University of Chicago, IL); 6E10 (Signet
Laboratories, Dedham, MA, USA) against APPsa; 192 Wt
(S. Sinha, Elan Pharmaceuticals, San Francisco, CA, USA)
against APP residues 591–596, detecting only b-secretasecleaved soluble APP (APPsb) antibodies against the C-termini of human ADAM10 and 17 (Chemicon, Temecula,
CA, USA). Overexpressed proteinases were detected with
the anti-HA serum Y-11 (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) or anti-Flag serum M2 (Stratagene, La

Jolla, CA, USA).

Constructs and mutagenesis
The cDNAs of murine TACE [50] and bovine ADAM10 [14]
were fused with a DNA-sequence coding for a hemagglutinin
epitope (YPYDVDDYA), and dominant negative ADAM10
was tagged with a Flag-epitope (DYKDDDDK) as described previously [14]. Expression of the tagged proteinases
was performed by using the vector pcDNA3 (Invitrogen,
Carlsbad, CA, USA).

Cell culture and transfections
HEK293 cells stably overexpressing either HA-tagged
ADAM10, Flag-tagged dominant negative ADAM10 or

5816

Western blot analysis of TACE and ADAM10
Cell pellets were washed with NaCl ⁄ Pi and dissolved in
Laemmli buffer containing 100 mm dithiothreitol, heated to
95 °C for 10 min, separated by SDS ⁄ PAGE on 7.5% gels
and transferred to poly(vinylidene difluoride) (PVDF) membranes. Bound antibodies against the endogenous or overexpressed proteinases were visualized by applying alkaline
phosphatase coupled antibodies and the chemiluminescence
substrate CDPstar (Tropix, Foster City, CA, USA). Emitted light was detected by using a digital camera and quantified with the software aida 3.50 (Raytest, Straubenhardt,
Germany).

Western blot analysis of APP, APLP2 and their
processing products
Cells were grown close to confluency, washed with serumfree culture medium and incubated for 4.5 h in serum-free
culture medium containing 2 mm glutamine, 100 mL)1
penicillin, 100 mgỈmL)1 streptomycin, 10 lgỈmL)1 fatty

acid-free bovine serum albumin and activators or inhibitors as indicated. PMA (1 lm) was added directly to the
serum-free harvesting medium (with 2 mm glutamine,
100 mL)1 penicillin, 100 lgỈmL)1 streptomycin and
10 lgỈmL)1 fatty acid-free bovine serum albumin) for
4.5 h. The inhibitors GM6001, its negative control and
GI254023X (10 lm) were added to the cells 18 h prior harvesting and also to the harvesting medium. For the dose–
response curve of GI254023X SH-SY5Y cells were pre-

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS


K. Endres et al.

incubated for 30 min with varying amounts of the inhibitor followed by a harvesting period of 4 h with freshly
added inhibitor. Proteins of the culture medium were precipitated with 10% trichloroacetic acid and collected by
centrifugation. The pellets were washed twice with ice-cold
acetone, dried and dissolved in Laemmli buffer containing
100 mm dithiothreitol and heated to 95 °C for 10 min.
Aliquots corresponding to equivalent protein contents of
cells were separated by SDS ⁄ PAGE on 7.5% gels and
blotted onto PVDF membranes. Soluble APLP2 was
detected with antibody D2II (1 : 2500), followed by incubation with anti-rabbit serum either coupled to alkaline
phosphatase (Tropix) or 35S labeled (Amersham Biosciences,
Arlington Heights, IL, USA). Shed APPsa and APPsb
were detected by using the antibodies 6E10 and 192 Wt,
respectively, in combination with secondary antibodies
either coupled to alkaline phosphatase or 35S-labeled.
Bound antibodies were visualized by using a digital camera
or the BAS Reader (Fujilm, Dusseldorf, Germany), and
ă

quantied as described above. For detection of full-length
APLP2 and its membrane-bound C-stubs, cells were centrifuged for 3 min, 960 g, 4 °C. An aliquot of the cells was
taken for quantification of the protein content. The residual cells were dissolved in an adequate volume of 1.5 ·
Nu-PAGE buffer (Invitrogen) containing 100 mm dithiothreitol, heated to 70 °C for 10 min, separated on 4–12%
Nu-PAGE gels (Invitrogen) and transferred to PVDF
membranes. As primary antibody we used CT12. Detection
of APLP2 protein fragments was performed as described
above for the soluble proteins.

Preparation of mouse brain homogenates from
transgenic mice
The generation of transgenic mice with neuron-specific
overexpression of bovine ADAM10 has been described previously [16]. Transgenity of mice was confirmed by PCR
and by detection of the overexpressed HA-tagged
ADAM10 proteins by western blotting. Mice were chosen
for the experiments with a 1.3-fold increase in the amount
of ADAM10 compared with their wild-type litter-mates.
Brains of 10-week-old mice (ADAM10 or wild-type nontransgenic littermates) were dissected and homogenized in
200 mm Tris ⁄ HCl (pH 8.4) in the presence of proteinase
inhibitors (complete mini, Roche, Mannheim, Germany).
Homogenates were centrifuged at 135 000 g for 1.75 h at
4 °C for sedimentation of cellular membranes. The supernatants containing the soluble proteins were removed and the
membrane pellet was suspended in NaCl ⁄ Tris. The protein
concentrations of both fractions were determined. Proteins
were separated on polyacrylamide gels and blotted onto
PVDF membrane as described above. As secondary antibody we used 35S-labeled secondary antibodies. For quantification the BAS Reader (Fujifilm) and the software
aida 3.50 were used.

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS

APLP2-shedding by disintegrin-metalloproteinases

Real-time RT-PCR
Total RNA was isolated using the RNeasy Kit (Qiagen, Hilden, Germany). RNA concentration and quality was determined by spectrophotometry. Aliquots of the RNAs were
dissolved in RNAse-free water (Sigma) to a concentration of
50 ngỈlL)1. Real-time RT-PCR primers were designed for
human GAPDH, ADAM10, BACE and APLP2 from Gene
bank mRNA (cDNA) sequences utilizing the primer
express 1.5 software (Applied Biosystems, Foster City, CA,
USA).
GAPDH_for
5¢-GAAGGGCTCATGACCACAGTCC
AT-3¢, GAPDH_rev 5¢-TCATTGTCGTACCAGGAAAT
GAGCTT-3¢; ADAM10_for 5¢-CTGGCCAACCTATTTG
TGGAA-3¢, ADAM10_rev 5¢-GACCTTGACTTGGACTG
CACTG-3¢; BACE_for 5¢-GTTATCATGGAGGGCTTC
TACGTT-3¢, BACE_rev 5¢-GCTGCCGTCCTGAACTCA
TC-3¢; APLP2_for 5¢-CTCAGCGGATGATAATGAG
CAC-3¢, APLP2_rev 5¢-GGTTCTTGGCTTGAAGTTCT
GC-3¢.
Real-time RT-PCR was performed using the one-step
QuantiTectSYBRGreen RT-PCR-Kit (Qiagen), the ABIPrism 7000 (Applied Biosystems), 250 ng RNA and the
specific primer pairs (0.5 lm of each primer). Reverse
transcription was performed at 50 °C for 30 min. The
quantitative PCR was induced by heating to 95 °C, followed by 45 PCR cycles (one cycle contained the following
steps: 15 s at 95 °C; 30 s at 55 °C; 30 s at 72 °C). The specificity of each primer pair was confirmed by melting curve
analysis and agarose gel electrophoresis. The quantity of
mRNA was calculated using either the DDCt method, when
PCR efficiency was close to 100%, or a standard curve (e.g.
for BACE). The mRNA of the housekeeping gene GAPDH

was unchanged under differentiation conditions, and all
other mRNAs were normalized to it.

Acknowledgements
We thank A. Roth for excellent technical assistance;
R. Black for the murine TACE cDNA; C. Prinzen for
introduction of the HA-tag into the TACE cDNA,
and G. Thinakaran for providing the APLP2 cDNA
and the antibodies CT12 and D2II. We are grateful to
Dr I. Hussain, Glaxo SmithKline (Harlow, UK) for
putting the inhibitor GI254023X at our disposal. This
work was supported by the DFG priority program
1085 ⁄ 3-Cellular mechanisms of Alzheimer’s disease.

References
1 Coulson EJ, Paliga K, Beyreuther K & Masters CL
(2000) What the evolution of the amyloid protein precursor supergene family tells us about its function.
Neurochem Int 36, 175–184.

5817


APLP2-shedding by disintegrin-metalloproteinases

2 Slunt HH, Von Thinakaran GKC, Lo AC, Tanzi RE
& Sisodia SS (1994) Expression of a ubiquitous,
cross-reactive homologue of the mouse beta-amyloid
precursor protein (APP). J Biol Chem 269, 2637–
2644.
3 De Strooper B & Annaert W (2000) Proteolytic processing and cell biological functions of the amyloid precursor protein. J Cell Sci 113, 1857–1870.

4 Scheinfeld MH, Ghersi E, Laky K, Fowlkes BJ &
D’Adamio L (2002) Processing of beta-amyloid precursor-like protein-1 and -2 by gamma-secretase regulates
transcription. J Biol Chem 277, 44195–44201.
5 Paliga K, Peraus G, Kreger S, Durrwang U, Hesse L,
Multhaup G, Masters CL, Beyreuther K & Weidemann
A (1997) Human amyloid precursor-like protein 1 –
cDNA cloning, ectopic expression in COS-7 cells and
identification of soluble forms in the cerebrospinal fluid.
Eur J Biochem 250, 354–363.
6 Walsh DM, Fadeeva JV, LaVoie MJ, Paliga K, Eggert
S, Kimberly WT, Wasco W & Selkoe DJ (2003)
Gamma-secretase cleavage and binding to FE65 regulate
the nuclear translocation of the intracellular C-terminal
domain (ICD) of the APP family of proteins. Biochemistry 42, 6664–6673.
7 Gu Y, Misonou H, Sato T, Dohmae N, Takio K &
Ihara Y (2001) Distinct intramembrane cleavage of the
beta-amyloid precursor protein family resembling
gamma-secretase-like cleavage of Notch. J Biol Chem
276, 35235–35238.
8 von Koch CS, Zheng H, Chen H, Trumbauer M,
Thinakaran G, Van der Ploeg LH, Price DL & Sisodia
SS (1997) Generation of APLP2 KO mice and early
postnatal lethality in APLP2 ⁄ APP double KO mice.
Neurobiol Aging 18, 661–669.
9 Heber S, Herms J, Gajic V, Hainfellner J, Aguzzi A,
von Rulicke T, Von KHKC, Sisodia S, Tremml P, Lipp
HP, et al. (2000) Mice with combined gene knock-outs
reveal essential and partially redundant functions of
amyloid precursor protein family members. J Neurosci
20, 7951–7963.

10 Collin RWSD, Leunissen JA & Martens GJ (2004)
Identification and expression of the first nonmammalian
amyloid-beta precursor-like protein APLP2 in the
amphibian Xenopus laevis. Eur J Biochem 271, 1906–
1912.
11 Eggert S, Paliga K, Soba P, Evin G, Masters CL, Weidemann A & Beyreuther K (2004) The proteolytic processing of the amyloid precursor protein gene family
members APLP-1 and APLP-2 involves alpha-, beta-,
gamma-, and epsilon-like cleavages: modulation of
APLP-1 processing by n-glycosylation. J Biol Chem 279,
18146–18156.
12 Li Q & Sudhof TC (2004) Cleavage of amyloid-beta
precursor protein and amyloid-beta precursor-like protein by BACE 1. J Biol Chem 279, 10542–10550.

5818

K. Endres et al.

13 Pastorino L, Ikin AF, Lamprianou S, Vacaresse N,
Revelli JP, Platt K, Paganetti P, Mathews PM, Harroch
S & Buxbaum JD (2004) BACE (beta-secretase) modulates the processing of APLP2 in vivo. Mol Cell Neurosci
25, 642–649.
14 Lammich S, Kojro E, Postina R, Gilbert S, Pfeiffer R,
Jasionowski M, Haass C & Fahrenholz F (1999) Constitutive and regulated alpha-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin
metalloprotease. Proc Natl Acad Sci USA 96, 3922–3927.
15 Buxbaum JD, Liu KN, Luo Y, Slack JL, Stocking KL,
Peschon JJ, Johnson RS, Castner BJ, Cerretti DP &
Black RA (1998) Evidence that tumor necrosis factor
alpha converting enzyme is involved in regulated alphasecretase cleavage of the Alzheimer amyloid protein
precursor. J Biol Chem 273, 27765–27767.
16 Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt

U, Kojro E, Prinzen C, Endres K, Hiemke C, Blessing
M, et al. (2004) A disintegrin-metalloproteinase prevents
amyloid plaque formation and hippocampal defects in
an Alzheimer disease mouse model. J Clin Invest 113,
1456–1464.
17 Xu KP, Zoukhri D, Zieske JD, Dartt DA, Sergheraert C, Loing E & FS (2001) A role for MAP kinase
in regulating ectodomain shedding of APLP2 in corneal epithelial cells. Am J Physiol Cell Physiol 281,
C603–C614.
18 Koike H, Tomioka S, Sorimachi H, Saido TC, Maruyama K, Okuyama A, Fujisawa-Sehara A, Ohno S,
Suzuki K & Ishiura S (1999) Membrane-anchored
metalloprotease MDC9 has an alpha-secretase activity
responsible for processing the amyloid precursor protein. Biochem J 343 Part 2, 371–375.
19 Roghani M, Becherer JD, Moss ML, Atherton RE,
Erdjument-Bromage H, Arribas J, Blackburn RK,
Weskamp G, Tempst P & Blobel CP (1999) Metalloprotease-disintegrin MDC9: intracellular maturation and
catalytic activity. J Biol Chem 274, 3531–3540.
20 Weskamp G, Cai H, Brodie TA, Higashyama S, Manova
K, Ludwig T & Blobel CP (2002) Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident
major abnormalities during development or adult life.
Mol Cell Biol 22, 1537–1544.
21 Sahin U, Weskamp G, Kelly K, Zhou HM, Higashiyama S, Peschon J, Hartmann D, Saftig P & Blobel CP
(2004) Distinct roles for ADAM10 and ADAM17 in
ectodomain shedding of six EGFR ligands. J Cell Biol
164, 769–779.
22 Abel S, Hundhausen C, Mentlein R, Schulte A, Berkhout TA, Broadway N, Hartmann D, Sedlacek R,
Dietrich S, Muetze B, et al. (2004) The transmembrane
CXC-chemokine ligand 16 is induced by IFN-gamma
and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10. J Immunol 172,
6362–6372.

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS


K. Endres et al.

23 Sanderson MP, Erickson SN, Gough PJ, Garton KJ,
Wille PT, Raines EW, Dunbar AJ & Dempsey PJ
(2005) ADAM10 mediates ectodomain shedding of the
betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx. J Biol Chem
280, 1826–1837.
24 Reiss K, Maretzky T, Ludwig A, de Tousseyn TSB,
Hartmann D & Saftig P (2005) ADAM10 cleavage of
N-cadherin and regulation of cell–cell adhesion and
beta-catenin nuclear signalling. EMBO J 24, 742–752.
25 Ruiz-Leon Y & Pascual A (2003) Induction of tyrosine
kinase receptor b by retinoic acid allows brain-derived
neurotrophic factor-induced amyloid precursor protein
gene expression in human SH-SY5Y neuroblastoma
cells. Neuroscience 120, 1019–1026.
26 Beckman M & Iverfeldt K (1997) Increased gene expression of beta-amyloid precursor protein and its homologues APLP1 and APLP2 in human neuroblastoma cells
in response to retinoic acid. Neurosci Lett 221, 73–76.
27 Adlerz L, Beckman M, Holback S, Tehranian R, Cortes
TV & Iverfeldt K (2003) Accumulation of the amyloid
precursor-like protein APLP2 and reduction of APLP1
in retinoic acid-differentiated human neuroblastoma
cells upon curcumin-induced neurite retraction. Brain
Res Mol Brain Res 119, 62–72.
28 Cappai R, Mok SS, Galatis D, Tucker DF, Henry A,
Beyreuther K, Small DH & Masters CL (1999) Recombinant human amyloid precursor-like protein 2 (APLP2)
expressed in the yeast Pichia pastoris can stimulate neurite outgrowth. FEBS Lett 442, 95–98.

29 Thinakaran G & Sisodia SS (1994) Amyloid precursorlike protein 2 (APLP2) is modified by the addition of
chondroitin sulfate glycosaminoglycan at a single site.
J Biol Chem 269, 22099–22104.
30 Thinakaran G, Slunt HH & Sisodia SS (1995) Novel
regulation of chondroitin sulfate glycosaminoglycan
modification of amyloid precursor protein and its
homologue, APLP2. J Biol Chem 270, 16522–16525.
31 Ethell DW, Kinloch R & Green DR (2002) Metalloproteinase shedding of Fas ligand regulates beta-amyloid
neurotoxicity. Curr Biol 12, 1595–1600.
32 Ito N, Nomura S, Iwase A, Ito T, Kikkawa F, Tsujimoto M, Ishiura S & Mizutani S (2004) ADAMs, a
disintegrin and metalloproteinases, mediate shedding
of oxytocinase. Biochem Biophys Res Commun 314,
1008–1013.
33 Hundhausen C, Misztela D, Berkhout TA, Broadway
N, Saftig P, Reiss K, Hartmann D, Fahrenholz F,
Postina R, Matthews V, et al. (2003) The disintegrinlike metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates
CX3CL1-mediated cell–cell adhesion. Blood 102, 1186–
1195.
34 Budagian V, Bulanova E, Orinska Z, Ludwig A, RoseJohn S, Saftig P, Borden EC & Bulfone-Paus S (2004)

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS

APLP2-shedding by disintegrin-metalloproteinases

35

36

37

38

39

40

41

42

43

44

45

46

Natural soluble interleukin-15Ralpha is generated by
cleavage that involves the tumor necrosis factor-alphaconverting enzyme (TACE ⁄ ADAM17). J Biol Chem
279, 40368–40375.
Parvathy S, Hussain I, Karran EH, Turner AJ & Hooper NM (1999) Cleavage of Alzheimer’s amyloid precursor protein by alpha-secretase occurs at the surface of
neuronal cells. Biochemistry 38, 9728–9734.
Racchi M & Govoni S (1999) Rationalizing a pharmacological intervention on the amyloid precursor protein
metabolism. Trends Pharmacol Sci 20, 418–423.
Zimmermann M, Gardoni F, Marcello E, Colciaghi F,
Borroni B, Padovani A, Cattabeni F & Di Luca M
(2004) Acetylcholinesterase inhibitors increase ADAM10
activity by promoting its trafficking in neuroblastoma
cell lines. J Neurochem 90, 1489–1499.

Ludwig A, Hundhausen C, Lambert MH, Broadway N,
Andrews RC, Bickett DM, Leesnitzer MA & Becherer
JD (2005) Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17
that differentially block constitutive and phorbol esterinducible shedding of cell surface molecules. Comb
Chem High Throughput Screen 8, 161–171.
Kojro E, Gimpl G, Lammich S, Marz W & Fahrenholz
F (2001) Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha-secretase
ADAM 10. Proc Natl Acad Sci USA 98, 5815–5820.
Pan D & Rubin GM (1997) Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition
during Drosophila and vertebrate neurogenesis. Cell 90,
271–280.
Sandbrink R, Masters CL & Beyreuther K (1994) Similar alternative splicing of a non-homologous domain in
beta A4-amyloid protein precursor-like proteins. J Biol
Chem 269, 14227–14234.
Voigt A & Zintl F (2003) Effects of retinoic acid on
proliferation, apoptosis, cytotoxicity, migration, and
invasion of neuroblastoma cells. Med Pediatr Oncol 40,
205–213.
Prinzen C, Muller U, Endres K, Fahrenholz F & Postina R (2005) Genomic structure and functional characterization of the human ADAM10 promoter. FASEB J
[Epub ahead of print] PMID: 15972296.
Caille I, Allinquant B, Dupont E, Bouillot C, Langer A,
Muller U & Prochiantz A (2004) Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone. Development 131,
2173–2181.
Endres K, Anders A, Kojro E, Gilbert S, Fahrenholz F
& Postina R (2003) Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases
to its mature form which is degraded upon phorbol
ester stimulation. Eur J Biochem 270, 2386–2393.
Goodman AB & Pardee AB (2003) Evidence for
defective retinoid transport and function in late onset

5819


APLP2-shedding by disintegrin-metalloproteinases

Alzheimer’s disease. Proc Natl Acad Sci USA 100,
2901–2905.
47 Puchades M, Hansson SF, Nilsson CL, Andreasen N,
Blennow K & Davidsson P (2003) Proteomic studies of
potential cerebrospinal fluid protein markers for Alzheimer’s disease. Brain Res Mol Brain Res 118, 140–146.
48 Rinaldi P, Polidori MC, Metastasio A, Mariani E,
Mattioli P, Cherubini A, Catani M, Cecchetti R, Senin
U & Mecocci P (2003) Plasma antioxidants are similarly
depleted in mild cognitive impairment and in Alzheimer’s disease. Neurobiol Aging 24, 915–919.

5820

K. Endres et al.

49 Misner DL, Jacobs S, Shimizu Y, de Urquiza AM,
Solomin L, Perlmann T, De Luca LM, Stevens CF &
Evans RM (2001) Vitamin A deprivation results in
reversible loss of hippocampal long-term synaptic plasticity. Proc Natl Acad Sci USA 98, 11714–11719.
50 Valeva A, Walev I, Weis S, Boukhallouk F, Wassenaar TM, Endres K, Fahrenholz F, Bhakdi S &
Zitzer A (2004) A cellular metalloproteinase activates
Vibrio cholerae pro-cytolysin. J Biol Chem 279,
25143–25148.

FEBS Journal 272 (2005) 5808–5820 ª 2005 FEBS