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INTRODUCTION |
Neurotrophins are a family of growth factors that mediate the
survival, development, and death of specific populations of neurons and
glial cells (1). Many of the classic trophic signals elicited by
neurotrophins (nerve growth factor
(NGF),1 brain-derived
neurotrophic factor, and neurotrophin-3) require the presence of a
specific member of the Trk tyrosine kinase receptor family (Trk A, Trk
B, and Trk C, respectively (2-8)). After neurotrophin binding, Trk
receptors undergo dimerization and transphosphorylation on specific
tyrosine residues, a requisite step for activation of their intrinsic
tyrosine kinase activity and coupling to downstream signaling pathways
(9, 10). In contrast, neurotrophin-induced death signals (11, 12) may
be mediated in specific cell types through the interaction of NGF with
a transmembrane protein known as the low-affinity neurotrophin
receptor, p75NTR (13, 14). The pro-apoptotic effect of
p75NTR may be mediated by generation of the bioactive lipid
metabolite ceramide (11) that derives from the hydrolysis of
sphingomyelin (SM) (15-17).
An emerging theme in neurotrophin signaling is that reciprocal
interactions between Trk and p75NTR signaling pathways can
dictate cellular responses to neurotrophins (14). Although it is well
appreciated that p75NTR can regulate
Trk-dependent responses (14, 18-20), the role of Trk in
regulating p75NTR-dependent signaling is not as
well documented; nonetheless, it is supported by both biochemical and
biologic observations. Although brain-derived neurotrophic factor and
neurotrophin-3 effectively induced SM hydrolysis in PC12 cells, which
do not express endogenous Trk B or Trk C receptors, NGF was ineffective
(16). Significantly, the inhibition of Trk A tyrosine kinase activity
with K252a enabled NGF to stimulate SM hydrolysis. These studies
provided the first biochemical clue that Trk A activation can inhibit
some aspects of p75NTR signaling. Importantly, this
biochemical observation has functional correlates relevant to the
effect of neurotrophins on cell survival. An elegant study by Bamji
et al. (21) demonstrated clearly that Trk A activation
inhibited brain-derived neurotrophic factor-induced p75NTR-dependent death of sympathetic neurons.
Van der Zee et al. (22) reported that expression of Trk A
was required to counteract the death-inducing effects of
p75NTR in medial septum and neostriatal cholinergic
neurons. Lastly, retroviral expression of Trk A in mature
oligodendrocytes inhibited NGF-induced
p75NTR-dependent apoptosis and blocked the
stimulation of stress-activated protein kinases (23). These
observations suggest that endogenous factors that regulate Trk tyrosine
kinase activity would be expected to modulate interactions between
neurotrophin signaling pathways.
Caveolin-1 is a putative transformation suppressor protein that has
emerged as a candidate molecule that may regulate the activity of cell
growth pathways. Caveolin-1 is a member of a multigene family that
includes caveolin-2 and caveolin-3 and is a key structural protein in
the morphologic formation of caveolae (24, 25); for simplicity, our use
of caveolin will be synonymous with caveolin-1. Caveolae are
invaginations of the plasma membrane that are involved in the cellular
transport of small molecules and are also considered as potential
localized centers for signal transduction (24), especially through
tyrosine kinase receptors (26-28), sphingolipid (17, 29), and
phosphatidylinositol signaling pathways (30, 31). Additionally, most if
not all cells contain caveolae-related membrane domains (CRDs). CRDs
share a similar lipid constitution with caveolae but lack caveolin.
Caveolin can associate with G-proteins (32), ras (33), protein kinase C
isoforms (34), Src (35), nitric oxide synthase (36), and tyrosine
kinase receptors (37, 38) through direct protein-protein interactions.
Importantly, the interaction of these proteins with caveolin has
functional consequences on signaling because caveolin can inhibit the
activity of nitric oxide synthase (36, 39), protein kinase C isoforms
(34), and tyrosine kinases (37). Therefore, we hypothesized that the
interaction of caveolin with Trk may also have functional consequences
on regulating cross-talk between the Trk A and p75NTR
signaling pathways. We demonstrate that overexpression of caveolin in
PC12 cells prevents neurite outgrowth and blocks prolonged Trk A
autophosphorylation in response to NGF. Trk A interacts specifically
with caveolin, and this interaction dramatically inhibits NGF-induced
autophosphorylation of Trk A. Moreover, similar to pharmacologic
inhibition of Trk A (16), caveolin inhibition of Trk A signaling
enables NGF to induce SM hydrolysis through p75NTR. These
data raise the possibility that caveolin or caveolin-related proteins
may regulate signaling through Trk and p75NTR receptors in
specific neuronal populations.
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EXPERIMENTAL PROCEDURES |
Materials--
Mouse 2.5S NGF was obtained from Harlan
Bioproducts for Science. Polyclonal antibodies against
antiphosphotyrosine and the C terminus of caveolin were purchased from
Signal Transduction Laboratories. Polyclonal antibodies raised against
the N terminus of caveolin and against the intracellular domain of Trk
(pan-Trk and C-14) were obtained from Santa Cruz Biotechnology.
Polyclonal antibodies against the Trk A extracellular domain were
obtained from Upstate Biotechnology.
Cell Lines--
Mock-transfected NIH-3T3 fibroblasts were
maintained as described previously (17). 615-Trk A-PC12 cells were a
kind gift from Dr. B. Hempstead (Cornell University, New York, NY) and
were maintained in RPMI 1640 medium containing 10% horse serum, 5% fetal calf serum, and 0.2 mg/ml Geneticin. Wild type PC12 cells were
grown in the same medium lacking Geneticin. PC12 and Trk A-PC12 cells
were transfected with a C-terminal myc-tagged form of caveolin-1 (33)
using LipofectAMINE. To obtain stable clones expressing caveolin, the
transfected cells were grown in the presence of 0.5 mg/ml hygromycin B. Antibiotic-resistant colonies were isolated and expanded, and the
expression of caveolin was determined by immunoblot analysis. Several
PC12 colonies expressing caveolin (Cav-PC12) were isolated and
maintained in RPMI 1640 medium containing 10% horse serum and 5%
fetal calf serum with 0.2 mg/ml hygromycin B. Caveolin expression was
found to slow cell growth and gradually diminish as the cells were
passaged. In some experiments, Trk A-PC12 cells were transiently
transfected with caveolin as described above and used 48-72 h after transfection.
Isolation of Caveolae-enriched Membranes (CEMs)--
CEMs were
isolated by nondetergent extraction in sodium carbonate and
centrifugation over discontinuous sucrose gradients (17, 33) or by
using the Opti-Prep method (40). After centrifugation in sucrose
gradients, 15 × 0.8 ml fractions were removed, starting from the
top of the gradient. To concentrate the membranes within different
regions of the gradient, CEMs (fractions 4-7), NCM-1 (fractions
8-11), and NCM-2 (fractions 12-15) were pooled, diluted into MBS (25 mM 2-(N-morpholino)ethanesulfonic acid, 150 mM NaCl, and 5 mM EDTA), and spun at
100,000 × g for 30 min at 4 °C. The membrane
proteins were used for immunoprecipitation or analyzed directly by
SDS-PAGE. After transferring the proteins to nitrocellulose, caveolin
was detected by immunoblot analysis and identified by co-migration with
caveolin from a human endothelial cell lysate.
Co-Immunoprecipitation of Trk A with Caveolin--
One 15-cm
dish of Trk A-PC12 cells or Cav-Trk A-PC12 cells was washed twice with
ice-cold PBS, and the cells were resuspended in 0.3 ml of
immunoprecipitation (IP) buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Nonidet P-40, 60 mM
-octylglucoside, 1 mM phenylmethylsulfonyl fluoride, and
10 µg/ml each of leupeptin, aprotinin, and bestatin). The cells were
broken by sonication with three 15-s bursts, and the lysate was
centrifuged at 21,000 × g for 15 min. Samples
containing 1.5 mg of protein from the supernatant were precleared with
protein A-Sepharose, the supernatant was incubated for 2 h at
4 °C with 2 µg of polyclonal caveolin antibody, and caveolin was
immunoprecipitated with the addition of PAS beads. The PAS beads were
washed three times with 1 ml of ice-cold MBST (MBS plus 1% Triton
X-100), and the bound proteins were solubilized by boiling in sample
buffer for 5 min. After SDS-PAGE, the proteins were transferred to
nitrocellulose membranes, and the membranes were probed with a pan-Trk
antibody. Immunoreactive proteins were visualized using a horseradish
peroxidase-conjugated secondary antibody and enhanced chemiluminescence.
Association of GST-Caveolin Fusion Proteins with Trk A and
p75NTR--
Characterization of the fusion proteins used
in these experiments and the methods used for their expression and
purification have been described previously (17, 32, 41). In some
experiments, caveolin fusion proteins were isolated with
agarose-conjugated glutathione beads, and caveolin was released from
the GST affinity handle by thrombin cleavage. After cleavage, the
agarose-conjugated GST was removed by centrifugation, and the
concentration of the released recombinant caveolin present in the
supernatant was determined using the Bio-Rad reagent with bovine serum
albumin as the standard. To examine the interaction of
p75NTR with caveolin, an agarose-conjugated
GST-p75NTR cytoplasmic domain fusion protein was incubated
with equal amounts of the indicated recombinant caveolin protein (freed
from the affinity handle), and association was determined in the
co-sedimentation assay that we have described previously (17).
To analyze the association of Trk A with the GST-caveolin fusion
proteins, CEMs, NCM-1, and NCM-2 membrane fractions from Trk A-PC12
cells were resuspended in binding buffer (17). Equal amounts of protein
from each fraction were then incubated with the indicated amounts of
agarose-conjugated GST or GST-caveolin. After rotating the samples for
2 h at 4 °C, the beads were sedimented by a brief
centrifugation and washed with 3 × 1 ml of ice-cold MBST. The
bound proteins were eluted with 30 µl of freshly prepared 10 mM reduced glutathione (pH 8.0), and the eluate was boiled in sample buffer. The samples were analyzed for the presence of Trk A
as described above.
NGF Response in PC12 and Cav-PC12 Cells--
PC12 and Cav-PC12
cells were cultured on polylysine or collagen-coated 35-mm culture
dishes and plated at a density of ~5 × 105
cells/dish in RPMI 1640 medium-1% horse serum in the presence of 0 or
100 ng/ml NGF for 4 days. Fresh NGF was added after 2 days, and the
cells were photographed on day 4.
Autophosphorylation of Trk A--
PC12 and Cav-PC12 cells were
grown on 10-cm dishes to 70% confluence. The cells were washed twice
with ice-cold PBS and placed in 10 ml of 1% horse serum for 4 h
before the addition of 100 ng/ml NGF. NGF was added for 0-30 min, and
Trk A was immunoprecipitated as described after the addition of
ice-cold IP buffer (16). Equal amounts of protein were
immunoprecipitated using 2 µg of the polyclonal pan-Trk antibody. The
immunoprecipitates were analyzed by immunoblotting with a horseradish
peroxidase-conjugated antiphosphotyrosine antibody.
In Vitro Inhibition of Trk A Autophosphorylation by
GST-Caveolin--
Two 15-cm dishes of Trk A-PC12 cells were washed
twice with ice-cold PBS and resuspended in 1 ml of ice-cold IP buffer.
The cells were incubated on a rotating platform at 4 °C for 20 min and sonicated for 45 s on ice, and the lysate was centrifuged at
21,000 × g for 15 min at 4 °C. The supernatant was
precleared with PAS beads and subsequently incubated for 2 h at
4 °C with 5 µg of a polyclonal antibody against the Trk A
extracellular domain. PAS beads were added to bind the Trk A antibody;
after 1 h at 4 °C, the PAS beads were sedimented by a brief
centrifugation, washed three times with MBST, and washed three times
with kinase buffer (10 mM Tris, pH 7.4, and 10 mM MnCl2), and the beads were resuspended in a
final volume of 400 µl of kinase buffer. 0-120 µg of GST,
GST-caveolin, or an equal volume of the glutathione elution buffer (10 mM glutathione in kinase buffer) were added to 100-µl
aliquots of the Trk A immunoprecipitate. This mixture was incubated at
4 °C for 1 h to permit association of the proteins. The kinase
reaction was initiated by the addition of 40 µM ATP and
100 ng/ml NGF, and the reaction proceeded for 15 min at 37 °C. The
PAS beads were washed three times with ice-cold MBST and boiled in
sample buffer. Immunoprecipitated proteins were resolved by SDS-PAGE,
and after transferring to nitrocellulose, Trk A autophosphorylation was
examined using an antiphosphotyrosine antibody.
Measurement of Sphingomyelin Levels in the CEM
Pool--
Sphingomyelin levels were measured in Cav-PC12 cells as
described previously (17, 42). The amount of SM hydrolyzed was normalized to the total protein present in the sample.
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RESULTS |
Caveolin Overexpression Inhibits NGF-induced Differentiation of
PC12 Cells--
To determine whether caveolin could modulate
neurotrophin signaling, we stably transfected PC12 cells with
caveolin-1 cDNA and examined the cells for caveolin expression
using our standard fractionation and immunoprecipitation protocols. As
controls, mock-transfected PC12 and NIH-3T3 fibroblast cells were also
fractionated, and the three membrane pools were isolated. Similar to
our previous results, caveolin was not detectable in the untransfected
PC12 membranes using a polyclonal antibody raised against the C
terminus of caveolin (Fig. 1A)
(17). However, significant amounts of caveolin were observed in the
Cav-PC12 cells, and it had the same apparent molecular mass as caveolin
from a human endothelial cell lysate prepared as a caveolin standard
(Fig. 1B). Importantly, the distribution of caveolin in
Cav-PC12 cells was similar to that seen in fibroblasts (Fig.
1C), which express caveolin endogenously. Indeed, there was
little difference in the percentage distribution of caveolin between
the membrane pools from NIH-3T3 fibroblasts and the Cav-PC12 cells
(Fig. 1D). These results indicate that PC12 cells
efficiently express caveolin and target it to the appropriate membrane
domains.

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Fig. 1.
Cav-PC12 cells have a distribution of
caveolin similar to that of fibroblasts. The CRDs, NCM-1, and
NCM-2 membrane fractions from wild type PC12 cells (A),
Cav-PC12 cells (B), or NIH-3T3 fibroblasts (C)
were immunoprecipitated using a polyclonal antibody against the C
terminus of caveolin. Immunoblot analysis was performed using a
monoclonal caveolin antibody. The arrow indicates the
migration of caveolin relative to caveolin from a human endothelial
(HE) cell lysate. The results shown are from one
representative clonal population of Cav-PC12 cells. D, the
bands from each membrane pool in B and C were
quantified by densitometry, and the intensity was expressed as a
percentage of the total intensity of the three caveolin bands.
E, caveolin was immunoprecipitated from the
caveolin-enriched fraction of the second Opti-Prep gradient using an
antibody against the N terminus of caveolin, and immunoblot analysis
was performed with the same antibody.
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Because the flotation of membranes through a 38% sucrose cushion is
not an overly stringent criterion for the isolation of caveolar
membranes, we also fractionated Cav-PC12 cells using a second
detergent-free method as described by Smart et al. (40). Using this procedure, which may provide a more homogenous caveolae preparation, caveolin was readily detectable in Cav-PC12 cells and PCNA
cells, which are an L1 fibroblast cell line used as a positive control
(Fig. 1E). However, because we have not yet examined whether
caveolin expression in PC12 cells results in the morphologic formation
of caveolae, these domains will still be referred to as CRDs.
Interestingly, we observed a small amount of caveolin immunoreactivity
in wild type PC12 cell membranes isolated using the Opti-Prep protocol
(40), but only when using a polyclonal antibody raised against the N
terminus of caveolin (43). These results suggest that undifferentiated
PC12 cells express low levels of caveolin endogenously. Densitometric
analysis of these bands indicates that the Cav-PC12 cells express
approximately 3-fold more caveolin than wild type cells.
PC12 cells are a well-accepted model for studying neurotrophin
signaling, and NGF induces a well-characterized morphologic change of
these cells to a sympathetic neuron-like phenotype (44). Interestingly,
caveolin overexpression had a dramatic effect on NGF-induced
differentiation of the transfected cells. After 4 days of exposure to
100 ng/ml NGF, wild type PC12 cells developed extensive neurites (Fig.
2A). In contrast, the Cav-PC12
cells showed significantly fewer processes with greater than 95% of the cells showing no apparent neuritic response to NGF (Fig.
2B).

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Fig. 2.
Transfection of PC12 cells with caveolin
cells prevents differentiation in response to NGF.
Mock-transfected PC12 cells (A) and Cav-PC12 cells
(B) were plated at low density (~5 × 105 cells/dish) on polylysine-coated 35-mm
dishes. Both cell lines were maintained in RPMI 1640 medium-1%
horse serum in the presence of 100 ng/ml NGF and photographed after 4 days.
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Caveolin Diminishes Trk Autophosphorylation--
Trk A has been
demonstrated to be critical to NGF-induced differentiation of PC12
cells (45), suggesting that the presence of caveolin disrupts the
signaling pathway through Trk A leading to differentiation. Therefore,
we examined the effect of caveolin on Trk A autophosphorylation. PC12
and Cav-PC12 cells were maintained in RPMI 1640 medium containing 1%
horse serum for 4 h and then treated with 100 ng/ml NGF for 0-30
min. The cells were scraped into lysis buffer, and equal amounts of
total protein were immunoprecipitated using a polyclonal pan-Trk
antibody. The immunoprecipitates were subjected to SDS-PAGE, and Trk
autophosphorylation was determined by immunoblot analysis using an
antiphosphotyrosine antibody. Both PC12 and Cav-PC12 cells showed
little Trk autophosphorylation at time 0. However, Trk
autophosphorylation significantly increased after exposure to NGF for
15 min in both cell types (Fig. 3). Interestingly, whereas PC12 cells continued to exhibit tyrosine phosphorylation of Trk A for up to 30 min after the addition of NGF,
tyrosine phosphorylation of Trk A was rapidly abrogated after 15 min of
NGF stimulation in Cav-PC12 cells. Importantly, the lack of Trk A
autophosphorylation was not due to significant changes in the amount of
Trk A immunoprecipitated at any time point. These observations suggest
that the expression of caveolin in PC12 cells may inhibit Trk A
autophosphorylation and disrupt the duration of signaling necessary to
induce cell differentiation (10).

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Fig. 3.
Trk A activation is inhibited in Cav-PC12
cells. Mock-transfected PC12 cells and Cav-PC12 cells were grown
to 70% confluence on 10-cm dishes. The cells were placed in RPMI 1640 medium containing 1% horse serum for 4 h and then treated for the
indicated times with 100 ng/ml NGF. The cells were lysed in IP buffer
and immunoprecipitated using an anti-pan-Trk antibody. Trk
autophosphorylation was determined by immunoblot analysis using an
antiphosphotyrosine antibody.
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Trk A Associates with Caveolin--
We next sought to determine
whether caveolin may directly inhibit Trk tyrosine kinase activity. We
hypothesized that if a direct inhibition occurred, then caveolin and
Trk A may undergo a specific protein-protein interaction. To examine
this interaction, Trk A-PC12 cells were transiently transfected with
caveolin. We chose this approach because Trk A-PC12 cells express up to
a 20-fold greater level of Trk relative to wild type cells (46).
Cav-Trk A-PC12 cells, Trk A-PC12 cells, and fibroblasts were lysed in IP buffer, and the supernatants were immunoprecipitated using the
polyclonal caveolin antibody raised against the C terminus of caveolin.
The immunoprecipitates were washed with MBST and solubilized in sample
buffer, and the proteins were resolved by SDS-PAGE. Immunoblot analysis
revealed that there was a significant Trk A band that
co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells (Fig.
4). In contrast, only minor amounts of
Trk A were detected in the mock-transfected Trk A-PC12 cells. As
expected, no Trk immunoreactivity was detected in immunoprecipitates
from the fibroblasts. The co-immunoprecipitation of Trk from the
mock-transfected Trk-PC12 cells may have been due to the
immunoprecipitation of some endogenous caveolin by the C terminus
caveolin antibody (Fig. 1E). However, because the
immunoprecipitation of caveolin from any of our PC12 cell lines using
this antibody gave very equivocal results, the co-immunoprecipitation
of any Trk with the C terminus caveolin antibody raised the possibility
that Trk was interacting nonspecifically with the antibody and/or to
the protein A-Sepharose beads.

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Fig. 4.
Trk A co-immunoprecipitates with caveolin in
Trk A-PC12 cells transfected with caveolin. One 15-cm dish of 70%
confluent mock-transfected fibroblasts (PCMV), Trk A-PC12
cells, or Cav-Trk-PC12 cells was immunoprecipitated using an
anti-caveolin antibody raised against the C terminus of caveolin.
Immunoblot analysis was performed using an anti-pan-Trk antibody. Data
are representative of similar results obtained in at least three
experiments.
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To further examine the specificity of the interaction of caveolin with
Trk A, CRDs, NCM-1, and NCM-2 fractions were isolated from wild type
Trk A-PC12 cells (which had no exogenous caveolin expression) and
incubated with increasing concentrations of an agarose-conjugated
GST-caveolin fusion protein. This fusion protein expressed the
full-length form of caveolin comprising amino acids 1-178 (17, 47).
The membrane fractions were incubated with 10, 30, and 60 µg of an
agarose-conjugated GST-caveolin fusion protein or agarose-conjugated
GST. After the incubation, the beads were sedimented and extensively
washed, and the bound proteins were eluted with glutathione. The
eluates were subjected to SDS-PAGE, and the presence of Trk A was
determined by immunoblot analysis. Fig. 5
shows that Trk A interacted specifically with the GST-caveolin fusion
protein and did not associate with GST alone. The GST-caveolin fusion
protein bound approximately 29% of the Trk A present in the CRDs from
Trk A-PC12 cells and 20% of the Trk A from CRDs isolated from wild
type PC12 cells (data not shown).

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Fig. 5.
Trk A co-sediments with a GST-caveolin fusion
protein. The indicated amounts of the agarose-conjugated GST or
GST-caveolin were incubated with 30 µg of protein from CRDs, NCM-1,
and NCM-2 membranes in IP buffer at 4 °C for 2 h. The beads
were washed with MBST, and the fusion proteins were eluted from the
beads with 10 mM glutathione. Immunoblot analysis was
performed using an anti-pan-Trk antibody. The results presented are
from one experiment performed twice with a similar outcome.
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Interestingly, we have identified that an interaction of Trk with
caveolin could only be demonstrated with the full-length caveolin and
not with fusion proteins that span the oligomerization and scaffolding
domain 1 of caveolin (Fig. 6). In
contrast, the interaction of p75NTR with caveolin is
mediated through amino acids in the caveolin scaffolding domain 1 (amino acids 82-101) (Fig. 6). These results suggest that both
neurotrophin receptors interact with caveolin, but that these
interactions may be regulated by distinct domains of caveolin.

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Fig. 6.
Trk A and p75NTR show
differential binding to caveolin scaffolding domain 1. A, an
agarose-conjugated GST-p75NTR cytoplasmic domain fusion
protein (30 µg) was incubated with equal amounts of recombinant
full-length caveolin (1-178), caveolin (1-81), or caveolin (61-101).
p75NTR-associated proteins were isolated and subjected to
SDS-PAGE. After transferring the proteins to nitrocellulose, the
membrane was stained with Ponceau red and cut just below the migration
of the GST-p75NTR fusion protein, and the top half of the
blot was probed with anti-p75NTR antiserum, and the lower
half was probed with the C terminus polyclonal caveolin antibody. The
schematic shows the full-length and truncated caveolin proteins with
the caveolin transmembrane region depicted in black.
B, CRDs were isolated from Trk A-PC12 cells, and 30 µg of
protein were incubated with an equal amount of agarose-conjugated GST
or GST-caveolin fusion protein. After 1 h at 4 °C, the
caveolin-associated proteins were sedimented by centrifugation, the
beads were washed, and the proteins were subjected to SDS-PAGE. After
the transfer to nitrocellulose, the presence of Trk A was determined
using a pan-Trk antibody. The results are representative of those
obtained from at least three experiments.
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GST-Caveolin Prevents Trk A Autophosphorylation--
The
experiments described above support that there is a specific
interaction between caveolin and Trk A. However, these results did not
establish whether this interaction affected kinase activity. To more
directly address this question, the effect of recombinant caveolin on
the activation of Trk A was assessed in an in vitro kinase
assay. Trk A was immunoprecipitated from a Trk A-PC12 lysate using an
antibody directed against the extracellular domain of Trk A. After
incubation with PAS for 1 h at 4 °C, the immunoprecipitate was
washed and resuspended in kinase buffer. The indicated amount of
GST-caveolin or GST (both eluted from the beads) or an equal volume of
glutathione elution buffer was added to the reaction, and the proteins
were allowed to associate for 1 h at 4 °C. The kinase reaction
was then initiated with the addition of ATP and NGF. After incubation
at 37 °C, the PAS beads were sedimented and washed with MBST, and
the bound proteins were solubilized with sample buffer. After SDS-PAGE,
the activation of Trk A was evaluated using immunoblot analysis for
antiphosphotyrosine. Fig. 7 shows that
there was a marked activation of Trk A in samples treated with either
GST or elution buffer alone, indicating that NGF was effectively
inducing receptor autophosphorylation. In contrast, the addition of 60 or 120 µg of GST-caveolin totally abolished autophosphorylation,
strongly supporting that caveolin was specifically inhibiting
NGF-induced Trk autophosphorylation.

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Fig. 7.
GST-caveolin inhibits Trk A
autophosphorylation in an in vitro kinase assay. Trk A
was immunoprecipitated from Trk A-PC12 cells, and the final PAS beads
were resuspended in 400 µl of kinase buffer. 100-µl aliquots were
then incubated for 1 h at 4 °C with the indicated amounts of
GST, GST-caveolin, or glutathione elution buffer. The reaction was
initiated by the addition of 40 µM ATP and 100 ng/ml NGF
and stopped after 15 min at 37 °C. The PAS beads containing the
immunoprecipitated Trk A were washed with ice-cold MBST, and the
proteins were subjected to SDS-PAGE. Trk autophosphorylation was
determined by immunoblot analysis using an antiphosphotyrosine
antibody. Data are from a representative experiment performed three
times with identical results.
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Caveolin Modulates Trk A Inhibition of NGF-induced Sphingomyelin
Hydrolysis--
We have observed previously that the activation of Trk
A by NGF inhibits p75NTR-dependent SM
hydrolysis (16). Significantly, the inhibition of Trk activation with
the relatively specific Trk A inhibitor K252a enabled NGF to signal
through p75NTR. Because caveolin was essentially mimicking
the inhibitory effect of K252a on Trk A tyrosine kinase activity, this
suggested that caveolin may also enable NGF to signal through
p75NTR in the Cav-PC12 cells. Cav-PC12 cells were labeled
with [3H]choline for 3 days and placed in RPMI 1640 medium containing 1% horse serum. After 4 h, the cells were
treated with either 100 ng/ml NGF or vehicle for 0-30 min. CRDs were
isolated and analyzed for SM content. Interestingly, in the Cav-PC12
cells, NGF-induced SM hydrolysis was observed at nearly all time points except for the 15 min time point (Fig.
8), when Trk A phosphorylation was found
to be maximal (compare time course with Fig. 3). Importantly, essentially no NGF-induced SM hydrolysis was observed in CRDs from wild
type PC12 cells (16). These observations suggest that caveolin may
affect cross-talk between Trk A and p75NTR by regulating
the extent of Trk activation.

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Fig. 8.
Caveolin prevents Trk A inhibition of
NGF-induced SM hydrolysis. Cellular SM in wild type and Cav-PC12
cells was labeled with [3H]choline for 3 days. The cells
were washed with ice-cold PBS and placed in RPMI 1640 medium containing
1% horse serum for 4 h. Cells were then incubated for the
indicated times with 100 ng/ml NGF, washed with ice-cold PBS, and lysed
in 0.5 M Na2CO3; the CRDs were
isolated, and the protein was content measured. Total lipids from the
CRDs were then extracted, and the amount of [3H]SM was
determined. SM levels were normalized to total protein concentration.
The results shown are from a representative experiment performed
twice.
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DISCUSSION |
We have determined that caveolin can interact with both
p75NTR and Trk A, and that this interaction may regulate
neurotrophin responses. In this regard, Trk A co-immunoprecipitated
with caveolin in Trk A-PC12 cells that were transfected with caveolin.
Trk A also bound specifically and co-sedimented with a GST-caveolin fusion protein expressing the full-length form of caveolin. Caveolin expression resulted in decreased ligand-induced activation of Trk A
tyrosine kinase activity that markedly blunted the NGF-induced differentiation of PC12 cells transfected with caveolin. Furthermore, we have demonstrated that caveolin can directly block NGF-induced Trk A
autophosphorylation in vitro. Thus, our data support that the effect of caveolin expression on NGF-induced differentiation may be
due to changes in the magnitude and duration of ligand-induced receptor
autophosphorylation mediated through a direct and inhibitory interaction of caveolin with Trk A.
The lack of NGF-induced differentiation in Cav-PC12 cells cannot be
explained by simple variability in the amount of Trk A immunoprecipitated from the Cav-PC12 cells, because Trk A levels were
very comparable at all time points examined (Fig. 3). Additionally, because we observed some in vivo autophosphorylation of Trk
A in the Cav-PC12 cells, it is unlikely that caveolin is preventing the
binding of NGF to Trk A. However, we cannot rule out the possibility that caveolin may interfere with or destabilize the formation of stable
Trk A dimers, leading to transient phosphorylation in vivo
and blocking activation in vitro (9). Additionally, we cannot rule out the possibility that, in vivo, caveolin may
recruit tyrosine phosphatases that rapidly dephosphorylate Trk A. However, the ability of the GST-caveolin fusion protein to prevent the activation of immunoprecipitated Trk A would suggest that a tyrosine phosphatase is not necessary for the inhibition of ligand-induced receptor autophosphorylation.
A previous study has demonstrated that caveolin inhibited epidermal
growth factor receptor autophosphorylation, likely through a direct
interaction of an N-terminal region of caveolin termed scaffolding
domain 1 with the epidermal growth factor receptor catalytic domain
(37). Couet et al. (37) have identified that scaffolding
domain 1 of caveolin interacts with a specific caveolin binding domain
present in the conserved kinase domain of many receptor-linked tyrosine
kinases including Trk A. However, we were not able to demonstrate a
strong interaction between Trk A and a caveolin fusion protein
containing the scaffolding domain 1 of caveolin (less than 1% of the
added Trk A bound to the GST-caveolin 61-101 fusion protein in
contrast to a 29% binding to full-length caveolin). Thus, although
tyrosine kinase receptors share a very similar sequence in this
putative caveolin binding domain (37), differences may exist in how
caveolin interacts with these receptors and affects kinase activity.
Indeed, the idiosyncratic nature of the interaction of caveolin with
tyrosine kinase receptors is exemplified by the association of the
epidermal growth factor receptor and the insulin receptor with
caveolin. Although the interaction of both of these receptors with
caveolin is mediated by the interaction of each receptor's caveolin
binding domain with scaffolding domain 1 of caveolin, caveolin inhibits
epidermal growth factor receptor kinase activity (37) but stimulates
insulin receptor kinase activity (38).
Surprisingly, p75NTR, but not Trk A, was found to interact
with caveolin through scaffolding domain 1. It is interesting that the
cytoplasmic domain of p75NTR does not contain what would be
considered a strong match to sequences that were identified as putative
caveolin binding domains (41), although it does possess a related
juxtamembrane sequence that may be responsible for this interaction.
However, a peptide of this region was unable to totally compete the
binding of p75NTR to full-length caveolin (data not shown),
suggesting that other sequences within p75NTR may regulate
the interaction of this receptor with scaffolding domain 1 of caveolin
(Fig. 6). Thus, p75NTR and Trk receptors may interact with
distinct domains of caveolin.
The identification that both classes of neurotrophin receptors may
localize to the CRDs of synaptosomal membranes (48) and that caveolin
and caveolin-related proteins are expressed in specific neuronal
populations (49, 50) suggests that the interactions of these proteins
may be relevant to neurotrophin signaling pathways. Indeed, similar to
the pharmacologic inhibition of Trk A with K252a, caveolin expression
modulated the inhibition of p75NTR signaling. In the
absence of K252a, Cav-PC12 cells exhibited significant SM hydrolysis at
all time points except 15 min after treatment with NGF (Fig. 8). This
was the same time at which we observed a brief period of Trk A
activation in Cav-PC12 cells. This pattern suggests that Trk A
activation may quickly turn off SM hydrolysis in cells, and that
caveolin, in turn, can regulate the ability of Trk A to inhibit
p75NTR signaling. These results provide the first evidence
that caveolin may be an endogenous modulator of cross-talk between
tyrosine kinase and sphingolipid signaling pathways coupled to Trk
receptors and p75NTR, respectively.
It is important to point out that there is little or no data that
support an overlapping distribution between caveolin (49, 50) and Trk A
(51) in cells of the central nervous system. Therefore, the physiologic
consequence of the inhibition of Trk A by caveolin is uncertain but may
serve as a model for the interaction of caveolin with other Trk family
members. However, while this article was under review, it was reported
that PC12 cells may express caveolin endogenously, and that caveolin
expression increases during NGF-induced differentiation of PC12 cells
(43). In agreement with this report, we were able to detect some
caveolin in wild type PC12 cells, but only when using an antibody
raised against the N terminus of caveolin (Fig. 1E). At
first, these results would seem incongruous with our findings because
if PC12 cells express caveolin endogenously and caveolin inhibits Trk A
tyrosine kinase activity, how do PC12 cells undergo differentiation in response to NGF? It is intriguing that Galbiati et al. (43) reported that caveolin expression was up-regulated 2-3-fold by day 4 of the NGF-induced differentiation of PC12 cells. We achieved an
increase of similar magnitude by stable overexpression of caveolin in
undifferentiated PC12 cells (Fig. 1E). These results would be consistent with the hypothesis that increased expression of caveolin
or caveolin-related molecules in terminally differentiating neurons may
help to regulate signals originating from Trk receptors. Because the
process of differentiation in PC12 cells is likely to involve multiple
pathways, it is conceivable that caveolin overexpression in
undifferentiated PC12 cells may not permit Trk A to become sufficiently
activated to couple effectively to the various downstream signaling
molecules involved in differentiation (14). However, in
undifferentiated wild type cells, endogenous caveolin levels may be
insufficient to interfere with the prolonged Trk A activation induced
by NGF addition. As caveolin levels increase during the process of
differentiation, the interaction of caveolin with Trk A may increase.
Our data suggest that this interaction would promote more a transient
phosphorylation of Trk A by NGF (Fig. 3) that may be sufficient to
maintain survival. This would be consistent with the ongoing
requirement for NGF activation of Trk A for the survival of
differentiated PC12 cells (44).
In summary, the neurotrophin/p75NTR/Trk ligand-receptor
cassette is an excellent model system for elucidating the mechanisms whereby interactions between multiple ligands and receptors dictate cellular responses. This ligand-receptor cassette is unique from other
signaling systems in that both receptor components can signal individually or in pair in response to multiple ligands and direct very
distinct biologic outcomes. These complex interactions necessitate a
high degree of organization and regulation. The mutual interaction of
these receptors with a caveolar structural protein that organizes, sequesters, and regulates the activity of critical signaling molecules adds an additional level of complexity to this system. These
interactions may have important functions in dictating neurotrophin
responses in specific cell populations, i.e. regulating the
rate of receptor clustering, which may affect the activation of both
tyrosine kinase and sphingomyelinase. Elucidating the biochemical
mechanisms affecting the interactions between the sphingolipid and
tyrosine kinase signaling pathways and their biological consequences
represents a novel and emerging area of cell regulation.