The Role of Endocytosis in Regulating L1-mediated Adhesion*

Kristin E. LongDagger , Hiroaki Asou§, Martin D. Snider, and Vance LemmonDagger ||

From the Dagger  Department of Neurosciences and  Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106 and § Department of Neurobiology, Tokyo Metropolitan Institute of Gerontology 35-2, Itabashi-ku, Tokyo, 173-0015, Japan

Received for publication, July 26, 2000, and in revised form, September 20, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

L1 is a neural cell adhesion molecule critical for neural development. Full-length L1 (L1FL) contains an alternatively spliced cytoplasmic sequence, RSLE, which is absent in L1 expressed in nonneuronal cells. The RSLE sequence follows a tyrosine, creating an endocytic motif that allows rapid internalization via clathrin-mediated endocytosis. We hypothesized that L1FL would internalize more rapidly than L1 lacking the RSLE sequence (L1Delta RSLE) and that internalization might regulate L1-mediated adhesion. L1 internalization was measured by immunofluorescence microscopy and by uptake of 125I-anti-rat-L1 antibody, demonstrating that L1FL is internalized 2-3 times faster than L1Delta RSLE. Inhibition of clathrin-mediated endocytosis slowed internalization of L1FL but did not affect initial uptake of L1Delta RSLE. To test whether L1 endocytosis regulates L1 adhesion, cell aggregation rates were tested. L1Delta RSLE cells aggregated two times faster than L1FL cells. Inhibition of clathrin-mediated endocytosis increases the aggregation rate of the L1FL cells to that of L1Delta RSLE cells. Our results demonstrate that rapid internalization of L1 dramatically affects L1 adhesion.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

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 L1Delta 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 L1Delta RSLE. Using either immunofluorescence techniques or radioactively labeled antibodies, we found that L1FL is internalized significantly faster than L1Delta 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 L1Delta RSLE cells with similar L1 cell surface expression levels were allowed to aggregate over time. L1FL cells aggregated more slowly than L1Delta RSLE cells, but when clathrin-mediated endocytosis was inhibited, the L1FL cells aggregated more rapidly, at a rate indistinguishable from L1Delta RSLE cells. This demonstrates that alterations in L1 internalization can regulate L1-mediated adhesion.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

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 L1Delta 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 L1Delta 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 beta -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-beta -galactosidase antibody (Roche Molecular Biochemicals). Only hemagglutinin-positive or beta -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 L1Delta 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 gamma  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 L1Delta RSLE cells, K+-deprived L1FL versus L1FL cells, and K+-deprived L1Delta RSLE versus L1Delta RSLE cells.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Immunocytochemistry Demonstrates That L1FL Internalizes Faster Than L1Delta RSLE-- To study the time course of internalization of different L1 isoforms, L1FL cells or L1Delta 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 L1Delta RSLE cells had internalized anti-L1 antibody, seen as green punctate staining. However, L1FL cells had many more green-labeled vesicles than L1Delta 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 L1Delta RSLE cells (p < 0.01) (Fig. 2). After 60 min, both L1FL cells and L1Delta 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 L1Delta 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 L1Delta 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.

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 beta -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, L1Delta RSLE cells internalized 40% of the L1 of L1FL cells (Fig. 3). After infection with K44A dominant negative dynamin, L1FL cells and L1Delta RSLE cells internalized equal amounts of L1. This suggests that the difference between L1FL and L1Delta RSLE internalization was due to clathrin-mediated endocytosis of the L1FL isoform. Surprisingly, both the infected L1FL cells and L1Delta RSLE cells internalized approximately twice as much L1 as uninfected L1Delta 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 beta -galactosidase adenovirus that should not affect internalization. After 10 min at 37 °C, infected L1FL cells had internalized twice as much L1 as infected L1Delta 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 L1Delta RSLE cells. L1-expressing cells were infected with K44A dominant negative dynamin adenovirus, beta -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 L1Delta 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.

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 L1Delta RSLE. In contrast, K+ depletion did not affect the low level of internalization in L1Delta 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. L1Delta 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 L1Delta 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.

L1 Internalization Rates Differ between L1FL and L1Delta RSLE Cells Measured by 125I-anti-L1 Antibody Uptake-- To confirm the immunohistochemistry results demonstrating that L1FL internalizes more rapidly than L1Delta 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 L1Delta RSLE cells. At the earliest time (5 min), L1FL cells internalized nearly twice as much antibody as L1Delta RSLE cells. Analysis of three experiments revealed that the difference between L1FL and L1Delta 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 L1Delta 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 L1Delta RSLE as well as both K+-deprived samples (p < 0.05).

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 L1Delta RSLE cells. The effect of potassium deprivation on L1Delta RSLE cells was not statistically significant in the 5- or 10-min incubation samples compared with untreated L1Delta RSLE cells. However, the effect of K+ deprivation on L1Delta RSLE internalization was larger than expected. Furthermore, potassium deprivation reduced L1 internalization of L1Delta RSLE cells significantly (p < 0.01) after the 30-min incubation. The effect of K+ deprivation on L1Delta 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. L1Delta RSLE cells aggregated significantly more rapidly than L1FL cells (Fig. 6). At 20 and 30 min, the L1Delta 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 L1Delta RSLE cells. This represents a 2-fold increase in aggregation rate compared with control L1FL cells. The aggregation rate of L1Delta RSLE cells after K+ depletion was not different from the untreated L1Delta 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 L1Delta 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 L1Delta 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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

Our results demonstrate that L1FL is internalized more rapidly than L1Delta 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 L1Delta 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 L1Delta 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 L1Delta 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 L1Delta RSLE explains the slower aggregation of L1FL versus L1Delta RSLE cells. It was shown previously that L1FL cells aggregate more slowly than L1Delta 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 L1Delta RSLE cells. However, as expected, inhibition of endocytosis does not affect L1Delta RSLE cell aggregation rate. The deletion of the RSLE sequence in L1Delta 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 L1Delta RSLE. Although momentarily unbound, more L1FL molecules would be endocytosed from the cell surface in response to CAM trans-binding events. L1Delta 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 L1Delta 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.


    CONCLUSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

We have shown that L1FL internalizes faster than L1Delta 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 L1Delta 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.


    ACKNOWLEDGEMENTS

We acknowledge the helpful comments of H. Kamiguchi and S. K. Lemmon.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants EY-05285 and P30EY11373 (to V. L.).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.

|| To whom correspondence should be addressed: Dept. of Neurosciences, Case Western Reserve University School of Medicine, Rm. E661, 2109 Adelbert Rd., Cleveland, OH 44106-4975. Tel.: 216-368-3039; Fax: 216-368-4650; E-mail vxl@po.cwru.edu.

Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M006658200


    ABBREVIATIONS

The abbreviations used are: CAM, cell adhesion molecule; L1FL, full-length L1; L1Delta RSLE, L1 with deletion of RSLE cytoplasmic sequence; PBS, phosphate-buffered saline.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES


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