From the Differentiation and Cell Cycle Group,
Laboratoire de Biologie Moleculaire et Cellulaire, UMR 5665 CNRS, Ecole
Normale Supérieure de Lyon and ¶ LaboRetro, INSERM U412,
Ecole Normale Supérieure de Lyon, 46 Allée d'Italie,
69364 Lyon Cedex 07, France and
Howard Hughes Medical Institute,
University of California, San Francisco, California 94143
Received for publication, March 12, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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A chimera of the nerve growth factor (NGF)
receptor, TrkA, and green fluorescent protein (GFP) was engineered by
expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP
becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK
activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared
as GFP chimerae in order to identify which part of the receptor
intracellular domain is involved in its trafficking. Immunofluorescence
observations show that TrkA-GFP is found mainly in cell surface
membrane ruffles and in endosomes. Biochemical analysis indicated that
the cytoplasmic domain of TrkA is not necessary for correct maturation
and cell surface translocation of the receptor. An antibody against the
extracellular domain of TrkA (RTA) was used as ligand to stimulate
internalization and phosphorylation of TrkA. Co-localization studies
with anti-phosphorylated TrkA antibodies support a role for such
complexes in the propagation of signaling from the cell surface,
resulting in the activation of TrkA in areas of the endosome devoid of
receptor-ligand complexes. Confocal time-lapse analysis reveals that
the TrkA-GFP chimera shows highly dynamic trafficking between
the cell surface and internal locations. TrkA-positive vesicles were
estimated to move 0.46 ± 0.09 µm/s anterograde and 0.48 ± 0.07 µm/s retrograde. This approach and the fidelity of the
biochemical properties of the TrkA-GFP demonstrate that real-time
visualization of trafficking of tyrosine kinase receptors in the
presence or absence of the ligand is feasible.
Growth factors of the neurotrophin family are involved in the
survival and differentiation of certain neurons in the peripheral nervous system (1). Signaling by these proteins is believed to be
mediated by binding to the neurotrophin receptor p75NTR
and/or to one member of the Trk family. p75NTR binds to
each neurotrophin with similar affinity. By contrast, Trk receptors
exhibit differential affinity toward neurotrophins: TrkA binds
preferentially to NGF,1 TrkB
to BDNF (brain-derived neurotrophic
factor) and NT4-5, and TrkC to NT-3 (2, 3).
Trk family members are tyrosine kinase receptors. Signaling by the
NGF-TrkA complex is the best characterized. Upon NGF binding to the
receptor, the tyrosine kinase domain of TrkA is activated, resulting in
the autophosphorylation of two major tyrosines in the intracellular
domain of the receptor (4). These phosphotyrosines are docking sites
for several adaptor proteins that are involved in signal transduction
pathways leading to the activation of
MAPK, phosphatidylinositol 3-kinase, and
phospholipase C- In the case of neurons, additional specific spatial constraints for
neurotrophin receptor signaling must be considered. The neurotrophin
hypothesis postulates that the source of NGF for neurons is restricted
to the target field of innervation (10). Most often, cell bodies of
neurons are located far from their target cells. Thus, neurotrophin is
available only at the growth cone of the neuron. NGF-induced survival
requires transmission of a signal from the axon tip to the cell body
where transcriptional modifications are induced. Numerous studies
indicate that the transmission of this NGF survival effect in neurons
of the peripheral nervous system is supported by retrograde transport
of NGF-TrkA complexes from the neurite tip to the cell body (11-14).
This points to the importance of intracellular trafficking of
neurotrophin receptors in the implementation of a response to their
ligands. Indeed, NGF neuronal responsiveness is highly dependent upon
NGF receptor targeting to where the growth factor is provided, namely the axon tip. Once complexed to its ligand, activated receptor is
transported back to the cell body. Some of the intracellular trafficking steps underlying this process have been elucidated. Retrograde transport is initiated by internalization via the coated pit
pathway (11, 15-17). After this step, TrkA is believed to undergo a
microtubule-supported vesicular retrograde transport to the cell body
(10, 12).
Internalization by coated pits appears to require trafficking motifs in
the intracellular domain of internalized proteins. Two major
trafficking motifs have been identified (18, 19). These motifs are
short stretches of amino acids corresponding to either the dileucine
type (two adjacent leucines or a leucine and an isoleucine) or to the
tyrosine-based type (YXX The aim of the present study was to gain insight into TrkA trafficking
steps prior to and during exposure to NGF and the resulting differentiation process. The intracellular domain of TrkA contains numerous potential trafficking motifs of the tyrosine-based and dileucine types. Involvement of these motifs in TrkA trafficking has
been investigated using receptor chimerae designed with TrkA progressively truncated from the carboxyl terminus in phase with GFP. Data are presented indicating that fusion of GFP to full-length TrkA does not modify the expected functioning of the receptor. Cell
surface translocation together with NGF-induced internalization of
receptor chimera have also been analyzed to ascertain which parts of
the TrkA cytoplasmic domain are involved in trafficking of the
receptor. Confocal time-lapse analysis of TrkA-GFP trafficking during
NGF-induced differentiation was undertaken to visualize and evaluate
the kinetics of the actual trafficking of the receptor. A description
of TrkA localization at different phases of differentiation is
presented together with the major fluxes occurring between cellular
compartments during this process.
Reagents--
Sulfo-NHS-biotin and streptavidin-agarose were
purchased from Pierce. Anti-TrkA extracellular domain (RTA) was
prepared as described previously (22). Anti-transferrin receptor
(HTR68-4) was kindly provided by I. Trowbridge (The Salk
Institute). Anti-phospho-490 Trk (E6) and anti-phosphotyrosine
(PY99) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFP
(JL-8) was from Clontech. Anti-active MAPK
(anti-ERK 1/2 pAb, catalog No. V1141) was from Promega.
R-phycoerythrin-conjugated Affinipure F(ab')2 fragment donkey anti-rabbit IgG, rhodamine-conjugated anti-mouse IgG,
cyanine-5-conjugated anti-mouse antibody, and phalloidin-rhodamine were
from Jackson Immunoresearch laboratories, Inc. NGF from mouse
submaxillary glands was from Quality Controlled Biologicals. Protein
A-Sepharose 4 fast flow was from Amersham Biosciences. K252-a was from
Calbiochem, and Polybrene was from Sigma.
Constructs--
DC971 retroviral vector encoding rat neuronal
TrkA has been described (22). An EGFP insert was prepared by PCR
reaction using GFP-ol5' and GFP-ol3' primer, allowing the introduction of ClaI site in 3' of EGFP stop codon and StuI
and NsiI site in 5' of EGFP start codon
(GFP-ol5', 5'-ATAGGCCTTG ATGCATATGGTGAGCA AGGGCGAGG-3';
GFP-ol3', 5'-CCATCGATTT ATCTAGATCC GGTGGATC-3'). The Cell Surface Biotinylation and Immunoprecipitation--
Intact
cells were incubated for 45 min in ice-cold PBS containing 0.5 mg/ml
sulfo-NHS-biotin. Cells were then washed four times in PBS supplemented
with 2 mM lysine to remove unbound reactive biotin. After
cell surface biotinylation, proteins were extracted using ice-cold
lysis buffer (20 mM Tris-HCl, pH 8, 137 mM
NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40, 20 µM leupeptin, 1 mM sodium vanadate, 1 mM Pefabloc, 0.15 units/ml aprotinin, 1 mM Flow Cytometric Analysis--
Cells were collected at 37 °C
in PBS supplemented with 0.1 mM CaCl2, 1 mM MgCl2, and 5 mM EDTA. After
centrifugation cells were resuspended in culture medium and subjected
to 50 ng/ml NGF treatment at 37 °C on a rotating wheel. Cells were
then washed twice in ice-cold PBS and used as "live intact cells"
for immunolabeling. All of the subsequent labeling steps were performed
on ice. Cells were washed twice in blocking buffer (PBS with 0.5%
bovine serum albumin and 0.02% sodium azide) and incubated for 30 min
in the same buffer containing an antibody directed against RTA. After two washes in blocking buffer, cells were exposed to
phycoerythrin-labeled secondary antibody for 30 min and washed three
more times. Cells were then analyzed using a FACScan flow cytometer (BD
Biosciences) equipped with an argon ion laser tuned to 488 nm. Emission
fluorescence was measured with a 585-42-nm dichroic filter
(phycoerythrin fluorescence) or a 530-30 bandpass filter (EGFP
fluorescence). Data acquisition and analysis were performed with
CellQuestTM software (BD Biosciences). Internalization
efficiency was calculated from the amplitude of the shift in the
geometric mean of the phycoerythrin signal between treated and
untreated GFP-positive cells.
Cell Culture--
PC12 cells, PC12 nnr5 cells (obtained from Dr.
P. Barker; Montreal Neurological Institute), and PC12 6-24 cells
over-expressing human neuronal TrkA (provided by Dr. D. Martin-Zanca,
Instituto de Microbiologia Bioquemica, Universida de Salamanca, Spain)
were grown as described previously (23).
Transfection and Infection--
Transfections were performed
using the calcium phosphate precipitation (23). Helper cell lines
(TE-FLY (53), generously provided by Dr. F.-L. Cosset, Lyon, France)
were transfected with retroviral vector encoding TrkA-GFP constructs
and selected with G418 for 3 weeks. Stably transfected helper cells, in
the exponential phase of growth, were incubated for 12-16 h in
complete PC12 cell medium. Vectors were prepared by filtering the
medium on a 45-µm filter. PC12 nnr5 or HeLa cells were pretreated
for1 h with 12 µg/ml Polybrene and then incubated for 8 h in the
presence of filtered vector. After three rounds of infection, cells
were selected for neomycin resistance.
Confocal Microscopy--
PC12 cells were transfected with
TrkA-GFP vector. At 24 h post-transfection, cells were spread on
collagen poly-L-lysine-coated coverslips (24). At 48 h
post-transfection, cells were fixed for 10 min in PBS, 3.7%
formaldehyde and permeabilized for 1 min in PBS, 0.5% Triton X-100.
After being washed with PBS, cells were blocked with PBS, 0.5% bovine
serum albumin for 30 min and incubated with primary antibody for 30 min. Cells were washed three times in PBS and incubated for an
additional 30 min with secondary antibody. After washing, cells were
mounted in Moviol. Scanning fluorescence images were acquired using the
MRC1000 confocal laser unit (Bio-Rad) coupled to a Zeiss Axioplan
microscope equipped with a Zeiss X40, C-APO, 1.3 NA oil immersion objective.
Confocal Time-lapse Microscopy--
Confocal time-lapse
fluorescence images were acquired using the MRC1000 confocal laser unit
(Bio-Rad) coupled to a Zeiss Axiovert microscope equipped with a Zeiss
X40, C-APO, 1.3 NA oil-immersion objective. Cells were maintained at
37 °C, and analyses were performed in a CO2-independent
medium (Invitrogen) to avoid medium acidification in a
CO2-free atmosphere. GFP bleaching experiments were
performed using the photobleaching module of LSM 510 software (Zeiss)
with a 488-nm argon laser set to 100% power. In vesicle tracking
experiments, pinholes of the confocal unit were set so that the optical
slice obtained encompassed the entire thickness of the structure
studied. Unless specified, pinhole settings were ~1
airy/unit.
Fusion of GFP to TrkA Carboxyl Terminus and Truncated
Mutants--
A TrkA-GFP chimera, named Trafficking Properties of TrkA-GFP--
The expression and
cellular localization of the various constructs were initially
characterized in the absence of NGF. Particular attention was given to
the analysis of cell surface targeting of TrkA because this event is
crucial in determining cellular responsiveness to NGF.
Intracellular Localization--
Confocal microscopy observations
indicate that Synthesis, Maturation, and Cell Surface Translocation--
In PC12
cells, synthesis and cell surface translocation of TrkA involve the
sequential production of two forms of the receptor. The receptor is
synthesized initially in the rough endoplasmic reticulum as a 110-kDa
form, which contains a 30-kDa N-linked sugar moiety (25,
26). During the time course of receptor targeting to the cell surface,
this gp110TrkA precursor is matured to a 140-kDa form,
gp140TrkA (Fig.
4A) The additional apparent
30-kDa molecular mass shift between mature and precursor forms
of TrkA is believed to be the result of modification in the
carbohydrate moiety of the receptor (54). Synthesis and maturation of
TrkA-GFP chimerae were evaluated by Western blot (Fig. 4A).
All of the constructs tested, with the exception of the soluble
Cell surface targeting of TrkA-GFP constructs was observed by both flow
cytometric analysis and confocal immunofluorescence microscopy (Figs. 2
and 4C). Cell surface localization of TrkA-GFP chimerae was
observed at the molecular level by cell surface biotinylation. In PC12
cells, the mature form of wild type TrkA is the only form translocated
to the cell surface (Fig. 4B, left panel). As
shown in Fig. 4B, NGF-induced Down-regulation--
Intracellular trafficking of
TrkA-GFP in response to NGF was analyzed. NGF binding to wild type TrkA
induces internalization and degradation of the receptor. Flow
cytometric analysis of NGF-induced internalization of TrkA-GFP chimerae
from the cell surface was performed in transiently transfected PC12
nnr5 cells. Fig. 4C shows that the chimeric receptors can be
separated into three categories with regard to NGF-induced
internalization. The first category, which includes
Following internalization of the NGF-TrkA complex, the receptor is
targeted to the lysosome for degradation (11, 54). As shown in Fig.
4D, NGF-induced degradation of transiently transfected receptor is restricted to the Signaling Properties of the Chimerae--
We tested the ability of
TrkA-GFP chimerae to promote signaling events upon binding of NGF. In
PC12 cells, NGF treatment induces autophosphorylation of endogenous
TrkA (Fig. 5A). PC12 nnr5
cells, a variant cell line devoid of TrkA receptor, do not respond to NGF (27, 28). Phosphorylation of TrkA-GFP chimerae was tested in PC12
nnr5 cells stably expressing TrkA-GFP constructs. As shown in Fig.
2A, NGF induces tyrosine phosphorylation of the mature form
of
The kinetics of NGF-induced receptor autophosphorylation was also
investigated. Fig. 5A shows that in PC12 cells,
phosphorylation of endogenous TrkA in response to NGF is prolonged as
compared with that observed for epidermal growth factor receptor
in response to epidermal growth factor. The duration of NGF-induced
phosphorylation of
The addition of NGF to PC12 cells promotes neuronal differentiation.
PC12 nnr5 cells, devoid of endogenous TrkA, have lost this ability to
differentiate in response to NGF (27, 28). Introduction of Real-time Monitoring of Receptor Trafficking--
The trafficking
of TrkA was followed using confocal time-lapse analysis of PC12 cells
stably expressing Long-term Recording--
At the beginning of the NGF treatment,
PC12 cells exhibit their undifferentiated round shape. In this cell,
TrkA is found mainly at two locations. The bulk of the receptor appears
to be concentrated in an internal perinuclear location. A part of the TrkA cellular pool is also found at or close to the plasma membrane. Receptors present at this site are accumulated in discrete locations of
the cellular periphery, most often in the form of highly mobile cellular protrusions (Fig. 6, 18 h).
After 21 h of NGF treatment, cells begin to enlarge and to extend
neurites. During this process of neurite outgrowth, a large number of
vesicles containing TrkA are transported to and from the plasma
membrane. It can be noted that this flow of TrkA-containing structures to the cell surface at the beginning of neurite outgrowth is
not a uniform process. Indeed TrkA-positive vesicles seem to be
preferentially addressed to the plasma membrane locations from which
neurite extension is attempted (Fig. 6, inset,
34 h). On the other hand, TrkA-positive membrane ruffles appear
concentrated at the tip of potential neurites (inset,
34 h).
Neurite outgrowth appears to proceed by trial and error. As exemplified
in the picture compiled from images taken from 47 h to
47 h 20 min (Fig. 6), some secondary neurites are retracted (arrow), whereas others appear to modify their direction of
extension (asterisks). After 52 h of NGF treatment,
neurites have retracted secondary outgrowths and extension is reduced.
At this step of differentiation, TrkA localization and trafficking
exhibit the following properties: First, TrkA is located in numerous
internal fixed locations, moving from the initial perinuclear
accumulation (Fig. 6, 52 h, red asterisk) to
immobile structures in the proximal part of the neurite (Fig. 6,
52 h, green asterisks) and of the growth cone
(Fig. 8D). Second, between these two latter locations, retro- and anterograde movements of vesicles (Fig. 8C) are
detected. Third, the plasma membrane location of TrkA appears to be
concentrated essentially in membrane ruffles at the growth cone and
around the cell body (arrows in Fig. 6, 52 h).
After this stage of differentiation, the culture was treated with the
TrkA inhibitor K252-a (0.5 µM). Treatment with this drug
induces a dramatic change in cell morphology, and TrkA trafficking. K252-a induces rapid flattening of the cell body. This modification of
cell shape is accompanied by a modification of membrane movement at the
cell surface as shown by TrkA concentration in large yet discrete and
highly mobile patches at the plasma membrane. Finally, TrkA undergoes a
massive internal redistribution as assessed by anterograde vesicular
transport from the proximal neurite "stations" to the perinuclear
area (Fig. 7, inset at
72 h). After 7 h of drug treatment, a complete loss of
peripheral TrkA-GFP is observed. Prolonged exposure to the drug led to
apoptotic cell death (Fig. 7, 82 h 30 min).
Short-term Recording--
Long-term confocal time-lapse
observations highlight the importance of vesicle shuttling between
membrane ruffles and endosomal locations. Vesicle tracking studies were
undertaken in HeLa cells. These cells are highly adherent and, as a
consequence, are thinner than PC12 cells. These morphological
properties of HeLa cells make them more suitable for analysis of
vesicle tracking. Fig. 8A
shows the overlay of three images recorded with a 1-s interval in HeLa
cells stably expressing
Recording of TrkA-GFP labeling during the time course of NGF-induced
PC12 differentiation shows retrograde and anterograde vesicle movements
in neurites. Recording over short time intervals in the cellular
context of differentiated PC12 cells was also undertaken. The picture
in Fig. 8C corresponds to the recording of a neurite
emerging from a cell body, located to the left of the
picture. This neurite divides in two parts in the middle of the
picture. Nine seconds of tracking, at 1-s intervals, of a vesicle
undergoing anterograde transport in this neurite is presented. The
distance between the successive locations of the vesicle
(red, green, and blue positions) is
different in the three panels presented. In the first 3 s,
the apparent vesicular velocity is about 0.26 µm/s. whereas it
increases to about 0.4 µm/s and to about 1.3 µm/s in the next
6 s. This illustrates that linear vesicle velocity does not appear
constant in the short-term recording experiments. Vesicle movement
often appears irregular, with apparent pauses in the transport process.
Moreover, despite the fact that global direction of the transport is
maintained, it would appear that vesicles are undergoing rapid changes
in transport orientation. The mean of vesicular TrkA-GFP anterograde
transport was recorded at 0.46 ± 0.09 µm/s (as evaluated from
measurement of 12 vesicles from four independent experiments).
TrkA-positive membrane ruffle mobility was evaluated in the long-term
time-lapse recording experiment (Fig. 6). The images from the 72 to
72 h 10 min recording indicate that selected patches of plasma
membrane, rich in TrkA, are moved around the cell surface in a
concerted manner. It is conceivable that in the context of a
differentiated cell, retrograde transport of the receptor may occur via
such membrane ruffles. This hypothesis is supported by the results
presented in Fig. 8D. Observation of the growth cone reveals
that membrane ruffles move around the surface. Asterisks show the successive positions of a ruffle for which the velocity of
displacement, in this particular experiment, is ~0.3 µm/s.
Trafficking and Activation--
Confocal time-lapse analysis
indicated numerous bidirectional movements of TrkA-containing vesicles
between the cell surface and the perinuclear
endosomal compartment (Figs.
6-8). Experiments were designed
to evaluate the activation state of TrkA receptors involved
in these movements. Activation status, together with ligand-binding
status of TrkA, was evaluated using confocal microscopy (Fig. 9; and
see "Materials and Methods" for details). Expression of chimeric
TrkA-EGFP receptor in PC12 nnr5 cells together with the use of RTA
antibody as an NGF agonist (22) allow simultaneous monitoring of three
different pools of cellular TrkA in a single cell (Fig. 9A)
as follows. (i) "Total TrkA" is reflected in GFP fluorescence, which corresponds to all of the cellular localizations of
TrkA. (ii) "Cell Surface TrkA at t = 0" is
identified by incubation of cells at 4 °C for 30 min in the presence
of RTA, followed by permeabilization and labeling of cells with
rhodamine-conjugated anti-rabbit antibody. This allows the detection of
TrkA-RTA complexes, which are initially at the cell surface. Following
the shift to 37 °C, it is possible to monitor the fate of the
receptors that have moved from the cell surface to intracellular
locations during the incubation time. (iii) "Activated
TrkA" corresponds to immunolabeling of permeabilized cells using
anti-phospho-TrkA specific antibody.
As shown in Fig. 9A, incubation of cells in the presence of
RTA at 4 °C does not allow receptor internalization, because all RTA-TrkA complexes are located at the cell surface. Moreover, incubation at 4 °C does not permit TrkA activation, because no phospho-TrkA is detected at this temperature. By contrast, incubation of cells for 20 min at 37 °C in the presence of RTA led to the accumulation of TrkA-antibody complex in discrete structures within cells. Intracellular RTA-TrkA complexes are found both at the cell
periphery (arrows) and in the aforementioned perinuclear location (arrowheads). RTA-TrkA complexes located at
this latter location also appear to correspond to phosphorylated
receptor. It can be noted that activated receptor is detected in a
perinuclear area that is larger than the perinuclear location of
internalized RTA-TrkA complexes. Fig. 9B illustrates that
internalized TrkA is targeted to endosomes, because the perinuclear
RTA-TrkA complexes appear to co-localize with the endosomal marker,
Tnf-R.
This study validates the use of TrkA-GFP chimerae as a tool to
follow intracellular trafficking of TrkA. Data presented herein indicate that wt TrkA-GFP ( The intracellular domain of TrkA contains a tyrosine kinase, which is
flanked by short juxtamembrane and carboxyl-terminal domains, each
including a single tyrosine known to be involved in signal transduction
(4, 31). Fusion of the 26-kDa GFP protein to the carboxyl terminus of
the 40-kDa intracellular domain of TrkA does not perturb the cellular
and biochemical properties of the receptor as compared with
wt TrkA. The Numerous pathways for signal transduction have been described for NGF
signaling in PC12 cells, many of which are initiated from TrkA (30, 33,
34). Clearly, short of performing a catalogue of each, it is not
possible to ensure that all are fully functional in the TrkA-GFP
chimera. However, the PC12 nnr5 model, devoid of functional TrkA,
allows the evaluation of the global cellular response as reflected by
the morphological differentiation accompanied by the anti-mitogenic
response to NGF, which are observed subsequent to expression of the
The presence of the GFP on the carboxyl terminus of TrkA does not
modify the turnover of this protein. Furthermore, both Western blot
analysis and pulse-chase experiments failed to detect any preferential
cleavage of GFP from the chimera. Thus, GFP appears to be a valuable
marker to follow expression and fate of TrkA in cells.
In the absence of NGF, The internal accumulation of neurotrophin receptors has been described
previously for both TrkA (24, 40) and TrkB (41). The observed
accumulation of TrkA-GFP in an endosomal compartment in the absence of
NGF treatment is consistent with these observations. Bidirectional
vesicular receptor transport between endosomal structures and the cell
surface indicates a highly dynamic equilibrium within these two
receptor locations. Calculation of the average velocity of anterograde
and retrograde vesicle transport (respectively 0.46 ± 0.09 µm/s
and 0.48 ± 0.07 µm/s) suggests that the vesicular transport
mechanism is of the fast axonal type (42). These values are in
agreement with the in vivo retrograde transport velocity of
NGF (from 0.7 to 0.8 µm/s (43, 44)).
Confocal time-lapse analysis highlights the fact that a significant
proportion of TrkA is located intracellularly at the level of
endosomes, even in the absence of growth factor, in agreement with
published results concerning the expression of endogenous TrkA (24).
The availability of antibodies specific to the extracellular domain of
rat TrkA, with a well characterized NGF agonist function, allows
monitoring of ligand-receptor complex movement from the cell surface
(22). This analysis shows that internalized TrkA is indeed targeted to
the endosome, as suggested by several groups (16, 17, 45). The studies
presented herein take these observations even further and clearly
illustrate that the internalized, activated TrkA is located mainly in
endosomes (54). A striking finding of this analysis is the fact that
activated TrkA is present in areas of the endosome devoid of the
internalized receptor-ligand complexes. This suggests that internalized
receptor may serve to propagate activation from the cell surface to the
fraction of TrkA accumulated in the endosome, thereby serving to
amplify the intracellular signal even though these receptors are not
occupied with ligand. This concept has been discussed by numerous
investigators in the context of neurotrophic factor signaling from the
nerve ending to the cell body and is at the heart of lively debate (14, 46-48, 52). It is expected that molecular analysis of
XFP-labeled neurotrophic factor receptors, coupled with the
multi-label time-lapse and confocal microscopy approaches described
here, could facilitate the clarification of this and other questions
concerning receptor trafficking and signal transduction.
In the context of differentiated PC12 cells, internal distribution of
the receptor appears not to be restricted to the perinuclear endosomal
location. For example, TrkA-GFP-positive structures have been observed
in the neurite tip. Hendry and colleagues (49, 50) have recently
proposed the existence of a sorting compartment located in the growth
cone responsible for the targeting of internalized NGF to retrograde
transport. The observed internal accumulation of TrkA-GFP inside the
neurite tip could represent this sorting station. This hypothesis is
strengthened by the observed bidirectional vesicular transport between
both the neurite tip cell surface and internal structures and between
these latter locations and the cell body.
TrkA targeting into NGF-dependent and -independent
trafficking pathways appears to rely on different regions of the
receptor cytoplasmic domain. Maturation and cell surface targeting of
TrkA are apparently not regulated via motifs within
cytoplasmic domain. Anchoring of the receptor in the
membrane appears to be the only requirement for correct maturation of
the receptor ectodomain. As mentioned above, at steady state, in the
presence or absence of NGF, TrkA-GFP is accumulated in the endosome,
yielding a strong fluorescence. It would appear therefore that
activation of the tyrosine kinase is not required for TrkA to traffic
to this endosomal location. However, the amount of TrkA-GFP present in
the endosome is significantly decreased when amino acids 788-673 are
deleted. Again, NGF has no effect on this distribution. These forms of TrkA are kinase-dead because of the absence of the activation loop in
the kinase domain. By contrast, inactivation of kinase activity via a
single substitution in the TrkA ATP binding site ( NGF-induced trafficking steps include receptor internalization followed
by receptor degradation. Tyrosine kinase activity of TrkA does not
appear to be necessary for NGF-induced internalization. Receptor
internalization is actually enhanced for the kinase-dead truncated
constructs The reagents and observations presented herein should offer a powerful
means for investigating the relation between trafficking of and
signaling via TrkA as well as other receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(5). By contrast, p75NTR does
not present any known catalytic activity in its cytoplasmic domain.
Recent studies have reported the identification of several proteins
that bind to the cytoplasmic domain of p75NTR (6-9).
Depending on the interactor considered, binding of a given effector to
p75NTR has been shown to be either enhanced or decreased by
NGF binding. The precise roles of these proteins in the mediation of
NGF signaling remain to be elucidated.
or NPXY type, where
X is any amino acid, and
is a hydrophobic amino acid). In addition to their involvement in the internalization processes, these trafficking motifs appear to mediate the recruitment of a receptor along numerous trafficking pathways ranging from targeting to specific cell surface domains to accumulation in defined
intracellular sites (19-21). Signal-mediated protein targeting is
believed to be supported by recognition of these motifs by members of
the adaptor complex family.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
chimera is the result of EGFP ligation between the ClaI and
StuI sites of DC971. This ligation create an in-frame fusion between the EGFP amino terminus and a truncated TrkA carboxyl terminus
missing 4 amino acids of TrkA carboxyl terminus. Full-length TrkA-EGFP
fusion (
0) was reconstituted by ligating prehybridized End1 and End2
complementary oligonucleotides between the NsiI and
StuI sites of
1 (End1, 5'-CCTTGGCACA
GGCGCCACCG AGTTACCTGG ACGTTCTGGG CATGCA-3'; End2,
5'-TGCCCAGAAC GTCCAGGTAA CTCGGTGGCG CCTGTGCCAA GG-3'). Other truncated
constructs were produced using the kit Erase-a-Base (Promega).
ExoIII digestion was performed on an
NsiI/StuI-digested
0 plasmid, and all
subsequent steps were performed following user guide instructions.
Kinase-dead
0 (
0-KD) corresponds to a
0
construct carrying a point mutation, K547N, which inactivates
the kinase activity of TrkA. This construct was obtained by mutating
pLNCX-
0 using the Transformer mutagenesis kit
(Clontech) with the following primers:
Select-ol, 5'-CATCATTGGAA AACGCGTTTC GGGGCGAAAA CTC-3';
K547N-ol, 5'-GCTGGTGGCT GTCAACGCAC TGAAGGAGAC
ATC-3'. All of the constructs were verified by sequencing (ABI Prism
kit, Applied Biosystems, Foster City, CA). pcDNA3-
ECTO was
generously provided by Dr. A. Pandiella (Instituto de Microbiologia Bioquimica, Salamanca, Spain).
ECTO insert was extracted from pcDNA3 vector and subcloned into the pIRES-2 EGFP vector from Clontech.
-glycero-phosphate, 6 mM sodium-fluoride). The extract
was clarified by centrifugation at 12,000 × g for 10 min. Cleared lysate was then subjected to precipitation with
streptavidin-agarose beads to obtain the cell surface fraction. In
TrkA-GFP immunoprecipitation experiments, cleared lysates were
precipitated using the JL-8 antibody directed against EGFP.
Immunocomplexes were collected using protein A-Sepharose beads and
eluted by boiling for 10 min in sample buffer. Proteins were then
subjected to SDS-PAGE and Western blot analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0, was engineered by the
addition of EGFP to the carboxyl terminus of rat neuronal TrkA (Fig.
1A). Deletion mutants of
TrkA-GFP with sequential truncation of the cytoplasmic domain of the
receptor were constructed to test the potential involvement of the
dileucine and tyrosine-based motifs in trafficking of TrkA (Fig.
1A). Fig. 1B indicates the terminal amino acid of
each TrkA deletion mutant, the potential trafficking motif that was
removed with respect to the previous construct, the theoretical kinase
activity based on published information on structure activity, and
finally, the tyrosine involved in the known signaling pathways still
present in the construct. In the following presentation, TrkA-GFP will
be used to refer to all of the truncated chimeric receptors, whereas
n will refer to the fusion of GFP with a specific truncated TrkA
mutant missing n potential trafficking motifs (Fig.
1A).
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Fig. 1.
TrkA-EGFP chimerae and associated mutant.
A, schematic representation of TrkA-GFP constructs.
Extra, extracellular domain; TM, transmembrane
domain; Intra, intracellular domain. L and
Y are, respectively, dileucine and tyrosine-based
trafficking motifs. The number attributed to each deletion
refers to the number of potential trafficking motifs deleted.
B, characteristics of truncated TrkA-GFP mutants. All
mutants have the signal peptide at the amino terminus. For each mutant,
the first amino acid of the remaining TrkA carboxyl terminus is
specified. Potential trafficking motifs eliminated by each deletion are
also indicated. The tyrosines Tyr794 and Tyr
499 of rat TrkA are believed to support the NGF-induced
recruitment of signaling molecules to the receptor. The presence of
these tyrosines in the deletion mutants is also indicated. The kinase
activity of the receptor is deduced from the presence of an
intact kinase domain in which the phosphorylation loop includes
Tyr764, Tyr 765, and Tyr 777 or
from published data ( ECTO (55)).
0 is targeted essentially to two cellular locations at
steady state in the absence of NGF.
0 accumulates mainly at an
internal perinuclear location and is also found at the cellular
periphery in the form of discrete bright punctate structures
(arrows in Fig. 2,
D, G, and J, respectively). Double
labeling experiments with the endoplasmic reticulum marker calreticulin
indicate that the relatively faint diffuse intracellular TrkA labeling
corresponds to receptors located in the endoplasmic reticulum
(ER) (arrows in Fig. 2, A-C). The intracellular
perinuclear compact labeling of
0 may be attributed, at least in
part, to an endosomal location, as shown by co-localization with the
endosomal marker Tnf-R (Fig. 2, D-F). Cell surface TrkA has
been detected by immunolabeling unpermeabilized cells with an antibody
directed against the TrkA extracellular domain. The cell surface TrkA
labeling co-localizes with peripheral
0-positive dots (Fig. 2,
G-I). Thus, it appears that the
0 peripheral labeling reflects the localization of cell surface TrkA. These discrete
0
cell surface structures also appear to co-localize with peripheral actin, a well known marker of membrane ruffles (Fig. 2,
J-L). The same kind of confocal microscopy analysis was
undertaken with TrkA-GFP deletion constructs. Characteristic patterns
of distribution are presented in Fig. 3.
0 and kinase-dead
0 (
0-KD) exhibit strong endosomal
expression,
2 more diffuse cellular endoplasmic reticulum
distribution, and
8 very weak endosomal location with strong
membrane expression. The results of these experiments are summarized in Table I. This study shows
that
0,
1, and
ECT appear to accumulate in the perinuclear
endosomal compartment at much higher levels than other constructs. It
appears that all of the truncated TrkA constructs, with the exception
of
TM and
ECT, are targeted to the cell surface membrane ruffles
of PC12 cells. A biochemical study was undertaken to analyze the
maturation and cell surface targeting of TrkA constructs at a molecular
level.
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Fig. 2.
Cellular localization of TrkA-GFP
( 0) chimera. Intracellular
localization of TrkA was investigated in transiently transfected PC12
cells by confocal microscopy. Double labeling experiments were
undertaken to analyze putative co-localization between TrkA-GFP (
0,
green) and several intracellular compartments (in
red). The endoplasmic reticulum and endosomes were
visualized with anti-calreticulin (B) and anti-transferrin
receptor (E) antibodies, respectively. Cell surface TrkA was
revealed by immunolabeling of intact nonpermeabilized cells with an
antibody directed against RTA (in H). Actin was stained with
rhodamine-conjugated phalloidin (in K). Bars
represent 10 µm.
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Fig. 3.
Cellular localization of TrkA-GFP
chimera. Intracellular localization of TrkA was investigated in
transiently transfected PC12 cells by confocal microscopy. Double
labeling experiments were undertaken to analyze putative
co-localization between TrkA-GFP construct (GFP fluorescence shown in
green) and endosome (Tnf-R in red).
Bars represent 10 µm.
Summary of co-localization analysis via confocal microscopy of the
various TrkA-GFP constructs and markers of intracellular compartments
, barely detectable; +, low expression; +++, highest expression;
ND, not determined.
TM
mutant, exhibited two different molecular mass forms of the receptor.
The molecular mass of the smallest forms appears to be about 30 kDa
higher than the calculated molecular mass of the polypeptide backbone.
This difference is similar to that observed for the endogenous TrkA of
PC12 cells, suggesting that these extra 30 kDa correspond to the
N-linked oligosaccharides normally added to the receptor.
The molecular mass difference between the two forms of a chimeric
receptor also appears to be close to 30 kDa, which is also comparable
with the difference seen between gp110TrkA and
gp140TrkA. These data suggest that synthesis and maturation
of the TrkA-GFP chimera are similar to that observed with the wild type
receptor. Pulse-chase analysis confirmed the precursor to mature
protein relationship between the two forms of each chimeric receptor
(not shown). It can be noted further from this experiment and from Western blot analysis that no preferential cleavage of GFP from the
chimeric receptor was observed. These observations confirm the fidelity
of GFP as a tag to follow the expression and fate of the TrkA receptor.
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Fig. 4.
Intracellular trafficking of TrkA-GFP fusion
proteins. A, processing: Western blot analysis of total
protein samples from PC12 nnr5 cells transiently transfected with
TrkA-GFP constructs ( 0-
TM) or
wt TrkA (TrkA). The left and
right panels are probed with RTA antibody directed against
TrkA extracellular domain and JL-8 antibody directed against GFP,
respectively. B, cell surface targeting: Western blot
analysis of total (T) versus cell surface
(S) protein samples from PC12 nnr5 cells stably expressing
TrkA-GFP constructs (see "Materials and Methods" for details). The
left and right panels show blots probed with
antibody directed against TrkA extracellular domain and GFP,
respectively. C, NGF-induced internalization: PC12 nnr5
cells were transiently transfected with TrkA-GFP vector or
wt TrkA along with a GFP expression vector. 48 h
post-transfection, cells were collected and treated or not with 50 ng/ml NGF for 15 min. After being labeled with antibody against TrkA
extracellular domain and phycoerythrin-conjugated secondary antibody,
cells were used for flow cytometric analysis. Internalization
efficiency was calculated on GFP-positive cells as described under
"Materials and Methods." D, NGF-induced degradation:
Western blot analysis of total protein sample from PC12 nnr5 cells
transiently transfected with TrkA-GFP or wt TrkA constructs
and treated or not with 50 ng/ml NGF for 5 h. Membranes were
probed with antibody directed against the extracellular domain of
TrkA.
0,
1,
2,
4, and
8 are also
correctly targeted to the plasma membrane because only the mature forms
of the chimeric receptors are labeled by biotinylation of cell surface
proteins. Thus, neither exogenous receptor expression nor the addition
of the GFP tag appear to affect maturation and cell surface targeting of TrkA in PC12 cells.
0 and
1,
demonstrated internalization properties similar to those of wild type
TrkA.
2,
3,
4, and
6 showed slightly enhanced NGF-induced
internalization as compared with the first group. Finally, the most
deleted construct (
8) has greatly reduced internalization
capability. This observation suggests that there are at least two kinds
of trafficking determinants in the cytoplasmic domain of TrkA,
which regulate NGF-induced internalization. A negative regulator of
this phenomenon is lost in the
2-
6 mutants, whereas a positive
regulator of NGF-induced internalization is lost in
8.
0 and
1 chimeric receptors and to
wtTrkA. It suggests that the process of NGF-induced TrkA
degradation requires trafficking information in the carboxyl terminus
of the receptor, which is missing in the
2-
8 constructs.
0 and
1 chimerae. As expected, the
2 construct, which is
lacking crucial amino acids in the catalytic domain of the kinase, is
not phosphorylated in response to the growth factor. This observation
suggests that the NGF-induced tyrosine phosphorylation of
0 and
1
is indeed the result of autophosphorylation of the receptor rather than
a transphosphorylation of the chimera by an NGF-activated endogenous
kinase.
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Fig. 5.
TrkA-GFP chimerae signaling. A,
NGF-induced TrkA-GFP phosphorylation. PC12 nnr5 cells stably expressing
TrkA-GFP constructs were treated for different periods of time with 50 ng/ml NGF or EGF. Cellular proteins were then extracted and
immunoprecipitated with an antibody directed against phosphotyrosine
(IP: P-Y). Western blot (WB) analysis of total
cellular extracts (C) or immunoprecipitated samples was then
carried out with an antibody directed against TrkA extracellular domain
or epidermal growth factor receptor (EGF-R)
intracellular domain. B, cells were treated as described in
A and subjected to Western blot analysis. Membranes were
probed with an antibody directed against the phosphorylated active form
of MAPK. C, PC12 nnr5 cells stably expressing
0,
1, or
2 chimerae were treated or not for 2 days (2d) with 50 ng/ml NGF. Photomicrographs of cells were then acquired with
phase contrast microscopy.
0 and
1 also appears to be longer than that of
epidermal growth factor receptor. Downstream of TrkA phosphorylation,
activation of MAPK plays a key role in the response to NGF (29).
Activation of these kinases is achieved by phosphorylation. Fig.
4B shows that NGF treatment of nnr5 cells stably expressing
0 and
1, but not
2, induces phosphorylation and thus
activation of MAPK.
0 and
1 into PC12 nnr5 cells restores NGF-induced differentiation, as
shown in Fig. 4C. By contrast, the more deleted mutant,
2, does not induce differentiation. These data indicate that the
fusion of GFP to the carboxyl terminus of TrkA does not alter the
signaling properties of the receptor. Moreover they show that deletion
of the 11 carboxyl-terminal amino acids of the receptor, which include
the phosphotyrosine involved in phospholipase C-
recruitment,
does not affect NGF-induced morphological differentiation of PC12 cells
(30).
0. The experiments presented in Fig. 5 represent a
subset of data extracted from a single cell recording over a period of
5 days. Two representations are used in this figure. Black and white
pictures represent recording at a single time point. Three-color
pictures are compiled from the overlaying of three sequential
recordings; the red channel is attributed to the first
recording, the green channel to the second recording, and
blue channel to the last recording. Thus, GFP labeling of
structures that are immobile over the three recordings appears
white (overlay of red, green, and
blue channels), whereas successive positions of a moving
structure appear as respectively colored marks.
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Fig. 6.
Long-term confocal time-lapse analysis of
TrkA-GFP trafficking. PC12 cells expressing 0 chimera were
grown on collagen-polylysine-coated plastic for 24 h. Live cells
were then observed under a confocal microscope. Cells were treated with
50 ng/ml NGF, and pictures were recorded every 10 min, other than the
period between 34 and 34 h 10 min, during which images were
collected every 30 s. For explanations of colored
versus black and white representations see the text.
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Fig. 7.
Long-term confocal time-lapse analysis of
TrkA-GFP trafficking (continued from Fig. 6).
PC12 cells expressing the 0 chimera were grown on
collagen-polylysine-coated plastic for 24 h. Live cells were then
observed under a confocal microscope. Cells were treated with 50 ng/ml
NGF, and pictures were recorded every 10 min for the first 52 h of
differentiation (see Fig. 5). After 52 h of NGF treatment, the
tyrosine kinase inhibitor K252-a was added to a concentration of 0.5 mM. Images were then acquired every 5 min.
0. As seen in Fig. 8A,
TrkA localization in this cell resembles the endosomal and cell surface
location observed in PC12 cells. Immobilization of TrkA in these
structures is revealed by the white labeling (Fig.
8A). By contrast, vesicles in transit between these
two compartments undergo rapid movement (presence of colored vesicles).
The multiplicity of vesicles going to and from the endosome does not
allow single vesicle tracking. To overcome this problem, photobleaching
of the endosome was performed (Fig. 8B). In this manner,
fluorescence of
0-positive vesicles emerging from endosomes is
eliminated. Under such conditions, the tracking of vesicles moving from
the cell periphery to the endosome is possible. The representative
velocity of this endocytic, retrograde-like movement is 0.48 ± 0.07 µm/s (as evaluated from the measurement of 20 vesicles from six
independent experiments).
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Fig. 8.
Short interframe confocal time-lapse analysis
of TrkA-GFP trafficking. A and B, plasma membrane
to endosome vesicle shuttling. HeLa cells expressing 0 were cultured
as described under "Materials and Methods." Confocal time-lapse
analysis was undertaken with a 1-s interframe. The same cell is
presented in A and B. In B, the
internal endosomal
0 fluorescence was photobleached, and NGF, 50 ng/ml, was added 5 min prior to the recording. C, vesicular
transport in neurites. PC12nnr5 cells expressing
0 were treated with
NGF for 2 days at 50 ng/ml. Confocal time-lapse analysis was undertaken
with a 1-s interframe. The cell body is located 200 µm to the
left of the picture. The observed neurite divides in two
parts. D, filopodial movement at the neurite tip.
PC12nnr5 cells expressing
0 were treated for 2 days with 50 ng/ml
NGF. Confocal time-lapse analysis of a neurite tip was undertaken with
a 7.5-s interframe. The cell body is located ~150 µm below the
picture.
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Fig. 9.
Trafficking and activation of TrkA-GFP
chimerae. Internalization of the TrkA-GFP chimerae was provoked by
exposure of PC12 cells to RTA as described under "Materials and
Methods." After fixation and permeabilization of the cell, RTA
localization was evaluated by exposure to secondary antibody labeled
with rhodamine at the indicated times. Thus, rhodamine fluorescence
indicates the location of TrkA-RTA complexes that were at the cell
surface at the beginning of the experiment (Cell Surface TrkA at
t = 0 min). GFP fluorescence reflects all cellular
TrkA (Total TrkA). Simultaneous immunolabeling with
anti-phospho-TrkA antibody (A) or anti-transferrin antibody
(B) allows detection of "Activated TrkA"
or the "Endosome," respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0) fulfills most of
the trafficking and signaling properties of wt TrkA.
Analysis of receptor location in the absence of NGF shows that this
transmembrane receptor is accumulated in specific areas of the
plasma membrane and in endosomes within the cell.
Monitoring of chimeric receptor trafficking in live cells sheds new
light on the biosynthetic and endocytic trafficking pathways followed
by TrkA. Observation of truncated TrkA-GFP constructs permitted
identification of those parts of the receptor intracellular domain that
are necessary for different steps of receptor trafficking. Receptor
maturation and cell surface translocation are not affected by the
removal of the entire TrkA cytoplasmic domain. Intracellular distribution of the receptor is modified by deletion of amino acids
788-793. All chimerae encompassing this deletion exhibit a reduction
in the amount of receptor that is retained intracellularly in the
absence or presence of NGF. Down-regulation events induced by NGF also
appear to be modulated by sequences within the intracellular domain.
0 chimera is activated in response to NGF,
leading to MAPK activation and differentiation comparable with that
observed with the wt receptor. The activation pattern of
TrkA is not modified by the addition of GFP, because
0 exhibits
activation kinetics similar to that of TrkA. This latter point is
believed crucial for the cellular response to tyrosine kinase
activation in PC12 cells. Indeed proliferative versus
differentiative responses induced, respectively, by EGF and NGF are
correlated with transient versus sustained signaling by
their respective tyrosine kinase receptor (32). Data presented herein
indicate that
0 also shows a sustained activation similar to
that of wt TrkA.
0 and
1 chimera.
0 trafficking appears similar to that of wt TrkA. The
transit of
0 through the biosynthetic pathway allows correct
receptor maturation and cell surface translocation. Moreover,
NGF-induced down-regulation of
0, namely internalization and
degradation, mimic that observed for wt TrkA. Taken
together, these data suggest that TrkA-GFP mimics wt TrkA behavior.
0 is distributed between actin-rich cell
surface membrane ruffles and internal endosomal locations. Confocal
time-lapse analysis allows the monitoring of trafficking of the
receptor between these structures. During the process of differentiation, TrkA-positive membrane ruffles appear to be
concentrated at the growth cone and around the cell body. It has been
shown that actin is involved in the retrograde transport of NGF (35). In light of these studies, this requirement may rely on two kinds of
retrograde transport processes. First, actin may participate in an
internalization-dependent retrograde transport of TrkA. The
observation that NGF treatment causes membrane protrusion (35) and the
present observation that TrkA is specifically enriched in these plasma
membrane areas raise the possibility that TrkA internalization could be
achieved by macropinocytosis. Indeed, this internalization process, as
contrasted with coated pit-mediated endocytosis, does not require
membrane invagination but membrane protrusion (36, 37). Alternatively,
actin may be required in a retrograde transport mechanism of TrkA,
which involves membrane ruffle transport (38). The localization of TrkA
in the membrane ruffle at the growth cone is also consistent with its
involvement in neurite tip oriented growth. Indeed, ruffle movements at
the growth cone are believed to "taste" extracellular medium (39). Local TrkA signaling in ruffles could support this function of the receptor.
0-KD chimera) does
not affect endosomal localization. Together, these results would
suggest that the difference in localization between these deletion
mutants and the full-length construct is due to the absence of a
trafficking motif such as YRKF rather than the lack of kinase activity.
2,
3,
4, and
6. This observation suggests that
the tyrosine kinase activity domain of the receptor may be a negative
regulator of NGF-induced internalization. It also suggests that the
mechanism of internalization does not require conformational modification of the receptor cytoplasmic domain, believed to be achieved by tyrosine kinase activation. Thus, NGF-induced
internalization may be triggered by NGF-mediated TrkA dimerization.
With regard to this hypothesis, adaptor protein binding to trafficking
signals in the coated pit pathway has been shown to be enhanced by
dimerization of the internalized protein (51). Further deletion of
amino acids 565-477 in the cytoplasmic domain of TrkA (
8 mutant)
greatly reduces NGF-induced internalization. Thus, a positive regulator of NGF-induced internalization, such as the putative trafficking motifs
531ECYNLL or 496NPQY, is probably present in
this portion of the TrkA cytoplasmic domain. A tempting hypothesis to
link these two types of internalization regulators is the competitive
binding of signaling and trafficking effectors on a single motif of the
TrkA cytoplasmic domain. Activation of the tyrosine kinase by NGF may
inhibit internalization of the receptor by favoring the recruitment of
signaling rather than trafficking effectors on the phosphorylated
496NPQY motif. With regard to this hypothesis, abolishing
receptor tyrosine kinase activity would invert the
signaling/trafficking binding balance on the motif, and deletion of the
motif would abolish the binding of both effector types.
![]() |
ACKNOWLEDGEMENT |
---|
We are extremely grateful to Dr. Pierre Colas of our group (Lyon, France) for advice in molecular biology.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Ligue Nationale Contre le Cancer, the committee of the Ligue from the Rhône, the Rhône-Alpes Region, the Association for Research against Cancer (ARC), and the Fondation de France.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.
§ Supported by grants from the Ministère de l'Enseignement Supérieur et de la Recherche and fellowships from the Association for Research against Cancer.
** An investigator of the Howard Hughes Medical Institute; work in his laboratory was also supported by United States Public Health Service Grant NS 16033.
To whom correspondence should be addressed. Tel.:
334-72-72-81-96; Fax: 334-72-72-80-80; E-mail:
bbrudkin@ens-lyon.fr.
Published, JBC Papers in Press, November 15, 2002, DOI 10.1074/jbc.M202401200
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ABBREVIATIONS |
---|
The abbreviations used are:
NGF, nerve growth
factor;
MAPK, mitogen-activated protein kinase;
GFP, green fluorescent
protein;
EGFP, enhanced green fluorescent protein;
PBS, phosphate-buffered saline;
RTA, antibody against extracellular
domain of TrkA;
wt, wild type;
TM, transmembrane domain deletion
construct;
ECT, extracellular domain deletion construct.
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