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INTRODUCTION |
Development of the nervous system is a complex process that
requires coordination of many cellular events including cell migration, axon outgrowth, and synapse formation. Many cell adhesion molecules (CAMs)1 participate in these
events. Although CAM expression appears to be rather static in the
adult nervous system, CAM expression is both spatially and temporally
dynamic during development. An important question in developmental
neurobiology is how expression of CAMs is regulated to allow for the
precise adhesive events necessary for proper neural development. The
CAMs are responsible for mediating homophilic and heterophilic binding
events and, in turn, cell-cell or cell-substrate interactions (1). Many CAMs interact with signaling molecules (2) and the cytoskeleton (3-5).
Therefore, CAM-CAM binding can produce intracellular signals that
regulate adhesion and trigger other events such as migration, proliferation, and synapse formation (6-11).
L1 is an immunoglobulin superfamily CAM important in vertebrate neural
development. L1 participates in neurite outgrowth (12) as well as
neuronal migration (13, 14). It binds both homophilically and
heterophilically with a number of CAMs including axonin-1 (15), CD9
(16), and integrins (17-19). The importance of proper L1 function is
demonstrated by the severe complications resulting from mutations in
the human L1 molecule (20, 21), causing X-linked hydrocephalus
which is characterized by varying degrees of corticospinal tract and
corpus callosum agenesis, retardation, adducted thumbs, spastic
paraplegia, and hydrocephalus (20). Mutations in L1 can disrupt proper
L1 adhesive function, which in turn causes mistakes in cell migration
and axon extension.
Alternative RNA splicing results in two L1 isoforms (22). Neurons
express the full-length form, L1FL. Non-neuronal
L1-positive cells express L1 lacking the cytoplasmic sequence, RSLE, as
well as a short extracellular sequence (22). The presence of RSLE in
L1FL creates the sequence YRSL in the cytoplasmic domain.
This corresponds to a tyrosine-based clathrin recognition motif,
YXXA, where A is any hydrophobic amino
acid (23). The YRSLE sequence is necessary for proper
sorting of L1 to axons (24). The YRSLE sequence in
L1FL is recognized by the µ2 subunit of AP-2 (25), a
clathrin-associated adaptor protein used in clathrin-mediated endocytosis. L1FL, but not
L1
RSLE, is internalized via
clathrin-mediated endocytosis (25).
Clathrin-mediated endocytosis is the most rapid form of
receptor-mediated endocytosis. We hypothesized that L1FL is
internalized more rapidly than L1
RSLE. Using
either immunofluorescence techniques or radioactively labeled
antibodies, we found that L1FL is internalized
significantly faster than L1
RSLE. Using dominant negative dynamin adenovirus infection or intracellular potassium deprivation, we showed that the difference in internalization is due to clathrin-mediated endocytosis of L1FL. Next we
examined how endocytosis influences L1 adhesiveness.
L1FL and L1
RSLE cells with
similar L1 cell surface expression levels were allowed to aggregate
over time. L1FL cells aggregated more slowly than L1
RSLE cells, but when clathrin-mediated
endocytosis was inhibited, the L1FL cells aggregated more
rapidly, at a rate indistinguishable from
L1
RSLE cells. This demonstrates that
alterations in L1 internalization can regulate L1-mediated adhesion.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
L cells were cultured in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 5% fetal
bovine serum (Life Technologies, Inc.), sodium pyruvate (Sigma),
antibiotic-antimycotic solution (Life Technologies, Inc.), 2 mM glutamine and maintained in a 37 °C, 5%
CO2, 95% humid air incubator in plastic tissue culture
flasks. Stably transfected L cells expressing full-length rat
L1FL (L3-2 cells) or L1
RSLE
cells lacking the RSLE sequence (L4-2 cells) were obtained (26, 27).
Expression of L1 in the transfected L cells was maintained using 60 µg/ml G418 (CLONTECH). Populations of
L1FL or L1
RSLE cells expressing
equal levels of surface L1 were obtained by flow cytometery based on
immunofluorescence of cells labeled on ice with rabbit anti-rat-L1
antibody (28).
Immunohistochemistry of L Cells--
Cells were plated in 2-well
slide chambers (Nalge-Nunc Labtek) 12-24 h before the experiment. Warm
culture medium containing a 1:250 dilution of rabbit anti-L1 antibody
was added, and dishes were incubated at 37 °C. After 10 or 60 min,
cells were placed on ice and rinsed three times with 4 °C culture
medium and fixed with 4% formaldehyde (Sigma). Cell surface antibody
was detected with a 1:500 dilution of Texas Red goat anti-rabbit
antibody (Molecular Probes) for 30 min at room temperature. Then cells
were rinsed with PBS and incubated with 0.25 mg/ml of unlabeled goat
anti-rabbit antibody (Molecular Probes) to block cell surface anti-L1
antibody. The cell surface secondary antibody was post-fixed for 5 min
using formaldehyde. After washing, cells were blocked and permeabilized with 10% horse serum, 0.1% Triton X-100 in PBS for 30 min at room temperature. To label internalized L1, cells were incubated with a
1:500 dilution of Oregon Green goat anti-rabbit antibody (Molecular Probes) in 5% horse serum, PBS, 0.02% Triton X-100. Then slides were
rinsed three times with PBS and mounted with Slow Fade Light Antifade
kit (Molecular Probes). Slides were imaged on a Zeiss LSM 410 confocal
microscope using a 100× lens. To standardize the collection of images,
the cell z-axis heights were measured, and optical slices
were collected through the middle of the cells. The average brightness
of at least 30 cells from each experimental group was measured using
the Metamorph image analysis program. Each experiment was performed a
minimum of three times. All cells in randomly selected images were
analyzed. Average brightness measurements were statistically analyzed
to determine the mean, S.D., and outcomes of relevant two-sided
t tests. Average brightness measurements were normalized to
the brightness value of L1FL cells incubated for 10 min at
37 °C.
Adenovirus Production and Infection--
Adenovirus expressing
either hemagglutinin-tagged dominant negative mutant K44A dynamin or
-galactosidase were the gift of J. E. Pessin (29). L cells were
incubated with medium containing virus for 1.5 h and then cultured
in fresh medium for 12-16 h. Cells were incubated with anti-rat-L1
antibody and stained as described above except that unconjugated goat
anti-rabbit IgG was bound to cell surface anti-L1 antibody. To identify
virus-infected cells, fixed-permeabilized cells were labeled with
anti-hemagglutinin (Sigma) antibody to detect dominant negative dynamin
or anti-
-galactosidase antibody (Roche Molecular Biochemicals). Only
hemagglutinin-positive or
-galactosidase-positive cells were
analyzed for internalized L1.
Potassium Depletion of L Cells--
Cells were rinsed three
times with K+-free HEPES-buffered saline (HBSK
; 140 mM NaCl, 20 mM HEPES, pH 7.4, 1 mM
CaCl2, 1 mM MgCl2, and 1 g/liter
D-glucose). Then cells were hypotonically shocked by
incubating them for 10 min in a 1:1 solution of HBSK
and water. Cells
were rinsed three times with HBSK
and allowed to recover in HBSK
for 10 min at 37 °C (30, 31).
Internalization of 125I-anti-L1
IgG--
Affinity-purified rabbit anti-rat-L1 antibody was iodinated
with 125I (32). L1FL or
L1
RSLE cells were plated on 0.01%
poly-L-lysine-coated 24-well tissue culture plates (Falcon)
at 1-2 × 105 cells/well. Cells were washed once with
Dulbecco's modified Eagle's medium, and then 0.5 ml was added of
either HBSK+ (140 mM NaCl, 20 mM HEPES, pH 7.4, 1 mM CaCl2, 1 mM MgCl2,
1 g/liter glucose, 5 mM KCl) with 5% fetal bovine serum
(Life Technologies, Inc.) or HBSK
with 5% fetal bovine serum
dialyzed against 1 M NaCl to remove potassium. Plates were
placed in a 37 °C water bath and incubated with 13.4 nM
125I-anti-rat-L1 IgG (876 cpm/fmol). Cells were washed
three times with ice-cold HBSK+ or HBSK
. To determine the amount of
cell-associated radioactivity, sextuplicate samples were dissolved in
0.5% sodium deoxycholate, 0.5 N NaOH and then collected
and counted in a
counter. To determine the amount of internalized
radioactivity, sextuplicate samples were incubated for 15 s with
ice-cold 0.25 M acetic acid, 0.5 M NaCl, pH
2.7, to strip cell surface radioactivity. Cells were rinsed three times
with HBSK+, and samples were dissolved and counted to determine
radioactivity. Data are presented as percent radioactivity
internalized, which represents the internalized radioactivity divided
by the total cell-associated radioactivity.
Aggregation of L Cells--
Cells were incubated for 10 min at
37 °C in calcium- and magnesium-free, HBSK+ or HBSK
supplemented
with 1 mM EDTA. Next, 0.002% trypsin-EDTA (Life
Technologies, Inc.) was added for 5 min at 37 °C. Cells in medium
containing trypsin were collected in the presence of 2% dialyzed
K+-free fetal bovine serum and centrifuged at 180 × g for 5 min. Cells were chilled on ice and suspended briefly
in 1 ml of 150 mM NaCl, 1 mM MgSO4
containing DNase I (Sigma). Cells were further diluted in ice-cold
calcium- and magnesium-free HBSK+ or HBSK
and divided into
scintillation vials chilled on ice. At time 0, cells were placed in a
37 °C incubator and rotated at 55 rpm. At various times, samples
were fixed in 1.5% glutaraldehyde in PBS. Fixed samples were counted
in a Coulter Counter to determine particle number. Percent aggregation
was determined from the particle number at time zero
(N0) and the particle number at time t
(Nt) using the formula ((N0
Nt)/(N0)) × 100. Paired t tests of the mean were performed on samples
incubated for 20 and 30 min for the following groups: L1FL
versus L1
RSLE cells,
K+-deprived L1FL versus
L1FL cells, and K+-deprived
L1
RSLE versus
L1
RSLE cells.
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RESULTS |
Immunocytochemistry Demonstrates That L1FL Internalizes
Faster Than L1
RSLE--
To study the time course of
internalization of different L1 isoforms, L1FL cells or
L1
RSLE cells were incubated in the presence
of rabbit anti-L1 antibody for 10 or 60 min at 37 or at 4 °C for 30 min. At the end of the incubations, cells were fixed but not
permeabilized, and their cell surface L1 was stained with a Texas
Red-conjugated anti-rabbit antibody followed by unconjugated anti-rabbit antibody. Then cells were permeabilized, and internalized L1 was labeled with Oregon green-conjugated goat anti-rabbit secondary antibody. The experiment was also performed at 4 °C, which inhibits all forms of endocytosis (33). Cells incubated at 4 °C showed no
green internal fluorescence after a 30-min incubation (Fig. 1). This demonstrates that our
immunofluorescence method discriminates between cell surface and
internalized L1. After 10 min at 37 °C, both L1FL cells
and L1
RSLE cells had internalized anti-L1 antibody, seen as green punctate staining. However, L1FL
cells had many more green-labeled vesicles than
L1
RSLE cells (Fig. 1, C and
D). At 60 min, both L1-expressing cell types contained similar amounts of green-labeled vesicles (Fig. 1, E and
F). Image analysis revealed that after a 10-min incubation,
L1FL cells had internalized approximately 3 times more L1
than L1
RSLE cells (p < 0.01) (Fig. 2). After 60 min, both
L1FL cells and L1
RSLE cells had
similar amounts of internalized L1 (Fig. 2). The internalized L1 is
probably similar at 60 min because the internalized antibody in both
cell lines has reached steady state, balancing uptake and recycling to
the cell surface.

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Fig. 1.
Confocal sections of L1FL cells
(A, C, and E) and
L1 RSLE cells (B,
D, and F) labeled for internalized L1
and cell surface L1 reveal more internalized L1 in L1FL
cells at 10 min. Live L cells were incubated with rabbit
anti-rat-L1 antibody for 30 min at 0 °C (A and
B) to inhibit L1 endocytosis or 10 min (C and
D) and 60 min (E and F) at 37 °C to
allow L1 endocytosis to occur. Superimposed images show internalized L1
in green and cell surface L1 in red. The
scale bar represents 10 µm.
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Fig. 2.
Average brightness (pixel intensity) of
internalized L1 in confocal micrographs from the experiment shown in
Fig. 1. After 10 min at 37 °C, there was a statistically
significant difference (p < 0.01) between
L1FL and L1 RSLE cells
(asterisk). After 60 min, no statistically significant
difference was seen between the two L1-transfected cell lines. Data is
from one representative experiment. Error bars indicate
S.E.
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Dominant Negative Mutant Dynamin Abrogates the Difference in
Internalization of L1 Isoforms--
To determine whether L1 was
internalized via clathrin-coated vesicles, the effect of infection with
adenovirus expressing the K44A dominant negative dynamin mutant was
examined. Dynamin is required for clathrin-mediated endocytosis (34,
35), and K44A dominant negative dynamin expression blocks
clathrin-mediated endocytosis specifically (29, 36). Cells were
infected with adenovirus expressing mutant dynamin or with adenovirus
expressing
-galactosidase as a control. Twelve to sixteen hours
after virus infection, the uptake of anti-L1 antibody over 10 min at
37 °C was assessed by immunofluorescence microscopy. As expected,
L1
RSLE cells internalized 40% of the L1 of
L1FL cells (Fig. 3). After infection with K44A dominant negative dynamin, L1FL cells
and L1
RSLE cells internalized equal amounts
of L1. This suggests that the difference between L1FL and
L1
RSLE internalization was due to
clathrin-mediated endocytosis of the L1FL isoform. Surprisingly, both the infected L1FL cells and
L1
RSLE cells internalized approximately
twice as much L1 as uninfected L1
RSLE cells
(Fig. 3.) This effect has been reported previously and has been
attributed to a compensatory up-regulation of clathrin-independent endocytosis (36, 37). We also analyzed cells infected with
-galactosidase adenovirus that should not affect internalization. After 10 min at 37 °C, infected L1FL cells had
internalized twice as much L1 as infected
L1
RSLE cells, similar to uninfected cells.
These results indicate that adenovirus infection itself did not affect
internalization rates.

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Fig. 3.
Dominant negative dynamin attenuates the L1
internalization difference between L1FL cells and
L1 RSLE cells. L1-expressing
cells were infected with K44A dominant negative dynamin adenovirus,
-galactosidase adenovirus, or no virus. After 12-16 h, cells were
incubated for 10 min at 37 °C in the presence of anti-rat-L1
antibody. Cells were then fixed, processed to reveal intracellular
antibody, and visualized by confocal microscopy. The
asterisks (*) indicate statistically significant differences
(p < 0.01) between L1FL cells and
L1 RSLE cells. Infection with K44A dominant
negative dynamin adenovirus attenuated the difference in
internalization. Measurements of the different samples were normalized
to the brightness of uninfected L1FL cells. Data is from
one representative experiment. Error bars represent
S.E.
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K+ Deprivation of Cells Inhibits Endocytosis of
L1FL--
To further examine whether L1 was internalized
via clathrin-mediated endocytosis, we deprived cells of potassium by
hypotonic shock in K+-free medium. Depletion of
K+ has been shown to inhibit clathrin-coated pit formation
and receptor-mediated endocytosis (31) as well as some forms of
clathrin-independent endocytosis (38). We found that K+
deprivation of L1FL cells reduced internalization of L1 by
~40% compared with untreated L1FL cells during a 10-min
incubation at 37 °C (Fig. 4), equaling
that of L1
RSLE. In contrast, K+
depletion did not affect the low level of internalization in L1
RSLE cells. The inhibition of
L1FL uptake by K+ depletion suggests that this
molecule is internalized by clathrin-mediated endocytosis. The low
level of uptake in K+-depleted L1FL cells
suggests that L1FL can also be taken up by a
clathrin-independent process. L1
RSLE is also
taken up by this process, since uptake of this protein was low and was
not inhibited by K+ depletion.

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Fig. 4.
K+ depletion inhibits rapid
internalization of L1FL. Average brightness (pixel
intensity) of internalized anti-L1 antibody. Control and
K+-depleted cells were incubated with anti-L1 antibody for
10 min at 37 °C. Internalized antibody was visualized by confocal
microscopy. Measurements were normalized to untreated L1FL
cells. K+ deprivation causes L1FL cells to
internalize statistically significantly less L1 than untreated
L1FL cells. K+ deprivation does not
significantly change L1 internalization in
L1 RSLE cells. *, p < 0.01 for t tests of L1FL compared with the other
three samples. Data is from one representative experiment. Error bars
represent S.E.
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L1 Internalization Rates Differ between L1FL and
L1
RSLE Cells Measured by 125I-anti-L1
Antibody Uptake--
To confirm the immunohistochemistry results
demonstrating that L1FL internalizes more rapidly than
L1
RSLE in transfected L cells, we quantified
the uptake of radioactive anti-L1 antibody. Cells were incubated at
37 °C in the presence of 125I-anti-L1 antibody. At
various times, both cell-associated and intracellular radioactivity
were determined. Levels of cell-associated IgG showed similar
time-dependent increases in the two cell lines (data not
shown). However, the internalization of antibody was different (Fig.
5). Antibody internalization was more
rapid in L1FL cells than in
L1
RSLE cells. At the earliest time (5 min),
L1FL cells internalized nearly twice as much antibody as L1
RSLE cells. Analysis of three experiments
revealed that the difference between L1FL and
L1
RSLE cells was statistically significant
(p < 0.05) at the 5- and 10-min times.

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Fig. 5.
The L1 internalization rate is greater in
L1FL cells than L1 RSLE
cells, and the difference is abolished by K+
depletion. Cells were incubated at 37 °C in the presence of
125I-anti-L1 antibody. Internalized and total
cell-associated radioactivity were determined as described under
"Experimental Procedures." The graph shows the percentage
internalized (internalized radioactivity divided by total
cell-associated radioactivity). Graphed data are the mean ± S.E.
of three experiments. The asterisk indicates that the
L1FL value is statistically significantly higher than
L1 RSLE as well as both
K+-deprived samples (p < 0.05).
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The effect of K+ deprivation on internalization of
125I-anti-L1 antibody was evaluated.
K+-depleted L1FL cells internalized less than
half as much L1 as untreated cells after 5 and 10 min (Fig. 5).
K+ depleted L1FL cells internalized similar
amounts of L1 as K+-deprived
L1
RSLE cells. The effect of potassium
deprivation on L1
RSLE cells was not
statistically significant in the 5- or 10-min incubation samples
compared with untreated L1
RSLE cells.
However, the effect of K+ deprivation on
L1
RSLE internalization was larger than
expected. Furthermore, potassium deprivation reduced L1 internalization of L1
RSLE cells significantly
(p < 0.01) after the 30-min incubation. The effect of
K+ deprivation on L1
RSLE
internalization is probably because potassium deprivation also inhibits
some forms of clathrin-independent endocytosis (38).
Inhibition of Endocytosis Increases L1FL Cell
Aggregation Rate--
To examine the role of L1 endocytosis on
adhesiveness, cells were tested for their ability to aggregate via
L1-L1 homophilic binding. Cells were removed from tissue culture
plates, and single cell suspensions were placed in a rotary shaker to
aggregate and then counted in a Coulter Counter to assess particle
number. L1
RSLE cells aggregated
significantly more rapidly than L1FL cells (Fig. 6). At 20 and 30 min, the
L1
RSLE aggregation was approximately twice
that of L1FL cells. The 2-fold difference in aggregation rates between the two L1-expressing cell types was statistically significant (Fig. 6.) To examine the role of endocytosis on L1-mediated adhesion, the aggregation of K+-depleted cells was examined
(31). Interestingly, inhibition of endocytosis dramatically increased
the aggregation rate of L1FL cells. After 20 and 30 min,
K+-deprived L1FL cells aggregated almost
identically to L1
RSLE cells. This represents
a 2-fold increase in aggregation rate compared with control
L1FL cells. The aggregation rate of
L1
RSLE cells after K+ depletion
was not different from the untreated L1
RSLE
cell aggregation rate. To rule out the possibility that inhibition of
endocytosis affected the aggregation rate by changing cell surface L1
levels (39), these levels were measured by confocal microscopy.
L1FL and L1
RSLE cells with and
without K+ depletion all had similar levels of cell surface
L1 (data not shown), demonstrating that measurable changes in surface
L1 expression are not responsible for the effect of K+
depletion on cell adhesion. Consequently, we conclude that
clathrin-mediated endocytosis directly regulates L1-mediated cell
adhesion.

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Fig. 6.
L1FL cells aggregate more slowly
than L1 RSLE cells, but
L1FL cell aggregation increases after
K+-depletion. The aggregation of cells in suspension
at 37 °C was measured as described under "Experimental
Procedures." The data are the mean ± S.E. for three independent
experiments. The asterisk (*) indicates that control
L1FL cells aggregate statistically significantly less
(p < 0.05) than each of the other three groups.
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DISCUSSION |
Our results demonstrate that L1FL is internalized more
rapidly than L1
RSLE in transfected L cells
and that inhibition of L1 clathrin-mediated endocytosis results in
elimination of the differences in internalization rates of the two L1
isoforms. Blocking clathrin-mediated endocytosis also completely
eliminates the difference in aggregation between L1FL and
L1
RSLE cells.
The differences in internalization and adhesion of the two L1 forms are
due to the alternatively spliced cytoplasmic sequence. The RSLE
sequence is the only difference between the L1FL and the
L1
RSLE used in our experiments. The sequence
that is spliced out of the extracellular sequence in non-neuronal
L1-positive cells (27) was present in the
L1
RSLE cells. The complete amino acid
conservation in the cytoplasmic domain among mammalian forms of L1
indicates that it has a critical function. The fact that mutations in
the cytoplasmic domain of human L1 cause mental retardation and axon
guidance errors further supports this idea (21). Previous studies have
demonstrated that the L1 cytoplasmic domain is phosphorylated by
several kinases (40-42) and that it interacts with both actin (43) and
ankyrin (3). Studies of integrins have shown that phosphorylation of
the cytoplasmic domains can influence CAM adhesion by altering the
conformation of the extracellular domain (44). Our studies show that a
different mechanism may work to regulate L1-mediated adhesion.
Clathrin-mediated endocytosis after L1FL binding could
reduce L1-mediated adhesion.
The current results indicate that differences in L1 internalization
rate between L1FL and L1
RSLE
explains the slower aggregation of L1FL versus
L1
RSLE cells. It was shown previously that
L1FL cells aggregate more slowly than
L1
RSLE cells (27). The difference in
aggregation rate was difficult to explain for two reasons. First, the
cells expressed similar amounts of cell surface L1. Not surprisingly,
reduced L1FL cell surface expression has been shown to
reduce cell adhesion in other aggregation studies (45). Second, two
earlier reports demonstrated that the cytoplasmic domain is not
required by L1 or by the Drosophila homologue neuroglian for
homophilic adhesion (45, 46). Since the extracellular domain is
necessary and sufficient for L1-mediated adhesion (27), it was
difficult to explain how a sequence difference in the cytoplasmic domain affects L1 adhesion.
We found that inhibiting endocytosis of L1 molecules increases the
L1FL cell aggregation rate to that of
L1
RSLE cells. However, as expected,
inhibition of endocytosis does not affect L1
RSLE cell aggregation rate. The deletion
of the RSLE sequence in L1
RSLE eliminates
the YRSL sequence, which is required for L1 clathrin-mediated
endocytosis (25). These two observations indicate that L1 adhesion is
regulated by clathrin-mediated endocytosis. Clathrin-mediated
endocytosis has been shown to regulate the cell surface expression of
many proteins including the epidermal growth factor receptor (47) and
low density lipoprotein receptor (48) as well as CAMs (49). For
example, cell surface expression of E-cadherin, another CAM, is
regulated by clathrin-mediated endocytosis (30).
How might a difference in L1 internalization rate regulate its
adhesion? Early modeling studies of homophilic CAM binding suggested
that after CAM binding, individual pairs of bound molecules rapidly
cycle between the bound and unbound states (50). Other studies indicate
that L1 homophilic binding events trigger internalization (42, 51). An
increased internalization rate for L1FL would cause it to
be more rapidly removed from the membrane after L1 homophilic binding
than L1
RSLE. Although momentarily unbound, more L1FL molecules would be endocytosed from the cell
surface in response to CAM trans-binding events.
L1
RSLE would remain on the surface longer
because of slower internalization and reengagement in L1 trans binding.
This effect of internalization is consistent with studies of the
transferrin receptor, where its endocytosis occurs at a rate similar to
transferrin binding. As a consequence, the apparent
Kd for transferrin binding is 20-fold higher at
37 °C, when endocytosis can occur, than at 0 °C, when endocytosis is blocked (52).
Our studies raise the possibility that L1 adhesion can be regulated by
modulation of clathrin-mediated endocytosis of L1FL. Phosphorylation or dephosphorylation has been shown to control clathrin-mediated endocytosis of many proteins and to be necessary for
initiation of cell signaling events (53, 54). Numerous reports
demonstrate features of the L1 cytoplasmic domain that might affect L1
adhesion via regulation of clathrin-mediated endocytosis. For example,
casein kinase II, a ubiquitous serine/threonine kinase, phosphorylates
the L1 cytoplasmic domain at serine 1181 in rat brain extracts (41).
The serine is located directly C-terminal to the tyrosine-based motif
YRSL in the L1 cytoplasmic domain. It is known that casein kinase II
phosphorylation at serine residues C-terminal to tyrosine-based motifs
is required for or enhances binding of µ2 of AP-2, a clathrin adaptor
protein, before endocytosis (55, 56). Previous studies of furin
endocytosis demonstrate that casein kinase II phosphorylation on two
different serines C-terminal to its tyrosine-based sorting signal
enhances its internalization (57), regulating sorting to degradation or
recycling pathways (58). Phosphorylation of the critical tyrosine in
the CTLA-4 receptor tyrosine-based sorting signal by src kinase
prevents µ2 binding and, therefore, inhibits endocytosis (59). L1
clathrin-mediated endocytosis also might be inhibited by
phosphorylation of tyrosine 1176 in the YRSL signal.
Rapid clathrin-mediated endocytosis of L1FL may participate
in the adhesive changes necessary for proper cellular migration and
axon outgrowth. Modeling studies demonstrate that rapid changes in
adhesion are necessary for axon outgrowth (60). We previously reported
that L1 is internalized at the rear of growth cones (25). This
internalization may allow for rear de-adhesion of the growth cone.
After internalization, L1 is recycled to the front of the growth cone
and reinserted into the membrane for adhesion (25, 51). The more rapid
internalization of L1FL is likely responsible for the
increased cell migration rate of L1FL cells on an L1
substrate compared with L1
RSLE cells (27).
Upon L1-L1 binding during migration, L1FL cells can migrate
faster because, after adhesion to the substrate, the rear of the cell
can de-adhere. Testing this in neurons is challenging due to
difficulties of expressing mutated proteins on nondividing cells and
combining this with neurite outgrowth studies. However, this may be
possible with recent advances in transfection methods.
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CONCLUSION |
We have shown that L1FL internalizes faster than
L1
RSLE in transfected L cells due to
clathrin-mediated endocytosis of L1FL. Blocking
clathrin-mediated endocytosis eliminated the difference in L1
internalization between the two isoforms. Inhibition of endocytosis by
depletion of intracellular potassium dramatically increases
L1FL cell adhesion but has no effect on
L1
RSLE cell adhesion. Blocking endocytosis
did not affect overall L1 cell surface expression. Rather, we
hypothesize that rapid clathrin-mediated endocytosis affects the dwell
time of L1FL on the cell surface and causes it to be
internalized more rapidly after L1-L1 binding. Restricting the location
of endocytic machinery in growth cones (25) and phosphorylation of
critical residues in the L1 cytoplasmic domain may provide key
mechanisms for regulating L1-dependant axon growth. Internalization of
CAMs via clathrin may be a general mechanism by which cell migration
and axon growth is regulated.