(Received for publication, November 11, 1994; and in revised form, January 4, 1995)
From the
Amyloid precursor protein (APP), a transmembrane precursor of
-amyloid, possesses a function whereby it associates with G
through its cytoplasmic
His
-Lys
. Here we demonstrate that APP
has a receptor function. In phospholipid vesicles consisting of
baculovirally made APP
and brain trimeric G
,
22C11, a monoclonal antibody against the extracellular domain of APP,
increased GTP
S binding and the turnover number of GTPase of
G
without affecting its intrinsic GTPase activity. This
effect of 22C11 was specific among various antibodies and was observed
neither in G
vesicles nor in APP
/G
vesicles. In APP
/G
vesicles, synthetic
APP
, the epitope of 22C11, competitively
antagonized the action of 22C11. Monoclonal antibody against
APP
, the G
binding domain of
APP
, specifically blocked 22C11-dependent activation of
G
. Therefore, APP has a potential receptor function whereby
it specifically activates G
in a ligand-dependent and
ligand-specific manner.
Alzheimer's disease (AD) ()is a progressive
neurodegenerative disorder causing dementia (Katzman, 1986). There are
two pathological hallmarks of depositions: extracellular amyloid
plaques and intracellular paired helical filaments. The former
structures consist of
-amyloid. In AD,
-amyloid is inferred
to deposit by being cleaved off from a transmembrane precursor, termed
APP. After the identification of APP
by Kang et
al.(1987), which consists of 695 residues, at least 10 isoforms of
APP have been identified as a result of alternative splicing of a
single gene (Sandbrink et al., 1994). APP
is
preferentially expressed in the brain.
In patients with dominantly
inherited AD (FAD), point mutations have been discovered in the
transmembrane domain of APP. The Val of APP
is converted to Ile (Goate et al., 1991; Naruse et
al., 1991; Yoshioka et al., 1991), Phe (Murrell et
al., 1991), or Gly (Chartier-Harlin et al., 1991). They
co-segregate with the disease phenotype (Karlinsky et al.,
1992), indicating that structural alteration of APP is a cause of AD.
APP structurally resembles a cell surface receptor possessing a single
transmembrane region (Dyrks et al., 1988) and is actually
localized in cellular membranes (Schubert et al., 1991).
Although these findings suggest that APP functions as a cell surface
receptor, this theory was difficult to prove because of the lack of
information about both the extracellular ligand and the intracellular
signal of APP. We have, however, found that
His
-Lys
of APP
selectively activates G
and that intact APP
forms a complex with the heterotrimeric G protein G
through this domain in a GTP
S-inhibitable manner (Nishimoto et al., 1993). Given the established role of heterotrimeric G
proteins in receptor signaling, these observations provide the first
evidence that APP encodes a signaling receptor.
Based on these
findings, we demonstrate here by reconstituting purified proteins into
phospholipid vesicles that APP mimics a functional
G
-coupled receptor regulated by a ligand. Since the natural
ligand for APP is unknown, we have used 22C11 as a potential agonist.
22C11 is a mAb raised against E. coli-made APP (Weidemann et al., 1989), whose epitope region has been assigned to
APP
in the ectoplasmic Cys-containing domain
(Hilbich et al., 1993). Although the recognition potency is
not very high, antibody absorption (Milward et al., 1992) and
immunoprecipitation (Nishimoto et al., 1993) indicate that
this mAb does recognize a native form of APP. This study, in which we
conclude that APP
serves as a normal
G
-coupled receptor, provides a novel insight into both
physiological and pathological roles of APP.
Baculovirus encoding normal APP gene has been
described previously (Nishimoto et al., 1993). APP was
purified from the membranes of Sf21 insect cells infected with this
baculovirus using two-step chromatography, as described (Moir et
al., 1992) with slight modification. Briefly, after washing insect
cells with phosphate-buffered saline and centrifuging them, the
precipitates were solubilized with homogenization buffer consisting of
10 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 2 mM phenylmethylsulfonyl
fluoride, 0.2 TIU/ml aprotinin, and 2 mM leupeptin with mixing
every 5 min for 1 h at 4 °C. The lysate was centrifuged at 3000 rpm
for 1 h at 4 °C. After adjusting the NaCl and EDTA concentrations
to 350 mM and 5 mM, the supernatant was loaded onto a
Q-Sepharose column. The column was then washed with 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, 350 mM NaCl, and
1% Triton X-100. Bound proteins were eluted with 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, and 1 M NaCl. The
peak protein fractions from the Q-Sepharose column were pooled and
applied to a G-25 Sepharose desalting column, through which the buffer
containing 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, and
175 mM NaCl was prompted. The protein fraction from the G-25
column was applied to a heparin-Sepharose column. After the column was
washed with homogenization buffer, bound protein was eluted with 50
mM Tris/HCl (pH 7.4), 5 mM EDTA, and 650 mM NaCl. Trimeric G
(Katada et al., 1986) and
G
(Katada et al., 1987) purified from bovine
brain to homogeneity were provided by Dr. T. Katada, stored in 20
mM Hepes/NaOH (pH 7.4), 0.1 mM EDTA, and 0.7% CHAPS,
and diluted
10-fold for reconstitution. APP and trimeric G
were reconstituted using the gel filtration method Nishimoto et al., 1989). During reconstitution, premature activation of
G
was strictly prohibited by cooling vesicles at 4 °C
as well as by removing Mg
from the solution.
Reconstitution of APP with G
was done in a similar manner.
Like vesicles consisting of APP and G
, these vesicles only
consisted of APP and G
(data not shown). 22C11 and
anti-
-adrenergic receptor antibody were from Boehringer Mannheim
and Oncogene Science, respectively. AC-1 was kindly provided by Dr. K.
Yoshikawa (Yoshikawa et al., 1992). 4G5 and 1C1 were described
(Nishimoto et al., 1993). The epitope peptide
APP
for 22C11 (KEGILQYCQEVYPELQ) was synthesized
and purified to >97% purity.
Vesicles were incubated with
stimulation at 37 °C, and either GTPase activity or GTPS
binding activity was measured. The turnover number of GTPase activity
was assayed as described (Okamoto et al., 1991). Unless
otherwise specified, the turnover number of GTPase activity was
measured by incubating G proteins with 100 nM [
-
P]GTP in the presence of 20
µM Mg
for 20 min. GTP
S binding to G
proteins was assayed in the presence of 20 µM Mg
, and the rate constant k
was calculated as described (Okamoto and Nishimoto, 1992).
Intrinsic GTPase activity was measured as described (Strittmatter et al., 1991). All assays were performed independently at
least three times, usually six times, with similar results.
Intact APP was purified from insect cell
homogenate infected with the recombinant baculovirus encoding
APP
cDNA and was reconstituted with purified trimeric
G
into phospholipid vesicles. Silver staining revealed that
APP
and G
were virtually the only proteins
that were contained in the reconstituted vesicles (Fig. 1A). Since soluble forms of APP are possible
contaminants, the membrane precipitate of infected insect cells was
used as a starting material to ensure the purification of full-length
APP
. The preparation of APP used for reconstitution only
contained a 130-kDa protein. This protein was immunoreactive with
anti-APP mAb 22C11 (data not shown), verifying that the target of 22C11
in reconstituted vesicles is only APP
. It has been
reported that insect cells infected with baculovirus encoding APP cDNA
secrete a truncated APP lacking the C terminus (Knops et al.,
1991). However, the purified APP was a full-length APP
,
as it reacted with both the extreme C-terminal antibody AC-1 and the
ectoplasmic domain antibody 22C11 in immunoblot analysis (data not
shown).
Figure 1:
Anti-APP antibody 22C11
activates G in APP
/G
vesicles. A, silver staining of APP
/G
vesicles. APP
(40 pmol) was reconstituted with
trimeric form of G
(20 pmol) into phospholipid vesicles.
The reconstituted vesicles were applied to SDS-polyacrylamide gel
electrophoresis and silver-stained. B, the
APP
/G
vesicles were incubated with 5
µg/ml 22C11 (
) or vehicle (
) for the indicated
periods, and the turnover number of GTPase activity of G
was measured as assessed with radioactive phosphate released from
[
-
P]GTP. All values represent means
± S.E. of at least three independent experiments. C, the APP
/G
vesicles were
incubated with 5 µg/ml 22C11 (
) or vehicle (
) for the
indicated periods in the presence of
[
-
S]GTP
S, and GTP
S binding to
G
was measured, as described previously (Okamoto and
Nishimoto, 1992). The total amount of G
in the vesicles was
determined after reconstitution, based on the measurement of maximal
GTP
S binding in the presence of 10 mM Mg
under the same condition, which yielded the consistent results
with the assay reported (Northup et al., 1982). D, The APP
/G
vesicles (rightpanel) or the vesicles reconstituted with
trimeric G
alone (leftpanel) were
incubated with or without several immunoglobulins (each 5 µg/ml),
and the turnover number of GTPase activity was measured. In G
vesicles, trimeric G
(20 pmol) was similarly
reconstituted into phospholipid vesicles.
When the APP/G
vesicles were
incubated with 5 µg/ml 22C11, the turnover number of GTPase
activity of G
was stimulated to 200-250% of the basal
G
activity of the vesicles incubated without 22C11 (Fig. 1B). The rate of GTP
S binding to G
was also promoted to
250% of the basal binding rate by the
same concentration of 22C11 (Fig. 1C). In vesicles
reconstituted with trimeric G
alone, little stimulation by
22C11 was observed (Fig. 1D), suggesting that the
mediator of 22C11 is APP
. The action of 22C11 was
immunoglobulin-specific (Fig. 1D). None of the
nonspecific IgG, 4G5, 1C1, or anti-
-adrenergic receptor antibody
stimulated G
in the same vesicles that allowed 150%
stimulation of G
by 22C11. 4G5 and 1C1 are antibodies
against two distinct cytoplasmic domains of APP
. A slight
but detectable difference in the basal G
activities between
G
vesicles and APP
/G
vesicles
provides additional evidence that APP
behaves like a
G
-linked receptor, as there could be a basally active
fraction in G-coupled receptors (Samama et al., 1993).
Coupling of APP seemed to be selective to
G
. When trimeric G
was similarly reconstituted
with APP
and stimulated by 22C11, no significant
stimulation of G
was observed (Fig. 2A).
This is highly consistent with our previous study, which detected
specific activation of G
by APP
as
well as specific association of intact APP
with G
(Nishimoto et al., 1993).
Figure 2:
Characterization of 22C11-dependent
G activation by APP. A, effect of 22C11
in APP
/G
vesicles. APP
(40
pmol) was reconstituted with trimeric G
(20 pmol) into
phospholipid vesicles. The turnover number of GTPase activity of
G
was measured by incubating the vesicles with or without
5 µg/ml 22C11. All values represent means ± S.E. of at least
three independent experiments, unless otherwise specified. B, dose-response effect of 22C11 and competitive
inhibition by the 22C11 epitope peptide. The APP
/G
vesicles used in Fig. 1were incubated with increasing
concentrations of 22C11 that had been pretreated with water (hatchedbar) or the 22C11 epitope peptide
(APP
, finally 1 mM) (blackbar), and the turnover number of GTPase activity was
measured. Treatment of 22C11 with the epitope peptide or water was
carried out for 40 min at room temperature. The values corresponding to
100% and 0% stimulations are 0.39 and 0.17 (min
),
respectively. C, effect of 4G5 on 22C11-induced
G
activation by APP
. The
APP
/G
vesicles were incubated with 5
µg/ml 22C11 in the presence of increasing concentrations of 4G5 (black bar) or 1C1 (hatchedbar), and the
turnover number of GTPase activity was measured. 4G5 or 1C1 had no
effect on the G
activity without 22C11. D, effect of 22C11 on the intrinsic GTPase activity
of G
in APP
/G
vesicles. The
APP
/G
vesicles were incubated with
[
-
P]GTP in the Mg-free buffer consisting of
20 mM Hepes/NaOH (pH 7.4) and 0.1 mM EDTA at 20
°C for 16 min. Reaction was initiated by adding 1.3 mM MgSO
and 20 µM GTP with (
) or
without (
) 5 µg/ml 22C11 at 37 °C, and was terminated
with ice-cold charcoal after indicated periods of incubation. These
experiments were done three times independently and showed similar
results. This figure shows a representative result. E and F, Mg
dependence of the 22C11 action on the
turnover number of GTPase activity (E) or GTP
S binding
activity (F) of APP
/G
vesicles. The
APP
/G
vesicles were incubated with
[
-
P]GTP for 10 min (for GTPase) or with
[
-
S]GTP
S for 2 min (for GTP
S
binding) at various Mg
concentrations in the absence
or presence of 5 µg/ml 22C11. The concentration of free
Mg
was determined according to Iyengar and
Birnbaumer(1982).
To verify the mediation of
APP in the action of 22C11, we examined the interfering
effect of APP
, the epitope peptide for 22C11. As
shown in Fig. 2B, 22C11 dose-dependently promoted the
turnover number of GTPase activity of G
in
APP
/G
vesicles. When premixed with 22C11 for
40 min at room temperature, APP
shifted the
dose-response curve to the right with the potency of 22C11 being
attenuated by severalfold and its efficacy being unaltered (Fig. 2B). This shows that APP
competitively antagonizes the action of 22C11 in this system,
being consistent with the fact that APP
is the
epitope for 22C11. The requirement for high concentrations of
APP
may be attributed to the low affinity with
which the synthetic short epitope binds to the antibody relative to the
affinity of the entire protein as well as to the short preincubation
period. This provides strong evidence that 22C11 acts on APP
to stimulate G
in APP
/G
vesicles.
4G5, mAb against APP, had no
effect in APP
/G
vesicles by itself (Fig. 1D). Even at 200 µg/ml, neither 4G5 nor 1C1
affected the G
activity in these vesicles (data not shown).
However, when APP
/G
vesicles were incubated
with 4G5 in addition to 22C11, the 22C11 effect on G
activity was inhibited (Fig. 2C). The inhibition
was dose-dependent for 4G5 and nearly complete at 200 µg/ml.
Whereas the effective concentration of 4G5 was high, 1C1, a mAb that
recognizes APP
(the 19-residue region adjacent
to the 20-residue epitope for 4G5), failed to inhibit activity. These
data offer excellent support to the idea that APP
activates G
through
His
-Lys
, which is a demonstrated
G
binding and activating domain (Nishimoto et al.,
1993).
In an effort to further characterize the mechanism for
G activation by 22C11 in APP
/G
vesicles, we measured intrinsic GTPase of G
(Fig. 2D). Although steady state GTPase activity,
which represents the turnover number of the G protein activation cycle,
was promoted by 22C11, intrinsic GTPase activity was not affected by
this antibody. This indicates that 22C11 stimulates GDP/GTP exchange of
G
without altering its intrinsic GTP hydrolysis in
APP
/G
vesicles. Ligand stimulation of
APP
thus causes G
activation in a manner
highly similar to that of receptors.
We also measured the
Mg dependence of the 22C11 effect on G
activity in APP
/G
vesicles (Fig. 2, E and F). Both assays of GTP
S
binding rate, which was assessed with the rate constant k
, and of the turnover number of GTPase activity
revealed that 22C11 most effectively stimulated G
over a
wide range of Mg
concentrations between 10 µM and 1 mM, showing the close similarity of the action of
APP
to that of conventional G-coupled receptors.
APP has been shown to complex with G
in a
receptor-like manner, suggesting that it encodes a potential
G
-coupled receptor (Nishimoto et al., 1993). The
first significance of the present work is to demonstrate the
ligand-dependent function in APP
, which provides direct
evidence of a receptor-like function of APP
. Here it is
shown that anti-APP mAb 22C11 specifically activates G
in
APP
/G
vesicles with the absolute requirement
of APP
for this effect of 22C11. Although 22C11 does not
have high potency to interact with intact APP, as shown in Fig. 2B, this mAb does recognize a native form of APP,
as has been shown by immunoprecipitation (Nishimoto et al.,
1993) and antibody absorption (Milward et al., 1992).
Inhibition of the 22C11 action by APP
provides
additional evidence that this antibody recognizes APP
in
APP/G
vesicles. It is also likely that this mAb, which
belongs to divalent IgG1, activates the function of APP
by facilitating its dimerization, as has been the mechanism
whereby ligands turn on the functions of all the single-spanning
receptors that have been investigated (Ullrich and Schlessinger, 1990;
Fuh et al., 1992; Watowich et al., 1994; Spencer et al., 1993). The mode of G
activation by
APP
characterized here is highly similar to that of
receptor-stimulated G protein activation. The function of APP
is likely mediated by the His
-Lys
region. The finding that APP
could not activate
G
in response to 22C11 clearly shows the action
specificity of APP
for the recognition of G proteins.
This G protein specificity provides additional evidence that
APP
couples to G
through a specific
mechanism.
The natural ligand for APP has not been
established. However, the function of this protein is studied here
through an activating mAb. Such a strategy has been successfully used
in the anti-CD3 antibody/T cell receptor system (Imboden and Stobo,
1985), the Fas antigen system (Suda et al., 1993), and others.
There are many precedent receptors in which antibodies mimic the
function of native ligands. Given the fact that activation of
APP/G
coupling by the agonistic antibody is virtually
identical to the known receptor/G protein coupling by receptor ligands,
it is highly likely that natural ligands also exist for APP. Several
proteins such as transforming growth factor-
(Bodmer et
al., 1990) bind to APP. Our system is suitable for the search of
the physiological APP ligands, which should not only bind to APP but
also activate its function.
The present study points to a potential
role for G as the mediator of normal function of
APP
. There is growing evidence suggesting that APP is
normally involved in synaptic contact (Schubert et al., 1991)
and cell adhesion (Ueda et al., 1989; Chen and Yankner, 1991;
Mönning et al., 1992). Multiple reports
also indicate that G
is involved in signaling of neuronal
adhesion (Schuch et al., 1989; Doherty et al., 1991),
neurite outgrowth (Strittmatter et al., 1994), and growth cone
regulation (Strittmatter et al., 1990). Our conclusion that
APP has functional properties of a cell surface receptor coupled to
G
is in excellent agreement with these observations.
The
mediation of APP signals by G also suggests that G
could contribute to the pathophysiology of AD. Indeed, the tissue
distribution of G
in the brain corresponds well to the
areas severely afflicted by AD. Thus, it is very important to examine
whether the APP
/G
coupling is abnormal in
AD-derived tissues or in FAD-linked mutants of APP. Based on the
extreme similarity of the point mutations found in the FAD-linked APPs
to the oncogenic Neu mutation of c-ErbB2, a receptor tyrosine kinase
(Bargmann et al., 1986), we assume that G
is
constitutively activated by these APP
mutants linked to
FAD. There are multiple lines of evidence suggesting that G proteins,
which could be G
, are activated in AD tissues (Smith et
al., 1989; Flynn et al., 1991; Gibson and Toral-Barza,
1992; Ito et al., 1994; Cowburn et al., 1992; Cowburn et al., 1993; Huang and Gibson, 1993). This study should
therefore provide the basis for both physiological and pathological
functions of APP.
There are many isoforms of APP except APP (Sandbrink et al., 1994). Regarding the novel function
that this study has identified, difference or identity should be
clarified between APP
and other isoforms. Some of them
have the extracellular domain identical to that of APP
with a truncated cytoplasmic domain and could thereby work as a
dominant-negative receptor for APP
. Conversely, both
APP
and APP
, which have the cytoplasmic
domain identical to that of APP
with different
extracellular domains, could interfere the signaling function of
APP
inside the cells. Cells may respond to the putative
APP ligand as a result from the whole body of molecular functions of
these isoforms.