(Received for publication, December 26, 1995; and in revised form, February 26, 1996)
From the
-Amyloid protein precursors (APPs, 695-770 amino
acids) are the source of the 39-43-amino acid
-amyloid
(A
) peptides that comprise diffuse and fibrillar deposits in the
cerebral cortex and vasculature of Alzheimer's disease brains.
A
is thought to play a role in the pathogenesis of
Alzheimer's disease, and, hence, considerable effort has been
invested in defining the means by which A
is generated from the
APPs. Knowledge of the normal function of the APPs is sure to provide
insights into the genesis and pathological persistence of A
in
Alzheimer's disease. APP is a cell surface protein with a large
extracellular amino-terminal domain, a single transmembrane segment,
and a short cytoplasmic tail. Its location and structural features
characteristic of a receptor for signal transduction led us to search
for potential effector proteins capable of binding and interacting with
its cytoplasmic domain. Here, we report the cloning of a cDNA encoding
one such protein. This ubiquitously expressed 59-kDa APP-binding
protein, called APP-BP1, is 61% similar to a protein encoded by the Arabidopsis AXR1 gene, required for normal response to the
hormone auxin, and is a relative of the ubiquitin activating enzyme E1.
The human -amyloid precursor protein (APP) (
)gene (Kang et al., 1987; Goldgaber et
al., 1987; Tanzi et al., 1987a; Robakis et al.,
1987) encodes a set of APPs (695, 714, 751, and 770 amino acids) that
are derived from alternatively spliced mRNAs. These APPs were initially
identified as precursors of
-amyloid (A
, 39-43 amino
acids), which forms abnormal extracellular deposits in the cerebral
cortex and blood vessel walls in the Alzheimer's disease brain
(for a recent review, see Ashall and Goate(1994)). The chain of events
culminating in the deposition of these pathological fragments remains
ob-scure. Knowledge of the normal activity, trafficking, and cleavage
of APP is an essential precursor to our understanding of how one or
more of these processes goes awry to create aberrant amyloidogenic
fragments.
APP is a member of a family of proteins that also
includes two human amyloid precursor-like proteins, APLP1 (Wasco et
al., 1992) and APLP2 (Wasco et al., 1993). The amino acid
sequences of APP and APLPs are highly conserved, and the protein
structures of APP and APLPs are similar. APLPs, like APP, are located
on the cell surface. They have a large extracellular amino-terminal
domain, a single transmembrane region, and a short cytoplasmic tail.
Within the extracellular domain, a cysteine-rich region, a zinc-binding
motif, an acidic region, and N-glycosylation sites are
conserved in all members of this gene family. Some forms generated from
alternatively spliced mRNA possess a Kunitz class protease inhibitor
domain in the extracellular portion of the molecule. However, only APP
has the A segment, whose amino terminus is extracellular and whose
carboxyl terminus is within the cell membrane.
APP is ubiquitously
expressed, but the relative levels of different APP isoforms vary among
cell types, with APP-695 expressed preferentially in the brain. The
biological function of the membrane-bound form of APP is unknown. Its
extracellular domain can be enzymatically cleaved either within or
upstream of the A sequence, to release secreted forms of APP. The
Kunitz class protease inhibitor-containing secreted forms may act as
inhibitors of extracellular serine proteases (Oltersdorf et
al., 1989) and of the platelet coagulation factor XIa (Smith et al., 1990). Secreted APPs have been shown to participate in
cell adhesion (Schubert et al., 1989; Breen et al.,
1991; Small et al., 1992; Jin et al., 1994), neurite
outgrowth (Koo et al., 1993; Small et al., 1994; Jin et al., 1994), and synaptic plasticity (Mattson et
al., 1993). Deletion of the Appl gene in Drosophila (Luo et al., 1992) or partial inactivation of the APP
gene in mice by gene targeting (Müller et
al., 1994) results in impairment of learning and memory in these
organisms. Mice with complete deletion of the APP gene exhibited a
compromised neuronal function (Zheng et al., 1995). Very
little is known about the role of membrane-bound APP in cellular
function. Notably, APP has structural features characteristic of cell
surface receptors (Kang et al., 1987) with signal-transducing
properties (Nishimoto et al., 1993). To learn more about the
intracellular signals propagated by APP, we sought proteins with the
potential to bind and interact with the cytoplasmic domain of this
molecule. Here we report the cloning and sequencing of a cDNA encoding
an APP-binding protein homologous to a protein encoded by the auxin
hormone-resistant gene AXR1 in the plant Arabidopsis (Leyser et al., 1993). Our data suggest that this binding
protein, called APP-BP1, may transduce signals mediated by the APP.
A human 20-22-week
fetal brain cDNA library in gt11 (Neve et al., 1986) was
plated (2
10
plaque-forming units) and screened at
4 °C. Replica filters were blocked overnight in phosphate-buffered
saline (PBS) containing 5% milk, 1 mM MgCl
, 1.2
mM CaCl
, 0.2 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol. The APP-C100His
probe (10
cpm) was added to the filters, and
incubation was continued overnight. The filters were then washed (in
PBS for 3 min, in PBS containing 0.1% Nonidet P-40 for 3 min, and in
PBS for 3 min), dried, and exposed to x-ray films. Phage plaques that
gave duplicated signals were purified and subcloned into pBluescript
(pBS, Stratagene).
The APP-BP1/pBS was used as a template for
PCR with primers complementary to the gt10 arms. The resulting PCR
fragments were cloned into pBS and used for in vitro transcription (Ambion) and translation (wheat germ lysate system,
Ambion). [
S]Methionine-labeled APP-BP1 was
incubated with control GST or GST-fusion proteins on beads in PBS in
the presence or absence of competitor proteins for 1 h at room
temperature. Complexes were washed with 5
1 ml of PBS
containing 0.5% Nonidet P-40, immediately boiled in SDS sample buffer,
and analyzed by PAGE. The gels were dried and exposed directly to film
overnight. The quantity of fusion protein on beads and competitor
proteins was estimated by SDS-PAGE followed by Coomassie Blue staining.
The amounts of GST-fusion proteins used in each reaction were adjusted
to equality by normalizing the quantity of beads added to each
reaction. Densitometric analysis was with a Macintosh
computer and a desktop scanner as described by Shea(1994).
COS cells
were cotransfected with Myc-tagged APP-BP1 and APP-695 in pcDNA I by
using LipofectAMINE (Life Technologies, Inc.). Three days after
transfection, the cells were lysed in lysis buffer (20 mM Tris, pH 7.5, 1 mM MgCl, 125 mM NaCl, 1% Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10
µg/ml aprotinin. After centrifugation, the supernatant was
incubated at 4 °C with monoclonal anti-Myc antibody (ATCC) or
anti-APP antibody 10D5 (Hyman et al., 1992) for 1 h. Protein
G-Sepharose (Pharmacia Biotech Inc.) was than added, and the incubation
was continued for another hour at 4 °C. The immunocomplexes were
washed 5 times in lysis buffer, resuspended in SDS sample buffer,
resolved by SDS-PAGE, and blotted. The blots were incubated with
anti-Myc or affinity-purified anti-APP antibody 369 (Buxbaum et
al., 1990), followed by enhanced chemiluminescence detection
(Tropix) and autoradiography.
Figure 1: Nucleotide and the predicted amino acid sequence of the APP-BP1 cDNA. The underlined region indicates the sequence found in the partial cDNA clone detected by initial expression cloning. ATG of the putative initiation codon and the TAG of the termination codon are in boldface.
Figure 2:
Specific binding of APP-BP1 to the
cytoplasmic domain of APP. A, S-labeled APP-BP1 (lane 1) was incubated with GST (lane 2), GST-GAP-43 (lane 3), GST-C100 (lane 4), GST-
/A4 (lane
5), or GST-C57 (lane 6) proteins that were noncovalently
coupled to glutathione-agarose beads.
[
S]Methionine-labeled APP-BP1 identified two
bands with apparent molecular sizes of
66 kDa on SDS gel. Both
forms of APP-BP1 are specifically retained by GST-C100 and GST-C57.
Protein size markers (Bio-Rad) are shown at the left. B,
S-labeled APP-BP1 was incubated with GST-C100 (lanes 1 and 3) or GST-C57 (lanes 2 and 4)
immobilized on beads in the presence of MBP (lanes 1 and 2) or free APP-C57 (lanes 2 and 4). The
amounts of competitor protein in each reaction were about the same as
the GST-fusion protein on beads. C, binding of
S-labeled APP-BP1 to GST-C100 is a linear function of the
APP-BP1 concentration in the reaction (R
=
0.994). In vitro binding assays were performed with various
amounts of APP-BP1 ranging from 2 to 12% (v/v) in final concentration.
The result shown was calculated from two sets of
assays.
Figure 3: Coimmunoprecipitation of APP-695 with APP-BP1. COS cells were transiently transfected with APP-695 and Myc-tagged APP-BP1 expression constructs. The lysates from control cells (lanes 1-3) and from cotransfected cells (lanes 4-6) were incubated with no antibody (lanes 1 and 4) and with antibodies 10D5 (lanes 2 and 5), or anti-Myc (lanes 3 and 6). Immunocomplexes were analyzed by Western blot, and the filters were incubated with antibodies 369 to APP (A) or anti-Myc to the tagged APP-BP1 (B).
Figure 4: Alignment of amino acid sequences of APP-BP1 with related proteins. Alignment was performed using the Pileup program of the GCG package. Pairwise comparisons between the proteins were done using a Gap program, and the results are shown in the text. Hyphens indicate gaps introduced to maximize alignment. A, APP-BP1, AXR1, and the predicted open reading frame of a C. elegans gene on chromosome III. Amino acid residues identical in at least two of the three sequences are displayed as white letters in black boxes. B, APP-BP1 and the amino-terminal 600 amino acids of ubiquitin-activating enzyme E1 from human, wheat, and mouse. Residues identical in at least four of the six E1 proteins are boxed, and the residues in APP-BP1 identical with these highly conserved amino acids are shown as white letters in black boxes.
APP-BP1 and AXR1
are relatives of ubiquitin-activating enzyme (E1), which is a highly
conserved molecule with a mass of 110 kDa (Handley et
al., 1991; McGrath et al., 1991; Hatfield et
al., 1990), which binds to ubiquitin prior to its conjugation to
cellular proteins that are targeted for rapid degradation. Alignment of
APP-BP1 with the amino-terminal 600 amino acids of E1 from human (Kok et al., 1993), wheat (Hatfield et al., 1990; Hatfield
and Vierstra, 1992), and mouse (Imai et al., 1992) reveals a
46-47% similarity (Fig. 4B).
Figure 5:
Southern blot analysis of the APP-BP1
sequence in human DNA. DNA was digested with EcoRI (lane
1), BamHI (lane 2), PstI (lane
3), and HindIII (lane 4). HindIII-digested DNAs were used as size markers as
indicated at the left of the blot. DNA was hybridized with a
full-length APP-BP1 probe. kb,
kilobases.
The APP-BP1 was localized to human chromosome 16 band q22 using fluorescent in situ hybridization (Fig. 6). Two independent experiments were performed, and more than 100 metaphase cells were evaluated. Signals were clearly visible on two chromatids of at least one chromosome in 45% of the cells. No other chromosomal sites with consistent signals were detected in more than 1% of the cells.
Figure 6: Fluorescence in situ hybridization mapping of the APP-BP1 gene to chromosome 16q22. A, a human chromosome preparation was hybridized with biotinylated phage cDNA. Fluorescein isothiocyanate signals are clearly visible in the chromomycin/distamycin reverse banded chromosome region of 16q22. B, a human chromosome 16 ideogram showing the location of APP-BP1 in the region of 16q22.
Figure 7: Northern blot analysis of human APP-BP1 RNA. A, RNA from various human fetal tissues: spleen (lane 1), thymus (lane 2), muscle (lane 3), kidney (lane 4), liver (lane 5), lung (lane 6), small intestine (lane 7), heart (lane 8), adrenal gland (lane 9), and brain (lane 10). B, RNA from subregions of an adult human brain: NbM, nucleus basalis of meynert (lane 1); BG, basal ganglia (lane 2); A20,21, temporal association cortex (lane 3); Hi, post hippocampus (lane 4); pHG, parahippocampal gyrus (lane 5); and A17, striate cortex (lane 6). The blots were hybridized with an APP-BP1 probe generated from an EcoRI fragment (bp 479-799) of APP-BP1 cDNA. The same filters were hybridized to a cDNA probe for cyclophilin to normalize the amount of RNA loaded in each lane.
In situ hybridization histochemistry was used to localize APP-BP1 mRNA in the rat brain. The data revealed that APP-BP1 mRNA is expressed throughout the brain. In the hippocampus, APP-BP1 showed robust expression in granule cells of the dentate gyrus and in the pyramidal cell layer (Fig. 8, A and B). Within the cerebral cortex, APP-BP1 riboprobe hybridized strongly to cells in the piriform cortex (Fig. 8, C and D). In the cerebellum, the APP-BP1 probe labeled only the Purkinje cells (Fig. 8, E and F).
Figure 8: Expression of APP-BP1 RNA in rat brain. Sagittal sections of rat brain were hybridized with an APP-BP1 antisense probe. Adjacent sections probed with the corresponding sense riboprobe did not show specific labeling (data not shown). Three regions with high levels of APP-BP1 signal are shown: hippocampus (A and B), piriform cortex (C and D), and cerebellum (E and F). Sections were viewed by dark field microscopy at low magnification for the localization of APP-BP1 signal (A, C, and E). Bright field higher magnification of the sections counterstained with Nissl shows silver grains over cells (B, D, and F). Arrows indicate either the areas that are magnified (A, C, and E) or the cells that have silver grains over them (B, D, and F). Bars, 200 µm (A and C), 100 µm (E), and 20 µm (B, D, and F).
A human cDNA encoding a 59-kDa protein was isolated by screening expression cDNA libraries with the radiolabeled carboxyl-terminal region of APP. This protein, termed APP-BP1, was shown to interact with the carboxyl-terminal region of APP in in vitro binding assays and with the full-length APP in immunoprecipitation assays. APP-BP1, like APP, is ubiquitously expressed in neural and non-neural tissues. Comparison of the deduced amino acid sequence with protein sequences in the data base revealed that APP-BP1 is highly homologous (61%) to AXR1, a protein encoded by the Arabidopsis auxin resistance gene. Southern blot analysis of human genomic DNA suggested that APP-BP1 is a single-copy gene, and fluorescence in situ hybridization was used to localize the gene to chromosome 16q22.
Although the function of cell-surface APP remains obscure, its structural features, a large extracellular amino-terminal domain, a single transmembrane region, and a short cytoplasmic tail, are reminiscent of those of integrins (reviewed by Dustin and Springer (1991)), which are the major receptors for extracellular matrix proteins. In addition to these features, the carboxyl terminus of APP shares with the integrins and with other known plasma membrane receptors (Tamkun et al., 1986) a highly conserved stretch of five amino acids (683-688 in the APP-695 sequence) representing the consensus sequence surrounding a tyrosine that is autophosphorylated in the epidermal growth factor receptor (Downward et al., 1984).
If the membrane-bound APP does
function as a cell-surface receptor, by what mechanism are signals
transduced from the cell surface to its interior? For many hormone and
growth factor receptors, signal transduction across the cell membrane
occurs by direct interaction of the receptor cytoplasmic domain with
its intracellular target protein. In this study, we report the
identification of such a target protein for APP, APP-BP1, supporting
the idea that APP may function as a cell surface receptor. Numerous
effects of integrin activation have been described. They include
reorganization of the cytoskeleton (Burridge et al., 1988),
alterations in tyrosine phosphorylation (Kornberg et al.,
1991; Schaller et al., 1992), activation of calcium-dependent
proteases (Fox et al., 1985), activation of the
Na/H
antiporter (Banga et
al., 1986), and changes in the subcellular distribution of
phosphoinositide 3-kinase (Zhang et al., 1992). Although
direct involvement of APP has not been demonstrated in such cellular
pathways, the known role of membrane-bound APP in neurite extension
(Qiu et al., 1995; LeBlanc et al., 1992; Milward et al., 1992), its synaptotrophic effects (Mucke et
al., 1994), and its possession of an RHDS sequence that promotes
cell adhesion and is blocked by an anti-(
1-integrin) antibody
(Ghiso et al., 1992) are consistent with an integrin-like
function for APP. Breen et al.(1991) have shown that
membrane-bound APP mediates cell-cell binding and cell adhesion to a
collagen substrate in a manner similar to that of the neural cell
adhesion molecule, which modulates both process outgrowth and synaptic
plasticity (Rutishauser and Jessell, 1988). Membrane-bound neural cell
adhesion molecule is associated with cell-surface heparan sulfate
proteoglycans in a complex in which heparan sulfate proteoglycan
mediates cell-substratum adhesion (Kallapur and Akeson, 1992). Several
studies suggest that the membrane-bound form of APP similarly is
associated with a heparan sulfate proteoglycan (Schubert et
al., 1988; Kalaria et al., 1992; Su et al.,
1992) which may also, by analogy, regulate cell-extracellular matrix
interactions.
The Arabidopsis AXR1 is thought to mediate the action of the plant hormone auxin, which is required for cell division, elongation, and differentiation (Leyser et al., 1993). Mutations in the AXR1 gene result in a variety of morphological defects presumably due to reduced auxin sensitivity (Lincoln et al., 1990). AXR1 may interact directly with auxin-binding site(s) (for a review, see Goldsmith(1993)) or indirectly via binding to intermediate protein(s) in the signal transduction pathway. The fact that APP-BP1 is a human homologue of AXR1 is consistent with the idea that APP functions as a membrane receptor and that APP-BP1 plays a role transducing APP-mediated signaling into the cell.
The biochemical activities of APP-BP1 and AXR1 are still unknown. Despite the sequence similarity between APP-BP1 and ubiquitin-activating enzyme E1, APP-BP1, AXR1, and the putative C. elegans protein probably define a new family of proteins. The overall degree of protein sequence similarity within the members of this family is greater than that between any member and E1, and, conversely, two Arabidopsis E1 proteins are more similar to E1 proteins from other species than they are to AXR1 (Leyser et al., 1993). It is unlikely that AXR1 and APP-BP1 have the same activity that E1 does, because a conserved cysteine at position 626, required for ubiquitin-conjugation activity in wheat E1 (Hatfield and Vierstra, 1992), is located outside the region of similarity, and because two consensus ATP-binding domains identified in yeast E1 (McGrath et al., 1991) are missing in AXR1 and in APP-BP1. These data do not, however, rule out the possibility that APP-BP1 with an activity different from E1 plays a role in a cellular ubiquitination pathway. Several cell surface receptors, such as the platelet-derived growth factor receptor (Mori et al., 1992), the T cell antigen receptor (Cenciarelli et al., 1992), and the immunoglobulin E (IgE) receptor (Paolini and Kinet, 1993), are ubiquitinated in response to ligand binding. Ubiquitination of proteins marks the selective degradation for many polypeptides. Activation-induced ubiquitination of the platelet-derived growth factor and the T cell antigen receptor may lead to a rapid degradation of ligand-receptor complexes. However, the activation-induced ubiquitination of the IgE is rapidly reversible. It suggests that the function of the IgE receptor may be regulated by ubiquitination. By analogy, cell surface APP might be ubiquitinated upon ligand binding by a mechanism in which the APP-associated APP-BP1 is involved directly or indirectly.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U50939[GenBank].