From the Neurodegeneration Research Group, The John
P. Robarts Research Institute, London, Ontario N6A 5K8, Canada and
the § Department of Biochemistry, the ¶ Graduate
Program in Neuroscience, and the
Department
of Physiology, University of Western Ontario, London,
Ontario N6A 5C1, Canada
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ABSTRACT |
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We have isolated a human cDNA for the
signaling adapter molecule FRS-2/suc1-associated neurotrophic factor
target and shown that it is tyrosine-phosphorylated in response to
nerve growth factor (NGF) stimulation. Importantly, we demonstrate that
the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus
proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the
SH2 domain containing tyrosine phosphatase SH-PTP-2, the
cyclin-dependent kinase substrate p13suc1, and the
Src homology 3 (SH3) domain of Src, providing a functional link between
TrkA, cell cycle, and multiple NGF signaling effectors. Importantly,
overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA
receptor mutant reconstitutes the ability of NGF to stop cell cycle
progression and to stimulate neuronal differentiation.
The molecular mechanisms regulating proliferation
versus differentiation in the developing nervous system
depend, in part, on the availability of the neurotrophins (nerve growth
factor (NGF),1 brain-derived
neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5
(NT-4/5)) (1). Increasingly, it is apparent that the neurotrophins not
only act as target-derived survival factors but can also regulate
mitotic activity and promote differentiation of neuronal progenitors in
both the peripheral (2, 3) and central (4-6) nervous systems. The
biological effects of the neurotrophins are mediated by 2 classes of
cell surface receptors: a low affinity p75NTR receptor and
a high affinity tyrosine kinase Trk receptor. As a low affinity
receptor, p75NTR modulates ligand binding (7, 8) and some
signaling pathways stimulated by TrkA (9, 10) and, as a member of the
Fas antigen family, stimulates sphingomyelin hydrolysis and ceramide
synthesis leading to apoptosis in some cellular contexts (11-13).
In contrast, the Trk receptors are essential to proliferation,
differentiation, and cell survival (14). TrkA is specifically activated
by NGF, whereas TrkB and TrkC are the primary receptors for BDNF and
NT-3, respectively (15, 16). Upon stimulation, the Trk receptors
activate numerous intracellular signaling molecules, including the Shc
adapter proteins, Grb-2/Sos, Ras, Map kinase (MAPK),
phosphatidylinositol 3-kinase, Src, phospholipase C SNT has been considered a likely candidate to regulate a decision
between cell cycle progression and cell cycle arrest based on its
tyrosine phosphorylation by factors that induce differentiation (NGF,
BDNF, NT-3, and fibroblast growth factor (FGF)) and by its ability to
bind to the cyclin-dependent kinase substrate
p13suc1. The role of SNT in neurotrophin signaling is
underscored by the observation that two independent Trk receptor
mutants that do not affect either mitogenic signaling and/or cell
survival responses, do selectively affect the tyrosine phosphorylation of SNT and NGF-dependent cell cycle arrest/neuronal
differentiation (20, 21). The identity of the SNT proteins(s) has
remained elusive until recently, when a murine FGFR signaling molecule, mFRS-2, was cloned and found to share properties comparable to SNT
(22).
To better understand the role of FRS-2/SNT in neurotrophin signaling
and cell cycle arrest, we have cloned and characterized human FRS-2.
Amino acid sequence comparisons between murine and human FRS-2 indicate
that FRS-2 has been evolutionarily conserved between mouse and human
(96%). FRS-2 contains an amino-terminal myristylation site to
facilitate tight association with the membrane, an amino-terminal
phosphotyrosine binding (PTB) domain, four putative binding sites for
Grb-2, two putative binding sites for the SH2 domain-containing
tyrosine phosphatase SH-PTP-2, and numerous potential binding sites
(PXXP) for SH3 domain-containing proteins. We demonstrate
that FRS-2 binds p13suc1 and the SH3 domain of Src
constitutively but shows NGF-dependent tyrosine
phosphorylation and association with the SH2 domains of Grb2, Crk, and
SH-PTP-2. Importantly, unlike signaling by the FGFR (22, 23), we show
that FRS-2 binds directly to neurotrophin-activated Trk receptors in a
phosphotyrosine-dependent manner at a site that also
regulates the binding of Shc. In vitro binding assays indicate that Shc and FRS-2 compete for binding to TrkA, suggesting a
model in which competition between signaling proteins may regulate neurotrophin-dependent proliferation and/or
differentiation. The role of FRS-2 expression in regulating cell cycle
arrest and neuronal differentiation is further supported by the
observation that FRS-2 overexpression in nnr5 cells expressing a
differentiation minus TrkA receptor mutant (TrkAS3 cells) (21)
reconstitutes NGF-dependent cell cycle arrest and neuronal differentiation.
Antibodies and Growth Factors--
Anti-phosphotyrosine antibody
(PY20) and the horseradish peroxidase (HRP)-conjugated derivative of
PY20 (RC20) were from Transduction Laboratories. Hybridoma 12CA5 cells
(anti-HA) (24) and 9E10 (anti-c-Myc) (25) were grown as ascites tumors
in 4-6-week-old female Balb/c mice. Rabbit anti-SH-PTP-2 antibody was
from Santa Cruz. HRP-goat anti-rabbit and HRP-goat anti-mouse secondary
antibodies were from The Jackson Laboratory. p13suc1 agarose
was from Upstate Biotechnology or was the gift of D. Litchfield
(University of Western Ontario). Cell Lines--
The nnr5 cell line expressing hemagglutinin
(HA)-tagged rat TrkA (B5 cells) and TrkAS3 mutant receptors
( FRS-2 cDNA Cloning, Baculovirus, and Constructs--
Using a
degenerate sequence based on the published mouse FRS-2 protein sequence
(22), we isolated a human genomic fragment from GenBankTM
and designed a 5' primer (5' GAA GAA GCC ATG GGT AGC TGT TGT3') and a
3' primer (5' TGC TGT CGA CGC TCA CAT GGG CAG ATC 3'). A 1.5-kb
polymerase chain reaction (PCR) product was generated, subcloned into
pCr2.1 (Invitrogen), and sequenced. hFRS-2 (residues 1-503) was cloned
into pcDNA3.1/Myc-His (Invitrogen) inserting a human c-Myc tag at
the carboxyl terminus. The rat TrkA K547A inactive kinase receptor was
generated by PCR overlap mutagenesis and sequenced. HA-tagged rat TrkB
and TrkC were generated by PCR overlap as described previously for TrkA
(26). The hemagglutinin epitope was inserted just 3' to the signal
sequence on rat TrkB and rat TrkC. HA-TrkB, HA-TrkC, and HA-TrkA
K547A-expressing baculoviruses were generated as described (27).
Glutathione S-transferase (GST)-hFRS-2 was generated by
subcloning hFRS-2 as a 1.5-kb EcoRI/SalI fragment
from pCr2.1/FRS-2 into pGEX-4T-2 (Amersham Pharmacia Biotech). The
GST/hFRS-2(PTB) construct contains the amino-terminal 240 residues of
hFRS-2. All other GST fusion constructs were the gifts of numerous
investigators: Shc (J. McGlade, The Hospital for Sick Children,
Toronto, Ontario, Canada); Grb-2, Crk, Ras-Gap, PLC Northern Blot--
A rat multiple tissue Northern blot
containing approximately 2 µg of poly(A)+ RNA per lane
(CLONTECH) was probed with the hFRS-2 cDNA that was radiolabeled with [ Yeast--
The yeast strain PJ694A (MAT In Vitro Binding Assays--
HA-tagged Trk receptors were
expressed in baculovirus-infected insect High Five cells (21). Cells
were stimulated (100 ng/ml
For competition experiments, GST-hFRS-2(PTB)- and
GST-PLC Neurite Response and 5-Bromo-2-deoxyuridine (BrdUrd)
Assays--
The neurite response assay was performed on poly
D-lysine-coated 24-well clusters. Fresh neurotrophin was
added every other day. On day 5, cells were assayed for changes in
proliferation by measuring the incorporation of the thymidine analogue
BrdUrd as described previously (21). The percentage of BrdUrd-positive cells was scored from 10-12 independent frames. The S3-FRS-2 sample represents as average of two clones expressing FRS-2 (clones 34 and 40)
and is thus an average of 20 independent frames.
Isolation of a Human FRS-2 cDNA and Northern Blot
Analysis--
An hFRS-2 cDNA (1.5 kb) was isolated from
fetal brain cDNA by PCR techniques with degenerate primers based on
the protein sequence of mFRS-2 (22). The cDNA sequence predicts a
protein with a primary sequence of 508 amino acids and a molecular mass of approximately 57 kDa. Amino acid comparison between hFRS-2 and
mFRS-2 (Fig. 1A) indicates
that FRS-2 has been evolutionarily conserved between mouse and human
(96% identity). hFRS-2, like mFRS-2, contains an amino-terminal
myristylation signal (MGXXXS) and PTB domain, 15 potential
sites of tyrosine phosphorylation, 4 Grb-2 binding sites, 2 potential
binding sites for the tyrosine phosphatase SH-PTP-2, and putative
binding sites for SH3 domain-containing proteins (PXXP).
Northern blot analysis of FRS-2 expression indicates low levels of two
transcripts of approximately 7.2 and 7.5 kb in all tissues examined
with slightly higher levels (2-5-fold) detected in brain, testes, and
lung (Fig. 1B). The ratio of the two transcripts is
comparable between tissues with slightly higher levels of the upper
transcript present in lung. The difference between our 1.5-kb cDNA
and the native transcripts represent untranslated regions of
approximately 1.5 and 4.2 kb, respectively (Fig. 1B).
FRS-2 Is Tyrosine-phosphorylated by NGF and Binds p13suc1
Constitutively--
To facilitate transfection studies, hFRS-2 was
tagged at the carboxyl terminus (c-Myc) without interfering with
NGF-dependent tyrosine phosphorylation and/or its signaling
properties. As shown in Fig.
2A, following transfection
into NGF-responsive cells, hFRS-2/c-Myc (80-90 kDa) was
tyrosine-phosphorylated in response to NGF stimulation and was
immunoprecipitated with the anti-Myc antibody (9E10) (top
panel, compare lanes 2 and 4). Because
hFRS-2/c-Myc predicts a protein of approximately 57 kDa, this suggests
that FRS-2 is posttranslationally modified by mechanisms that include myristylation (22) and both serine/threonine and tyrosine
phosphorylation. Similar to endogenous FRS-2, FRS-2/c-Myc bound
p13suc1, although the transfected gene product migrated more
slowly than the endogenous protein consistent with the addition of the
c-Myc epitope (approximately 3 kDa) to the carboxyl terminus (Fig.
2A, top panel, compare lanes 6 and 8).
Stripping and re-probing the blot with anti-Myc antibodies indicated
that hFRS-2/Myc can bind p13suc1 constitutively and that the
binding is not regulated by tyrosine phosphorylation (Fig. 2A,
bottom panel, lanes 7 and 8). Moreover, more than one
form of FRS-2 was consistently detected, indicating multiple forms of
posttranslational modifications. Typically, only the highest molecular
mass form of FRS-2 was tyrosine-phosphorylated in response to NGF.
Analysis of FRS-2-binding Proteins--
As outlined above, the
primary sequence of FRS-2 predicts that phosphorylation of specific
tyrosines will generate binding sites for the SH2 domains of the
adapter protein Grb-2 and the tyrosine phosphatase SH-PTP-2. To assay
FRS-2/Grb-2 interactions, FRS-2/c-Myc was transfected into TrkA
overexpressing nnr5 cells, termed B5 (26). As shown in Fig.
2B, GST fusion proteins corresponding to both full-length
Grb-2 and the central SH2 domain bound NGF-induced, tyrosine-phosphorylated hFRS-2/Myc. Neither the amino-terminal nor the
carboxyl-terminal SH3 domains of Grb-2-bound hFRS-2. Stripping and
reprobing the blot with anti-Myc antibodies confirmed that the 80-90
kDa tyrosine-phosphorylated protein bound by Grb-2(SH2) corresponds to
the fully processed hFRS-2/c-Myc and co-migrates with FRS-2/c-Myc
immunoprecipitated with 9E10 (Fig. 2B, bottom panel).
As stated above, FRS-2 is also predicted to contain binding sites for
SH-PTP-2 (also known as Shp-2, syp, PTP1D, and PTP2C (31)). As shown in
Fig. 2C (left panel), immunoprecipitation of
hFRS-2/Myc with anti-Myc antibodies, from hFRS-2/Myc-transfected B5
cells, indicates that SH-PTP-2 also co-immunoprecipitates with FRS-2
following NGF stimulation (compare lanes 2, 4, and
5). Because SH-PTP-2 has been shown to be transiently
associated with TrkA (32), we next examined the kinetics of association
between FRS-2 and SH-PTP-2 in response to NGF stimulation. As shown in
Fig. 2C (right panel), both p13suc1 and
anti-SH-PTP-2 immunoprecipitations indicate tyrosine phosphorylation of
FRS-2 within 2 min of NGF stimulation (lanes 7 and
10). The tyrosine phosphorylation of SH-PTP-2 was maximally
observed 2 min following NGF stimulation (lane 10).
Interestingly, tyrosine phosphorylation is only required to initiate
FRS-2/SH-PTP-2 binding because SH-PTP-2 was rapidly de-phosphorylated
by 5 min (lane 11) without altering FRS-2 binding.
Moreover, the anti-SH-PTP-2 immunoprecipitations identified an
approximately 120-kDa protein that is tyrosine-phosphorylated in
response to NGF stimulation. The identity of this protein is presently
unknown, but it may correspond to a member of the signal-regulatory
protein (SIRP) family of SH-PTP-2-binding proteins that are both
substrates of receptor tyrosine kinases and negative regulators of
receptor tyrosine kinase signaling (33). Importantly, stripping the
blots and reprobing with anti-SH-PTP-2 antibodies indicates that
SH-PTP-2 co-precipitates with p13suc1 from NGF-stimulated cells
(Fig. 2C, lanes 7 and 8) comparable to
the SH-PTP-2 co-immunoprecipitation observed with the transfected hFRS-2/c-myc gene (lanes 4 and 5).
From the primary sequence, it is also predicted that SH3
domain-containing proteins can interact with FRS-2 and facilitate alternate signaling pathways. To this end, we screened a panel of SH3
domains (Grb-2, Nck, Crk, Ras-Gap, c-Abl, PLC FRS-2 Directly Binds the Neurotrophin Trk Receptors--
As
described above, hFRS-2 is modular in nature and contains an
amino-terminal PTB domain that in many molecules mediates protein-protein interactions involving phosphotyrosine. Whereas FRS-2
does not readily co-immunoprecipitate with either the FGFR (22) or TrkA
(data not shown), FRS-2 has been shown to interact with the FGFR
in vitro and in yeast (23). These observations suggest that
FRS-2/receptor interactions are labile and/or weak in cells but that
direct binding can be observed by alternative methods. These
observations are reminiscent of weak interactions between the insulin
receptor
Accordingly, we used in vitro binding assays and yeast
interaction-trap assays to determine whether hFRS-2 could interact with
TrkA and if so, where the site of interaction might be. HA-tagged TrkA,
TrkB and TrkC were expressed in baculovirus-infected insect cells and
lysates containing equivalent amounts of expressed Trk protein were
used in in vitro binding experiments with purified GST
fusion proteins. As shown in Fig.
3A, GST-hFRS-2(PTB), like the
Shc(PTB) domain and the PLC
To determine whether the FRS-2/TrkA interaction is also
phosphorylation-dependent in the in vitro binding
assays, baculoviruses expressing wild type TrkA and the kinase inactive
TrkA were assayed for binding to GST-FRS-2(PTB) in comparison with
GST-Shc(PTB) as a positive control. Shc interactions with TrkA have
previously been shown to be phosphotyrosine-dependent (37). As
shown in Fig. 4A, the
TrkAK547A receptor expresses high levels of receptor (anti-HA blot)
that is not catalytically active (anti-Tyr(P) blot). In
vitro binding assays with purified GST fusion proteins indicated that the PTB domain of FRS-2 interacts with the kinase active TrkA, as
does GST-Shc(PTB), and not the catalytically inactive form (Fig.
4B). Thus, as we observed in yeast (Fig. 3), FRS-2 interaction with TrkA is phosphotyrosine-dependent.
FRS-2 Binds TrkA at the Shc Binding Site,
Tyr499--
Thus far, we have demonstrated that hFRS-2
interacts with the Trk receptors through an amino-terminal PTB domain
in a tyrosine phosphorylation-dependent manner. To map the
binding site(s) on TrkA for hFRS-2(PTB) interaction, we took advantage
of a series of rat TrkA receptor mutants that selectively affect
specific signaling pathways. In this respect, NGF induces receptor
dimerization and tyrosine phosphorylation at five intracellular
tyrosine residues in rat TrkA (Tyr499, Tyr679,
Tyr683, Tyr684, and Tyr794). SHC
and PLC FRS-2 and Shc Compete for Binding to TrkA at
Tyr499--
The observation that both Shc and hFRS-2 bind
to the same phosphotyrosine residue on TrkA suggests a model in which
Shc and FRS-2 may compete for binding to TrkA in vivo).
Because Shc/Grb-2/Ras/Map kinase activation facilitates mitogenic
signaling (39) and a decrease in FRS-2 phosphorylation correlates with
an inability of TrkA to support NGF-dependent neurite
outgrowth (20, 21), this suggests that competition between Shc and
FRS-2 for binding to Tyr499 may, in part, regulate a
cellular switch between cell cycle progression/mitogenesis and cell
cycle arrest/differentiation.
To this end, competition between Shc and hFRS-2 binding to TrkA was
assayed in vitro. GST-Shc (5 µg) was used to precipitate baculovirus expressed HA-TrkA in the presence of increasing
concentrations of soluble hFRS-2(PTB). As shown in Fig. 5C,
hFRS-2 competes for binding to TrkA with an EC50 requiring
a 2-5-fold excess (between 10 and 25 µg). Complete competition was
obtained with a 20-fold excess (100 µg). In comparison, increasing
concentrations of the SH2 domain of PLC FRS-2 Overexpression Reconstitutes NGF-dependent
Neurite Outgrowth by a Differentiation Minus TrkA Receptor
Mutant--
As shown above, FRS-2 showed a dramatic reduction in
binding to both the Y499A TrkAS8 mutant and the
In summary, we have cloned the human FRS-2 adapter protein
and begun to characterize its role in NGF-dependent
signaling through TrkA. Although TrkA can stimulate both a mitogenic
response and differentiation and cessation of cell cycle progression in
nonneuronal and neuronal cells, respectively, the molecular
mechanism(s) underlying this differential response is not well
understood. Here, we provide evidence of a novel mechanism by which
competitive binding between FRS-2 and Shc may regulate a cellular
switch between cell cycle progression and cell cycle
arrest/differentiation. Moreover, we have begun to address the
mechanisms by which FRS-2 may regulate these processes. hFRS-2 is
tyrosine-phosphorylated in response to NGF-stimulation and binds
directly to TrkA through its amino-terminal PTB domain in a
phosphotyrosine-dependent manner. hFRS-2 also binds to
neurotrophin-stimulated TrkB and TrkC receptors, indicating that it is
probably involved in an analogous role in BDNF and NT-3 signaling as
well. Recently, the PTB domain of FRS-2 has also been shown to bind the
juxtamembrane region of the FGFR (23) by both yeast two-hybrid assays
and in vitro binding assays using recombinant fusion
proteins. In these assays, FRS-2 binding to the FGFR is independent of
kinase activation and has been localized to a juxtamembrane region that
lacks both asparagine and tyrosine residues (23). Although these data
indicate that FRS-2 binding to the FGFR is constitutive, the
possibility that FGF may regulate FRS-2 recruitment in vivo
through an alternative mechanism has not yet been determined. Moreover,
the precise residues within the FGFR juxtamembrane region that are
essential to FRS-2 recruitment and activation have not yet been determined.
Thus, FRS-2 recruitment by the FGFR appears to be
phosphotyrosine-independent, whereas we have shown that FRS-2 binding
to TrkA requires both an active kinase and is
phosphotyrosine-dependent. Tyrosine-phosphorylated FRS-2 binds the SH2 domains of Grb-2 and the
tyrosine phosphatase SH-PTP-2. SH-PTP-2 is transiently
tyrosine-phosphorylated in response to NGF stimulation, which
correlates with an initial ability to bind FRS-2. Because SH-PTP-2 also
binds Grb-2 through carboxyl-terminal phosphotyrosines (22, 40), FRS-2
facilitates the Shc-independent recruitment of Grb-2 into the NGF-Trk
signaling complex both directly and indirectly.
FRS-2 also binds the SH2 and SH3 domain-containing adapter protein Crk,
in an NGF and phosphotyrosine-dependent manner. The relevance of this pathway to neuronal differentiation is exemplified by
the fact that Crk/C3G/Rap1, but not Grb-2/Sos/Ras, appears to regulate
prolonged MAPK activation (17) and by the fact that Crk overexpression
induces constitutive PC12 cell differentiation (41). Consistent with
these data, the Grb-2 binding sites on FRS-2 are dispensable for
FGF-induced differentiation of PC12 cells (40). Importantly, the recent
observation that prolonged MAPK activation stimulates expression of the
cell cycle inhibitor p21CIP/Waf-1 (42) provides an
important functional link between FRS-2, Crk, cell cycle arrest, and
neuronal differentiation. Collectively, these data highlight the
functional differences between Shc and FRS-2-dependent
mechanisms to activate MAPK (Grb-2/Sos/Ras and Crk/C3G/Rap1) and
emphasize the need to assay site-directed FRS-2 mutants incapable of
binding Crk on NGF-dependent cell cycle arrest and neuronal differentiation.
By comparison, recent studies indicate that SH-PTP-2 activation and
binding to FRS-2 are essential to FGF-induced differentiation of PC12
cells (40). Although the mechanism is not yet fully understood, given
the arguments outlined above that Crk is essential to prolonged MAPK
activation and neuronal differentiation, it is possible that SH-PTP-2
may inactivate an antagonist of MAPK activation, such as Ras-Gap or a
MAPK phosphatase, and thereby positively regulate neuronal
differentiation. Although overexpression of SH-PTP-2 in PC12 cells does
not enhance NGF-dependent differentiation (43), as observed
with FGF (40), overexpression of a catalytically inactive SH-PTP-2
decreases NGF-dependent differentiation (43), consistent
with the model suggested above that inactivation of a MAPK antagonist,
rather than recruitment of Grb-2, is essential to the role of SH-PTP-2
in neuronal differentiation.
FRS-2 also contains several potential binding sites for SH3
domain-containing proteins (PXXP). Of the several proteins
that we tested, only the SH3 domain of Src bound FRS-2 constitutively in our in vitro binding assays. It has long been known that
constitutive activation of Src generates NGF-independent neuronal
differentiation (34) and that the activation of Src precedes the
activation of Ras in NGF-dependent TrkA signaling (44). Our
demonstration that FRS-2 binds Src in vitro provides the
first biochemical evidence of how the Trk receptors recruit Src into
their signaling cascades in vivo and further supports the
importance of Trk-dependent binding of FRS-2 to the process
of NGF-dependent neuronal differentiation.
By taking advantage of a series of specific TrkA signaling mutants, we
demonstrate that the FRS-2 binding site on TrkA involves tyrosine 499, as well as the amino acids just 5' of this residue. Interestingly,
these same residues (around the NPXY motif) also bind the
PTB domain of Shc (37, 38, 45). The possibility that more than one
protein could bind to TrkA at this site was previously suggested (21)
and is consistent with the observation that Shc, IRS-1, and IRS-2
compete for binding to the NPXY motif of the Although overexpression of many components of the Shc/MAPK pathway
(Shc, MAPK kinase (MEK), and MAPK) (50, 51) is sufficient to stimulate
constitutive neurite outgrowth, this does not imply that
Shc-dependent activation of MAPK is the physiologically
relevant pathway correlated with prolonged MAPK activation and neuronal differentiation in vivo. Rather, overexpression studies
simply saturate one pathway at the expense of another, and if driving prolonged MAPK stimulation is required for neuronal differentiation, then it is perhaps not surprising that overstimulation of
Shc-dependent MAPK can drive constitutive neurite
outgrowth. Importantly, however, FRS-2 overexpression in nnr5 cells
expressing mutant TrkA S3 (
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-1, SH-PTP-1,
SH-PTP-2, and the suc1-associated neurotrophic factor target protein
(SNT) (14). Phosphorylation of the Shc adapter proteins, and the
concomitant recruitment of Grb-2/Sos, results in the
Ras-dependent transient activation of MAPK (17), which is
correlated with mitogenic and proliferative cell signaling. In
contrast, phosphorylation of SNT and prolonged activation of MAPK
correlate with neurotrophin-dependent cell cycle arrest and differentiation (18, 19). Interestingly, prolonged activation of MAPK
has recently been shown to be regulated by a parallel pathway involving
Crk/C3G-dependent activation of a Ras-like GTPase, Rap-1
(17).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-NGF was from Harlan Bioproducts
for Science. BDNF and NT-3 were the gifts of A. A. Welcher (Amgen
Inc.).
493IMENP497; S3A3) have been previously
described (21). B5 and S3A3 cells express approximately 100-fold higher
levels of TrkA than PC12 cells. B5 cells were transfected with
FRS-2/c-Myc by electroporation (26), and 3-4 days later, cells were
stimulated with 100 ng/ml
-NGF (2-5 min) as described below.
Untreated cells and
-NGF-treated cells (50 ng/ml, 5 min) were lysed
in Nonidet P-40 lysis buffer, and equivalent amounts of clarified
lysates were immunoprecipitated with 5 µg of 9E10. Samples were
analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting
with anti-Tyr(P) (RC20) as described below. TrkAS3A3 cells were
transfected with internal ribosome entry site (pIRES)/enhanced green
fluorescent protein (EGFP)-FRS-2/Myc and cells were selected in 0.05 mg/ml G418 and 0.2 mg/ml hygromycin (Boehringer Mannheim) for 3-4
weeks. Clones positive for EGFP expression were identified by
epifluorescence, picked, and assayed for FRS-2 expression by Western
blotting as described below. Briefly, naive and 50 ng/ml
-NGF-treated cells (5 min) were lysed in Nonidet P-40 lysis buffer,
and equivalent amounts of clarified lysates were immunoprecipitated
with 5 µg of 9E10. Samples were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting with anti-Tyr(P) (RC20) as
described below.
-1, and v-Src (T. Pawson, Lunenfeld Research Institute, Toronto, Ontario, Canada); and
Abl and Nck (P. P. DiFiore, European Institute of Oncology, Milan,
Italy). GST fusion proteins were purified according to standard
procedures. The FRS-2/c-Myc cDNA was also cloned into pIRES-EGFP
(CLONTECH) in which transfected cells can be easily
identified based on low levels of enhanced green fluorescent protein
expression from an internal ribosomal initiation site.
-32P]dCTP (Amersham Pharmacia
Biotech) by random priming to a specific activity greater than 1 × 109 cpm/µg. The blot was washed to a final stringency
of 42 °C, 1× SSC, 0.1% SDS. The blot was stripped and reprobed
with actin (55 °C, 0.1× SSC). Densitometry was performed with a
Bio-Rad imaging densitometer (Model GS-700) and Multi-Analyst software.
Trp1-901 Leu2-3, 112 ura3-52 His3-200 gal4
gal80
Lys2::Gal1-His3 Gal2-Ade2 Met2::Gal7-lacZ) was the gift
of Philip James (University Wisconsin) and was cultured and transfected
as described (28). A 1.5-kb NcoI-SalI fragment of
FRS-2 was cloned into the NcoI-SalI sites of
pAS2-1 (CLONTECH). Wild type rat TrkA, the kinase
inactive rat TrkA kinase (K547A), and p13suc1 were subcloned
into pGAD424. A unique EcoRI site was inserted at nucleotide
1381 of rat TrkA (29) by PCR overlap, allowing the entire intracellular
domain (residues 443-779) to be cloned in-frame into pGAD. The yeast
p13suc1 cDNA was subcloned from pRK172-cdc13+ (gift of D. Litchfield, University of Western Ontario (30)) into the
NdeI-SmaI sites of pAS-1 and then re-cloned into
the EcoRI/BamHI sites of pGAD424. Yeast
expressing pAS-FRS-2 were transformed with pGAD vectors and plated onto
nonselective media (YPD), synthetic complete (sc) medium minus Trp (to
select for expression of the pAS vector), sc minus Leu and Trp (to
select for both pAS and pGAD vectors), and finally sc minus Leu, Trp,
His, and Ade (HALT medium) with 3-amino triazole. Typically, 15-20
mM 3-amino triazole was sufficient to eliminate
self-activation of pAS-FRS-2.
-NGF, BDNF, or NT-3) and lysed in Nonidet
P-40 buffer as described (21). Protein concentrations of clarified
lysates were determined with the Bio-Rad DC kit. The level
of expressed Trk receptor was determined by titrating lysates by
SDS-polyacrylamide gel electrophoresis and blotting with anti-HA.
Lysates containing equal amounts of expressed Trk receptors or lysates
prepared from hFRS-2-transfected B5 cells were used in binding assays
containing 10 µg of Sepharose-bound GST proteins. Samples were
assayed by Western blotting with RC20 (1:2500), anti-HA (1 µg/ml),
anti-c-Myc (2 µg/ml), or anti-SH-PTP-2 (1:2000). HRP-goat anti-rabbit
and HRP-goat anti-mouse were used at a dilution of 1:10,000. The
chemiluminscence detection kit was from NEN Life Science Products.
-1(SH2)-bound Sepharose was digested with 10 units of
thrombin for 30 min at room temperature. The digests were washed twice
with PBS containing protease inhibitors and 1 mM EDTA and
pooled, and the eluate was used directly.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence alignment between mouse (22) and
human FRS-2. A, myristylation signal at the amino
terminus is shown in lowercase letters, and the PTB domain
is boxed. Tyrosine residues are shown in boldface
type. Four putative Grb-2 binding sites are underlined,
and two putative SH-PTP-2 binding sites are underlined in
boldface type (53). Putative SH-3 domain binding sites
(PXXP) are shown in italics. Subsequent to our
isolation of hFRS-2, the sequence was deposited in
GenBankTM (accession number AF036718). B,
Northern blot analysis. A rat multiple tissue Northern blot
(CLONTECH) was probed sequentially with hFRS-2 and
actin. Arrows indicate the position of FRS-2 and actin
transcripts.
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Fig. 2.
Analysis of FRS-2 binding. A,
hFRS-2/c-Myc was expressed in HA-TrkA expressing B5 cells (26) and
lysates precipitated with control antibodies (lanes 1 and
3), 9E10 (anti-Myc) (lanes 2 and 4),
and p13suc1 agarose (lanes 7 and 8).
Untransfected B5 cells were analyzed for NGF-dependent
phosphorylation of endogenous FRS-2 by precipitation with
p13suc1 agarose (lanes 5 and 6). Samples
were analyzed by Western blotting with anti-Tyr(P) and anti-c-Myc
antibodies. Arrows indicate the positions of endogenous and
transfected FRS-2 (80-90 kDa). B and D,
hFRS-2/c-Myc-transfected B5 cells (unstimulated and NGF-stimulated)
were assayed in an in vitro binding assay. Equivalent
lysates were precipitated with GST; GST fusion proteins
corresponding to Grb-2 (full-length), Grb-2(SH2), Grb-2(NSH3),
Grb-2(CSH3), c-Abl(SH3), v-Src(SH3), Crk (full-length and SH3
domain), Nck1(SH3), Ras-Gap(SH3), PLC -1(SH3); control
antibodies; and anti-Myc antibodies (9E10). Arrows indicate
the positions of hFRS-2/c-Myc. C, equivalent lysates from
untransfected (right panel) and hFRS-2/c-Myc-transfected
(left panel) B5 cells were precipitated with control
antibodies, 9E10, p13suc1, and anti-SH-PTP-2 antibodies.
Samples were analyzed by Western blotting with RC20 and anti-SH-PTP-2.
Arrows indicate the positions of the FRS-2, SH-PTP-2, and
p120.
-1, and v-Src), as well
as full-length Crk, for interaction with hFRS-2. As shown in Fig.
2D, the only SH3 domain-mediated interaction observed was
with v-Src. As predicted for SH3 domain interactions, the v-Src
interaction occurred constitutively and was independent of NGF
stimulation (lanes 19 and 20). The constitutive
FRS-2/Src interaction is not surprising and may help explain the
previous observation that v-Src expression in PC12 cells generates
NGF-independent neurite outgrowth (34). Full-length Crk also interacts
with hFRS-2, but because the binding is not SH3 domain-mediated (lanes 11, 12), and the interaction is NGF-dependent, it likely
involves the Crk SH2 domain.
-chain and its major signaling component IRS-1 in cells
relative to readily detectable interactions detected in yeast (35).
-1(SH2) domain, effectively precipitates TrkA as well as TrkB and TrkC, indicating that hFRS-2 is a signaling component common to neurotrophin signaling by NGF, BDNF and NT-3. To
assay direct interaction between TrkA and hFRS-2 in vivo, we utilized the yeast two-hybrid assay. Specifically, hFRS-2 was expressed
as a fusion protein with the DNA binding domain of Gal 4 (pAS2-FRS-2;
Trp+) and cells were transfected and tested for interaction with either
p13suc1 (pGAD-p13; Leu+) or the intracellular domain of either
kinase-active TRKA or kinase-inactive TrkAK547A (pGAD-TrkA; Leu +) in a
standard two-hybrid assay (36). Untransfected yeast, yeast expressing pAS-FRS-2, and yeast co-expressing pAS and pGAD vectors were plated on
nonselective medium (plate 1), sc medium minus Trp (plate 2), sc minus
Leu and Trp (plate 3), and HALT medium containing 20 mM
3-amino triazole to assay two-hybrid interaction (plate 4). As shown in
Fig. 3B, only functional Gal 4 transcription factor activity, and thus growth under selective conditions (HALT medium), was
detected in yeast co-expressing hFRS-2 and either p13suc1 or
kinase-active TrkA (lanes 3 and 4 on plate
4). The interaction between hFRS-2 and p13suc1 was
constitutive, indicating that interaction occurred independently of
hFRS-2 tyrosine phosphorylation and is consistent with the constitutive
interaction observed in the transfection studies (Fig. 2A).
In contrast, hFRS-2-TrkA interaction in the yeast two-hybrid assay is
tyrosine phosphorylation-dependent. Dimerization of the Gal
4 transcription factor is sufficient to stimulate the Trk kinase
activity and to assay activity-dependent interactions (data not shown). Accordingly, despite a high level of TrkA ICD expression in
yeast, the kinase inactive TrkA (K547A) does not interact with hFRS-2
(Fig. 3B, plate 4, lane 5).
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Fig. 3.
FRS-2 binds Trk receptors. A,
HA-tagged Trk receptors were assayed for binding to GST,
GST-hFRS-2(PTB), GST-Shc(PTB), and GST-PLC -1(SH2). Arrows
indicate the position of the Trks. B, yeast two-hybrid
interaction between hFRS-2, p13suc1, and the intracellular
domain of TrkA. Yeast (PJ694A (lane 1)) expressing pAS-FRS-2
(Trp+ (lane 2)) was re-transfected with pGAD-p13 (Leu+
(lane 3)), pGAD-TrkA kinase (Leu+ (lane 4)), and
pGAD-TrkA kinase dead (K547A) (Leu+ (lane 5)). Cells were
plated on nonselective sc medium (plate 1), sc medium minus
Trp (plate 2), sc medium minus Leu and Trp (plate
3), and sc medium minus leucine, tryptophan, histidine, and
adenine plus 20 mM 3-amino triazole (AT)
(plate 4).
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Fig. 4.
FRS-2/TrkA binding is
phosphotyrosine-dependent. A, HA-tagged Trk
receptors were expressed in baculovirus-infected insect cells. Lysates
prepared from a wild type baculovirus and Trk baculovirus-infected
cells were assayed for tyrosine phosphorylation and receptor expression
by anti-Tyr(P) and anti-HA Western blots, respectively. B,
lysates containing equivalent amounts of expressed Trk receptors were
assayed for binding to GST fusion proteins and Western blotted with
anti-HA antibodies. Arrows indicate the position of TrkA
(gp140).
-1 interact at phosphotyrosine residues Tyr499
and Tyr794, respectively (Tyr490 and
Tyr785 in human TrkA) through intrinsic PTB and SH2
domains. A total of four independent TrkA signaling mutants have been
previously described and were used in the present study (see Table
I and Fig.
5A). Trk receptors were
HA-tagged at the amino terminus and expressed in baculovirus-infected
insect cells. Lysates containing comparable levels of expressed Trk
receptors were used in in vitro binding assays with purified
GST fusion proteins. As shown in Fig. 5B, GST fusion
proteins corresponding to Shc(PTB), PLC
-1(SH2), and hFRS-2(PTB)
effectively precipitate wild type TrkA to levels significantly greater
than GST alone. As predicted, the TrkAS9 mutant showed a loss of
PLC
-1 binding (Fig. 5B, lane 20) but retained both
Shc(PTB) and hFRS-2(PTB) domain binding, indicating that tyrosine 794 is not involved in hFRS-2-TrkA binding. Analysis of the KFG minus
TrkAS17 mutant indicates that it retained PLC
-1 and Shc(PTB) binding
but showed a reduction in hFRS-2(PTB) domain binding. This suggests
that the KFG deletion reduced hFRS-2 binding but did not completely
abolish the interaction. In contrast, both the TrkAS3 deletion mutant
(
493IMENP497) and the Shc binding site
mutant (Y499F) retained PLC
-1 binding but showed a complete loss of
both Shc(PTB) and hFRS-2(PTB) binding. Because Tyr499 is
still phosphorylated in the TrkAS3 mutant (21), these data indicate
that both tyrosine 499 and the amino acids 5' of Tyr499 are
critical for hFRS-2(PTB)/TrkA binding and are consistent with the
observation that residues 5' of the phosphotyrosine residue are also
critical for high affinity Trk-Shc PTB domain binding (38).
Importantly, we also found that a low, but detectable, amount of
TrkA-FRS-2 binding could be recovered when very high concentrations of
either TrkAS3 or TrkAS8 were used (data not shown), indicating that
overexpression of either TrkA mutant can facilitate some
protein-protein interaction.
Summary of TrkA receptor mutants
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Fig. 5.
FRS-2 competes with Shc for binding to rat
TrkA at Tyr499. A, schematic of TrkA
mutants: S17 ( 450KFG452) (20), S3
(
493IMENP497) (21), S8 (Y499F) (37, 52), and
S9 (Y794F) (37). Shc binds Tyr499 and PLC
-1 binds
Tyr794. Arrows indicate the signaling molecules
affected (decreased activation and/or receptor binding) by each mutant.
B, HA-tagged rat TrkA (lanes 1-4), TrkAS17
(lanes 5-8), TrkAS3 (lanes 9-12), TrkAS8
(lanes 13-16), and TrkAS9 (lanes 17-20) were
assayed for binding to GST, GST-hFRS-2(PTB), GST-Shc(PTB), and
GST-PLC
-1(SH2). Western blotting was performed with anti-HA
antibodies. Arrows indicate the positions of the Trks.
C, HA-tagged rat TrkA was assayed for binding to
GST-Shc(PTB) in the presence of increasing concentrations of soluble
hFRS-2(PTB), PLC
-1(SH2), or bovine serum albumin (BSA).
Western blotting was performed with anti-HA antibodies.
Arrows indicate the position of TrkA (gp140).
-1 or bovine serum albumin
(up to 100 µg) did not compete, indicating that the hFRS-2
competition both is specific and involves tyrosine 499.
493IMENP497 TrkAS3 deletion mutant,
consistent with the decrease in stoichiometry of FRS-2/SNT tyrosine
phosphorylation observed in TrkAS3 expressing nnr5 cells (21). To
determine whether the reduced FRS-2/SNT tyrosine phosphorylation and
receptor binding ability in TrkAS3 expressing nnr5 cells underscores
the NGF nonresponsive phenotype, FRS-2 was ectopically expressed in
TrkAS3A3 cells (21) on a vector that co-expresses EGFP from an internal
ribosome entry site. Cells expressing EGFP were selected and assayed
for NGF-dependent tyrosine phosphorylation of FRS-2, as
well as an NGF-dependent decrease in proliferation and a
concomitant increase in neuronal differentiation. As shown in Fig.
6A, two independent clonal
lines demonstrate high levels of NGF-induced tyrosine-phosphorylated FRS-2/Myc in comparison to either the parental S3A3 cells or cells expressing EGFP alone. To determine whether ectopic expression of
FRS-2/Myc can stop cell cycle progression and facilitate neuronal differentiation, cells were grown for 5 days in the absence or presence
of NGF and scored for neurite outgrowth. As shown in Fig.
6B, S3-FRS-2 expressing cells (clone 40 is shown, but a
similar response was also found in clone 34; data not shown)
demonstrated significant neurite outgrowth 5 days poststimulation with
100 ng/ml NGF (approximately 80-90% of the culture) relative to the parental S3A3 cells or cells expressing EGFP (approximately 2%). Thus,
FRS-2 overexpression in S3 cells reconstitutes
NGF-dependent differentiation that is morphologically
similar to wild type TrkA-expressing nnr5 cells (B5). To address
whether the rescue of process outgrowth was accompanied by a
corresponding decrease in the mitotic index, S3A3 cells, S3 cells
expressing FRS-2 or EGFP, and wild type TrkA-expressing B5 cells were
assayed for changes in BrdUrd incorporation. As shown in Fig.
6C, B5 cells showed an NGF-dependent decrease in BrdUrd incorporation of approximately 70%, whereas the levels of
BrdUrd incorporation in S3A3 cells and cells expressing EGFP were
unaffected by NGF. In contrast, S3 cells co-expressing FRS-2/Myc showed
an NGF-dependent decrease in BrdUrd incorporation of
approximately 50%.
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Fig. 6.
FRS-2 Expression in nnr5 cells
expressing the TrkA-S3 deletion (S3A3 cells). A, cells
(unstimulated and -NGF (100 ng/ml)-stimulated, 5 min) were analyzed
for NGF-dependent tyrosine phosphorylation of stably
expressed FRS-2/c-Myc by precipitation with anti-Myc antibodies 9E10.
Samples were analyzed by Western blotting with anti-Tyr(P) antibodies
(RC20). Arrows indicate the positions of FRS-2/c-Myc. Two
independent S3A3 lines expression FRS-2/c-Myc (clones 34 and 40) and
cells expressing EGFP were analyzed. B, neurite response
assay of mutant S3A3 cells, S3 cells expressing FRS-2/c-Myc or EGFP,
and nnr5 cells expressing wild type TrkA, B5 cells. Cells were cultured
for 5 days in the absence and presence of NGF (100 ng/ml).
C, BrdUrd assay. S3A3 cells, S3 cells expressing FRS-2/c-Myc
or EGFP, and B5 cells were assayed for proliferation in the absence and
presence of NGF (100 ng/ml, 5 days). Proliferation was assayed by
determining the percentage of cells incorporating BrdUrd. Values
represent the mean of 10-12 independent frames. The S3-FRS-2 samples
reflect a pool of two independent clones (clones 34 and 40), and the
values were calculated from counting 10 independent frames from each
clone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-chain of
the insulin and insulin-like growth factor-1 receptors (46-48).
Overlapping binding sites have also been demonstrated for Nck and PI-3
kinase (p85) binding to the platelet-derived growth factor receptor
(49). Thus, the possibility that Shc and FRS-2 can compete for binding
to the Trk receptors is not unprecedented. Importantly, we provide
direct evidence that Shc and FRS-2 do, in fact, compete for
binding to TrkA in vitro. Using a constant amount of
GST-Shc(PTB), we found that soluble FRS-2 was able to compete with Shc
for binding to TrkA with an EC50 requiring an approximately
2-5-fold excess of FRS-2. Collectively, these data provide a
novel model by which competition between Shc and FRS-2 may occur
in vivo and, possibly, regulate a switch between cell cycle
progression/mitogenesis and cell cycle arrest/differentiation.
493IMENP497)
receptors (S3A3 cells) (21) reconstitutes
NGF-dependent differentiation and cessation of
cell cycle progression. TrkAS3 receptors retain the ability to
phosphorylate Shc, as well as mitogenic signaling in transfected
fibroblasts, yet show a dramatic reduction in the stoichiometric
phosphorylation of FRS-2/SNT, as well as the ability to support neurite
outgrowth in S3A3 cells (21). Because high concentrations of mutant
TrkAS3 receptors can stimulate low levels of FRS-2 binding in
vitro (discussed above), it is likely that stable overexpression
of FRS-2 in TrkAS3 cells either reconstitutes sufficient TrkA binding
or permits sufficiently high levels of FRS-2 tyrosine phosphorylation
to facilitate NGF-dependent cell cycle arrest and
differentiation. The role of FRS-2 in NGF-dependent differentiation is also supported by mutant Trk receptors in which Tyr499 is replaced with phenylalanine (TrkAS8). Cells
expressing TRK Y499F do not stimulate NGF-dependent Shc
tyrosine phosphorylation and/or Shc/Grb-2 binding (21, 37). Moreover,
Trk Y499F receptors show a reduction in the stoichiometry of
NGF-dependent FRS-2 phosphorylation (data not shown).
However, as described above for TrkAS3, we have found that
overexpression of Trk Y499F receptors can stimulate both FRS-2
phosphorylation and low levels of neuronal differentiation, which may
account for the observations by Obermeier et al. (52) and
Stephens et al. (37) in which cells expressing high levels of Y499F Trk receptors retain NGF-dependent SNT
phosphorylation, as well as varying degrees of neurite outgrowth. The
important relevance of the FRS-2 overexpression studies described here
are that they reconstitute NGF-dependent
neuronal differentiation, suggesting that FRS-2 may be the
physiologically relevant substrate essential to
NGF-dependent mitotic arrest. The competition between Shc
and FRS-2 for binding to NGF-activated TrkA suggests a novel mechanism
by which proliferation and differentiation may be regulated in response
to neurotrophin stimulation.
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ACKNOWLEDGEMENTS |
---|
We acknowledge T. Pawson (Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada) and P. P. Di Fiore (European Institute of Oncology, Milan, Italy) for the gifts of numerous GST constructs, J. McGlade (The Hospital for Sick Children, Toronto, Ontario, Canada) for GST-Shc, and P. James (University of Wiscosin) for the yeast strain PJ694A.
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FOOTNOTES |
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* This research was supported by operating grants from the Medical Research Council of Canada (to S. O. M. and J. M. V.) and the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to S. O. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF036717.
Research scholar of the Medical Research Council of Canada and
recipient of an EJLB Foundation Scholar Research Program Grant. To whom correspondence should be addressed: Research Scientist, The
John P. Robarts Research Institute, 100 Perth Dr., London, Ontario N6A 5K8, Canada. Tel.: 519-663-5777, ext. 34304; Fax: 519-663-3789; E-mail: smeakin{at}rri.on.ca.
** Recipient of a studentship from the Cancer Research Society.
§§ Research scholar of the Medical Research Council of Canada.
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ABBREVIATIONS |
---|
The abbreviations used are: NGF, nerve growth factor; p75NTR, low affinity neurotrophin receptor; MAPK, mitogen-activated protein kinase; SNT, suc1-associated neurotrophic factor target; FGF, fibroblast growth factor; FGFR, FGF receptor; GST, glutathione S-transferase; BrdUrd, 5-bromo-2-deoxyuridine; SH, Src homology; BDNF, brain-derived neurotrophic factor; NT, neurotrophin; sc, synthetic complete; HA, hemagglutinin; HALT medium, sc minus Leu, Trp, His, and Ade; EGFP, enhanced green fluorescent protein; HRP, horseradish peroxidase; PCR, polymerase chain reaction; PTB, phosphotyrosine binding; kb, kilobase(s).
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REFERENCES |
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