From the Departments of Pathology and Laboratory
Medicine, UCLA, Los Angeles, California 90095-1732, the Departments
of § Physiology,
Pediatrics and Neurology, and the
§§ Howard Hughes Medical Institute, University
of California, San Francisco, California 94143-0723, and the
Department of Neurology and Neurological
Sciences, Stanford University Medical Center,
Palo Alto, California 94305
Received for publication, June 14, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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The TrkA receptor is activated
primarily by nerve growth factor (NGF), but it can also be activated by
high concentrations of neurotrophin 3 (NT-3). The
pan-neurotrophin receptor p75NTR strongly
inhibits activation of TrkA by NT-3 but not by NGF. To examine the role
of p75NTR in regulating the specificity of TrkA signaling,
we expressed both receptors in Xenopus oocytes. Application
of NGF or NT-3 to oocytes expressing TrkA alone resulted in efflux of
45Ca2+ by a phospholipase
C- Nerve growth factor
(NGF),1 brain-derived
neurotrophic factor (BDNF), neurotrophin-4 (NT4), and neurotrophin-3
(NT-3) initiate their actions by binding to Trk receptors and
p75NTR (1). NGF binds to TrkA; BDNF and NT-4 bind to TrkB,
and NT-3 binds to TrkC. NT-3 also binds TrkA (and TrkB), although with significantly lower affinity (1), and can activate TrkA signaling and
cellular responses (2-7). In vivo, NT-3 signaling through other Trk receptors is required for nervous system development, since
NT3 p75NTR also binds each of the neurotrophins (11) and can
signal independently of TrkA in response to neurotrophin stimulation (12-14). In addition, p75NTR regulates Trk receptor
signaling. p75NTR potentiates NGF activation of TrkA at low
ligand concentrations (15, 16) and collaborates with TrkA to form high
affinity binding sites (17-19). Furthermore, the signaling pathways
initiated by p75NTR block Trk receptor signaling in certain
contexts (20, 21) and synergize with Trk receptor signaling in other
contexts (22). These opposing actions suggest a complex interaction
between p75NTR and Trk receptors in regulating neurotrophin signaling.
NT-3 does not activate TrkA signaling in PC12 cells (4, 23), unless
p75NTR levels are reduced (23), suggesting that
p75NTR directly suppresses the ability of TrkA to respond
to NT-3 (4). In support of this hypothesis, isolated sympathetic
neurons from p75NTR We used a Xenopus oocyte microinjection assay to study NGF
and NT-3 signaling through TrkA and to assess the role of
p75NTR in regulating this signaling. We showed that
p75NTR prevented the signaling of NT-3 through TrkA but not
that of NGF. We demonstrated that the extracellular domain of
p75NTR, but not the cytoplasmic domain, was necessary to
inhibit NT-3 signaling through TrkA. Furthermore, p75NTR
binding to NT-3 was not required to inhibit NT-3 activation of TrkA.
Finally, we showed that p75NTR and TrkA could be
coimmunoprecipitated from Xenopus oocytes, suggesting that
interaction of these receptors mediates the inhibition of NT-3
signaling through TrkA.
Growth Factors and Antibodies--
Purified recombinant human
neurotrophins were obtained from Amgen Inc. (Thousand Oaks, CA) and
Genentech Corp. (South San Francisco, CA). Rabbit polyclonal
anti-p75NTR antibody was raised against the extracellular
domain of rat p75NTR (25). Rabbit polyclonal antibody
raised against the cytoplasmic domain of p75NTR was
purchased from Promega (Madison WI). Rabbit polyclonal antibody (26)
was raised to the C terminus of human TrkA. M2 monoclonal anti-FLAG
antibody was purchased from Stratagene (La Jolla, CA), and monoclonal
anti-EGFR antibody (LA22) was purchased from Upstate Biotechnology,
Inc. (Lake Placid, NY).
cDNA Constructs and Plasmid Generation--
cDNAs
encoding the full length rat TrkA and rat p75NTR receptors
were subcloned into PGEMHE vector, which contains 5' and 3' Xenopus beta-globin untranslated sequences for RNA stability
(gift from Dr. E. R. Liman) (27). Truncated p75NTR
receptor was generated from the rat p75NTR cDNA using
PCR with forward primer 5'GAA TTC ATG AGG AGG GCA GGT GCT GCC and
reverse primer 5'CAC GGG TCT AGA CTA GGC GCC TTG TTT ATT TTG TTT GCA
GCT G to amplify the extracellular and transmembrane domains, and the
first nine amino acids of the cytoplasmic domain, flanked by an 5'
EcoRI site and a 3' XbaI site. The amplified product was subcloned into PGEMHE. All constructs used in these experiments were verified by sequencing.
A chimeric EGF receptor/p75NTR cDNA encoding the
extracellular domain of the human EGF receptor with the transmembrane
and cytoplasmic domains of human p75NTR (gift of Dr. Moses
Chao) (28) was subcloned from pCMVEN10 into the PGEMHE vector. This was
done using PCR with forward primer 5' GCG CGC GTC GAC GCG ATG CGA CCC
TCC GGG ACG GCC and reverse primer 5' GCG CGC TCT AGA GGC TCA CAC CGG
GGA TGA GGC AGT GG to amplify the chimeric EGFR/p75NTR
construct flanked by a 5' SalI site and a 3' XbaI
site. The amplified product was digested with SalI and
XbaI and subcloned into PGEMHE.
The C-terminal FLAG-tagged TrkA receptor was generated by PCR using
forward primer 5'CA GGG ACT AGT GGT CAA GAT GAT TGG A and reverse
primer 5' CTT ATC ATC ATC ATC CTT GTA ATC GCC CAG AAC GTC CAG G to
amplify a fragment encoding the last 405 base pairs of rat TrkA
receptor coding sequence (minus the stop codon) including the 5'
SpeI site (part of the coding sequence) and the first FLAG
epitope on the 3' end. This product was then used as a template for a
second PCR using the same forward primer and a reverse primer encoding
the second FLAG tag followed by a stop codon and an XhoI
restriction site 5'CTG CTC GAG CTA CTT ATC ATC ATC ATC CTT GTA ATC CTT
ATC ATC ATC. The amplified product was digested with SpeI
and XhoI and subcloned into similarly digested full-length
TrkA in PGEMHE.
In Vitro Transcription of cRNA and Expression in Xenopus
Oocytes--
DNA templates were linearized and transcribed in
vitro using the T7 RNA polymerase promoter with mMessage in
vitro transcription kit from Ambion (Austin, TX). Transcripts were
purified using an RNAeasy kit purchased from Qiagen Inc. (Valencia, CA)
and then analyzed on agarose gels and quantified by spectrophotometry
(as well as by densitometric comparison of bands to known RNA standards).
Xenopus laevis and Preparation of Oocytes--
Mature
Xenopus oocytes (Dumont stage V-VI) were harvested and
defolliculated enzymatically with 1-2 mg/ml collagenase (type I or II)
for 2-3 h (Worthington). Oocytes were maintained at 17-18 °C in
ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5) plus 1 µg/ml ciprofloxacin, 6 µg/ml
ceftazidime, and 1 µg/ml gentamicin.
45Ca2+ Efflux
Assays--
For calcium efflux assays, oocytes were injected with a
total of 0.2 ng of cRNA per oocyte. When cRNA encoding a single
receptor was injected, 0.1 ng of GFP cRNA was coinjected to equalize
the total amount of injected cRNA. Efflux experiments were conducted according to methods published previously (29, 30). In brief, 2 days
after injection, oocytes were incubated in
45Ca2+ (from Amersham Pharmacia Biotech), 100 mCi/ml for 2.5 h in calcium-free ND96. Groups of 8 (or 4) oocytes
were washed and transferred to 24-well dishes with 0.5 ml of ND96 per
well. Medium was collected and replaced every 10 min, and radioactivity
was measured by liquid scintillation counting. After stabilization of
background 45Ca2+ efflux, 100 ng/ml either NT-3
or NGF was added to each group of oocytes. Data were collected from
each of the experimental conditions by using 4-8 oocytes per condition
(same number of oocytes in each experiment), with each condition run in
triplicate for each experiment. The initial NT-3 dose-response curve
(Fig. 2) was run in duplicate. Each experiment was repeated two or
three times, using oocytes from two or three different frogs.
Therefore, every data point for each condition in one experiment
consisted of the average of three measurements obtained from two or
three groups of four to eight oocytes. Signaling curves were graphed using Microsoft Excel, and mean and standard errors of measure were
determined using Microsoft Excel. For analysis of dose-response curve,
mean peak counts per min were compared using analysis of variance
methods and allowing for unequal variances. The Tukey-Fisher criterion
was used to compute post hoc t statistics and their corresponding p values. Similar results were obtained using
a log scale transformation of the data where equal variances could be assumed.
Immunoprecipitations and Immunoblotting--
For analysis of
TrkA, p75NTR, and truncated p75NTR
For analysis of the chimeric EGFR/p75NTR receptor,
Xenopus oocytes were injected with 5 ng per oocyte of
EGFR/p75NTR cRNA. Thirty six hours post-injection, 25 oocytes were manually homogenized in ice-cold homogenization buffer (65 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM sodium orthovanadate, and 10 mM sodium fluoride), and lysates were centrifuged at
735 × g for 5 min at 4 °C to separate a heavy
fraction (containing the plasma membrane and cortical granules) from
the cytoplasmic fraction. The heavy fraction was solubilized in
250 µl of RIPA buffer (with 2.5% Triton X-100) for 1 h at
4 °C and centrifuged at 18,370 × g for 20 min at
4 °C. Samples were pre-cleared with protein G-Sepharose beads.
EGFR/p75NTR receptors were immunoprecipitated with
anti-EGFR monoclonal antibody (clone LA22, Upstate Biotechnology, Inc.,
Lake Placid, NY) overnight at 4 °C. Precipitated samples were washed
four times with 1× solubilization buffer, after which precipitated
proteins were removed from the beads by boiling for 5 min in 3×
Laemmli sample buffer, and SDS-PAGE was performed on 7.5%
polyacrylamide gels. After transfer to nitrocellulose membranes
(Hybond, Amersham Pharmacia Biotech), samples were blocked in TBST with
nonfat dried milk and incubated with anti-EGFR antibody.
For coimmunoprecipitation experiments, equal amounts of cRNA for
FLAG-tagged TrkA and p75NTR (5 ng of each cRNA) were
microinjected into each oocyte. After 48 h, oocytes were incubated
with NT-3 (100 ng/ml), NGF (100 ng/ml), or no neurotrophin for 20 min
at room temperature, after which they were placed on ice. Oocytes were
manually homogenized in ice-cold homogenization buffer, and samples
were centrifuged at 735 × g for 5 min at 4 °C. The
pellet (containing the membrane) was solubilized in 500 µl of a
buffer containing 130 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2% Triton X-100, and 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate. The
cytoplasmic fraction was solubilized by adding a 2× concentration of
this solubilization buffer to the 250 µl of supernatant. Fractions
were solubilized for 15 min at 37 °C and centrifuged at 18,370 × g for 20 min at 4 °C. Samples were pre-cleared with
protein A/G-Sepharose beads and immunoprecipitated with
anti-p75NTR (REX) (25). After SDS-PAGE and transfer,
nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech) were
blocked in TBST with nonfat dried milk and incubated with primary
antibodies against p75NTR extracellular domain (REX) (25)
or anti-FLAG M2 monoclonal antibody (Stratagene, La Jolla, CA).
Expression of TrkA and p75NTR in Xenopus
Oocytes--
The Xenopus oocyte
45Ca2+ efflux assay has been used to quantify
receptor activation and signal transduction (29, 30). Mature oocytes
are capable of translating injected cRNA, targeting proteins to correct
subcellular locations, and inserting receptors into the plasma membrane
(31). Because the phospholipase C-
To determine whether TrkA and p75NTR protein expression
correlate with the amount of each cRNA injected, we microinjected
Xenopus oocytes with 0, 1, 2.5, or 5 ng of TrkA per oocyte
and determined the level of TrkA protein expressed. There was a
proportional increase in the amount of TrkA receptor protein expressed
by oocytes in response to increasing amounts of injected TrkA cRNA
(Fig. 1A). Similarly, oocytes
injected with 0, 1, or 2.5 ng of p75NTR cRNA demonstrated
that p75NTR protein expression correlated with the amount
of p75NTR cRNA injected (Fig. 1B). To determine
whether coexpression of p75NTR interferes with TrkA
receptor expression, oocytes were coinjected with TrkA cRNA (1 ng per
oocyte) and increasing amounts of p75NTR cRNA (0, 1, or 2.5 ng per oocyte). Injection of increasing amounts of p75NTR
cRNA resulted in increasing p75NTR expression; however, the
amount of TrkA expressed was unaffected by p75NTR
coexpression (Fig. 1C). Thus, the amount of TrkA and
p75NTR protein expressed correlated with the amount of each
cRNA injected, similar to other proteins exogenously expressed in
Xenopus oocytes (29, 30, 36).
NT-3 Activates Signaling through TrkA Receptors--
Having
determined that TrkA and p75NTR receptor expression
correlate with the amount of each cRNA injected, we next studied the signaling of TrkA-expressing Xenopus oocytes in response to
a range of NT-3 concentrations. NT-3 activated TrkA signaling through the phospholipase C- p75NTR Specifically Inhibited NT-3 Signaling through
TrkA--
To determine whether p75NTR inhibited NT-3
signaling through TrkA and to assess whether this inhibition was
specific to NT-3, we coexpressed TrkA and p75NTR in
oocytes. Equal amounts of cRNA encoding p75NTR and TrkA
were injected. Oocytes that expressed TrkA alone were also injected
with an equal amount of green fluorescent protein (GFP) cRNA to control
for total RNA injected. (GFP had no effect on the signaling response to
neurotrophins, data not shown.) After loading the oocytes with
45Ca2+ and allowing for stabilization of
45Ca2+ efflux, 100 ng/ml of either NGF or NT-3
was added, and 45Ca2+ efflux was measured every
10 min. Application of NGF or NT-3 to TrkA-expressing oocytes elicited
45Ca2+ efflux. NGF signaling was unaffected by
coexpression of p75NTR, and p75NTR had no
effect on the timing or peak level of NGF signaling (Fig. 3A). In contrast,
p75NTR completely inhibited NT-3 signaling through TrkA
(Fig. 3B). The inhibitory effect of p75NTR on
NT-3 signaling through TrkA increased with increasing expression of
p75NTR (Fig. 3C). Remarkably, almost full
potency of TrkA signaling was recovered when the
p75NTR:TrkA ratio was decreased from 1:1 to 1:2. These data
are evidence that rather small changes in p75NTR expression
have marked effects on TrkA signaling. Increased expression of
p75NTR relative to TrkA did not impair the response of
oocytes to subsequent NGF stimulation (Fig. 3C). These data
demonstrate that p75NTR specifically inhibits NT-3
signaling through TrkA and that the level of inhibition depends on the
ratio of p75NTR relative to TrkA.
The Extracellular Domain of p75NTR Inhibited NT-3
Signaling through TrkA--
To determine which structural domain of
p75NTR mediated the inhibitory effect on NT-3 signaling
through TrkA, we constructed a mutant p75NTR receptor with
a truncated cytoplasmic domain (containing only the first nine amino
acids of the cytoplasmic domain). We coexpressed this truncated
p75NTR with TrkA to assess its effect on NT-3 signaling.
Expression of the truncated p75NTR receptor was confirmed
by immunoblotting (Fig. 4B),
and surface expression was further confirmed by 125I -NT-3
binding and cross-linking studies (data not shown). After stabilization
of 45Ca2+ efflux, 200 ng/ml of NT-3 was added,
and 45Ca2+ efflux was measured every 10 min.
The truncated p75NTR completely inhibited NT-3 signaling
through TrkA (Fig. 4A). This suggested that the cytoplasmic
domain of p75NTR (excluding the juxtamembrane region) is
not required to inhibit NT-3 signaling through TrkA. To determine
whether the extracellular domain of p75NTR is necessary, we
coexpressed TrkA along with an EGFR/p75NTR chimera,
containing the extracellular domain of the EGFR fused to the
transmembrane and cytoplasmic domains of p75NTR. This
chimeric receptor has been used to study p75NTR signaling
in PC12 cells and has been shown to be expressed on the cell surface
(28). Expression in oocytes was confirmed by the presence of a 130-kDa
band, consistent with the predicted size (28) (Fig. 4D).
NT-3 signaling in Xenopus oocytes expressing the
EGFR/p75NTR receptor along with TrkA was as robust as in
oocytes expressing TrkA alone (Fig. 4B). From these
experiments, we conclude that the extracellular domain of
p75NTR is necessary for inhibition of NT-3 signaling
through TrkA. We also conclude that the transmembrane and cytoplasmic
domains of p75NTR are not sufficient to inhibit NT-3
signaling through TrkA.
Antagonistic interactions between the signaling pathways of Trk
receptors and p75NTR have been described (21, 37, 38).
However, our finding that the extracellular domain of
p75NTR inhibited NT-3 signaling, whereas most of the
cytoplasmic domain was dispensable, suggests that events occurring
between the receptors on the plasma membrane and between their
extracellular domains are critical for inhibiting NT-3 signaling
through TrkA.
p75NTR Inhibited NT-3 Activation of TrkA Even When It
Did Not Bind NT-3--
This raised two possibilities as follows:
either p75NTR interacts with NT-3 in a manner that inhibits
its ability to activate TrkA or p75NTR interacts with TrkA
to inhibit activation by NT-3. The first possibility requires
p75NTR binding to NT-3 to suppress signaling. Under the
second possibility, NT-3 binding to p75NTR is dispensable.
To distinguish between these possibilities, we performed a
45Ca2+ efflux assay using oocytes coexpressing both
receptors, in the presence or absence of anti-p75NTR
antibody or excess BDNF (1 µg/ml), each of which prevents NT-3 binding to p75NTR. Oocytes expressing TrkA and
p75NTR or TrkA alone (along with GFP) were loaded with
45Ca2+ as described above. Oocytes expressing
both receptors were incubated for 2 h prior to the addition of
NT-3 with 1 µg/ml of BDNF, or with 50 µg/ml of
anti-p75NTR antibody (REX), or in medium alone. BDNF and
anti-p75NTR antibody at these concentrations nearly
abolished NT-3 binding to p75NTR in PC12 cells
overexpressing TrkA (data not shown), and the ability of the
anti-p75NTR antibody to block NT-3 binding to
p75NTR in oocytes was verified by binding and cross-linking
studies (data not shown). 200 ng/ml of NT-3 was added to the oocytes, and 45Ca2+ efflux was measured every 10 min. The
data show that blocking NT-3 binding to p75NTR had no
effect on the ability of this receptor to block TrkA activation (Fig.
5). No response to NT-3 was seen in the
presence of the p75NTR antibody or BDNF. This experiment
shows that p75NTR inhibited NT-3 signaling through TrkA,
even in the absence of direct binding to NT-3. Because BDNF and the
anti-p75NTR antibody may act as p75NTR ligands,
this experiment does not exclude the possibility that p75NTR liganding may alter TrkA responsiveness to NT-3.
However, it does indicate that direct interaction of p75NTR
with NT-3 is not required to inhibit TrkA activation.
p75NTR and TrkA Physically Interacted in Xenopus
Oocytes Coexpressing Both Receptors--
Our results suggested that
TrkA and p75NTR may physically interact in surface
membranes. To address this possibility, we coexpressed a C-terminal
FLAG-tagged TrkA receptor along with p75NTR in
Xenopus oocytes. The TrkA construct consisted of full-length TrkA with two FLAG epitopes in tandem at the C terminus. Expression of
the FLAG-tagged receptor was verified by homogenizing 15 oocytes microinjected with cRNA encoding the receptor, immunoprecipitating with
a monoclonal antibody against the FLAG epitope (M2), and immunoblotting
with the same anti-FLAG antibody. A specific doublet corresponding to
TrkA was identified, which was not present in uninjected oocytes (Fig.
6).
We next asked if the FLAG-tagged TrkA receptor functionally responded
to neurotrophin stimulation. NGF activation of TrkA results in
activation of the phospholipase C-
To determine whether p75NTR physically interacted with
TrkA, we performed coprecipitation studies in microinjected oocytes.
Equal amounts of cRNA for FLAG-tagged TrkA and p75NTR (5 ng
of each cRNA) were injected, and after 48 h, oocytes were exposed
to NT-3 (100 ng/ml), NGF (100 ng/ml), or no neurotrophin for 20 min at
room temperature, after which they were placed on ice (to prevent
further membrane trafficking events).
Oocytes were manually homogenized and fractionated into a heavy
fraction, containing the membrane and cortical granules, and a
cytoplasmic fraction (see "Experimental Procedures"). Fractions were solubilized in a buffer containing 2% Triton X-100 (and protease inhibitors) for 15 min at 37 °C and were then centrifuged.
Supernatants were pre-cleared with protein A/G-Sepharose beads and
immunoprecipitated with a polyclonal antibody to p75NTR
(25). SDS-PAGE was performed on samples (see "Experimental Procedures"). After transfer to nitrocellulose membranes, samples were immunoblotted with the monoclonal antibody to the FLAG epitope (Stratagene, La Jolla, CA). As seen in Fig.
7, TrkA was coprecipitated with
p75NTR from the membrane fractions of oocytes injected with
TrkA and p75NTR (lanes 2-4) but not from
uninjected oocytes (lane 5) or from the antibody plus
protein A/G bead control sample (lane 1). p75NTR
and TrkA were coprecipitated in oocytes expressing these receptors regardless of whether they were treated with 100 ng/ml of NT-3 (lane 4), NGF (lane 3), or with no neurotrophin
(lane 2). The anti-p75NTR serum has previously
been shown to bind p75NTR and to lack detectable binding to
TrkA (25). We further verified that the anti-p75NTR serum
did not immunoprecipitate TrkA from oocytes expressing TrkA alone and
that the anti-FLAG monoclonal antibody did not recognize a specific
band in oocytes expressing only p75NTR (data not shown).
Because high levels of expression of both p75NTR and TrkA
were required to observe this interaction, and only a relatively small
percentage of each receptor appears to be in this complex, we cannot
fully determine the role of this physical interaction at physiological
concentrations. However, these results do suggest that
p75NTR and TrkA can physically interact in the plasma
membrane and that this interaction appears to be independent of the
presence of neurotrophins.
In this paper, we examined the role of p75NTR in
inhibiting NT-3 signaling through TrkA. By using a calcium efflux assay
to assess signaling in Xenopus oocytes microinjected to
express both receptors, we demonstrated quantitatively that
p75NTR completely suppressed NT-3 signaling through TrkA.
The inhibition was specific to NT-3, because NGF signaled equally well
through TrkA, regardless of whether or not p75NTR was
present. Our data are consistent with studies in PC12 cells which
demonstrate that p75NTR suppresses NT-3 signaling through
TrkA (4, 23) and with the observation that TrkA-expressing sympathetic
neurons from p75NTR In addition to inhibiting TrkA signaling, p75NTR may also
inhibit signaling through TrkB. In transfected A293 cells expressing TrkB, coexpression of p75NTR decreased TrkB phosphorylation
in response to NT-3 and NT-4 but not in response to BDNF (41). In
contrast to this finding, another group (42) has recently demonstrated
that coexpression of p75NTR with TrkB inhibited
phosphorylation in response to BDNF and NT-4 but not NT-3. The full
effect of p75NTR on TrkB signaling in response to BDNF,
NT-4, and NT-3 signaling through TrkB will need to be further
elucidated. However, the mechanism by which p75NTR
suppresses TrkB activation may be similar to the way p75NTR
suppresses NT-3 signaling through TrkA.
Structural Domains of p75NTR Involved in the Inhibition
of NT-3 Signaling through TrkA--
In these experiments, we showed
that the extracellular domain of p75NTR was necessary to
inhibit NT-3 signaling through TrkA. NT-3 could not signal through TrkA
in Xenopus oocytes coexpressing a mutant p75NTR
receptor that lacks all but the first nine amino acids of its cytoplasmic domain. In contrast, oocytes coexpressing an
EGFR/p75NTR chimeric receptor, which contained full
p75NTR transmembrane and cytoplasmic domains, did not
inhibit NT-3 signaling through TrkA. Based on these findings, we
conclude that the extracellular domain of p75NTR is
necessary to inhibit NT-3 signaling through TrkA. These experiments also clearly showed that the transmembrane and cytoplasmic domains of
p75NTR are not sufficient to inhibit NT-3 signaling through TrkA.
The truncated p75NTR receptor contains the juxtamembrane
region of p75NTR, including cysteine 279, which is critical
for palmitoylation of p75NTR (43). Because palmitoylation
of p75NTR appears to be important for targeting
p75NTR (as well as TrkA) to caveolae-like membranes where
much of TrkA receptor signaling is initiated (44), it is possible that
the juxtamembrane region of p75NTR may be important for
inhibiting NT-3 signaling through TrkA. However, our experiments
clearly show that this region, in the absence of the extracellular
domain, is not sufficient to inhibit NT-3 signaling through TrkA.
p75NTR and Trk receptors may engage in cross-talk through
the interaction of their cytoplasmic signaling pathways. To date, much of this interaction appears to be inhibitory. NGF activation of TrkA
can block the p75NTR-mediated cell death pathway (21), and
activation of p75NTR by BDNF suppresses cellular responses
to NGF activation of TrkA (37, 38). Our data do not entirely exclude
the possibility that p75NTR initiates a cytoplasmic signal
through its juxtamembrane region, either directly or by interacting
with another cytoplasmic protein. However, our observation that the
p75NTR receptor, lacking almost the entire cytoplasmic
domain, inhibited NT-3 signaling through TrkA suggests that
p75NTR did not act downstream from the receptor to suppress
TrkA signaling.
Interaction of p75NTR and TrkA to Mediate the
Inhibition of NT-3 Signaling through TrkA--
By coexpressing TrkA
and p75NTR in oocytes and occupying p75NTR with
either BDNF of anti-p75NTR antibody, we determined that
p75NTR inhibited activation of TrkA even in the absence of
direct binding to NT-3. Because BDNF and REX may act as
p75NTR ligands, these results do not exclude the
possibility that liganding of p75NTR alters the ability of
TrkA to respond to NT-3. More importantly, these results suggest
strongly that events occurring between p75NTR and TrkA,
either directly or indirectly, are critical for inhibiting TrkA
response to NT-3.
In oocytes coexpressing both receptors at significantly higher levels
than used in the 45Ca2+ efflux assays, we
coimmunoprecipitated p75NTR and TrkA from the membrane
fraction of oocytes. Although we could only detect this interaction
when both receptors were expressed at these relatively higher levels,
these findings suggest that p75NTR and TrkA can physically
interact in the surface membrane and that this interaction does not
require the presence of neurotrophins.
The structural basis for p75NTR and TrkA interactions has
not been defined. Evidence to support their interaction has been
presented (41, 45-49), and most reports have focused on the role of
p75NTR in increasing the affinity of TrkA for NGF (19, 48,
49). Our results suggest a potential role for the physical interaction of p75NTR and TrkA in regulating the specificity of TrkA
for NGF relative to NT-3. The coimmunoprecipitation data suggest the
existence of either a direct physical interaction between
p75NTR and TrkA or the formation of a complex bridged by
one or more mutually interacting proteins. p75NTR and TrkA
are both significantly enriched in caveolae-like domains at the plasma
membrane, and the two receptors may interact (at least functionally)
within this micro-environment (44). Furthermore, indirect interaction
between p75NTR and TrkA via the mutually interacting
protein caveolin-1 has been suggested (50).
It is also possible that p75NTR inhibits NT-3 signaling by
inhibiting NT-3 binding to TrkA. However, preliminary data from our laboratory using 125I -NT-3 binding and cross-linking in
PC12 cells that overexpress TrkA indicate that NT-3 can still bind TrkA
in the presence of p75NTR.3 In these
cells, occupying p75NTR with BDNF or REX did not alter the
amount of NT-3 bound to TrkA.3 However, since BDNF and REX
may act as p75NTR ligands, it is possible that liganded
p75NTR alters the conformation of the TrkA-binding site. An
allosteric model for p75-TrkA interactions has been proposed that may
account for this property (51). In the future, it will be important to
examine NT-3 binding in a cell line that expresses TrkA alone, or in
combination with p75NTR, to fully determine the effect of
p75NTR on NT-3 binding to TrkA.
In summary, our experiments have shown that p75NTR
interacts with TrkA to inhibit NT-3 signaling. Our work demonstrates
that the extracellular domain of p75NTR is necessary for
this inhibition and suggests that this interaction occurs at the level
of p75NTR-TrkA interaction on the surface membrane. Our
study has important implications for understanding the regulation of
NT-3 signaling through TrkA in vivo. During late embryonic
and early postnatal development, nearly 50% of sympathetic neurons
that are NGF-dependent develop an additional requirement
for NT-3, mediated by TrkA (8, 9, 52, 53). These neurons express low
levels of p75NTR relative to TrkA (53), which our data
suggest would enable NT-3 to promote survival by activating TrkA (2, 5,
53). As targets are reached, p75NTR expression increases
and responsiveness to NT-3 declines (2). In vivo studies of
the neonatal sympathetic ganglia show that p75NTR regulates
the survival of TrkA-expressing sympathetic neurons by regulating the
responsiveness of these neurons to NT-3 (40). In fact, it appears that
one of the most important roles for p75NTR in the
developing sympathetic ganglia is to regulate TrkA responsiveness to
NT-3 (40). In addition to sympathetic neurons, a subset of late
embryonic TrkA-expressing sensory neurons that are
NGF-dependent also develop a requirement for NT-3 that is
mediated by TrkA (5, 10). Unlike postnatal sympathetic neurons,
postnatal sensory neurons lose their ability to respond to NT-3 via
TrkA. It will be interesting to determine whether p75NTR
plays a role in decreasing the responsiveness of postnatal sensory neurons to NT-3. Finally, although our work has focused on the role of
p75NTR in regulating NT-3 signaling through TrkA,
p75NTR may interact with TrkB to regulate its
responsiveness. It will be important in the future to determine whether
p75NTR regulates TrkB signaling by a similar mechanism.
-dependent pathway. Coexpression of p75NTR
with TrkA inhibited 45Ca2+ efflux in response
to NT-3 but not NGF. The inhibitory effect on NT-3 activation of TrkA
increased with increasing expression of p75NTR.
Coexpression of a truncated p75NTR receptor lacking all but
the first 9 amino acids of the cytoplasmic domain inhibited NT-3
stimulation of 45Ca2+ efflux, whereas
coexpression of an epidermal growth factor receptor/p75NTR
chimera (extracellular domain of epidermal growth factor receptor with
transmembrane and cytoplasmic domains of p75NTR) did not
inhibit NT-3 signaling through TrkA. These studies demonstrated that
the extracellular domain of p75NTR was necessary to inhibit
NT-3 signaling through TrkA. Remarkably, p75NTR binding to
NT-3 was not required to prevent signaling through TrkA, since
occupying p75NTR with brain-derived neurotrophic factor or
anti-p75 antibody (REX) did not rescue the ability of NT-3 to activate
45Ca2+ efflux. These data suggested a physical
association between TrkA and p75NTR. Documenting this
physical interaction, we showed that p75NTR and TrkA could
be coimmunoprecipitated from Xenopus oocytes. Our results
suggest that the interaction of these two receptors on the cell surface
mediated the inhibition of NT-3-activated signaling through TrkA.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice exhibit significantly greater neuronal loss than
TrkC
/
mice (8). Recently, NT-3 has been shown to be required for
the survival of TrkA-expressing postmitotic sympathetic neurons (9) and
sensory neurons (10) in vivo.
/
mice are more responsive to NT-3
in culture than are wild type sympathetic neurons (24). These data
suggest that p75NTR can prevent NT-3 signaling through
TrkA, but the molecular nature of this interaction remains to be characterized.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors,
Xenopus oocytes were injected with each cRNA (1-5 ng per
oocyte of each cRNA). Thirty six hours post-injection, 25 oocytes from
each condition were manually homogenized in 250 µl of ice-cold
solubilization buffer (65 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 2% Triton X-100, 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml
leupeptin, 1 mM sodium orthovanadate, and 10 mM
sodium fluoride). Lysates were incubated at 4 °C for 1 h, and
the soluble phase was partitioned from the insoluble and lipid phases
by three rounds of centrifugation at 18,370 × g for 5 min at 4 °C. 250 µl of 2× IP buffer (solubilization buffer with
10 mg/ml of bovine serum albumin) was added to each sample and
pre-cleared with protein A- or protein G-Sepharose beads for 1 h.
TrkA receptors were immunoprecipitated with the anti-FLAG monoclonal
antibody (M2), and P75NTR and p75
receptors were
precipitated using wheat germ lectin-agarose beads (Amersham Pharmacia
Biotech). Precipitated samples were washed four times with 1×
solubilization buffer, after which precipitated proteins were removed
from the beads by boiling for 5 min in 3× Laemmli sample buffer, and
SDS-PAGE was performed on 7.5% polyacrylamide gels. After transfer to
nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech), samples
were blocked in TBST with nonfat dried milk and incubated with primary
antibodies against the FLAG epitope of TrkA (Stratagene, La Jolla CA),
the extracellular domain of p75NTR (REX), or the
cytoplasmic domain of p75NTR (Promega, Madison, WI)
overnight at 4 °C. After washing, blots were incubated with
horseradish peroxidase-conjugated goat anti-mouse (Promega, Madison, WI
or New England Biolabs, Beverly, MA) or horseradish
peroxidase-conjugated goat anti-rabbit (Promega, Madison, WI or New
England Biolabs, Beverly, MA) at a dilution of 1:5000 for 1-2 h at
room temperature. After washing, bands were visualized by incubating
with Supersignal (Pierce) or ECL (Amersham Pharmacia Biotech), followed
by exposure to Kodak BioMax film (Eastman Kodak Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pathway is conserved in oocytes,
activation of receptor tyrosine kinases, such as Trk receptors,
stimulates phospholipase C-
and phosphatidylinositol turnover and
stimulates intracellular calcium release (32, 33). The increased
intracellular calcium activates a chloride current in
Xenopus oocytes and causes cytoplasmic calcium to be
released into the extracellular medium (32, 34, 35). Receptor
activation and signal transduction through the phospholipase C-
pathway can therefore be quantified by using a
45Ca2+ efflux assay (29, 30). For this reason,
we chose to express TrkA (with or without p75NTR) in
Xenopus oocytes to quantitatively study NT3 activation of TrkA and the role of p75NTR in regulating it.
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Fig. 1.
TrkA and p75NTR receptor
expression are highly correlated with the amount of cRNA injected into
Xenopus oocytes, and coexpression of
p75NTR does not interfere with TrkA receptor
expression. A, the amount of TrkA receptor protein
expressed correlates with the amount of TrkA receptor cRNA injected.
Xenopus oocytes were injected with 0, 1, 2.5, or 5 ng of
TrkA cRNA, and the level of TrkA protein expressed was determined by
immunoprecipitation followed by immunoblotting. There is a proportional
increase in the amount of TrkA receptor, 140- and 110-kDa doublet,
expressed in response to increasing amounts of TrkA cRNA injected.
B, the amount of p75NTR protein expressed
correlates with the amount of p75NTR cRNA injected. Oocytes
were injected with 0, 1, or 2.5 ng of p75NTR cRNA, and
p75NTR protein expression was determined by precipitation
with wheat germ lectin-agarose followed by immunoblotting. A
proportional increase in p75NTR protein, 75-80 kDa, is
seen with increasing p75NTR cRNA injection. C,
increasing p75NTR expression does not alter the level of
TrkA receptor expressed. Oocytes were injected with 1 ng of TrkA and
increasing amounts of p75NTR (0, 1, and 2.5 ng per oocyte).
p75NTR expression is noted to increase proportionally;
however, TrkA expression is not altered by p75NTR
coexpression.
pathway in a dose-dependent fashion
(Fig. 2). Peak response was seen 30 min
after addition of 10 ng/ml NT-3, 20 min after addition of 25 or 50 ng/ml of NT-3 and 10 min after addition of 100, 200, or 300 ng/ml NT-3
(data not shown). The peak response to NT-3 plateaued at about 100 ng/ml, suggesting that 100 ng/ml was functionally saturating. Similar
results were obtained using a log scale transformation of the data
where equal variances could be assumed. In addition, differences in
mean peak signaling were compared using one-way analysis of variance.
There were no statistically significant mean differences in NT-3
signaling at 100, 200, and 300 ng/ml (p > 0.52 or
larger for the three comparisons). However, the mean response at 100 ng/ml was significantly different from the response at 50 ng/ml
(p = 0.002). All subsequent experiments were done using
100 or 200 ng/ml of NT-3.
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Fig. 2.
Dose-response curve of 45Ca
efflux after NT-3 application to TrkA-expressing oocytes. Oocytes
expressing TrkA (0.1 ng of cRNA/oocyte) were loaded with
45Ca2+ and washed (see "Experimental
Procedures"). After 45Ca2+ efflux levels
stabilized, NT-3 was added (at time 0) to the medium at final
concentrations ranging from 10 to 300 ng/ml. Peak mean counts/min were
plotted against the dose of NT-3. Peak mean response to NT-3 was seen
after 30 min in response to 10 ng/ml NT-3, after 20 min for 25 and 50 ng/ml of NT-3, and 10 min after addition of 100, 200, or 300 ng/ml of
NT-3 (data not shown).
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Fig. 3.
p75NTR inhibits NT-3 signaling
through TrkA but not NGF signaling. A, oocytes
expressing TrkA alone or in combination with p75NTR (see
"Experimental Procedures") were loaded with
45Ca2+. After stabilization of
45Ca2+ efflux, 100 ng/ml NGF (A) or
100 ng/ml NT-3 (B) was added at time 0 (indicated by
arrow), and calcium efflux was measured every 10 min.
C, inhibitory effect of p75NTR on NT-3 signaling
through TrkA increases with increasing expression of
p75NTR. Oocytes injected with TrkA receptor cRNA (0.2 ng/oocyte) and p75NTR (ranging from 0.04 to 0.2 ng/oocyte)
were loaded with 45Ca2+. After stabilization of
45Ca2+ efflux, 100 ng/ml of NT-3 was added at
time 0 (indicated by arrow), and calcium efflux was measured
every 10 min. Subsequent response of oocytes to application of 100 ng/ml of NGF (arrow) demonstrates that the oocytes are
capable of responding to NGF stimulation.
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Fig. 4.
The extracellular domain of
p75NTR is necessary to prevent NT-3 signaling through TrkA,
and the transmembrane and cytoplasmic domains are not sufficient to
inhibit signaling. A, a truncated mutant
p75NTR receptor with a minimal cytoplasmic domain (9 amino
acids) inhibits NT-3 signaling through TrkA. Oocytes expressing TrkA
alone in combination with truncated p75NTR were loaded
with 45Ca2+. After stabilization of
45Ca2+ efflux levels, 200 ng/ml of NT-3 was
added to the medium (indicated by arrow), and
45Ca2+ efflux was measured every 10 min.
B, oocytes injected with p75NTR
cRNA express
the truncated p75NTR receptor protein. Lysates from oocytes
injected with p75NTR
cRNA (+) and from uninjected oocytes
(
) were precipitated with wheat germ lectin-agarose followed by
immunoblotting with an antibody against the extracellular domain of
p75NTR (REX). C, a chimeric
EGFR/p75NTR receptor containing the extracellular domain of
the EGF receptor fused to the transmembrane and cytoplasmic domains of
p75NTR does not inhibit NT-3 signaling through TrkA.
Oocytes expressing TrkA alone or in combination with
EGFR/p75NTR were loaded with
45Ca2+. After stabilization of
45Ca2+ efflux levels, 200 ng/ml NT-3 was added
to the medium (indicated by arrow), and calcium efflux was
measured every 10 min. D, oocytes injected with the
EGFR/p75NTR cRNA express the chimeric receptor protein.
Lysates from oocytes injected with EGFR/p75NTR cRNA (+) and
from uninjected oocytes (
) were immunoprecipitated with monoclonal
antibody that recognizes the extracellular domain of the EGF receptor
followed by immunoblotting using the anti-EGF receptor antibody.
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Fig. 5.
p75NTR does not need to directly
bind to NT-3 to inhibit activation of signaling through TrkA.
Oocytes expressing TrkA alone or in combination with p75NTR
were loaded with 45Ca2+. Oocytes expressing
TrkA and p75NTR were treated for 2 h with 50 µg/ml
of anti-p75NTR antibody (REX), 1 µg/ml BDNF, or were left
untreated as indicated. The anti-p75NTR antibody and BDNF
at this concentration have both been shown to prevent NT-3 binding to
p75NTR (see "Results"). After stabilization of
45Ca2+ efflux, 200 ng/ml of NT-3 was added at
time 0 (indicated by arrow), and calcium efflux was measured
every 10 min.
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Fig. 6.
Expression of FLAG-tagged TrkA receptor in
microinjected Xenopus oocytes. Fifteen oocytes
were injected with 1 ng of TrkA-FLAG cRNA (+) or were left uninjected
( ). Thirty six hours after injection, oocytes were lysed,
immunoprecipitated with anti-FLAG antibody, separated by 7.5%
SDS-PAGE, and immunoblotted with the anti-FLAG antibody (see
"Experimental Procedures"). Bands of 140 and 110 kDa are seen in
the TrkA-injected oocytes.
pathway with resultant intracellular calcium release (39). Because a calcium-activated chloride current occurs in Xenopus oocytes in response to
release of intracellular calcium (32, 33, 35), we performed
electrophysiologic recordings from single oocytes in the presence of
NGF to confirm that FLAG-tagged TrkA was functional. Oocytes expressing
this receptor responded specifically to 100 ng/ml of
NGF,2 demonstrating that this
receptor is indeed functional.
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Fig. 7.
p75NTR and TrkA can be
coimmunoprecipitated from the membrane fractions of microinjected
Xenopus oocytes expressing both receptors.
Oocytes were injected with TrkA-FLAG and p75NTR (5 ng
each). Thirty six hours later, 12 oocytes per condition were treated
with 100 ng/ml NGF (lane 3), 100 ng/ml NT-3 (lane
4), or no neurotrophin (lane 2) for 20 min at room
temperature (see "Experimental Procedures"). Oocytes were lysed,
immunoprecipitated with anti-p75NTR antibody, and
immunoblotted with anti-FLAG antibody. TrkA is coprecipitated with
p75NTR in oocytes injected with TrkA and p75NTR
cRNA (lanes 2-4), but not in uninjected oocytes (lane
5), or in antibody and protein A/G-Sepharose beads only control
(lane 1). Anti-p75NTR serum has previously been
shown to bind p75NTR and to lack detectable binding to TrkA
(25). We have also observed that anti-p75NTR anti-serum
does not precipitate TrkA from oocytes expressing TrkA only, nor does
the anti-FLAG monoclonal antibody recognize a specific band from
oocytes expressing p75NTR alone (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice are more responsive to NT-3
than wild type sympathetic neurons (24). Our findings are also
consistent with the recent evidence that p75NTR can inhibit
the ability of NT-3 to activate TrkA in vivo (40). Compared
with control littermates, NGF+/
mice have a 50% deficit in
sympathetic neurons. This deficit is rescued by NT-3 in the absence of
p75NTR (NGF+/
and p75NTR
/
) but not in
mice that express normal levels of p75NTR (40). The rescue
of these TrkA-expressing sympathetic neurons depends on NT-3, since
many of these neurons are lost in NGF+/
, p75NTR
/
,
NT-3+/
mice (40).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Moses Chao for the EGFR/p75NTR construct. We also thank Drs. Eric Huang, Song Hu, and Ardem Patapoutian for helpful discussions and Kan Lu for help with preparation of the figures.
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FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Research Grants MH48200 (to L. F. R.), NRSA NS10301 (to P. S. M.), and NS24054 (to W. C. M.), the Howard Hughes Medical Institute, a Stein Oppenheimer award (to P. S. M.), the McGowen Charitable Fund (to W. C. M.), the Adler Foundation (to W. C. M.), and the John Douglas French Alzheimer's Foundation (to W. C. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Conducted this research as a Pfizer Scholar. To whom reprint requests and correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, UCLA Medical Center, 10833 Le Conte Ave., Los Angeles, CA 90095-1732. Tel.: 310-794-5223; Fax: 310-206-0657; E-mail: pmischel@mednet.ucla.edu.
** Present address: Dept. of Neurology and Neurological Sciences, Stanford University Medical Center, Palo Alto, CA 94305.
¶¶ Investigator of the Howard Hughes Medical Institute.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M005132200
2 P. S. Mischel and J. Umbach, S. Eskandari, S. G. Smith, C. B. Gundersen, and G. A. Zampighi, submitted for publication.
3 P. S. Mischel, J. S. Valletta, W. C. Mobley, and L. F. Reichardt, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: NGF, nerve growth factor; NT-3, neurotrophin-3; BDNF, brain-derived neurotrophic factor; NT-4, neurotrophin-4; p75NTR, p75 neurotrophin receptor; EGFR, epidermal growth factor receptor; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
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