Transactivation of Trk Neurotrophin Receptors by G-Protein-Coupled Receptor Ligands Occurs on Intracellular Membranes

Abstract

Neurotrophins, such as NGF and BDNF, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and intracellular signaling. It has been shown that activation of Trk receptor tyrosine kinases can also occur via a G-protein-coupled receptor (GPCR) mechanism, without involvement of neurotrophins. Two GPCR ligands, adenosine and pituitary adenylate cyclase-activating polypeptide (PACAP), can activate Trk receptor activity to increase the survival of neural cells through stimulation of Akt activity. To investigate the mechanism of Trk receptor transactivation, we have examined the localization of Trk receptors in PC12 cells and primary neurons after treatment with adenosine agonists and PACAP. In contrast to neurotrophin treatment, Trk receptors were sensitive to transcriptional and translational inhibitors, and they were found predominantly in intracellular locations particularly associated with Golgi membranes. Biotinylation and immunostaining experiments confirm that most of the transactivated Trk receptors are found in intracellular membranes. These results indicate that there are alternative modes of activating Trk receptor tyrosine kinases in the absence of neurotrophin binding at the cell surface and that receptor signaling may occur and persist inside of neuronal cells.

Full text

Cellular/Molecular
Transactivation of Trk Neurotrophin Receptors by
G-Protein-Coupled Receptor Ligands Occurs on
Intracellular Membranes
Rithwick Rajagopal,
1
Zhe-Yu Chen,
2
Francis S. Lee,
2
and Moses V. Chao
1
1
Molecular Neurobiology Program, Skirball Institute of Biomolecular Medicine, Departments of Cell Biology and Physiology and Neuroscience, New York
University School of Medicine, New York, New York 10016, and
2
Department of Psychiatry, Weill Medical College of Cornell University, New York, New
York 10021
Neurotrophins, such as NGF and BDNF, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed
by autophosphorylation and intracellular signaling. It has been shown that activation of Trk receptor tyrosine kinases can also occur via
a G-protein-coupled receptor (GPCR) mechanism, without involvement of neurotrophins. Two GPCR ligands, adenosine and pituitary
adenylate cyclase-activating polypeptide (PACAP), can activate Trk receptor activity to increase the survival of neural cells through
stimulation of Akt activity. To investigate the mechanism of Trk receptor transactivation, we have examined the localization of Trk
receptors in PC12 cells and primary neurons after treatment with adenosine agonists and PACAP. In contrast to neurotrophin treatment,
Trk receptors were sensitive to transcriptional and translational inhibitors, and they were found predominantly in intracellular locations
particularly associated with Golgi membranes. Biotinylation and immunostaining experiments confirm that most of the transactivated
Trk receptors are found in intracellular membranes. These results indicate that there are alternative modes of activating Trk receptor
tyrosine kinases in the absence of neurotrophin binding at the cell surface and that receptor signaling may occur and persist inside of
neuronal cells.
Key words: NGF; tyrosine phosphorylation; basal forebrain; adenosine; PACAP; Golgi apparatus
Introduction
Neurotrophins are essential regulators of neuronal development,
growth, and differentiation in the vertebrate nervous system
(Huang and Reichardt, 2001). The actions of neurotrophins are
dictated by two classes of cell surface receptors, the Trk receptor
tyrosine kinase and the p75 neurotrophin receptor, a member of
the tumor necrosis factor (TNF) receptor superfamily (Hemp-
stead, 2002; Chao, 2003a; Huang and Reichardt, 2003). Although
NGF binds exclusively to TrkA, BDNF and neurotrophin-4
(NT-4) bind to TrkB, and NT-3 binds to TrkC, all neurotrophins
bind to p75. After binding, neurotrophins and each of their re-
ceptors undergo internalization and transport from axon termi-
nals to neuronal cell bodies (Hendry et al., 1974; DiStefano et al.,
1992; Bronfman et al., 2003), in which a number of transcrip-
tional and enzymatic activities are activated. Engagement of Trk
receptors results in increases in cAMP response element-binding
protein (CREB) and extracellular signal-related kinase 5 (Erk5)
activities (Riccio et al., 1997; Watson et al., 1999b, 2001), as well
as phosphoinositide lipid phosphorylation and activation of GT-
Pases, such as Ras and Rap1 (York et al., 1998).
Another mode of receptor activation is through transactiva-
tion of receptor tyrosine kinases in response to G-protein-
coupled receptor signaling (Daub et al., 1996; Luttrell et al., 1999;
Fischer et al., 2003). For example, receptors for epidermal growth
factor (EGF), insulin-like growth factor-1, and platelet-derived
growth factor can be transactivated through G-protein-coupled
receptors to give proliferative and mitogen-activated protein ki-
nase (MAPK) responses. Also, signaling through dopamine re-
ceptors leads to PDGF receptor activation that results in changes
in synaptic transmission (Kotecha et al., 2002). Hence, transacti-
vation can lead to effects on cell proliferation, differentiation, and
synaptic plasticity; however, the cellular mechanisms that ac-
count for these transactivation effects are not well understood.
We reported previously that TrkA and TrkB receptors can be
activated by ligands of the G-protein-coupled receptor (GPCR)
family of transmembrane receptors in the absence of NGF or
BDNF (Lee and Chao, 2001; Lee et al., 2002b). The ligands in-
clude the nucleoside adenosine or adenosine agonists, such as
CGS 21680, and the neuropeptide pituitary adenylate cyclase-
activating polypeptide (PACAP). Adenosine and CGS 21680 rec-
ognize the A
2A
receptor (Jarvis et al., 1989), whereas PACAP
binds to the PAC1 receptor (Ishihara et al., 1992). Not only do
adenosine and PACAP activate Trk receptors, but effectors of the
Received Jan. 2, 2004; revised June 8, 2004; accepted June 14, 2004.
This work was supported by National Institutes of Health Grants NS21072 and HD23315 (M.V.C.) and the White-
hall Foundation and the National Alliance for Research on Schizophrenia and Depression (F.S.L.). We thank Juan
Carlos Arevalo for generating Trk antibodies, Lani Mustacchi for primary cultures, Daniela Pereira for advice, and
Wen-biao Gan for advice on confocal microscopy.
Correspondence should be addressed to Moses V. Chao, Skirball Institute of Biomolecular Medicine, New York
University School of Medicine, 540 First Avenue, New York, NY 10016. E-mail: chao@saturn.med.nyu.edu.
DOI:10.1523/JNEUROSCI.0010-04.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/246650-09$15.00/0
6650 •The Journal of Neuroscience, July 28, 2004 •24(30):6650 – 6658

Trk tyrosine kinase are also phosphorylated in response to these
ligands. Adenosine treatment of PC12 cells promotes the phos-
phorylation of Shc adaptor proteins, as well as phospholipase C
␥
,
analogous to the induction observed with NGF. Activation of
phosphoinositide (PI)-3 kinase and Akt accounted for neuropro-
tective effects afforded by transactivation by adenosine and
PACAP. The effects of adenosine and PACAP can be specifically
blocked by K252a, an inhibitor of Trk tyrosine kinases.
In the present study, we report that Trk activation by adeno-
sine and its agonist CGS 21680 or PACAP occurs exclusively in an
intracellular location and partly involves the Golgi apparatus.
Biotinylation assays indicate that CGS 21680 activates an intra-
cellular pool of Trk receptors. Indirect immunofluorescence ex-
periments confirm that only intracellular Trk receptors are acti-
vated by CGS 21680. Furthermore, colocalization of activated
Trk receptors and markers of the Golgi apparatus, such as
TGN-38 and GM130, suggest that Trk receptors can reside and be
activated in Golgi membranes. Taken together with previous ob-
servations, these results suggest that intracellular membranes
may potentially serve as a platform for Trk receptors to mediate
cell survival through the PI3K–Akt pathway.
Materials and Methods
Reagents. Adenosine and CGS 21680 were obtained from Sigma (St.
Louis, MO). PACAP 38 was from Peninsula Laboratories (San Carlos,
CA). K252a and brefeldin A were purchased from Calbiochem (La Jolla,
CA). Cycloheximide and actinomycin D were purchased from Sigma.
Sulfo-NHS-LC-biotin, sulfo-NHS-acetate, and streptavidin-agarose
were purchased from Pierce Biotechnology (Rockford, IL). NGF was
obtained from Harlan Bioproducts (Indianapolis, IN), and BDNF was
from PeproTech (Rocky Hill, NJ). Anti-pan-Trk rabbit antiserum raised
against the C-terminal region of the Trk receptor was from Barbara
Hempstead (#45, Weill Medical College, New York, NY), and C-14 pan-
Trk was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse
monoclonal anti-Trk antibodies against the C terminus of Trk were ob-
tained from Santa Cruz Biotechnology (B-3), and the TrkE7 antibody
against the extracellular domain of TrkA was from Zymed (San Fran-
cisco, CA). Phosphotyrosine antibodies (pY99), monoclonal Akt anti-
bodies (Akt B-1), and Erk 1/2 antibodies were from Santa Cruz Biotech-
nology. Antibodies to Y490 and Y674/Y675 of Trk as well as antibodies to
phosphorylated Akt (pAkt S473) and phosphorylated ERK1/2 were pur-
chased from Cell Signaling Technology (Beverly, MA). Mouse monoclo-
nal antibodies to Golgi matrix 130 (GM130) and early endosome
antigen-1 (EEA-1) were from BD Biosciences (San Diego, CA). Mouse
monoclonal antibodies to trans Golgi network-38 (TGN-38) were from
Affinity Bioreagents (Golden, CO).
Immunoprecipitation and immunoblotting. PC12-615 cells (Hemp-
stead et al., 1992) were maintained in DMEM containing 5% fetal bovine
serum, 10% horse serum supplemented with 2 mMglutamine plus 250
␮
g/ml G418. For all experiments, cells were serum-starved by washing
twice with PBS and placing in serum-free media for 24 hr. In some cases,
cells were pretreated with 5
␮
g/ml brefeldin A, 20
␮
g/ml cycloheximide,
200 ng/
␮
l actinomycin D, or vehicle. Cell lysates from PC12 cells were
prepared by incubating in lysis buffer (1% Nonidet P-40, 150 mMNaCl,
1m
MEDTA, 10 mMTris, pH 8.0, 10% glycerol, 10 mMNaF, 1 mMsodium
orthovanadate, 2
␮
g/ml aprotinin, 1
␮
g/ml leupeptin, and 25
␮
g/ml
phenylmethylsulfonyl fluoride) for 30 min on ice. Clarified lysates were
immunoprecipitated by incubating overnight at 4°C with anti-pan-Trk
polyclonal antibody (Trk C-14) followed by incubation with protein
A-Sepharose beads. Equivalent amounts of protein were analyzed for
each condition. The beads were washed three times with lysis buffer, and
the immune complexes were boiled in SDS-sample buffer and loaded on
SDS-PAGE gels for immunoblot analysis. The immunoreactive protein
bands were detected by enhanced chemiluminescence (Amersham Bio-
sciences, Arlington Heights, IL).
RT-PCR analysis. Total RNA from PC12-615 cells or from subman-
dibular gland dissected from adult Sprague Dawley rats was isolated
using TRIZOL (Invitrogen, Carlsbad, CA). Five hundred nanograms of
total RNA were subjected to reverse transcription using avian myeloblas-
tosis virus reverse transcriptase (Roche Applied Science, Penzberg, Ger-
many) for 30 min at 50°C. Total RNA not incubated with reverse tran-
scriptase was used as a negative control. cDNA products were incubated
with TaqDNA polymerase (Roche Applied Science) and primers for rat
NGF or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Primers used for NGF amplification were designed as described previ-
ously (Donovan et al., 1995): NGF, 5⬘-CGCTCATCCACCCACCCAGT-
CTTC, 3⬘-CTTGACAAAGGTGTGAGTCGTGGT. These primers
yielded a product of 267 base pairs. After an initial 95°C hot start, 35
cycles of 94°C for 30 sec, 60°C for 1 min, and 68°C for 1 min were
performed, followed by a final extension of 68°C for 5 min. Products
were resolved by electrophoresis using a 2% agarose gel and visualized
using ethidium bromide.
Cell surface biotinylation. Serum-starved cells were rinsed twice with
PBS containing 1 mMCaCl
2
and 0.5 mMMgCl
2
(PBS-Ca-Mg) and then
incubated with 0.5 mg/ml sulfo-NHS-LC-biotin dissolved in biotinyla-
tion buffer (0.01 MTEA, pH 7.4, 2 mMCaCl
2
, 150 mMNaCl) for 30 min
at 4°C. Cells were then quenched with PBS-Ca-Mg containing 100 mM
glycine for 20 min at 4°C. After warming to 37°C, cells were treated with
10 nMCGS 21680, 100 ng/ml NGF, or vehicle media for the indicated
times. Cells were then lysed in RIPA buffer (1% NP-40, 0.1% SDS, 0.1%
deoxycholate, 150 mMNaCl, 1 mMEDTA, 10 mMTris, pH 8.0, 10 mM
NaF, 1 mMsodium orthovanadate, 2
␮
g/ml aprotinin, 1
␮
g/ml leupeptin,
and 25
␮
g/ml phenylmethylsulfonyl fluoride) for 30 min on ice. Clarified
lysates were then split into two equal portions, one that was immunopre-
cipitated with streptavidin-agarose and the other with Trk C-14.
Receptor insertion assay. Serum-starved PC12 cells were rinsed twice
with PBS-Ca-Mg and incubated with 1.5 mg/ml sulfo-NHS-acetate in
blocking buffer (0.1 Msodium phosphate buffer, pH 7.4, 150 mMNaCl)
for 30 min at 4°C. The blocking reaction was quenched for 20 min at 4°C
using 100 mMglycine dissolved in PBS-Ca-Mg. Cells were subsequently
allowed to warm to 37°C and then treated with CGS 21680 or NGF as
indicated. After treatment, surface biotinylation and lysis were per-
formed as outlined above. Cell lysates were subjected to immunoprecipi-
tation with streptavidin-agarose. Material obtained from this immuno-
precipitation was labeled “cell surface pool.” Supernatants from the
initial immunoprecipitation were subjected to a second immunoprecipi-
tation with streptavidin-agarose to clear the lysate of biotinylated mate-
rial. The second supernatant was confirmed to be free of biotinylated
proteins by blotting with streptavidin-HRP. TrkA was then immunopre-
cipitated from the biotin-cleared lysate using Trk C-14 antibodies and
labeled “internal pool.” Immunoprecipitates were analyzed by blotting
with phosphotyrosine antibodies, phospho-Trk antibodies, or pan-Trk
antibodies.
Generation and affinity purification of antibodies against phosphorylated
TrkA. A peptide containing phosphorylated tyrosine 794 of rat TrkA,
previously shown to be a site for phospholipase C-
␥
(PLC-
␥
) recruitment
in response to NGF (Loeb et al., 1994), was used to immunize rabbits
(LQALAQAPPSpYLDVC). Crude serum was first passed through an af-
finity column made by coupling the nonphosphorylated version of the
peptide to cyanogen bromide-activated Sepharose beads. Flow through
from this column was then passed though a column made by coupling
the phospho-peptide to cyanogen bromide-activated Sepharose. The col-
umn was washed extensively with Tris-buffered saline containing 0.1%
Tween 20, and antibodies were then eluted with 0.1 Mglycine, pH 2.5.
Fractions were collected from the eluate, neutralized with Tris buffer, pH
9.5, and then tested for specificity by blotting NGF-treated PC12 cell
lysates. Specificity was further confirmed by a peptide competition assay
in which a fivefold weight excess of either phosphorylated or nonphos-
phorylated TrkA peptide was preincubated with antibody for 2 hr at 4°C
and then used for Western blotting.
Basal forebrain cell cultures. Dissociated primary cultures of basal fore-
brain neurons from embryonic day 18 (E18) rats were prepared from
timed-pregnant Sprague Dawley rats as described previously (Aibel et al.,
1998). Fetuses were removed under sterile conditions and kept in PBS on
ice for microscopic dissection of the basal forebrain. The meninges were
removed, and the tissue was placed in HBSS supplemented with 0.37%
Rajagopal et al. •Activation of Trk Receptors J. Neurosci., July 28, 2004 •24(30):6650 – 6658 • 6651

glucose. The tissue was minced briefly with fine forceps and then tritu-
rated with a fire-polished Pasteur pipette. Cells were counted and plated
on culture wells coated with 0.01 mg/ml poly-L-lysine overnight in Neu-
robasal media containing B27 supplement and L-glutamine (0.5 mM).
Experiments were conducted 10 d after plating.
Immunofluorescence and confocal microscopy. Cells were cultured on
Nunc Permanox eight-well chamber slides coated with poly-L-lysine.
Twenty-four hours after plating, cells were serum-starved by washing
twice with PBS and placing in serum-free media for 24 hr. Cells were
fixed directly after treatment in 4% paraformaldehyde for 10 min at
room temperature. In some cases, cells were pretreated with 5
␮
g/ml
brefeldin A. After two rinses in PBS, cells were permeabilized with 0.1%
Triton X-100 for 10 min on ice. Cells were blocked in 5% BSA/PBS for 30
min at room temperature before incubation for 3 hr at room temperature
with primary antibody diluted in blocking buffer. Cells were washed
three times with PBS and then incubated with secondary antibody
(FITC-conjugated anti-rabbit; Cy3-conjugated anti-mouse; Jackson Im-
munoResearch, West Grove, PA; 1:100) diluted in blocking buffer for 1
hr at room temperature. Cells were washed three times in PBS and then
mounted in Vectashield mounting medium containing 4⬘,6⬘-diamidino-
2-phenylindole (DAPI). Standard fluorescence microscopy was per-
formed using a Nikon Eclipse E800 microscope equipped with a Nikon
Plan Apo 60⫻, 1.4 numerical aperture (NA) oil immersion objective.
Images were acquired with a Zeiss AxioCam HRc digital camera. Confo-
cal images were acquired using a Bio-Rad Radiance 2000 confocal unit
coupled to an Olympus BX5OWI microscope equipped with an Olym-
pus PlanApo 60⫻, 1.4 NA oil immersion objective.
Results
GPCR ligands activate TrkA isoforms
Treatment of hippocampal neurons or
PC12 cells with adenosine resulted in an
activation of Trk activity and increased cell
survival in the absence of BDNF or NGF
(Lee and Chao, 2001). Transactivation of
Trk neurotrophin receptors leads to selec-
tive induction of the PI3-kinase–Akt path-
way that accounts for the survival re-
sponse. The increase in Trk activity was
inhibited by protein kinase inhibitors,
such as PP1, specific for the Src family
members. The Trk-dependent activation
of Akt could be blocked with LY294002, a
PI3-kinase inhibitor. PACAP also transac-
tivated Trk receptors in a manner very
similar to adenosine (Lee et al., 2002b), al-
though many other GPCR ligands failed to
transactivate Trk receptors.
To determine which residues of the Trk
receptor are phosphorylated after GPCR
stimulation, we performed phosphoryla-
tion analysis of Trk in PC12-615 cells
(Hempstead et al., 1992). These cells ex-
press elevated levels of TrkA and have been
widely used to study signal transduction.
TrkA was immunoprecipitated from cells
treated with 10
␮
Madenosine and ana-
lyzed with antibodies to specific Trk phos-
phorylation sites, including the activation
loop of the kinase domain phosphoty-
rosines (amino acids 683 and 684 of rat
TrkA) and the Shc-binding phosphoty-
rosine site (amino acid 490). Indeed, all of
these tyrosine residues were phosphory-
lated in response to adenosine treatment
(Fig. 1A). The increase in specific phos-
phorylation sites is consistent with the finding that Trk enzymatic
activity is enhanced after adenosine or PACAP treatment (Lee et
al., 2002b). Furthermore, the phosphorylation events occurred in
a similar time interval for all three residues, which involved at
least 60 min to observe the activation of TrkA.
Phosphorylation of the activation-loop and Shc-binding res-
idues was observed in both the 140 and 110 kDa isoforms of Trk.
The phosphotyrosine analysis clearly indicated that the imma-
ture 110 kDa form of Trk was activated first, followed by the
mature 140 kDa form (Fig. 1A). The Trk receptor exists in a
number of different glycosylation states within the cell, but only
the fully glycosylated 140 kDa receptor is thought to be targeted
to the plasma membrane (Watson et al., 1999a; Jullien et al.,
2002). At the cell surface, NGF engages only the fully mature
receptor found to initiate signaling. The appearance of immature,
underglycosylated forms of TrkA early in the time course of
transactivation suggests that intracellular processing events may
play an important role in the mechanism and that a distinct pool
of Trk receptors is involved in this process.
To assess whether the activation and processing of Trk recep-
tors depended on transcriptional or posttranscriptional events,
we performed analyses using inhibitors of transcription and
translation. PC12 cells were pretreated with either actinomycin D
(0.2
␮
g/
␮
l) or cycloheximide (20
␮
g/ml) for 30 min and then
Figure 1. Adenosine activates TrkA receptors at multiple phosphotyrosine residues. A, PC12 cells were treated with 10
␮
Madenosine for the indicated times. Trk was immunoprecipitated from cell lysates using a pan-Trk antibody. Phosphor-
ylation of Trk was assessed with antibodies against phosphotyrosine 499 of rat TrkA [pTrk 490 (Cell Signaling)] or
antibodies against phosphotyrosines 683 and 684 of rat TrkA [pTrk 674/675 (Cell Signaling)]. Immunoblot with pan-Trk
antibody (B3) was used to ensure equal loading. B, PC12 cells were pretreated with either 20
␮
g/ml cycloheximide or 200
ng/
␮
l actinomycin D for 30 min before incubation with 10 nMCGS 21680 for 1 or 3 hr. Trk was immunoprecipitated from
cell lysates and analyzed with phosphotyrosine antibodies. Trk protein levels were determined by blotting with pan-Trk
antisera. C, Quantitation of phospho-Trk and total Trk levels from lysates of CGS 21680-treated PC12-615 cells. Three
independent experiments were quantified using ImageJ analysis software. Levels of band intensity relative to untreated
control are represented on the y-axis. Error bars reflect SEM. D, RT-PCR for NGF mRNA was performed on total RNA
extracted from untreated PC12 cells or cells treated with 10 nMCGS 21680 for 3 hr. RNA extracted from adult rat subman-
dibular gland was used as a positive control; GAPDH was used as an internal loading control.
6652 •J. Neurosci., July 28, 2004 •24(30):6650 – 6658 Rajagopal et al. •Activation of Trk Receptors

treated with CGS 21680 (10 nM). CGS
21680 was used instead of adenosine be-
cause it is a specific and stable agonist that
interacts with the A
2A
receptor (Jarvis et
al., 1989). Previous experiments indicated
that CGS 21680 elicited identical re-
sponses as adenosine (Lee et al., 2001).
Phosphorylation of Trk was assessed by
immunoprecipitation from lysates using a
pan-Trk antibody followed by blotting
with a phosphotyrosine antibody, pY99.
After 1 and 3 hr of CGS 21620 treatment,
we consistently observed phosphorylation
of the 110 kDa TrkA species. Pretreatment
with actinomycin D, a potent inhibitor of
cellular transcription, or cycloheximide, a
translation inhibitor, prevented the trans-
activation of Trk by the adenosine agonist
CGS 21680 (Fig. 1B). Hence, GPCR-
mediated transactivation of Trk receptors
requires transcriptional and protein syn-
thesis events that may influence the pro-
cessing and activation of the receptor. An
increase in Trk receptor levels was ob-
served after 3 hr of treatment with CGS
21680; however, it should be noted that acti-
vation of TrkA occurred before this increase.
Quantitation of multiple experiments clearly
indicates that TrkA activity was induced af-
ter 1 hr of CGS 21680 treatment (Fig. 1C), a
time at which TrkA receptors were not sig-
nificantly upregulated.
To determine whether the gene up-
regulation requirement for transactivation
directly involved production of NGF, we
performed RT-PCR analysis on PC12 cells
treated with CGS 21680. As shown in Fig-
ure 1D, NGF mRNA was not detected in
either untreated or CGS-treated PC12
cells. In contrast, NGF mRNA was readily
detected in adult rat submandibular gland.
Previous experiments also failed to detect
any NGF biological activity in cells treated
with GPCR ligands (Lee et al., 2002).
Therefore, it is unlikely that transactiva-
tion of TrkA receptors involves NGF.
GPCR ligands activate an internal pool of Trk receptors
The finding that immature forms of Trk were activated preferen-
tially early during the time course of transactivation (Fig. 1A)
suggested than an intracellular pool Trk receptors is targeted. To
localize the site of Trk receptor transactivation, we performed a
cell surface biotinylation procedure, as outlined in Figure 2A.
PC12 cells were subjected to surface labeling with sulfo-NHS-
biotin and then treated with 10 nMCGS 21680 or 100 ng/ml NGF.
After 10 min of NGF treatment, a considerable amount of
activated TrkA receptors could be found in the biotinylated cell
surface pool of proteins (Fig. 2 B); however, at no point after CGS
21680 treatment was phosphorylated TrkA detected in the cell
surface pool, although transactivated Trk appeared in the total
Trk pool after 2 hr of CGS 21680 treatment (Fig. 2B). This de-
layed time course was consistent with previous observations (Lee
and Chao, 2001). The lack of receptors in the biotinylated frac-
tion suggests that most of the transactivated receptors did not
arise from the cell surface pool.
Although these biotinylation results revealed that preexisting
surface Trk receptors were not involved in the transactivation
mechanism, the possibility still remained that newly synthesized
Trk receptors induced by GPCR activity could be inserted into
the plasma membrane and become activated. To assess whether
the newly synthesized Trk receptors are correctly processed and
inserted into the cell membrane after CGS 21680 treatment, we
performed a membrane receptor insertion assay using a modified
cell surface biotinylation procedure (Peng et al., 2001). Cells were
first blocked from biotinylation using sulfo-NHS-acetate and
then treated with 10 nMCGS 21680 for the indicated times (Fig.
3A). After treatment, cells were biotinylated with sulfo-NHS-LC-
biotin and then subjected to immunoprecipitation. Nonbiotiny-
lated Trk was isolated by immunoprecipitation with pan-Trk an-
tibodies from the biotin-depleted lysate.
Figure 2. Transactivated Trk receptors are not detected at the cell surface. A, A schematic diagram of the biotinylation
procedureisshown.PC12 cells were biotinylated as described in Materials andMethodsandthentreated with 10 nMCGS 21680 for
the indicated times or with 100 ng/ml NGF for 10 min. Cell lysates were prepared and split into two equal portions. Half of the
lysate was subjected to immunoprecipitation with streptavidin-agarose, and the other half was immunoprecipitated with pan-
Trk antibodies. B, Immunoprecipitates were analyzed by blotting with phosphotyrosine antibodies and with pan-Trk antibodies.
Figure 3. TrkAistransactivatedon internal membranes. A, A receptor insertion assay wasperformedasdescribed in Materials
and Methods and is depicted in the schematic diagram. PC12 cells were treated with the biotinylation-blocking reagent S-NHS-
acetate and then treated with either 10 nMCGS 21680 or 100 ng/ml NGF for 3 hr. Cell lysates were immunoprecipitated with
avidin-agarose. Supernatants from the avidin-agarose immunoprecipitation that were depleted of biotinylated material were
subsequently subjected to immunoprecipitation with pan-Trk antibodies. B, Immunoprecipitates were analyzed by blotting with
phosphotyrosine antibodies, antibodies to phosphorylated Trk, or pan-Trk antibodies. Untreated cells that were biotinylated
immediately after block (lane 1) or 3 hr after block (lane 2) were used as controls for basal levels.
Rajagopal et al. •Activation of Trk Receptors J. Neurosci., July 28, 2004 •24(30):6650 – 6658 • 6653

This procedure allowed us to measure the newly inserted
membrane proteins during a treatment period after the blocking
step. The level of receptor insertion was then determined by sub-
sequent SDS-PAGE and immunoblotting with pan-Trk antibod-
ies. The basal level of receptor insertion was determined by com-
paring cells that were blocked and then immediately biotinylated
(Fig. 3B, lane 1) with cells that were blocked and left in serum-free
media for 3 hr before biotinylation (lane 2). All conditions of
treatment were compared with cells that were left untreated for 3
hr (lane 2).
Treatment with NGF gave rise to a detectable increase in acti-
vated Trk receptors at the cell surface (Fig. 3B, lane 7). An in-
crease in the amount of biotinylated Trk was observed with in-
creasing periods of incubation with CGS 21680 (top panel).
Insertion of receptors can be seen as early as after 1 hr of treat-
ment (lane 4), and maximal insertion is detected at 3 hr of treat-
ment (lane 6). Notably, NGF did not induce significant Trk re-
ceptor insertion after 3 hr of treatment (Fig. 3B, compare lane 7
and lane 2).
To determine whether newly inserted Trk receptors induced
by CGS 21680 treatment are activated, we reblotted the top panel
of Figure 3Bwith a phosphotyrosine antibody (pY). Surprisingly,
none of the Trk receptors at the cell surface were tyrosine phos-
phorylated after treatment with CGS 21680. After NGF treat-
ment, however, surface receptors were robustly activated. These
results were confirmed by blotting with an antibody specific to
phosphotyrosine 490 site of Trk (pTrk).
These results indicate that the activation of Trk receptors by
CGS 21680 is restricted to an internal pool of receptors that do
not reach the cell membrane. To confirm that Trk receptors
transactivated by CGS 21680 are found inside the cell, we ana-
lyzed the nonbiotinylated lysate by phosphotyrosine blotting. In-
deed, activation of TrkA by CGS 21680 was detected in the inter-
nal pool of receptors after 2 hr (Fig. 3B, bottom panel), which is
consistent with previous measurements with adenosine and
PACAP (Lee et al., 2001, 2002b).
Immunostaining with phospho-specific TrkA antibody
Previous experiments in PC12 cells and sympathetic neurons in
compartmentalized cultures have provided evidence that Trk re-
ceptor signaling occurs in intracellular locations (Grimes et al.,
1997; Tsui-Pierchala and Ginty, 1999; Jullien et al., 2002; Ye et al.,
2003). To address the intracellular localization of transactivated
Trk receptors, we subjected PC12 cells to indirect immunofluo-
rescence to determine the intracellular location of Trk activation
by GPCR ligands.
To follow activated Trk receptors, we generated a new
phospho-specific TrkA antibody for this purpose. The antibody
was made against a phosphopeptide spanning the PLC-
␥
site at
Y785. Western blot analysis of the affinity-purified antibody
showed that it specifically recognized the activated TrkA receptor
and not the unstimulated receptor in PC12 cells (Fig. 4). Speci-
ficity for the phosphorylated form of TrkA was confirmed using
peptide competition assays. As shown in Figure 4, the phos-
phopeptide, and not the unphosphorylated peptide, effectively
blocked binding of the pTrkA antibody to immunoblots from
NGF-treated PC12 cell lysates. This antibody did not recognize
TrkB receptors in transfected and hippocampal cells or other
tyrosine phosphorylated proteins (data not shown).
To image TrkA receptors by immunofluorescent microscopy,
we serum-starved PC12 cells for 24 hr before treatment with
either CGS 21680 or 100 ng/ml NGF for 5 min. Cells were fixed,
permeabilized, and stained with the phospho-TrkA-specific an-
tibody generated against the C-terminal phosphotyrosine. Cells
were simultaneously costained with pan-Trk antibodies. As seen
in Figure 5, untreated PC12 cells display Trk localization that
spans the entire cell. Trk (red) was detected uniformly at the
plasma membrane as well as in numerous small-diameter intra-
cellular vesicles and in a perinuclear compartment; however, un-
treated cells display only a negligible amount of phospho-Trk
immunoreactivity (pTrkA) that is mostly limited to the small
intracellular vesicles. On the other hand, cells treated with NGF
for 5 min displayed punctate staining for phospho-Trk along the
plasma membrane but not within the cell, where there is an ex-
tensive amount of nonactivated Trk as shown by the pan-Trk
staining in red.
The staining pattern of phospho-Trk that is observed after
CGS 21680 treatment is quite distinct from that observed in
NGF-treated cells. As seen in Figure 5A(bottom panels),
phospho-Trk staining in cells treated with CGS 21680 for 3 hr was
concentrated to closely apposed large vesicles in the perinuclear
region of the cell. Staining was absent along the plasma mem-
brane, consistent with our findings that GPCR-activated Trk is
limited to an intracellular pool of receptors.
The staining pattern that we observed was reminiscent of
Golgi staining from PC12 cells (Kim and von Zastrow, 2003). To
confirm the Golgi localization, we analyzed PC12 cells treated
with CGS 21680 by confocal microscopy using anti-phospho-Trk
(green) and antibodies against GM130 or TGN38 (red), both of
which are well established markers of the Golgi apparatus (Luzio
et al., 1990; Nakamura et al., 1995). As shown, phospho-Trk par-
tially colocalizes with both of these Golgi markers (Fig. 5B). On
the other hand, cells stained with a marker for early endosomes,
EEA-1 (Fig. 5B), or for late endosomes, LAMP1, displayed much
less colocalization with phospho-Trk after GPCR stimulation
(data not shown). These results suggest that GPCR-activated Trk
is restricted to an intracellular region that is not associated with
the endocytic trafficking pathway and suggests that part of the
activated pool is detected close to or contiguous with the Golgi
apparatus.
Brefeldin A treatment
To verify that the Golgi is involved in transactivation by GPCRs,
we disrupted cells with brefeldin A, a fungal metabolite that is a
Figure 4. Specificity of phospho-TrkA antibodies. Affinity purification of polyclonal anti-
bodies raised against a short peptide in the C-terminal region of rat TrkA containing tyrosine
794,whichisphosphorylated in response to NGF (Loeb etal.,1994),was performed as described
in Materials and Methods. To assess specificity of purified antibodies, lysates (40
␮
g each well)
obtained from PC12 cells treated with 50 ng/ml NGF for 10 min were probed with affinity-
purified serum as well as purified sera that was preincubated with either a phosphorylated or
nonphosphorylated TrkA peptide competitor.
6654 •J. Neurosci., July 28, 2004 •24(30):6650 – 6658 Rajagopal et al. •Activation of Trk Receptors

potent inhibitor of Golgi vesicle fusion (Pelham, 1991). PC12
cells were treated with either 5
␮
g/ml brefeldin A or vehicle for 3.5
hr. During this period of time, cells were simultaneously treated
with 10 nMCGS 21680 or 100 ng/ml NGF, such that the treatment
period would coincide with the end of brefeldin A treatment.
Untreated cells were used for control. Cell lysates were prepared
and used for Trk immunoprecipitation and subsequent analysis
by SDS-PAGE and blotting with phosphotyrosine antibodies. As
shown in Figure 6 (top panel), brefeldin A pretreatment com-
pletely abolished activation of Trk by CGS 21680 at both time
points of treatment, whereas NGF-mediated Trk activation was
left unimpaired.
In addition, Akt activation was also blocked when cells were
pretreated with brefeldin, indicating that activation and down-
stream signaling of Trk mediated by GPCRs requires an intact
Golgi apparatus. Interestingly, TrkA glycosylation was impaired
by brefeldin as indicated by the pan-Trk blot shown in Figure 6 A.
The 110 kDa isoform of Trk accumulated when cells were treated
with brefeldin. These results imply that an intact Golgi apparatus
is a potential location for Trk transactivation and that the positive
phospho-Trk staining observed in cells (Fig. 3A) may have arisen
from this organelle.
To determine how brefeldin A affected TrkA subcellular local-
ization, we subjected PC12 cells to indirect immunofluorescence
for TrkA and pTrkA after treatment with brefeldin A. As depicted
in Figure 6B, brefeldin A abolished perinuclear TrkA immuno-
reactivity in cells treated with CGS 21680 but did not affect the
ability of NGF to activate TrkA at the plasma membrane. This
indicated that Trk localization at the plasma membrane was not
greatly disrupted by brefeldin A.
Effect of transactivation on primary neurons
PACAP displays pronounced effects on the survival of septal cho-
linergic neurons (Takei et al., 2000) as well as many other neuro-
nal cells, such as mesencephalic dopaminergic neurons (Takei et
al., 1998) and sensory neurons (Lioudyno et al., 1998). PACAP
also is capable of triggering transactivation of Trk receptors in
primary neurons in a time course identical to adenosine or CGS
21680 treatment (Lee et al., 2002b). To determine the distribu-
tion of transactivated TrkA receptors in primary neurons, we
established E18 basal forebrain cultures. It was established previ-
ously that TrkA receptors are expressed by a subset of these neu-
rons (Bernd et al., 1988). After treatment with PACAP, the local-
ization of Trk receptors and phospho-Trk receptors was followed
using pan-Trk antibodies and the phospho-Trk-specific anti-
body. Similar to what was observed in cultured PC12 cells, the
phosphorylated form of Trk resided predominantly in an intra-
cellular location in basal forebrain neurons, whereas Trk recep-
tors could be found throughout the cell body and processes (Fig.
7). The phospho-Trk staining was heavily concentrated in a pe-
rinuclear region of the cell body, as illustrated by the DAPI and
phospho-Trk distribution. This pattern of staining was inhibited
by both K-252a and brefeldin A (Fig. 7), confirming that the
observed immunoreactivity represents activated TrkA in a Golgi
compartment. The finding of activated Trk receptors in response
to PACAP treatment in basal forebrain neurons confirms that
transactivation occurs in an intracellular localization in primary
neurons.
Discussion
Cross talk between GPCR ligands and receptor tyrosine kinases
has been described as a means of conveying proliferative signals
in cells (Downward, 2003; Fischer et al., 2003). In contrast, trans-
activation of neurotrophic receptors can result in survival re-
sponses and neuronal differentiation. In this study, we have
shown that Trk receptors undergo activation in intracellular
membranes and not at the cell surface. Biotinylation and immu-
nofluorescence measurements indicate that transactivation
events displayed a delayed time course and absence of involve-
Figure 5. Localization of transactivated TrkA receptors. Subcellular localization of transac-
tivated TrkA in PC12 cells was examined by confocal microscopy. A, Cells were treated with 10
nMCGS 21680 for 3 hr or with 100 ng/ml NGF for 5 min and then double labeled for phospho-
TrkA (rabbit polyclonal pTrkA; FITC-labeled goat anti-rabbit) and pan-Trk (monoclonal Trk B3;
Cy3-goat anti-mouse). B, To further characterize the specific location of transactivation events,
double labeling of CGS 21680-treated cells was performed with antibodies directed against
EEA-1, GM130, or TGN-38 (red) and phospho-TrkA (green).
Rajagopal et al. •Activation of Trk Receptors J. Neurosci., July 28, 2004 •24(30):6650 – 6658 • 6655

ment of cell surface receptors. These features are in direct con-
trast to activation of mitogenic growth factor receptors (Daub et
al., 1997; Luttrell et al., 1999; Prenzel et al., 1999).
The location of signaling events representing cross talk be-
tween GPCR ligands and Trk receptor tyrosine kinases plays a
crucial role in the kinetics and mechanism of transactivation.
Although EGF receptor transactivation by GPCR ligands is rapid
and requires endocytosis and close proximity of EGF receptor
and GPCRs (Maudsley et al., 2000; Pierce et al., 2001), the time
course of Trk receptor transactivation is longer and involves in-
tracellular processing events. For example, processing of imma-
ture forms of Trk is involved in transactivation.
Several other aspects of GPCR-mediated transactivation of
Trk receptors distinguish it from other transactivation and neu-
rotrophin signaling events. A noticeable difference between
adenosine and NGF Trk is that the phosphorylation of Trk sub-
strates, such as the Shc adaptor proteins PLC-
␥
and PI3 kinase,
requires an extended period of time, ⬎1 hr of adenosine treat-
ment (Lee and Chao, 2001), in contrast to NGF treatment, which
requires ⬍1 min. Second, GPCR-mediated activation of Trk re-
sults in downstream signaling to phosphoinositide 3-kinase and
Akt but not MAPK, unlike neurotrophin signaling, which can
robustly activate both pathways. The specific activation of Akt by
transactivated Trk receptors provides a survival response in neu-
rons (Lee et al., 2001, 2002a). MAPK activation is the hallmark of
Figure 6. The Golgi apparatus is involved in Trk transactivation. A, PC12 cells were pre-
treated for 30 min with 5
␮
g/ml brefeldin A or vehicle and then treated with CGS 21680 or NGF
as indicated. In all cases of brefeldin A treatment, cells were exposed to the drug for a total
duration of 3.5 hr. Trk immunoprecipitates were then analyzed by blotting with phosphoty-
rosine or pan-Trk antibodies. Phosphorylation of Akt was examined by blotting cell lysates with
either phospho-Akt or anti-Akt antibodies for activated and total Akt levels. B, PC12 cells pre-
treated with brefeldin A or vehicle were treated with 10 nMCGS 21680 for 3 hr or 100 ng/ml NGF
for 5 min and then stained for pTrkA (green) or total Trk (red) and imaged by standard immu-
nofluorescent microscopy. Cell nuclei were visualized with DAPI.
Figure 7. Localization of transactivated TrkA in basal forebrain neurons. Indirect immuno-
fluorescence analysis was used to examine localization of transactivated TrkA in primary basal
forebrain neuronal cultures. Cultures were treated with PACAP38 as indicated and labeled with
phospho-TrkA (green) and pan-Trk (red) antibodies. Where indicated, K-252a (100 nM)or
brefeldin A (5
␮
g/ml) was used to pretreat cells before addition of PACAP38. Cell nuclei were
stained with DAPI (blue).
6656 •J. Neurosci., July 28, 2004 •24(30):6650 – 6658 Rajagopal et al. •Activation of Trk Receptors

many growth factor receptors, such as EGF and PDGF receptors,
that provide proliferative signals after transactivation (Luttrell et
al., 1999; Wetzker and Bohmer, 2003).
Transactivation by adenosine or PACAP does not involve the
production or release of newly synthesized or processed neuro-
trophins, as has been observed for the EGF receptor (Prenzel et
al., 1999). One mechanism for transactivation of the EGF recep-
tor involves the action of matrix metalloproteases. Cleavage of
membrane-bound proHB-EGF by metalloproteases results in the
release of EGF that binds and activates the EGF receptor (Prenzel
et al., 1999). Experiments performed in PC12 cells indicate that
GPCR ligands did not result in an increase in the expression of
NGF that might contribute to TrkA receptor activation (Fig. 1C)
(Lee et al., 2002a). Also, binding experiments show that adeno-
sine and PACAP do not displace NGF from binding to TrkA
receptors, indicating that the effects of these GPCR ligands occur
through an intracellular pathway.
What is the relevance of these transactivation events? During
retrograde transport, complexes of NGF with TrkA in clathrin-
coated vesicles and endosomes have been envisioned, giving rise
to the model of NGF and TrkA as components of a retrograde
signal (Riccio et al., 1997; Senger and Campenot, 1997; Ginty and
Segal, 2002; Grimes and Miettinen, 2003). A number of signaling
molecules have been found to be associated with the TrkA recep-
tor during retrograde transport (Delcroix et al., 2003). This sug-
gests that signaling by trophic factors persists after internalization
of their receptors in intracellular locations. Implicit in this model
is that internalization of NGF is necessary for eliciting survival
signals at the cell body. The evidence comes from the activation of
the CREB transcription factor (Riccio et al., 1997) and Erk5
(Watson et al., 2001) by retrograde signals at distal terminals.
Retrograde signaling events, however, may also be transmit-
ted in the absence of NGF internalization and transport. Survival
of sympathetic neurons can be mediated by transported Trk re-
ceptors in the cell body after stimulation at nerve terminals by
NGF linked to beads (MacInnis and Campenot, 2002). This raises
the possibility that mechanisms other than endocytosed NGF
may propagate signals from distal axons to TrkA receptors at the
cell body (Campenot and MacInnis, 2004). A number of argu-
ments have been raised both for and against the hypothesis that
retrograde signaling can occur in the absence of NGF uptake
(Chao 2003b; Ye et al., 2003), and several diverse models have
been proposed to account for the discrepancies in retrograde
signaling mechanisms (Ginty and Segal, 2002; Heerssen and Se-
gal, 2002; Miller and Kaplan, 2003).
One common feature of retrograde transport of neurotrophin
signaling is the ability of internalized Trk receptors to signal in a
productive manner. Activation of tyrosine kinase receptors from
GPCR transactivation illustrates an alternative way in which sig-
nals may be transmitted by intracellular receptors in the absence
of ligand. This is exemplified by the findings in this study in
which adenosine is capable of activating Trk receptors in intra-
cellular membranes and not at the cell surface. Activation of in-
tracellular Trk receptors may depend on Src kinase activity, re-
cruitment of adaptor proteins, or an increase in the local
concentration of Trk, which could lead to constitutive activation.
The results demonstrate that internal Trk receptors may be found
in discrete organelles (Grimes et al., 1997; Jullien et al., 2002;
Delcroix et al., 2003), where they are fully active and promote Akt
survival signals (Lee et al. 2001, 2002a,b). Also, it has been estab-
lished that TrkA receptors found in sympathetic neurons may be
activated by engagement of the Ret tyrosine kinase by GDNF in
the absence of NGF (Tsui-Pierchala et al., 2002). Hence, these
observations solidify the growing realization that Trk receptors
function not only at the cell membrane but provide potent signals
from discrete intracellular locations without neurotrophin bind-
ing. Trafficking of neurotrophin receptors in different mem-
brane compartments must play a decisive role in regulating var-
ious fundamental signaling pathways in neurons.
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