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Biomedical Sciences Graduate Program, and Department of Medicine, Division of Endocrinology and Metabolism, School of Medicine, University of California, San Diego, La Jolla, California 92093
Address all correspondence and requests for reprints to: Jerrold M. Olefsky, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: jolefsky{at}ucsd.edu
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Introduction |
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NGF endocytosis has been characterized in neuronal cells and in the sympathetic neuronal-like PC12 cell line (23). It has been established that NGF internalization in neuronal cells occurs via retrograde transport of the growth factor from the nerve terminals to the cell body (24). Clearly, fibroblasts and other nonneuronal cells differ morphologically from neuronal cells due to the absence of axons. Retrograde transport, therefore, plays no role in NGF uptake in these cells. Since endocrine and immune functions of NGF have been established, we pursued the characterization of NGF internalization in a nonneuronal cell line, i.e. transfected fibroblasts stably expressing TrkA. Many neuronal cell lines coexpress Trk and p75. Internalization mediated through p75 in the absence of Trk has been examined in glial cell lines (21), in sympathetic neurons (24), and in mutant PC12 (PC12nnr) cell lines (7, 22), however, little information is published on NGF internalization and subsequent trafficking and processing events in a p75 minus cell line. Thus, our studies in CHO/TrkA cells address NGF internalization, processing, and trafficking in a nonneuronal context and in the absence of p75.
Endocytosis, a general and distinctive property of all eukaryotic cells, refers to the uptake of macromolecular material into a membrane-limited organelle in a living cell. The process of receptor-mediated endocytosis is comprised of noncovalent binding of ligand to cell-surface receptors, which induces the clustering of these complexes in clathrin-coated pits (25, 26, 27). The ligand-receptor complexes are internalized as coated pits invaginate and pinch off to form small intracellular coated vesicles. Clathrin is rapidly removed from the vesicles giving rise to endosomes (28, 29). In these structures, acidification takes place, followed by a sorting step which establishes the destination of receptor and ligand. Receptors and ligands can be recycled to the cell surface (30, 31), degraded by lysosomal enzymes, or sequestered in an intracellular compartment. We designate the intact ligand recycling process retroendocytosis (32, 33, 34, 35), sometimes referred to as recycling, exocytosis, diacytosis, transcytosis, or re-externalization in the literature.
TrkA endocytosis has not been well characterized, but recent evidence suggests that TrkA receptors are internalized by a clathrin-mediated mechanism (19). Our findings describe these events of 125I-NGF internalization, processing, and retroendocytosis mediated through the TrkA receptor in the absence of p75. We find that 125I-NGF is capable of efficient internalization and retroendocytosis, and propose that its ability to stay associated with the TrkA receptor under acidic conditions diverts it from lysosomal degradation.
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Materials and Methods |
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Antibodies
Anti-Trk antiserum 203 was generously provided by D. R.
Kaplan, NCI-Frederick Cancer Research and Development Center
(Frederick, MD). Polyclonal anti-Trk antibody 1087 was kindly donated
by W. C. Mobley (UCSF, San Francisco, CA). PY20 antibody was
bought from Transduction Laboratories, (Lexington, KY). Polyclonal
antihuman p75 antibody was purchased from Promega (Madison, WI).
Cell culture
CHO/TrkA cells were maintained in HAMS/F12 medium supplemented
with 10% FCS, 1 x GlutaMAX-1, 50 µg/ml gentamicin sulfate, 500
µg/ml G418 at 5% CO2. PC12 cells were routinely grown in
DME/high glucose supplemented with 10% horse serum, 5% FCS, 1 x
GlutaMAX-1, 50 µg/ml gentamicin sulfate at 7.5% CO2.
Generation of stable CHO/TrkA cells
pDM69 was digested with EcoRI to excise the human
TrkA cDNA insert. The ends of the insert were blunted and ligated into
the polylinker region of the EcoRV-digested CLDN vector. CHO
cells were transfected with this plasmid via the lipofectamine method
followed by G418 selection (500 µg/ml). Clonal cell lines were
established and individual clones were screened for TrkA expression by
125I-NGF binding.
125I-NGF equilibrium binding studies
1 x 105 CHO/TrkA cells per well were plated
into six-well plates 2 days before binding study, such that on the day
of the experiment, 4 x 105 cells/well were present.
After rinsing twice with ice-cold PBS, 900 µl KRP-HEPES binding
buffer including 1 ng/ml final (1 x 105 cpm)
125I-NGF were added per well, immediately followed by
addition of 100 µl of 10 x cold NGF of varying final
concentrations ranging from 1–1000 ng/ml. After overnight incubations
at 4 C, unbound ligand was removed by three PBS washes, cells were
lysed in 0.4 N NaOH, and cpms were determined in
-counter. Curve fitting and data analysis were performed with LIGAND
software version 4.1 (NIH) (36).
p75 Western blotting
Fifty micrograms of PC12 and CHO/TrkA protein were subjected to
7.5% SDS-PAGE. After transfer onto Immobilon, the membranes were
incubated for 1 h at RT in TBS/0.1% Tween20 (TBST) containing 1%
BSA, followed by a 2-h incubation with anti-p75 antibody (1:1000) in
TBST. After several washes with TBST, a 30-min incubation with goat
antirabbit IgG-HRP (1:1000) was performed at RT. Following washing with
TBST, enhanced chemiluminescence (ECL) was performed.
Western blotting with anti-Trk antibody 1087
CHO and CHO/TrkA cells plated in 60 mm plates were lysed in 200
µl lysis buffer (30 mM Tris, pH 7.5, 150 mM
NaCl, 1% TX-100, 0.5% deoxycholate, 10 mM EDTA, 0.1%
SDS, 1 mM PMSF, 800 KIU/ml aprotinin, 1 µM
leupeptin, 1 mM sodium orthovanadate, 160 mM
sodium fluoride). After centrifugation at 14,000 rpm, protein
concentrations were determined with the supernatants and 50 µg of
protein were subjected to 7.5% SDS-PAGE. After transfer of proteins
onto Immobilon, the membrane was incubated in TBST/2% BSA for 1 h
at RT followed by incubation with polyclonal anti-Trk antibody 1087
(1:2000) for 2 h in TBST/2% BSA at RT. After several washes in
TBST, goat antirabbit IgG-HRP (1:2000) was added for 30 min followed by
detection with ECL.
Immunoprecipitation with anti-Trk antiserum 203
CHO/TrkA cells were plated in 60 mm dishes and starved the
following day for 48 h in HAMS/F12/0.1% BSA. Cells were treated
for 5 min with or without 100 ng/ml NGF, immediately washed with
ice-cold PBS and lysed in 750 µl lysis buffer. After centrifugation
at 14,000 rpm, 4 µl anti-Trk antisera 203 were added to each
supernatant followed by rotation for 2 h at 4 C. Immunocomplexes
were precipitated with protein A sepharose beads. Beads were washed 4
times, boiled in 100 µl 1 x Laemmli’s buffer and 40 µl were
subjected to 7.5% SDS-PAGE, followed by Western blotting with
antiphosphotyrosine antibody PY20 (1:1000 in TBST/2.5% BSA). The same
membrane was stripped for 10 min in 0.5 M NaCl/0.5
M acetic acid at RT. After blocking in TBST/2.5% BSA, the
membrane was reprobed with polyclonal anti-Trk antibody 1087 (1:2000)
to verify equal protein loading.
125I-NGF internalization
CHO/TrkA cells were plated at 50% confluency the day before the
experiment in six-well plates. Cells were rinsed twice with ice-cold
PBS and once with KRP-HEPES binding buffer (pH 7.5). Cells were
incubated at 37 C in the presence of 1 ng/ml 125I-NGF in
binding buffer for various times, followed by aspiration of unbound
ligand and three washes with 2 ml ice-cold binding buffer. One
milliliter acidic binding buffer (pH 3.5) was added per well, and cells
were transferred to 4 C for 10 min to remove surface-bound ligand. This
acid wash plus one additional 1 ml wash were transferred to a
borosilicate culture tube. Cells were washed 3 more times with 2 ml
acidic binding buffer and lysed with 1 ml 0.4 N NaOH for 30
min. Lysates and one additional 1 ml wash were transferred to separate
tube. Acid-extractable and nonextractable cpms were measured in
-counter.
The method for determining the endocytic rate constant of occupied receptors was previously described (25, 26, 32). Briefly, internalized and surface radioactivity was measured as described above at 1-min intervals for 6 min. Surface values were integrated over time using the trapezoidal rule, and values were plotted against internalized cpms. The slope of the plot at any point is equal to the specific internalization rate of occupied receptors (Ke) at that time. Linear regression was performed using Statview, Abacus Concepts, Inc. (Berkeley, CA).
Internalized 125I-NGF degradation
Cells were treated for various times with 1 ng/ml
125I-NGF at 37 C. Acid-extractable 125I-NGF was
collected and counted, and acid-resistant 125I-NGF was
subjected to 7.5% TCA precipitation. Cells were lysed in 1 ml 0.4 N
NaOH/1%BSA for 15 min, and 500 µl of lysate were incubated with 500
µl 15% TCA on ice for 30 min. Samples were centrifuged at 14,000 rpm
and supernatants plus one 7.5% TCA wash were transferred to tube. Cpms
of pellet and supernatants were determined in a -counter. In our
hands, this method is consistently 70–80% efficient, as determined by
the ability of intact 125I-NGF to precipitate. The TCA
precipitation method has been used previously to monitor
125I-NGF degradation (21, 37). Treatment with 0.4 N NaOH
does not contribute to 125I-NGF degradation.
Retroendocytosis
CHO/TrkA cells were plated as described above 1–2 days before
the experiment. Cells were washed twice with ice-cold PBS and once with
binding buffer (pH 7.5). One milliliter of 3 ng/ml 125I-NGF
in binding buffer was added per well, and cells were incubated for 20
min at 37 C. Unbound ligand was immediately aspirated and surface-bound
ligand was removed by acid wash as described above. Cells were washed
two more times with acidic binding buffer, twice with PBS, and once
with binding buffer (pH 7.5). 1 ml of 37 C binding buffer was added per
well, and cells were transferred to 37 C for various times. At the
appropriate time, 500 µl media were transferred to an Eppendorf tube
containing 500 µl 15% TCA. TCA precipitation procedure was followed
as above. The cells were washed 3 times with PBS and lysed, and counted
as above.
Tris-Tricine gel electrophoresis of TCA precipitated
125I-NGF
TCA pellets from parallel 10- and 30-min media time-points in
the retroendocytosis experiment above were washed with ice-cold
acetone, and the pellet was solubilized in 50 µl 4 x
Tris-Tricine sample buffer (0.1 M Tris-HCl, pH 6.8, 24%
glycerol, 1% SDS, 2% ß-mercaptoethanol, 0.02% Coomassie G-250).
After boiling for 10 min and centrifugation at 14,000 rpm, the samples
were subjected to 16.5% Tris-Tricine gel electorphoresis at 100 V
using Bio-Rad’s Tris-Tricine Ready Gels for peptides and small
proteins. For molecular weight controls, 0.1 µCi of
125I-NGF, 7.5% TCA-precipitated 125I-NGF (0.1
µCi before TCA-precipitation), and Bio-Rad’s peptide and small
protein molecular weight marker were also included. The gel was placed
in 40% methanol/10% acetic acid fixative solution for 30 min. The
molecular weight lane was cut off from the rest of the gel and stained
for 1 h in 0.025% Coomassie Blue G-250 for 1 h and destained
in 10% acetic acid for 3 x 15-min washes. The rest of the gel
was wrapped in plastic and placed on film for 50 days and
developed.
pH binding studies
CHO/TrkA cells were incubated with 1 ng/ml 125I-NGF
in KRP-HEPES binding buffer, pH 6–8, at 4 C overnight in the presence
and absence of 330 ng/ml unlabeled NGF to determine specific
binding.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and Scatchard
analysis (Fig. 1B) performed with the LIGAND program yielded a
Kd of 4 x 10-10 M and
approximately 2.5 x 105 binding sites per cell. The
one binding site model was the preferential fit, which is consistent
with the fact that CHO cells do not express p75 (M. Chao, personal
communication). The absence of p75 in these clones was verified by
Western blotting experiments with an antihuman p75 antibody raised
against the highly conserved extracellular domain of p75. p75
expression is clearly detectable in PC12 cells, but no immunoreactivity
was observed even when 100 µg of cell lysates were used (Fig. 2). It is possible, however, that a high
number of low affinity binding sites do exist on these cells, as
suggested by the curvilinear Scatchard plot. The two site model
predicts Kd values of 3 x 10-10 and
4 x 10-8 M, with approximately 2 x
105 high- and 2 x 106 low-affinity
binding sites. This is consistent with published observations in
transfected NIH3T3 expressing Trk receptors (18, 38), where Scatchard
analysis also points to two binding sites, despite the absence of p75
in these cells. These binding studies clearly reveal that CHO/TrkA
cells express high affinity 125I-NGF binding sites.
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). The fainter
approximately 110-kDa band, which is also immunoreactive with the
anti-Trk antibody, is most likely the high-mannose precursor to mature
TrkA (19, 39). To assess TrkA function, we performed
immunoprecipitation experiments with protein obtained from
NGF-stimulated CHO/TrkA cells using anti-Trk antiserum 203 and analyzed
the tyrosine phosphorylation state of the immunoprecipitated protein.
Western blot analysis with antiphosphotyrosine antibody PY20 indicates
that a 140-kDa tyrosine phosphorylated protein is clearly detectable in
cells stimulated for 5 min with NGF but not in the basal state,
demonstrating TrkA’s ability to autophosphorylate in these cells (Fig. 3B, left panel). TrkA is maximally tyrosine phosphorylated
between 5 and 10 min, and even at 30 min continues to be phosphorylated
above basal levels (data not shown). To verify that similar amounts of
protein were immunoprecipitated with anti-trk antiserum 203 in the
basal and NGF stimulated state, the membrane was stripped and reprobed
with anti-trk antibody 1087. Clearly, equal amounts of TrkA are present
(Fig. 3B, right panel). The 25- and 50-kDa bands represent
the rabbit IgG light and heavy chains, respectively, as polyclonal
antibodies were employed for immunoprecipitation and Western blotting.
In addition, Western blot analysis of protein obtained from NGF
stimulated cells with the antiphosphotyrosine antibody PY20 revealed an
increase in the tyrosine phosphorylation states of several cellular
proteins, including a 140-kDa protein, presumably TrkA, as early as 1
min following NGF treatment (Fig. 3C), clearly demonstrating that TrkA
is capable of mediating further downstream tyrosine phosphorylation
events. Together, these ligand binding and phosphorylation studies
demonstrate that CHO/TrkA cells express functional TrkA receptors
capable of mediating autophosphorylation and tyrosine phosphorylation
of downstream signaling proteins.
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). We performed
kinetic studies, according to the method of Wiley and Cunningham (40, 41), to obtain a value for the endocytic rate constant
(Ke), the cellular constant that defines the probability of
an occupied receptor (i.e. TrkA) being internalized in 1 min
at 37 C. By integrating the amount of surface bound
125I-NGF (acid-released 125I-NGF) at early time
points and plotting each value vs. the amount of
internalized 125I-NGF at the same time-point, the
Ke is derived from the slope of the plot (Fig. 4B). The
Ke for the 125I-NGF-Trk complex is 0.098;
i.e. there is a 9.8% probability that an occupied Trk
receptor will internalize in 1 min at 37 C.
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shows the time-course
of the various components of cell associated radioactivity. The cells
were incubated with 125I-NGF and at each time point acid
extractable (cell surface) and nonextractable (internalized) cpms were
determined. The intracellular 125I-NGF was further
subdivided into degraded and intact components. As can be seen, during
the early part of the time course, most of the cell associated
radioactivity was surface bound, but at approximately 30 min, surface
binding of 125I-NGF reaches steady state while
internalization progressively continues. At 120 min 75% is
internalized and 25% is surface-bound, and of the internalized
125I-NGF 95% is intact and 5% degraded.
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). TCA-precipitable (intact) ligand is
already detectable 5 min after reincubation (i.e. 25 min
after the onset of internalization), whereas TCA-soluble (degraded)
125I-NGF does not appear until 10 min after reincubation.
After 90 min, approximately 60% of released ligand is
TCA-precipitable, indicating that 125I-NGF is
preferentially sorted into a recycling, rather than lysosomal
degradation pathway.
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, the molecular weight of the bands in lanes 3 and 4
(30- and 10-min time-points, respectively) comigrate with the 13-kDa
bands obtained from intact tracer 125I-NGF (lane 2) and
TCA-precipitated intact tracer 125I-NGF (lane 1). NGF
usually exists as a dimer of 26 kDa; however, the reducing conditions
in this experiment yield NGF monomers. No bands of molecular weight
less than 13,000 were visible, demonstrating that the TCA precipitation
method is valid for separating intact from degraded NGF. Together,
these studies demonstrate that NGF is capable of retroendocytosis and
that the TCA precipitation method is valid for separating intact from
degraded 125I-NGF.
The effect of pH on 125I-NGF binding
As the endosome undergoes maturation, the vesicular milieu becomes
increasingly acidic resulting in dissociation of ligand-receptor
complexes. Because 125I-NGF preferentially undergoes
retroendocytosis, rather than entering a degradation pathway, we
hypothesized that 125I-NGF stays bound to its receptor
despite the decreases in pH, and thereby returns to the cell-surface by
continuing association with a recycling receptor. To assess this idea,
we analyzed the effect of changes in pH on 125I-NGF binding
to whole cells. Although 125I-NGF binding decreases in
response to a fall in pH, this effect is much less than for other
peptide hormone receptor complexes (6), and even at pH 6, 40% of
maximal binding remained (Fig. 7). This
finding suggests a possible mechanism underlying the efficient NGF
recycling observed in these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings corroborate Jing et al.’s observation in
NIH3T3 cells that TrkA can mediate efficient and rapid NGF
internalization, in the absence of p75. Because we have previously
performed the same experiments with insulin and IGF-I in fibroblasts
(6), we are able to compare them to our findings in this study with
NGF. The endocytic rate constants for IGF-I, NGF, and insulin are 0.07,
0.1, and 0.2 min-1, respectively, indicating that
125I-NGF-bound TrkA’s probability of internalizing within
1 min at 37 C is approximately half that of insulin’s. Although these
studies do not address the mechanism underlying this observation,
various possibilities that aren’t mutually exclusive exist. The
binding affinities of the ligands for their receptors may differ,
permitting a higher affinity ligand to internalize more rapidly.
However, all these ligands bind their respective tyrosine kinase
receptors with affinities in the low nM range, thus
rendering this possibility an unlikely mechanism for the observed
differences in endocytic rate constants. Alternatively, the rates at
which the various receptors diffuse in the plasma membrane and pass
through a coated pit may differ, which could account for different
internalization kinetics. Furthermore, the efficiency with which
ligand-receptor-complexes become trapped in coated pits and the rate at
which coated pits give rise to endosomes may differentially affect
rates of receptor-mediated endocytosis. Potentially distinct
interactions of the ligand-bound tyrosine kinase receptors with
clathrin-associated proteins, such as 
-adaptin or ß-arrestin,
may further contribute to the observed differences in internalization
rates between NGF, IGF-I, and insulin. Clearly, there are many steps
which participate in the modulation of internalization kinetics. This
highlights the complexity and potential of regulation of growth factor
action before internalization.
The majority of internalized 125I-NGF escapes degradation, as evidenced by its absence in TCA-soluble fractions of internalized radioactivity. We propose that the ability of NGF to remain associated with its receptor in acidic conditions, indicative of the endosomal milieu, protects it from being targeted to the lysosome. The ability of NGF to remain receptor-associated is further supported by the appearance of TCA-precipitable NGF in the extracellular medium 25 min following internalization. Receptor recycling has been documented in several systems and recent cross-linking studies suggest that TrkA may recycle between 10–30 min after initiation of internalization (19). It is, therefore, likely, that intact NGF can return to the cell surface as the receptors are reincorporated into the plasma membrane. This is the first demonstration of NGF retroendocytosis in the absence of p75. Eveleth et al. (22) suggest that the observed retroendocytosis in PC12nnr5 cells could be due to p75 because Trk function in these mutant cells is defective. Although p75 may contribute to NGF retroendocytosis, we demonstrate that TrkA is sufficient for this process.
Other potential mechanisms may also contribute to NGF’s efficient retroendoctyosis. To our knowledge, no endosomal NGF degradation enzyme has been discovered, unlike is the case for insulin, which is degraded by an endosomal acidic thiol metalloprotease that functions optimally at low pH (43). Thus, the absence of endosomal degradation may serve to protect NGF’s integrity. An alternative mechanism by which NGF may be protected from degradation may be through its interaction with low affinity binding sites that, according to binding analysis, may be present on these cells. The identity of these binding sites is unknown; perhaps they are binding proteins that protect NGF from degradation.
Tyrosine kinase receptors and their respective ligands activate a
multitude of identical signal transduction molecules, yet their
biological effects are distinct. It has been suggested that specificity
of growth factor action can be achieved by differences in the kinetics
of activation of signaling proteins. Others have published that NGF
elicits a higher and more sustained level of ERK1/2 activation in
comparison to other growth factors (44, 45, 46, 47), and we have observed
similar findings in CHO/TrkA cells (unpublished observations). It is
possible that a ligand is able to mediate signaling events not only at
the plasma membrane but continues to do so from the endosome, provided
it is still bound to its receptor. The hypothesis that internalized
ligand-receptor complexes can extend the signaling process initiated at
the plasma membrane was proposed by Posner et al. (48, 49)
and several studies (50, 51) have addressed this in the insulin and EGF
receptor system. Furthermore, Grimes et al. (19) demonstrate
that endosomal TrkA receptors remain associated with NGF, are
autophosphorylated, and associate with PLC-, suggesting that
endocytosed ligand-receptor complexes can continue to participate in
signal transduction events. Thus, strong and sustained ERK1/2
activation in the case of NGF could be the direct result of prolonged
internalized ligand-receptor association and lack of NGF lysosomal
degradation. Perhaps NGF cannot fulfill its biological functions
adequately unless it is internalized. This can be tested by measuring
NGF’s biological effects in cell lines expressing Trk receptor
mutants, which lack the ability to internalize yet maintain their
tyrosine kinase activity upon ligand-binding. Because we observed very
efficient retroendocytosis of NGF, it is possible that retroendocytosed
NGF could bind once again to cell-surface receptors and thereby
continue to activate downstream molecules, such as ERK1/2. Thus, we
hypothesize that the processes following receptor-mediated endocytosis,
including ligand-receptor dissociation, ligand degradation, and ligand
trafficking, can play critical roles in imparting growth factor
specificity.
It has been previously suggested that functions of receptor-mediated ligand internalization include down-regulation of receptors and termination of signal transduction. However, recent evidence suggesting the ability of endosomal ligand-receptor pairs to contribute to signal transduction, modifies this original view. The function of receptor-mediated endocytosis may be to regulate, either positively or negatively, ligand initiated signaling events. NGF, a chronically required factor involved in cell survival and differentiation, may sustain signaling through relatively longer association with its receptor, its ability to bypass lysosomal degradation, and subsequent retroendocytosis.
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Acknowledgments |
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Footnotes |
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Received March 2, 1998.
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