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Activation of Trk neurotrophin receptors in the
absence of neurotrophins
Francis S. Lee* and Moses V. Chao
†‡
*Department of Psychiatry, Weill Medical College of Cornell University Medical College, New York, NY 10021; and †Molecular Neurobiology Program,
Skirball Institute of Biomolecular Medicine, Departments of Cell Biology and Physiology and Neuroscience, New York University School of Medicine,
New York, NY 10016
Communicated by Hans Thoenen, Max Planck Institute of Neurobiology, Martinsried, Germany, January 11, 2001 (received for review November 2, 2000)
Neurotrophins regulate neuronal cell survival and synaptic plas-
ticity through activation of Trk receptor tyrosine kinases. Binding
of neurotrophins to Trk receptors results in receptor autophos-
phorylation and downstream phosphorylation cascades. Here, we
describe an approach to use small molecule agonists to transacti-
vate Trk neurotrophin receptors. Activation of TrkA receptors in
PC12 cells and TrkB in hippocampal neurons was observed after
treatment with adenosine, a neuromodulator that acts through G
protein-coupled receptors. These effects were reproduced by using
the adenosine agonist CGS 21680 and were counteracted with the
antagonist ZM 241385, indicating that this transactivation event by
adenosine involves adenosine 2A receptors. The increase in Trk
activity could be inhibited by the use of the Src family-specific
inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to
other G protein-coupled receptor transactivation events, adeno-
sine used Trk receptor signaling with a longer time course. More-
over, adenosine activated phosphatidylinositol 3-kinase兾Akt
through a Trk-dependent mechanism that resulted in increased cell
survival after nerve growth factor or brain-derived neurotrophic
factor withdrawal. Therefore, adenosine acting through the A
2A
receptors exerts a trophic effect through the engagement of
Trk receptors. These results provide an explanation for neuropro-
tective actions of adenosine through a unique signaling mecha-
nism and raise the possibility that small molecules may be used to
elicit neurotrophic effects for the treatment of neurodegenerative
diseases.
Neurotrophins play a prominent role in the development of
the vertebrate nervous system by inf luencing cell survival,
differentiation, and cell death events (1, 2). Neurotrophins also
exhibit acute regulatory effects on neurotransmitter release,
synaptic strength, and connectivity (3, 4). In addition to pro-
moting axonal and dendritic branching, neurotrophins serve as
chemoattractants for extending growth cones in vitro (5). These
actions are mediated by neurotrophin binding to two separate
receptor classes, the Trk family of tyrosine kinase receptors and
the p75 neurotrophin receptor, a member of the tumor necrosis
factor receptor superfamily (6).
Mutations in Trk neurotrophin receptor function lead to
deficits in survival, axonal and dendritic branching, long-term
potentiation, and behavior (7–9). Nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3, and
neurotrophin-4 also bind to the p75 neurotrophin receptor, a
potential cell death receptor whose actions are negated by Trk
tyrosine kinase signaling (10, 11). Therefore, the ability to
regulate Trk tyrosine kinase activity is critical for neuronal
survival and differentiation.
Ligands for G protein-coupled receptors are capable of acti-
vating the mitogen-activated protein (MAP) kinase signaling
pathway, in addition to classic G protein-dependent signaling
pathways involving adenylyl cyclase and phospholipase C (12,
13). Induction of mitogenic receptor tyrosine kinase phosphor-
ylation also occurs through signaling from several G protein-
coupled receptors (14). In particular, receptors for epidermal
growth factor, platelet-derived growth factor, and insulin-like
growth factor 1 can be transactivated by G protein-coupled
receptors(12, 15, 16). Whether transactivation of neurotrophic
receptor tyrosine kinases occurs by means of G protein-coupled
receptors has not been demonstrated to date.
We have tested the possibility that ligands of G protein-
coupled receptors might activate neurotrophin receptors of the
Trk tyrosine kinase subfamily. Here, we report that adenosine
and adenosine agonists can activate Trk receptor phosphoryla-
tion, through a mechanism that requires the adenosine 2A (A
2A
)
receptor. The activation does not require neurotrophin binding
and is observed in PC12 cells, as well as primary cultures of
hippocampal neurons. Unlike the results obtained with other
tyrosine kinase receptors, increased Trk receptor activity pro-
vides increased cell survival over a prolonged time course that
requires Akt, and not MAP kinase, signaling. These findings
suggest alternative approaches of stimulating trophic functions
in the nervous system by linking different receptor signaling
pathways.
Materials and Methods
CGS 21680, CPA, A23187, and insulin-like growth factor-1 were
purchased from Sigma-RBI. ZM 241385 was from Tocris Neu-
rochemicals (Ballwin, MO), PP1 from Alexis Biochemicals (San
Diego, CA), LY294002 from Biomol, K252a from Calbiochem,
and PD98059 from New England Biolabs. NGF was obtained
from Harlan Bioproducts (Indianapolis, IN) and BDNF from
PeproTech (Rocky Hill, NJ). All other compounds were from
Sigma. An anti-pan-Trk rabbit antiserum raised against the
C-terminal region of the Trk receptor was from Barbara Hemp-
stead (Cornell University); anti-NGF antibody was obtained
from Chemicon. Anti-phosphotyrosine and anti-Akt antibodies
were from Santa Cruz Biotechnologies. Anti-phospho-Akt, anti-
MAP kinase, and anti-phospho-MAP kinase antibodies were
from New England Biolabs.
Immunoprecipitation and Immunoblotting. PC12 cells or PC12
(615) cells (17), were maintained in DMEM containing 10%
FBS supplemented with 100 units兾ml penicillin, 100
g兾ml
streptomycin, and 2 mM glutamine plus 200
g兾ml G418. Cells
were placed in low-serum medium (1% FBS, 0.5% horse serum)
overnight before experiments. Cell lysates from PC12, 615 cells,
or hippocampal cells were incubated in lysis buffer (1% Nonidet
P-40) for4htoovernight at 4°C with anti-pan-Trk polyclonal
antibody followed by incubation with protein A-Sepharose
beads. Equivalent amounts of protein were analyzed for each
condition. The beads were washed five times with lysis buffer,
and the immune complexes were boiled in SDS-sample buffer
Abbreviations: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; A2A,
adenosine 2A; PI3-kinase, phosphatidylinositol 3⬘-kinase; MAP, mitogen-activated protein;
LDH, lactate dehydrogenase; En, embryonic day n.
‡To whom reprint requests should be addressed. E-mail: chao@saturn.med.nyu.edu.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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and loaded on SDS-PAGE gels for immunoblot analysis. The
immunoreactive protein bands were detected by enhanced
chemiluminescence (Amersham Pharmacia).
125
I-NGF Binding Analysis. For equilibrium binding studies,
125
I-
NGF was prepared as described previously (18). PC12 cells
stably overexpressing TrkA (2 ⫻10
5
cells) and HEK 293 cells
expressing TrkA (2 ⫻10
5
) were incubated with
125
I-NGF in the
absence and presence of adenosine compounds for 30 min at
25°C. The cells were then washed twice with PBS, and
125
I-NGF
was stripped with an acid solution (0.2 M acetic acid, 0.5 M
NaCl). Nonspecific binding was assessed by adding unlabeled
NGF at a final concentration of 1000 ng兾ml and represented
⬍20% of total binding. Specific binding was defined as total
binding minus nonspecific binding. All conditions were carried
out in triplicate and SEM calculated.
Hippocampal Cell Cultures. Dissociated primary cultures of hip-
pocampal neurons from embryonic day 17 (E17) rats were
prepared from timed-pregnant Sprague–Dawley rats as de-
scribed previously (19). Fetuses were removed under sterile
conditions and kept in PBS on ice for microscopic dissection of
the hippocampus. The meninges were removed and the tissue
was placed in Neurobasal media (GIBCO兾BRL). The tissue was
briefly minced with fine forceps and then triturated with a
fire-polished pasteur pipet. Cells were counted and plated on
culture wells coated with 0.01 mg兾ml poly-D-lysine overnight.
Hippocampal cells were maintained in Neurobasal media, con-
taining B27 supplement and L-glutamine (0.5 mM). Experiments
were conducted 7–10 days after plating.
Cell Death Assay. PC12 cells were differentiated in DMEM
supplemented with 0.33% FBS, 0.67% heat-inactivated horse
serum, 2 mM L-glutamine, and NGF (50 ng兾ml) for 7 days.
Serum and NGF were then removed, and adenosine agonists or
growth factors were added to the media. After 48 h, cell death
was quantified by measuring lactate dehydrogenase (LDH)
released from injured cells into the media by using the Cytox 96
Cytotoxicity Assay Kit (Promega). LDH values were normalized
by subtracting the LDH released by cells maintained in NGF (50
ng兾ml) and scaling to a full kill (⫽100%) reference induced by
5 min of treatment with 1% Triton, an exposure that consistently
killed all PC12 cells.
Hippocampal neurons were maintained in Neurobasal media
containing B27 supplement and 0.5 mM L-glutamine for 10 days.
B27 was then removed, and adenosine agonists or BDNF (100
ng兾ml) were added to the media. MK-801 (1
M) was added to
all conditions to decrease the contribution of N-methyl-D-
aspartate-mediated cell death. After 48 h, cell death was assessed
by measurement of LDH released into media. LDH values were
normalized by subtracting the LDH released by cells maintained
in BDNF (100 ng兾ml) and scaling to full kill (⫽100%) reference
induced by 24 h of treatment with A23187 (30
M), a condition
that resulted in complete cell death of all neurons (20).
Results
Transactivation of mitogenic tyrosine kinase receptors through
G protein-coupled receptors has been described previously (12,
15, 16). To explore whether any G protein-coupled receptors
exert an effect on neurotrophin receptor signaling, several
ligands were tested for their ability to influence TrkA tyrosine
kinase activity in PC12 cells. Receptors for each ligand are found
on PC12 cells (21–24). TrkA receptors were immunoprecipitated
from PC12 cell lysates and then probed with an anti-
phosphotyrosine antibody. Activated TrkA receptors were ob-
served with adenosine treatment (10
M), but not with nucle-
otides such as ATP or GTP (Fig. 1). The TrkA doublet
represents an underglycosylated form of 110 kDa and the fully
glycosylated form of 140 kDa (17). Activation of TrkA receptors
was not observed with other G protein-coupled ligands, includ-
ing bradykinin and dopamine agonists, apomorphine and quin-
pirole (Fig. 1). The specificity of adenosine’s effects was also
confirmed by the use of CGS 21680, 2-[(4-(2-carboxyethyl)phe-
nylethyl)] aminoadenosine-5⬘-N-ethylcarboxamide, a selective
adenosine A
2A
agonist (25).
The effect of adenosine on TrkA receptor activity occurred in
a low concentration range (Fig. 2A). This response was verified
by the use of 1 nM CGS 21680. A time course of adenosine action
showed that the increase in TrkA activation was slow and
required at least 90 min (Fig. 2B), which is delayed compared
with NGF treatment. This increase was inhibited by K252a, a
known inhibitor of Trk tyrosine kinases (see below). It is
formally possible that adenosine treatment leads to the produc-
tion of NGF by PC12 cells that could act in an autocrine fashion
to stimulate TrkA receptors. This possibility was discounted by
the absence of neurite outgrowth activity of supernatants taken
from PC12 cells treated with adenosine and by a lack of effect
of anti-NGF antibody on adenosine’s action (data not shown).
Adenosine interacts with four different G protein-coupled
receptors, designated A
1
,A
2A
,A
2B
, and A
3
receptors (26). The
A
2
class of adenosine receptors are expressed in PC12 cells and
have been detected by radioligand binding (23). Adenosine does
not bind to the TrkA receptor. There was no displacement of
125
I-NGF binding with an excess of adenosine (1 mM) or CGS
21680 (1
M) in PC12 cells overexpressing TrkA (Table 1). As
PC12 cells express the p75 neurotrophin receptor, which also
binds
125
I-NGF, similar experiments were carried out in HEK
293 cells after transfection with TrkA. Again, excess concentra-
tions of adenosine or CGS 21680 did not displace
125
I-NGF
binding to HEK 293 cells that expressed TrkA (Table 1). The
concentrations of adenosine and CGS 21680 were approximately
100-fold greater than those normally used in A
2A
receptor
binding and signaling studies (27).
To verify adenosine interacted specifically with the A
2A
re-
ceptor, several adenosine analogs were used. A low concentra-
tion (10 nM) of CGS 21680 gave a similar increase in phosphor-
ylated TrkA receptors with the same time course as adenosine
(Figs. 2Aand 3A). In contrast, a selective A
1
receptor agonist,
CPA, N(6)-cyclopentyladenosine, had no effect (Fig. 3A). Incu-
Fig. 1. Activation of TrkA receptors with G protein-coupled receptor ligands.
Stably transfected PC12 cells expressing high levels of TrkA (615) were treated
with the indicated compounds for 2 h. The cells were subsequently harvested
in lysis buffer as described in Materials and Methods. Lysates were immuno-
precipitated with anti-pan Trk rabbit antiserum. Immunocomplexes were
analyzed by immunoblotting with anti-phosphotyrosine antibody (PY99).
Immunoprecipitation of TrkA receptors was then confirmed by immunoblot-
ting of the immunocomplex with anti-pan Trk antiserum.
3556
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.061020198 Lee and Chao
bation of PC12 cells with the A
2A
antagonist, ZM 241385, 4-
(2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a] (1, 3, 5)triazin-5-
ylamino]ethyl] phenol, that binds the A
2A
receptor with high
affinity (28) antagonized the effects of adenosine on the phos-
phorylation of TrkA receptors (Fig. 3A). These results are
consistent with the involvement of adenosine A
2A
receptors in
mediating the increase in Trk receptor activity.
The mechanism by which G protein-coupled receptors are
linked to the activation of receptor tyrosine kinases is not well
understood. Src family kinases have been implicated as media-
tors of mitogenic receptor tyrosine kinase transactivation by
several G protein-coupled receptor agonists, such as lysophos-
phatidic acid, angiotensin II, thrombin, and bradykinin (14, 29).
To test whether a Src family member is involved in the activation
of Trk receptors by adenosine, the inhibitor PP1 (30) was used.
Treatment of PC12 cells with 1
M PP1 resulted in a marked
decrease in the level of tyrosine phosphor ylated TrkA receptors
elicited by adenosine (Fig. 3B). Increasing concentrations of PP1
produced a progressively stronger inhibition. These results sug-
gest that the regulation of TrkA activity by adenosine may be
mediated by a Src family member. An involvement of Src was
previously implicated in NGF signaling downstream of its re-
ceptor (31). However, it is conceivable that members of the Src
tyrosine kinase activity may be activated by G proteins. This has
been demonstrated for Lck, which acts in thymocytes down-
stream of the

-adrenergic receptor and whose activity can be
increased in vitro by Gs (32).
Hippocampal Neurons. To extend the generality of adenosine
effects on Trk receptors, we established primary hippocampal
neuronal cultures from E17 rat embryos. Hippocampal neurons
predominately express the TrkB receptor and respond to BDNF
(Fig. 4). These neurons also express and A
1
and A
2A
receptors
(33), but not TrkA receptors. Treatment with 10
M adenosine
or 10 nM CGS 21680 for 2 h gave rise to phosphorylated TrkB
receptors in hippocampal neurons (Fig. 4 Aand B), similar to the
activation of TrkA receptors by adenosine. An A
1
-specific
agonist, CPA, however, did not activate TrkB receptors (Fig.
4B), confirming the specificity of this effect to the A
2A
receptor.
These results not only extend the effects of adenosine to
hippocampal neurons, but also demonstrate that TrkB can also
be activated by signaling through the A
2A
receptors.
Downstream Signal Transduction. To characterize the signaling
pathways activated by adenosine, further experiments were
carried out. Pretreatment with 100 nM K252a abolished ade-
nosine’s activation of TrkA tyrosine kinase activity (Fig. 5). This
concentration of K252a has been used to block NGF activation
of TrkA receptors (34) and the subsequent biological effects of
neurotrophins.
Many G protein-coupled receptors activate the MAP kinase
pathway. Indeed, within 10 min of adenosine treatment, a
marked increased in phosphorylated MAP kinase was detected
Fig. 2. Time course and dose of adenosine activation of TrkA receptors. (A)
Different concentrations of adenosine were administered to PC12 615 cells for
2 h or NGF (1 ng兾ml) for 10 min. Cells were also treated with CGS 21680 at the
indicated doses for 2 h. (B) PC12 cells (615) were treated with adenosine (10
M) for various times or with 5 ng兾ml NGF for 10 min. Phosphorylated TrkA
receptors were detected by immunoblot analysis using PY99 anti-phosphoty-
rosine antibodies. The amount of Trk receptors in each condition was verified
by immunoblotting.
Table 1. No effect of adenosine on
125
I-NGF binding
Condition Specific binding
PC12
Control 14316 ⫾350
Adenosine (1 mM) 14403 ⫾888
CGS 21680 (1
M) 14237 ⫾1055
293兾TrkA
Control 39078 ⫾2885
Adenosine (1 mM) 36618 ⫾4185
CGS 21680 (1
M) 39568 ⫾2032
Fig. 3. Adenosine activation of TrkA by A2A receptors. (A) PC12 cells (615)
were treated with the A2A agonist CGS 21680 (10 nM) and an A1agonist CPA
(10 nM) for 2 h. ZM 241385 (10 nM), an A2A antagonist, was incubated with the
cells for 15 min before treatment with adenosine (10
M)for2h.(B) PC12 cells
(615) were incubated with the indicated concentrations of PP1, a Src family
kinase inhibitor (30), for 30 min, and then treated with adenosine (10
M) for
2 h. Activation of TrkA was assessed by immunoprecipitation and immunoblot
analysis using PY99 anti-phosphotyrosine antibody.
Lee and Chao PNAS
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March 13, 2001
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NEUROBIOLOGY
in PC12 cells (Fig. 5), consistent with previous observations
(35–37). After 10 min, the levels of activated MAP kinase
declined to a baseline level. Activation of MAP kinases can be
achieved either by A
2A
receptors, or through Trk receptor
signaling. To distinguish between these alternatives, PC12 cells
were treated with adenosine in the presence and absence of
K252a. Using a concentration of K252a that specifically blocks
TrkA signaling (34), we found that MAP kinase activity was not
altered (Fig. 5). Thus, MAP kinase induction occurred quickly,
whereas Trk activation by adenosine followed a slower time
course and did not influence MAP kinase activity. This result is
in contrast to other examples of G protein-coupled receptor
transactivation, in which MAP kinase activities are directly
stimulated downstream of the tyrosine kinase receptor (14).
Another pathway activated by receptor tyrosine kinases is
phosphatidylinositol 3⬘-kinase (PI3-kinase)兾Akt. Interestingly,
adenosine (Fig. 5) or CGS 21680 treatment (data not shown) of
PC12 cells was also able to activate Akt as detected by a
phospho-specific antibody. This response has not been previ-
ously associated with adenosine action. The time course of Akt
activation was very similar to Trk autophosphorylation induced
by adenosine. This effect was eliminated either by pretreatment
with K252a (100 nM) or LY2494002 (10
M), a PI3-kinase
inhibitor. These results indicate that Akt activation by adenosine
is Trk- and PI3-kinase-dependent.
Trophic Effects. To test the functional consequences of adenosine-
activated Trk receptor activity, we assessed the ability of aden-
osine to maintain survival of differentiated PC12 cells after
withdrawal of NGF. After culture for 48 h in the absence of
NGF, cell survival was assessed by measuring LDH release.
Whereas cells grown without NGF underwent rapid cell death,
a one-time treatment with CGS 21680 effectively rescued nearly
50% of the cells (Fig. 6A). The action of CGS 21680 required
TrkA receptors, because K252a (100 nM) eliminated the positive
effects of CGS 21680 under similar conditions that blocked the
activation of TrkA receptors (Fig. 5). Likewise, a similar dose of
K252a reversed the survival effects of NGF, but not of insulin-
like growth factor 1, in this deprivation assay.
Similar survival results with CGS 21680 were obtained in
hippocampal neurons grown in the absence of BDNF (Fig. 6B).
The action of CGS 21680 was again dose-dependent and K-252a-
sensitive. Treatment with CGS 21680 effectively rescued ⬎60%
of the cells (Fig. 6B). Hence, a potent adenosine agonist at
nanomolar concentrations was able to reverse cell death in both
PC12 cells and hippocampal neurons initiated by withdrawal of
trophic support by neurotrophins.
The ability of K252a to block adenosine’s trophic effect as well
as induction of Trk receptor activity suggested that Trk receptor
downstream signaling was involved in this process. This was
confirmed by the ability of LY294002 to eliminate the trophic
effect of CGS 21680 after NGF withdrawal (Fig. 6), indicating
that the PI3-kinase兾Akt pathway was involved in the survival
effects of adenosine. Consistent with the lack of a MAP kinase
response, we did not find that the mitogen-activated kinase
kinase inhibitor PD98059 had any effect on survival imparted by
CGS 21680.
Discussion
Adenosine receptor activation leads to many modulatory effects
on neuropeptide and neurotransmitter systems (38). Here, we
report a property of adenosine in neuronal cells that affects
neurotrophin signaling. Through crosstalk with Trk receptor
tyrosine kinases, adenosine is capable of activating the PI3-
kinase兾Akt cascade, resulting in a survival response in PC12 and
hippocampal cells. This response is similar to the effect of NGF
and BDNF on their Trk receptors, but differs in the longer time
course.
Neurotrophin receptors and A
2A
receptors have considerable
overlap in their central and peripheral nervous system distribu-
tion. In the central nervous system, A
2A
receptors are expressed
in striatum, amygdala, and olfactory tubercles, and in cerebral
cortex, hippocampus, and cerebellum (39). All of these regions
express TrkB receptors. In the peripheral nervous system, A
2A
receptor expression has been localized primarily to dorsal root
Fig. 4. Adenosine activation of TrkB receptors in hippocampal neurons.
Primary cultures of E17 hippocampal neurons were prepared as described in
Materials and Methods and treated with (A) CGS 21680 (10 nM) or BDNF (1
ng兾ml) for various times and (B) adenosine (10
M), CGS 21680 (10 nM), CPA
(10 nM), or BDNF (10 ng兾ml) for 2 h. Activation of TrkB receptors was assessed
by immunoprecipitation and Western blotting with anti-phosphotyrosine
antibody.
Fig. 5. Effects of adenosine on MAP kinase and Akt activation. PC12 cells
(615) were treated with adenosine (10
M) for various times in the presence
or absence of K252a (100 nM) or LY294002 (10
M). The cells were subse-
quently harvested in lysis buffer; lysates and immunoprecipitated samples
were subsequently immunoblotted with anti-phospho-MAP kinase, anti-
phospho-Akt, and anti-phosphotyrosine. Reprobing with anti-MAP kinase
and anti-pan-Trk antibodies was carried out to ensure equal protein loading.
3558
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ganglion and superior cervical ganglion (40), two regions that
express TrkA receptors. Interestingly, mice deficient in the
adenosine A
2A
receptor display decreased sensitivity to thermal
stimulation (41). It is noteworthy that mice with mutations in
NGF or TrkA also display hypoalgesia to thermal and mechan-
ical stimuli.
What are the in vivo consequences of these events observed in
culture? During hypoxia or ischemic conditions, adenosine is
released in large amounts and can mediate cellular protection.
A
2A
receptor agonists, such as CGS 21680, have been shown to
be neuroprotective against ischemia (42, 43) and kainate-
induced neuronal damage (44) in animals. However, A
2A
an-
tagonists have been also reported to reduce hypoxic-ischemic
neuronal injury (43, 45). The differential effects of A
2A
receptor
ligands may reflect short term versus long term effects by
adenosine receptors (46). Acute effects of adenosine analogs
may lead to opposite effects on neuroprotection than chronic
treatment. Engagement of receptor tyrosine kinases such as the
Trk subfamily may account for differences in the functional
consequences of adenosine action. A distinctive feature of
adenosine’s transactivation of Trk is the longer time course of
Trk mediated signaling, which is similar to neurotrophin-induced
signaling.
Adenosine has been proposed as a potential treatment for a
wide number of neurological disorders, including cerebral isch-
emia, sleep disorders, hyperalgesia, Parkinson’s disease, and
other neurodegenerative conditions (47). Previous effects on
growth arrest and enzyme induction by adenosine in PC12 cells
(48, 49) might well be explained by transactivation of TrkA
receptors. Our findings on adenosine delineate a pathway for
activating the neurotrophin signaling system in the absence of
neurotrophins. In contrast to other transactivation events in-
volving receptor tyrosine kinases that lead to transient increases
in MAP kinase activity, G protein-coupled receptor signaling to
neurotrophin receptors leads to selective activation of the PI3-
kinase兾Akt pathway over a prolonged time course.
These findings provide a mechanism for the neuroprotective
actions of adenosine involving engagement of the A
2A
receptor,
transactivation of Trk tyrosine kinase receptors, and selective
induction of the PI3-kinase兾Akt pathway. A number of ap-
proaches have been taken to use neurotrophins to treat Alzhei-
mer’s dementia, amyotrophic lateral sclerosis, and peripheral
sensory neuropathy (50, 51). However, there are considerable
hurdles in the use of neurotrophic molecules that are related to
difficulties in their delivery and pharmcokinetics and unantici-
pated side effects (51). The selective and sustained effects of
adenosine on survival signaling pathways suggest that small
molecules may be used to target populations of neurons that
express both adenosine and Trk receptors. The identification of
small ligands in the G protein-coupled receptor family that
regulate tyrosine kinase activity in neural cells offers a strategy
for promoting trophic effects during normal and neurodegen-
erative conditions.
We thank B. Hempstead and A. Kim for advice and R. Rajagopal and
L. Aibel for assistance. F.S.L. was supported by the DeWitt–Wallace
Fund in the New York Community Trust and an American Psychiatric
Association Program for Minority Research Training in Psychiatry
Fellowship, and M.V.C. was supported by grants from the National
Institutes of Health.
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Materials and Methods. On NGF withdrawal, various concentrations of CGS
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