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The Journal of Neuroscience, June 15, 2002, 22(12):4918-4931
The Neural Cell Adhesion Molecule L1 Potentiates
Integrin-Dependent Cell Migration to Extracellular Matrix Proteins
Karsten
Thelen*,
Vishram
Kedar*,
Anitha K.
Panicker*,
Ralf-Steffen
Schmid,
Bentley R.
Midkiff, and
Patricia F.
Maness
Department of Biochemistry and Biophysics, University of North
Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260
 |
ABSTRACT |
The L1 adhesion molecule regulates axon growth and is mutated in
the X-linked mental retardation syndrome CRASH (acronym for corpus
callosum agenesis, retardation, aphasia, spastic paraplegia, hydrocephalus). A novel role for L1 as a potentiator of neuronal cell
migration to extracellular matrix proteins through 
1 integrins and
intracellular signaling to mitogen-activated protein (MAP) kinase was
identified. L1 potentiated haptotactic migration of B35 neuroblastoma
cells toward fibronectin, vitronectin, and laminin through the
signaling intermediates c-Src, phosphatidylinositol-3 kinase, and MAP
kinase. L1 potentiated migration toward fibronectin through 
51
integrin in human embryonic kidney 293 cells and depended on
determinants of L1 endocytosis: dynamin I, c-Src, and the AP2/clathrin
binding site (Arg-Ser-Leu-Glu) in the neuronal splice form of
L1. L1 clustering on the cell surface enhanced the internalization of
activated 1 integrins and L1 into distinct endocytic vesicles.
L1-potentiated migration, enhancement of 1 integrin endocytosis, and
activation of MAP kinase were coordinately inhibited by mutation of an
RGD sequence in the sixth immunoglobulin-like domain of L1. Moreover,
three CRASH mutations in the L1 cytoplasmic domain (1194L, S1224L,
Y1229H), two of which interfere with ankyrin association, inhibited
L1-potentiated migration and MAP kinase activation. Function-blocking
antibodies to L1 and 1 integrin retarded the migration of
5-bromo-2'-deoxyuridine-labeled mouse cerebellar granule cells in slice
cultures, underscoring the potential physiological relevance of these
findings. These studies suggest that L1 functionally interacts with
1 integrins to potentiate neuronal migration toward extracellular
matrix proteins through endocytosis and MAP kinase signaling, and that
impairment of this function by L1 cytoplasmic domain mutations may
contribute to neurological deficits in CRASH.
Key words:
L1; integrin; cell migration; endocytosis; MAP kinase; mental retardation
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INTRODUCTION |
The L1 cell adhesion molecule is an
integral membrane protein that promotes axon growth and guidance
critical to nervous system development (for review, see Schmid and
Maness, 2001
). L1 belongs to a family of Ig-like cell recognition
molecules including L1, CHL1, NrCAM, NgCAM, neurofascin, and
Drosophila neuroglian, which share common structural
elements and the ability to promote axon growth. The L1 gene (on
chromosome Xq28) is the target for mutations in the human mental
retardation syndrome CRASH (acronym for corpus callosum agenesis,
retardation, aphasia, spastic paraplegia, hydrocephalus) (Kenwrick et
al., 2000). L1 null mutant mice display aspects of the CRASH phenotype
including axon guidance errors in the corticospinal tract and corpus
callosum, cortical dendrite abnormalities, reduced numbers of
hippocampal neurons, defects in spatial memory, and enlarged ventricles
(Cohen et al., 1997; Dahme et al., 1997; Fransen et al., 1998;
Demyanenko et al., 1999).
The L1 extracellular region is composed of six Ig-like and five
fibronectin type III domains that engage in L1-L1 homophilic binding
and heterophilic interactions with other Ig-class cell recognition
molecules including axonin 1/TAG1, F11/F3/contactin, and NCAM (Kadmon
et al., 1990; Kuhn et al., 1991; Brummendorf et al., 1993). L1 has been
shown to interact functionally with some integrins, including the
fibronectin receptor 51 and vitronectin receptor v3, for
adhesion or neurite growth on L1 substrates (Moos et al., 1988, Ruppert
et al., 1995; Ebeling et al., 1996; Montgomery et al., 1996;
Felding-Habermann et al., 1997). The L1 intracellular domain bears a
binding site for the cytoskeletal linker protein ankyrin (Bennett and
Chen, 2001) and a neuron-specific sequence Arg-Ser-Leu-Glu (RSLE)
arising from alternative splicing, which together with a preceding
tyrosine residue, comprises a motif for axon targeting and clathrin-
and dynamin-mediated endocytosis (Kamiguchi and Lemmon, 1998; Kamiguchi
et al., 1998). Although originally identified as a neural cell adhesion
molecule, L1 is also expressed in Schwann cells, melanocytes,
hematopoietic cells of lymphoid and myelomonocytic lineages, and
epithelial cells (Takeda et al., 1996; Pancook et al., 1997; Kadmon et
al., 1998; Nolte et al., 1999). Potential involvement of L1 in tumor
development and metastasis is further suggested by its expression on
many tumor cell lines including neuroblastoma, melanoma, and carcinomas (Linnemann et al., 1989; Reid and Hemperly, 1992; Pancook et
al., 1997).
In addition to the known function of L1 in axon growth, there is
evidence that L1 can participate in the migration of neuronal precursors. L1 is expressed in dopaminergic neuronal precursors during
their tangential migration on L1-positive fiber tracts (Ohyama et al.,
1998), and their final location is altered in L1 knock-out mice
(Demyanenko et al., 2001). L1 is also expressed transiently in
migrating neuronal precursors within the developing cerebellum and
neocortex (Fushiki and Schachner, 1986; Beasley and Stallcup, 1987;
Chung et al., 1991; Demyanenko et al., 1999), and function-blocking L1
antibodies have been shown to perturb the inward migration of granule
cell precursors in cerebellar explant cultures (Lindner et al., 1986;
Chuong et al., 1987; Crossin et al., 1990). In addition, L1 can promote
haptotactic migration of several types of culture cells toward purified
L1 substrates (Montgomery et al., 1996; Gutwein et al., 2000).
L1 promotes neurite outgrowth in cell cultures through the central
signal integrator MAP kinase (Schaefer et al., 1999; Schmid et al.,
2000) by a pathway that includes the nonreceptor tyrosine kinase c-Src
(Ignelzi et al., 1994), phosphatidylinositol-3 (PI3) kinase, Rac1
GTPase (Schmid et al., 2000), and the guanine nucleotide exchange
factor Vav-2 (R.-S. Schmid and P. F. Maness, unpublished results). This pathway is closely related to an early-acting
signaling pathway induced by integrin activation that promotes membrane ruffling, lamellipodia, and migration of non-neural cells (Clark et
al., 1998; Meng and Lowell, 1998; Miranti et al., 1998). The common
features of signaling by L1 and integrins and evidence that L1 may
participate in regulating neuronal precursor migration lead us to
speculate that L1 might interact functionally with integrins to
stimulate neuronal cell migration on extracellular matrix molecules. In
this study, we report a new role for L1 as a potentiator of neuronal
migration to extracellular matrix proteins through 1 integrin and
intracellular signaling and show that CRASH mutations in the L1
cytoplasmic domain impair this function.
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MATERIALS AND METHODS |
Reagents. Plasmids subcloned into pcDNA3 included
human L1(+/
RSLE) (John Hemperly, BD Technologies, Research Triangle
Park, NC), hemagglutinin (HA)-tagged extracellular signal regulated kinase (ERK)2 (Melanie Cobb, University of Texas Southwestern Medical School, Dallas, TX), dynamin I (K44A) (Marc Caron, Duke University, Durham, NC), Src (K295M) (Sara Courtneidge, Sugen, San
Francisco, CA), and enhanced cyan fluorescence protein
(Invitrogen, San Diego, CA). All L1 mutants used here have been
described in our previous work (Needham et al., 2001). Mouse monoclonal
antibody against human L1 (Neuro4) and anti-L1 polyclonal rabbit
antibody 6096 were gifts of John Hemperly (BD Technologies). The
following antibodies were obtained commercially: polyclonal
anti-Active MAP kinase antibody (pTEpY) (Promega, Madison, WI),
antibody against MAP kinase protein (Santa Cruz Biotechnology, Santa
Cruz, CA), anti-human 1 integrin monoclonal antibody (clone P4C10)
against the RGD binding site (Invitrogen, Gaithersburg, MD), anti-human 5 integrin (clone P1D6) against the PHSRN synergy site (Invitrogen), anti-human v3 integrin monoclonal antibody 1976Z (Chemicon, Temecula, CA), and monoclonal antibody MAB 2000 that activates human
1 integrin (Chemicon). Human vitronectin, human fibronectin, murine
laminin, and peptides GRGDSP and GRGESP were
from Invitrogen or Peninsula laboratories; murine collagen type IV was
from Sigma (St. Louis, MO). Recombinant reelin secreted from
reelin-transfected human embryonic kidney (HEK) 293T cells and
control supernatant from untransfected cells were a gift from Eva Anton
(University of North Carolina, Chapel Hill, NC). The Src inhibitor
4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-D]pyrimidine (PP2)
(Hanke et al., 1996) and inactive analog
4-amino-7-phenylpyrazolo[3,4-D]pyrimidine
(PP3) were from Calbiochem (La Jolla, CA). MAP kinase kinase
(MEK) inhibitor U0126 (Promega), PI3 kinase inhibitor Ly294002
(Calbiochem), PP2, and PP3 were dissolved in dimethylsulfoxide (DMSO).
Cell culture procedures. Rat B35 neuroblastoma cell lines
stably expressing human L1(+RSLE) or L1 point mutants S1194L, S1224L, and S1229H in pcDNA3 have been described (Needham et al., 2001). B35
cell lines were maintained in DMEM, 10% fetal bovine serum, and
gentamycin/kanamycin, 250 µg/ml G418. HEK293 cells were maintained in
the same medium without G418 and transfected for transient expression
of pcDNA3 plasmids (10 µg/100 mm dish) using lipofectamine with
transfection efficiency of ~70% as described (Needham et al.,
2001).
Haptotactic cell migration assay. Haptotactic cell migration
assays were performed in modified Boyden chambers (Transwells; Corning/Costar 3422) in serum-free DMEM containing 0.4 mM
MnCl2·4H2O, 50 µg/ml
gentamicin, and 200 µg/ml kanamycin. Transwell chambers had
polycarbonate filters with 8.0-µm-diameter pores, the bottom sides of
which were coated with extracellular matrix proteins or bovine serum
albumin (BSA; fatty acid free, Sigma). Proteins in PBS were applied to
the bottom side of filters (3 µg of protein per filter) and air
dried. The bottom surfaces of filters were washed and blocked in 2%
BSA. Cells were detached using PBS/Na-EDTA (5 mM)
and 10,000-20,000 cells plated per chamber. Some cells were
preincubated with antibodies (2 µg/100 µl medium) for 10 min at
4°C or peptides (2 mM) for 1 hr at 4°C in
serum-free medium.
To score B35 cells, cells from the top side of the filter were removed,
and cells on the bottom side of the filter were stained for 10-30 min
with Gill's formula hematoxylin (Vector Laboratories, Burlingame, CA),
and at least 150 cells were scored within a 10 × 10 mm grid from
nine or more randomly selected fields using a 20× microscope
objective. The mean number of cells per field was multiplied by a
factor based on the number and size of fields scored and on a filter
diameter of 6.5 mm to obtain the total number of cells migrated.
Experiments were performed in duplicate or triplicate, and results were
averaged. The means of experimental and control samples and SEM were
compared for significant differences using Student's t
test. L1-expressing HEK293 cells were visualized by indirect
immunofluorescence staining with anti-L1 monoclonal antibody Neuro4 and
FITC-labeled goat anti-mouse IgG and scored on both top and bottom
surfaces of filters under epifluorescence illumination. The percentage
of L1-expressing cells that transmigrated was calculated by determining
the ratio of L1-positive cells on the bottom of the filter compared
with the total number of L1-positive cells on both sides of the filter.
MAP kinase assay. B35 cells were transfected for transient
expression with HA-tagged ERK2 plasmid together with L1 plasmids, and
phosphorylation of immunoprecipitated HA-ERK2 protein was measured by
Western blotting with anti-Active MAPK antibody against dually
phosphorylated, activated ERKs as described (Schmid et al., 2000). At
36-40 hr after transfection, the medium was replaced with OptiMEM-I,
and 8 hr later cells were treated with preformed complexes of either
nonimmune mouse IgG or L1 antibody Neuro4 and
F(ab')2 fragments of secondary antibodies against
Fc fragments of mouse IgG (Jackson ImmunoResearch, West Grove, PA) for
10 min at 37°C. Cells were lysed in buffer containing 1% NP-40,
0.25% Na-deoxycholate, 50 mM HEPES, pH 7.4, 137 mM NaCl, 1 mM Na-EDTA, 10 mM phenylmethylsulfonyl fluoride, 10 mM -glycerophosphate, 10 µg/ml leupeptin,
0.1 TIU/ml aprotinin, 1 µg/ml pepstatin, 2 nM
calyculin A, and 10% glycerol. HA-tagged ERK2 was immunoprecipitated from lysates with anti-HA antibody and protein G-Sepharose. Immune complexes were analyzed by immunoblotting with anti-Active MAPK antibody using enhanced chemiluminescence. Blots were stripped and
reprobed with antibodies against total ERK protein. Bands were
quantified by densitometric scanning, and levels of phosphorylated ERK2
were normalized to ERK2 protein. Each cell line was assayed in
duplicate, and results were averaged. L1-induced ERK2 phosphorylation was expressed as the fold-activation relative to normal IgG control. Experiments were repeated 3-10 times, and mean ERK2 phosphorylation relative to normal IgG and SEM was determined for each cell line. Some
amount of nonspecific ERK2 phosphorylation was elicited by nonimmune
IgG in cells expressing mutant or wild-type L1, presumably attributable
to binding of residual uncomplexed IgG to endogenous Fc receptors
(Schmid et al., 2000), but little variation in the normal IgG control
was observed for each cell line. Means for each L1 mutant or splice
form were compared with L1(+RSLE), and significant differences were
determined by the t test.
Immunofluorescence staining for endocytosis of L1 and 1
integrins. HEK cells were plated at 20,000 cells per well onto
LabTek II chamber slides (Nunc, Naperville, IL) coated with
poly-D-lysine (0.01%) and human fibronectin (5 µg/ml) and transfected with plasmid encoding human L1(+RSLE). After
16-18 hr cells were washed and incubated for 5-60 min at 37°C with
polyclonal rabbit antibody 6096 against L1 (50 µg/ml) and MAB2000, an
activating monoclonal antibody against human 1 integrin (40 µg/ml). Cells were fixed in 2% paraformaldehyde for 30 min and then
permeabilized with 0.05% Triton X-100 in PBS, 10% goat serum for 1 hr. Cells were incubated with FITC-conjugated goat anti-mouse IgG (750 µg/ml) in PBS, 10% goat serum for 1 hr to label surface and
internalized integrins. After washing, cells were incubated with
tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit IgG
(750 µg/ml) in PBS containing 10% goat serum for 30 min to label
surface and internalized L1. Slides were mounted with Vectashield, and
cells were analyzed using a Zeiss LSM10 confocal microscope with an argon laser. Images of 0.5 µm optical thickness through the middle of
the cells were captured at the University of North Carolina Microscopy
Services Facility, Department of Pathology (Dr. Robert Bagnell,
Director). L1 labeling was recorded using the 514 nm excitation filter
with an emission range of 575-645 nm. Integrin labeling was recorded
using a narrow band filter with 488 nm excitation range and 515-545 nm
emission range. NIH Image J software was used for pseudocolor. The mean
fluorescent pixel density of internalized integrins was measured in
18-28 randomly selected cells using Scion imaging for three
experimental groups: HEK293, L1(+RSLE)-HEK293, and mutant
L1(KGE)-HEK293 cells treated for 5 min at 37°C with L1 and 1
integrin antibodies as described above. The number of fluorescent
pixels inside each cell was measured and divided by the total number of
pixels inside the cell to obtain the fluorescent pixel density of each
cell. The mean fluorescent pixel density was calculated for all cells
within each experimental group. The mean fluorescent pixel densities of
L1 and L1(KGE)-expressing cells in three different experiments were
normalized to the corresponding mean fluorescent pixel densities of
HEK293 cells (no L1) in the same experiment and averaged (see Fig. 9).
Significant differences in means were determined by the t
test (one-tailed; p < 0.05).
Neuronal migration in cerebellar slices. Newly generated
neurons were labeled with 5-bromo-2'-deoxyuridine (BrdU), and the extent of their radial migration away from the external granular layer
(EGL) was measured after 21 hr in culture. Mice (C57BL/6) at postnatal
day 4 were injected intraperitoneally with BrdU (10 mg/kg body weight)
in saline (Boehringer Mannheim, Indianapolis, IN). After 45 min brains
were removed, cerebella were dissected out, and 200 µm coronal slices
were made. Adjacent slices were cultured on Millicell-CM 0.4 µm
membranes (Millipore, Milford, MA) in MEM supplemented with 10% horse
serum, 40 mM glucose, 1.8 mM glutamine, 24 mM
Na-bicarbonate, and 90 U/ml penicillin-streptomycin. In some samples,
medium with adjacent sections of cerebellar slices from different mice
was supplemented with purified antibodies; rabbit polyclonal antibody
6096 against L1 (1 mg/ml), hamster anti-rat CD29 monoclonal antibody
against 1 integrin (0.1 mg/ml) (BD PharMingen), or nonimmune rabbit
IgG (1.1 mg/ml). Slices were removed at 0 or 21 hr of incubation in
culture at 37°C in 5% CO2 and processed for
BrdU by immunofluorescence staining. Slices were fixed in 70% ethanol
for 12 hr at 4°C. After three rinses in PBS, slices were fixed in 2N
HCl for 1 hr at room temperature, washed in PBS, and incubated for 4 hr
with BrdU monoclonal antibody (1:75 in PBS, 0.5% Tween 20) for 4 hr
(Becton Dickinson, Research Triangle Park, NC). Slices were washed in
PBS and incubated with anti-mouse IgG/M conjugated to Cy3 (1:100;
Jackson ImmunoResearch) for 2 hr. Bis-benzimide (10 µM) was added 5 min before the end of the
incubation. Filters were rinsed in PBS, and slices were cut out and
mounted onto glass slides with Vectashield mounting medium. Slices were
visualized with a Zeiss confocal microscope using rhodamine or UV
filter sets. Images of ~3 µm optical sections were collected and
stored on a CD and then analyzed for cell migration using Scion Image
software or assembled into the panel of figures presented using Adobe Photoshop.
To determine the extent of migration of BrdU-labeled cells, the
shortest distance between the BrdU-labeled cells and the pial surface
was measured within at least four different slices at different
locations, and the average distance was calculated for at least 150 cells per experimental condition. The average distance was then
expressed relative to the respective thickness of the cerebellar cortex
within the same location measured from bis-benzimide images to yield
the migration index. Only cells with brightly labeled nuclei were
scored. Bis-benzimide labeling of nuclei demarcated the different
regions of the cerebellar cortex and white matter. The location of the
EGL, molecular layer (ML), and internal granular layer (IGL) were
confirmed by staining with hematoxylin-eosin. After such demarcation,
the number of BrdU-labeled cells within three randomly selected regions
of the cerebellar cortex from the pial surface to the bottom of the IGL
was compared with the number of labeled cells within the EGL of the
corresponding region for each experimental condition to estimate the
percentage of BrdU-labeled cells in the EGL. Statistical differences
between experimental groups was tested by Student's t test
(p < 0.05; one-tailed).
| |
RESULTS |
L1 potentiates haptotactic cell migration to extracellular matrix
molecules through integrins
L1 signaling and endocytosis have been analyzed previously in the
CNS-derived B35 neuroblastoma cell line that stably expresses the
neuronal form of human L1(+RSLE) (Schmid et al., 2000; Needham et al.,
2001). Haptotactic migration of L1(+RSLE)-B35 cells and parental B35
cells, which lack L1, was studied in Transwell assays using modified
Boyden chambers in which cells migrated from top to bottom chambers
through filters coated on the bottom side with extracellular matrix
protein. Parental B35 neuroblastoma cells displayed greater migration
in 16 hr toward fibronectin compared with their random migration toward
BSA (Fig. 1). Migration of L1(+RSLE)-B35
cells toward fibronectin was strongly stimulated (three- to fourfold)
compared with parental B35 cells. Migration of L1(+RSLE)-B35 cells
toward laminin and vitronectin was also stimulated compared with B35
cells not expressing L1. Neither B35 cells nor L1(+RSLE)-B35 cells
displayed significant migration toward collagen type IV or reelin, an
extracellular matrix-like molecule that regulates cortical neuron
migration on radial glia (Dulabon et al., 2000). The slight increase in
migration of L1(+RSLE)-B35 cells to control and reelin-containing
supernatants was probably caused by small amounts of fibronectin in the
conditioned medium.
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Figure 1.
L1 potentiates migration of B35 neuroblastoma
cells to extracellular matrix proteins. Haptotactic cell migration was
measured in B35 cells (20,000 cells per chamber) expressing no L1 or
L1(+RSLE) toward extracellular matrix proteins or BSA for 16 hr. Each
sample was assayed in triplicate, and experiments were repeated at
least twice with similar results.
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To determine whether L1-potentiated migration to fibronectin was
mediated through an integrin-dependent mechanism, HEK293 cells were
used, because unlike B35 cells, they express defined integrins for
which function-blocking antibodies are available. HEK293 cells express
51 integrin, which functions as an RGD-dependent fibronectin
receptor, and v1 integrin, which functions as a fibronectin or
vitronectin receptor (Bodary and McLean, 1990), but they lack v3
or v5 integrins (Simon et al., 1997) or L1 (Needham et al.,
2001). HEK293 cells were transfected for transient expression with a
plasmid encoding human L1(+RSLE) or the control pcDNA3 plasmid, and
haptotactic migration was assayed toward fibronectin for 4 hr.
L1(+RSLE)-HEK293 cells exhibited increased migration to fibronectin
compared with nonexpressing cells (Fig.
2). The migration of HEK293 cells after 4 hr was greater than that of B35 cells after 16 hr. Treatment of
L1-expressing cells with antibodies against the extracellular region of
L1 (anti-L1; Neuro4), at concentrations that did not induce clustering
of L1, reduced migration to the levels of untransfected cells, whereas
an equivalent amount of nonimmune IgG did not affect migration (Fig.
2). Antibody P1D6 specific for the 5 integrin subunit (anti-5)
inhibited haptotactic migration of both L1-expressing and nonexpressing
cells toward fibronectin (Fig. 2). This antibody maps to the binding
site for the fibronectin synergy sequence (PHSRN) in 5 integrin
(Burrows et al., 1999). Antibody P4C10 against the 1 integrin RGD
binding site also inhibited migration toward fibronectin, as did a
mixture of 5 and 1 integrin antibodies (Fig. 2). Monoclonal
antibodies specific for v3 integrin had no effect on migration.
These results demonstrated that in the absence and presence of L1,
haptotactic migration toward fibronectin occurred primarily through
51 integrins in HEK293 cells.
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Figure 2.
L1 potentiates migration of HEK293 cells through
integrins. Haptotactic migration of HEK293 (10,000 cells per chamber)
expressing no L1 or L1(+RSLE) toward fibronectin was measured for 4 hr.
Cells were pretreated as follows: none (None), nonimmune
mouse IgG (NIgG), anti-L1 monoclonal antibody Neuro4
(Anti-L1), anti-integrin 5 antibody
(Anti-5), anti-integrin 1 antibody
(Anti-1), a combination of antibodies
against 5 and 1 integrins
(Anti-5+1), or
anti-integrin v3
(Anti-v3) monoclonal
antibody (each at 20 µg/ml). *Statistically significant differences
in means of treated and untreated (None) samples using
the t test (p < 0.05).
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An RGD sequence within the cell binding domain of fibronectin is the
major binding site for 51 integrin. To investigate the
requirement for RGD binding in L1-potentiated migration to fibronectin,
HEK293 cells were preincubated with the RGD-containing hexapeptide
GRGDSP (2 mM) or an RGE-containing control peptide GRGESP
that does not bind to integrins and assayed for haptotactic migration
to fibronectin. Migration of both L1-expressing and nonexpressing
HEK293 cells was completely inhibited by RGD peptides but not by RGE
peptides, consistent with an 51 integrin mechanism (Fig.
3A). Haptotactic migration of
both L1-expressing and nonexpressing B35 neuroblastoma cells toward
fibronectin was also inhibited by RGD but not RGE peptides (Fig.
3B). However, RGD peptides did not fully block migration of
L1-B35 cells, suggesting the minor contribution of an RGD-independent
mechanism. As an example, L1 might increase 51 integrin affinity
for the fibronectin synergy sequence, or alternatively L1 might
activate a non-RGD-binding integrin, such as 41 integrin, which
binds the Glu-Ile-Leu-Asp-Val (EILDV) sequence in fibronectin. In any
case, adhesion of cells was not significantly altered by RGD peptides,
because there was no decrease in total cell number on the top and
bottom sides of the filters.
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Figure 3.
RGD peptides abrogate L1-potentiated migration
toward fibronectin. HEK293 (10,000 cells per chamber)
(A) or B35 cells (20,000 cells/chamber)
expressing no L1 or L1(+RSLE) (B) were incubated
with or without hexapeptide GRGDS (RGD) or inactive
GRGES (RGE) control peptides (2 mM) for 1 hr
on ice and then assayed for haptotactic migration toward fibronectin or
BSA for 4 hr. Each sample was assayed in triplicate, and experiments
were repeated at least twice with similar results. *Statistically
significant differences in means of treated and untreated (No
Peptide) samples using the t test
(p < 0.05).
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An RGD in the L1 Ig6 domain is required for
potentiating migration
There is only one RGD sequence in the Ig6 domain of human L1
(Hlavin and Lemmon, 1991). Conservative mutation of the L1 RGD to
Lys-Gly-Glu (KGE) reduced L1-potentiated migration of HEK293 cells
toward fibronectin to the level of HEK293 cells not expressing L1 (Fig.
4). This effect was highly specific for
the RGD
KGE mutation, because L1-potentiated migration was not
adversely affected by a number of different CRASH point mutations in
other extracellular domains of L1 (R184Q in Ig2, H210Q in Ig2, E309K in
Ig3, Y784C in FN2, Y1070C in FN5) (Fig. 4). Each of these mutants is
expressed at levels equivalent to wild type on the surface of HEK293
cells and is able to recruit ankyrin normally to the plasma membrane (Needham et al., 2001). Surface expression of some of these (R184Q, E309K, Y784C) has been reported to be impaired (29-72%) in Chinese hamster ovary (CHO) cells (DeAngelis et al., 2002) and thus the altered
surface expression appears to be cell-type dependent. The R184Q and
E309K mutations are known to interfere with L1 binding to heterophilic
partners F3/F11/contactin and axonin-1/Tag-1 (DeAngelis et al., 1999);
thus the ability of wild-type L1 to stimulate migration to fibronectin
was unlikely to be mediated through these molecules. In addition, the
R184Q mutation as well as the H210Q mutation perturb homophilic L1-L1
binding (DeAngelis et al., 1999) and neurite outgrowth on L1 substrates
(Zhao and Siu, 1996), suggesting that the mechanism of L1-potentiated
migration to extracellular matrix proteins also did not involve
homophilic L1-L1 interactions. These results indicated that the RGD
sequence in the Ig6 domain of L1 was a critical determinant of
L1-potentiated cell migration to extracellular matrix.
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Figure 4.
L1-potentiated migration to fibronectin requires
RGD and RSLE sequences in L1. HEK293 cells were transfected for
transient expression of L1(+RSLE), L1(RSLE), or L1(+RSLE) with
RGDKGE or extracellular CRASH mutations. Cells were assayed for
haptotactic migration toward fibronectin for 4 hr. Each sample was
assayed in duplicate or triplicate, and experiments were repeated at
least twice with similar results. *Statistically significant
differences in means of L1 mutants compared with L1(+RSLE) using the
t test (p < 0.05).
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Neuronal L1 enhances migration through dynamin I, c-Src, PI3
kinase, and MAP kinase
The neuronal splice form L1(+RSLE) is endocytosed rapidly by
clathrin and dynamin-mediated endocytosis required for MAP kinase signaling (Kamiguchi et al., 1998; Schaefer et al., 1999; Schmid et al., 2000), whereas the non-neuronal form lacking RSLE is
endocytosed more slowly by a clathrin-independent mechanism (Long et
al., 2001). Haptotactic migration of HEK293 cells toward
fibronectin was potentiated after 4 hr by L1(+RSLE), which binds the
AP2/clathrin adapter (Kamiguchi et al., 1998), and not by the
non-neuronal form of L1 lacking RSLE (Fig. 4). Expression of the
dominant negative dynamin I (K44A) mutant, which impairs
receptor-mediated endocytosis, abolished the migration-potentiating
effect of L1(+RSLE) (Fig. 5). This mutant
also produced a small but significant inhibitory effect on migration of
HEK cells to fibronectin in accord with evidence that dynamin-dependent
internalization of 1 integrins plays a role in cell motility (Ng et
al., 1999; Pierini et al., 2000). These results suggested that
L1-potentiated migration to fibronectin in HEK293 cells after 4 hr
occurred through a mechanism involving clathrin- and dynamin-mediated
endocytosis. Cells expressing L1(RSLE) might exhibit potentiated
migration after longer migration times because this form is
internalized more slowly (Long et al., 2001); however, HEK293 cells
have a rapid rate of migration that precluded examining this
possibility.
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Figure 5.
L1-potentiated migration is regulated by dynamin-1
and c-Src. HEK293 cells were transfected with or without human
L1(+RSLE) and one of the following plasmids: pcDNA3
(Control), dominant negative dynamin-1(K44A)
(DN-Dynamin), or dominant negative c-Src(K295M)
(DN-Src). Cells were assayed for haptotactic migration
toward fibronectin or random migration to BSA for 2 hr. Each sample was
assayed in triplicate and repeated at least twice with similar results.
*Statistically significant differences in means of mutant-expressing
and control cells using the t test
(p < 0.05).
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The nonreceptor tyrosine kinase c-Src is required for endocytosis of L1
(Schmid et al., 2000), regulation of neurite outgrowth on L1 (Ignelzi
et al., 1994), and L1-induced MAP kinase activation (Schmid et al.,
2000). Expression of the dominant negative Src (K295M) mutant in
L1(+RSLE)-transfected HEK293 cells decreased L1-potentiated migration
to the level of untransfected cells (Fig. 5). To confirm the role of
c-Src in L1-potentiated haptotactic migration in neuronal cells, B35
neuroblastoma cells stably expressing L1(+RSLE) and parental B35 cells
lacking L1 were treated with PP2, a pyrazolopyrimidine inhibitor
selective for Src family kinases, under conditions that inhibit Src in
cell culture (Hanke et al., 1996). PP2 substantially inhibited the
haptotactic migration of L1(+RSLE)-B35 cells to fibronectin but did not
significantly inhibit migration of parental B35 cells (Fig.
6). PP3, an inactive structural analog,
did not affect the migration of either L1(+RSLE)-expressing or parental
B35 cells, and the inhibitor solvent DMSO (0.05%) had no significant
effect. PP2 did not completely inhibit migration of L1-expressing
cells, suggesting that there was either an Src-independent component of
L1-potentiated migration or that sufficient levels of PP2 were not
sustained over the duration of the assay.
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Figure 6.
Src kinase, MAP kinase, and PI3 kinase are
required for L1-potentiated migration of B35 cells. Migration of B35
cells expressing no L1 or L1(+RSLE) was measured toward fibronectin for
16 hr. Cells were untreated or treated with 0.05% DMSO alone or with
Src kinase inhibitor PP2 (1 µM), inactive analog PP3 (1 µM), PI3 kinase inhibitor Ly294002 (25 µM),
or MEK inhibitor UO126 (10 or 20 µM). Each sample was
assayed in triplicate, and experiments were repeated at least twice
with similar results. *Statistically significant differences in means
of treated and untreated cells using the t test
(p < 0.05).
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MAP kinase and PI3 kinase are phosphorylated and activated during L1
endocytosis through Src (Schmid et al., 2000). To determine whether MAP
kinase was important for haptotactic cell migration to fibronectin, B35
cells and L1(+RSLE)-B35 cells were treated with an inhibitor (U0126) of
the dual specificity kinase MEK, which phosphorylates and activates MAP
kinase. The inhibitor was used under conditions that produce maximal
inhibition of L1-promoted neurite growth and phosphorylation of the MAP
kinases ERK1 and ERK2 in B35 cells (Schmid et al., 2000). The MEK
inhibitor effectively suppressed migration of both parental and
L1-expressing B35 cells to fibronectin, indicating that MAP kinase was
required for haptotactic migration to fibronectin (Fig. 6). To assess
the involvement of PI3 kinase, B35 cells and L1(+RSLE)-B35 cells were
treated with the PI3 kinase inhibitor LY294002 under conditions that
inhibit ERK1,2 phosphorylation in L1-B35 cells (Schmid et al., 2000). Ly294002 significantly inhibited migration of both L1-expressing and
parental B35 cells to fibronectin, indicating that PI3 kinase also
played a role in haptotactic migration to extracellular matrix proteins.
MAP kinase activation and haptotactic migration are impaired by L1
CRASH mutations
Three known mutations in the intracellular domain of L1 occurring
in the CRASH syndrome were assessed for adverse effects on potentiating
migration to fibronectin. Established B35 cell lines were studied that
expressed L1(S1194L), L1(S1224L), or L1(Y1229H) at levels equivalent to
wild-type L1 in L1(+RSLE)-B35 cells (Needham et al., 2001). All three
mutants displayed reduced ability to potentiate haptotactic migration
to fibronectin (Fig. 7). Similar results
were observed in HEK293 cells transiently expressing these mutants
(data not shown). Attachment of B35 or HEK293 cells to the filters was
unaffected by expression of L1 mutant proteins, because the total
number of cells on both sides of the filters was the same for mutant
and wild-type L1-expressing cells after the migration assay.
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Figure 7.
CRASH mutations in the L1 cytoplasmic domain
suppress L1-potentiated migration to fibronectin. B35 cell lines stably
expressing no L1 (B35), wild-type L1(+RSLE), or L1(+RSLE) with point
mutations S1194L, S1224L, or Y1229H were assayed for haptotactic
migration to fibronectin for 24 hr. Each sample was assayed in
triplicate, and experiments were repeated at least twice with similar
results. *Statistically significant differences in means compared with
L1(+RSLE) using the t test (p < 0.05).
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To determine whether the intracellular L1 CRASH mutants were
coordinately impaired for intracellular signaling to MAP kinase, ERK2
phosphorylation was assayed in B35 cells expressing L1(+RSLE) or L1
CRASH mutants. L1 was clustered on the B35 cell surface with complexes
of Neuro4 monoclonal antibodies against the L1 extracellular domain,
followed by immunoprecipitation and immunoblotting for dually
phosphorylated, activated ERK2 as described (Schmid et al., 2000). This
antibody recognizes all L1 mutants equivalently as shown by
immunoblotting and immunolabeling (Needham et al., 2001). L1(+RSLE)-B35
cells displayed levels of ERK2 phosphorylation approximately threefold
greater than parental B35 cells as expected (Schaefer et al., 1999;
Schmid et al., 2000), whereas B35 cells expressing L1 mutants S1194L,
S1224L, or Y1229H exhibited significantly lower levels of ERK2
phosphorylation (Fig. 8). The neuronal L1 RSLE sequence in the cytoplasmic domain was also required for maximal
MAP kinase activation in B35 cells, as shown by the substantial reduction in ERK2 phosphorylation in B35 cells expressing L1(RSLE) (Fig. 8) in accord with studies using other cell types (Schaefer et
al., 1999). To address whether the RGD sequence in the L1 Ig6 domain
was required for MAP kinase activation induced by L1 clustering, B35
neuroblastoma cells expressing the RGDKGE mutation were analyzed similarly for ERK2 phosphorylation. Cells expressing the L1(KGE) mutant
displayed a significantly reduced level of ERK2 phosphorylation compared with wild-type L1(+RSLE) (Fig.
9). Thus effective MAP kinase activation
and potentiated migration required determinants of the L1 cytoplasmic
domain, including the RSLE motif necessary for L1 internalization
through the clathrin pathway, and additionally the extracellular RGD
sequence in the L1 Ig6 domain.
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Figure 8.
MAP kinase activation is impaired by L1 CRASH
mutations. Parental B35 cells were cotransfected for transient
expression of HA-tagged ERK2 and plasmids encoding L1(+ or RSLE) or
indicated L1 mutants. Cells were stimulated for 10 min with NIgG or L1
monoclonal antibody Neuro4 complexed with F(ab')2 fragments
of anti-mouse IgG (Fc-specific). HA-tagged ERK2 was immunoprecipitated
with anti-HA antibody and blotted with anti-Active MAPK antibodies
(p ERK2), and then the amount of
immunoprecipitated ERK2 protein was analyzed by stripping and reprobing
blots with ERK2 antibodies (ERK2) as shown in a
representative experiment in the panels below. Densitometric scanning
determined the amount of ERK2 phosphorylation relative to ERK2 protein
in L1 antibody-treated and NIgG-treated cells. Results of multiple
experiments were averaged to obtain the mean values with SEM shown in
the panel above. ERK2 phosphorylation is expressed relative to basal
levels of ERK2 phosphorylation in nonimmune IgG-treated B35 cells
(NIg). *Statistically significant differences in the
mean ERK2 phosphorylation of L1 mutant-expressing B35 cells compared
with L1(+RSLE)-B35 cells were evaluated by the t test
(p < 0.02).
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Figure 9.
Antibody-induced endocytosis of L1 and 1
integrins in L1-expressing HEK cells. HEK293 cells were transfected for
transient expression with L1(+RSLE)
(A-C,
E-G,
I-K) or mutant L1(KGE)
(M-O) plasmids. Confocal microscopy images of
cells are labeled green for 1 integrin
(A, E, I,
M), red for wild-type or mutant L1
(B, F, G), and
yellow for colocalized 1 integrin and L1
(C, G, K,
O). Nontransfected cells were labeled
green for 1 integrin alone (D,
H, L). Live HEK cells were incubated with
antibodies against L1 and 1 integrin for 0 min
(A-D), 5 min
(E-H,
M-O), or 60 min
(I-L). After 5 min, increased internal staining
of 1 integrins was seen in L1-transfected cells (E,
arrowheads) compared with nontransfected cells
(H). Long arrow in
J indicated larger endocytotic vesicles of wild-type L1
resembling multivesicular bodies. L1(KGE) mutant cells
(M) did not show increased 1 integrin
internalization after 5 min. Images show representative cells from
multiple experiments. Scale bars, 10 µm.
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Endocytosis of L1 and 1 integrins
The results above raised the possibility that L1 might act by
stimulating the endocytosis of 1 integrins. To test this,
L1(+RSLE)-transfected and nontransfected HEK293 cells were treated with
L1 antibody 6096 under conditions sufficient to cluster cell surface L1
and also with the 1 integrin-activating antibody MAB2000, and then internalization was followed by double-label indirect
immunofluorescence. As seen by confocal microscopy, L1 and 1
integrins were initially present on the surface of L1-transfected cells
(Fig. 9A,B) (0 min). A similar
distribution of 1 integrin was seen in untransfected cells (Fig.
9D). In image overlays, L1 and 1 integrins appeared to
represent primarily distinct molecular populations on the cell surface
(Fig. 9C). After 5 min of antibody treatment, 1 integrins and L1 were observed in small cytoplasmic vesicles (Fig.
9E,F, arrows), which
were probably early endosomes as judged from their size, location, and
time of appearance (Ng et al., 1999). L1 and 1 integrins did not
colocalize to a major degree within these endocytic vesicles (Fig.
9G). Interestingly, substantially more 1
integrin-containing vesicles were seen after 5 min in L1-transfected cells (Fig. 9E) compared with nontransfected cells (Fig.
9H). Image analysis revealed that ~2.5-fold more
1 integrin accumulated in internalized vesicles in
L1(+RSLE)-expressing cells than in untransfected HEK cells (no L1)
(Fig. 10). After 60 min, L1 antibody complexes accumulated in larger endocytotic vesicles with the appearance of multivesicular bodies (Fig. 9J, long
arrow) in accord with previous results (Kamiguchi et al.,
1998; Needham et al., 2001). At this time 1 integrins were still
present in the small endocytic vesicles and few colocalized with L1
(Fig. 9I,K).
Nontransfected cells displayed less prominent internalization of 1
integrin than L1 transfected cells at 60 min (Fig. 9L) or 40 min (data not shown). These results were consistent with the
interpretation that L1 clustering induced an accumulation of 1
integrins in early endocytic vesicles.
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Figure 10.
Mean fluorescent pixel density of internalized
1 integrin in confocal micrographs from experiments of Figure 9.
After 5 min at 37°C there was a statistically significant increase in
internalized 1 integrins in L1(+RSLE)-HEK293 cells compared with
HEK293 cells not expressing L1 (*) and a decrease in internalized
1 integrins in L1(KGE)-expressing cells compared with
L1(+RSLE)-HEK293 cells (**) by the t test (one-tailed;
p < 0.05).
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This increase was dependent on the RGD sequence in the L1 Ig6 domain,
because clustering of the L1(KGE) mutant expressed in HEK293 cells did
not elicit enhanced 1 integrin accumulation at 5 min of incubation
(Fig. 9M), although the L1 mutant appeared to be
internalized (Fig. 9N). Image analysis revealed that
L1 (KGE)-expressing cells internalized approximately the same amount of
1 integrin as untransfected HEK293 cells (no L1) (Fig. 10). It
should be noted that a physical association between L1 and 1
integrin was also not detected by co-immunoprecipitation from 1%
Triton X-100 extracts of L1(+RSLE)-transfected HEK293 cells plated on
fibronectin with or without antibody-induced L1 clustering or from
extracts of postnatal day 7 mouse cerebelli (data not shown).
L1 and 1 antibodies perturb neuronal migration in acute
cerebellar slices
To evaluate the significance of L1 and 1 integrins in neuronal
cell migration in a bioassay, we tested the ability of function blocking L1 and 1 integrin antibodies to perturb migration of cerebellar granule neurons in mouse cerebellar slice cultures. L1 is
expressed on postmitotic premigratory and migrating cerebellar granule
neurons (Persohn and Schachner, 1987), and L1 antibodies have been
reported to impede granule cell migration in explants (Lindner et al.,
1986; Chuong et al., 1987; Crossin et al., 1990). Extracellular matrix
proteins such as laminin, thrombospondin, and tenascin are also present
in the developing cerebellum and participate in aspects of cell
migration (O'Shea et al., 1990; Husmann et al., 1992; Liesi et al.,
1992; Fishman and Hatten, 1993). To ask whether L1 and 1
integrins could cooperate in modulating granule cell migration under
conditions reflective of an in vivo environment, mouse
cerebellar neurons generated on postnatal day 4 were pulse labeled with
BrdU in vivo as described (Anton et al., 1996).
Labeled cerebella were sectioned coronally into 200 µm slices and
incubated in culture for 21 hr in the presence and absence of L1 and
1 integrin antibodies. After 21 hr the extent of radial migration of
BrdU-labeled neurons from the EGL into the IGL was assessed after
fixation by immunofluorescence staining with BrdU antibodies. At time
0, labeled cells were present almost exclusively in the EGL (Fig.
11A). After 21 hr,
many labeled cells had migrated into the IGL with some remaining in the
EGL (Fig. 11B). Nonimmune IgG had no effect on
granule cell migration (Fig. 11C). In the presence of either
polyclonal L1 antibodies (Fig. 11D) or monoclonal
1 integrin antibodies (E), there was little visible retardation in granule cell migration. However, in the presence
of both L1 and 1 integrin antibodies, migration was strikingly
inhibited, as manifested in an accumulation of labeled neurons in the
EGL (Fig. 11F). Cell migration was not inhibited completely, because labeled cells were also present in the top part of
the IGL, indicating the involvement of other factors. Quantitative
evaluation of the effect of antibodies on migration of labeled neurons
was made by measurement of the migration index; i.e., the radial
distance (perpendicular to the pial surface) that BrdU-labeled granule
cells migrated relative to the width of the cerebellar cortex in the
same region. L1 or 1 integrin antibodies alone produced a small but
significant decrease in the migration index. The inhibition of
migration by our L1 antibody (17%) after 21 hr in vitro was
in general accord with the inhibition (33%) reported by Lindner et al.
(1986) for slices from P10 mice using a different L1 antibody after
3 d in vitro. A combination of the two antibodies
decreased the migration index to a greater than additive extent (Table
1). The percentage of BrdU-labeled cells
in the EGL was also measured and was found to increase to a greater
extent in the presence of both antibodies compared with either antibody
alone (Table 1). By this measurement the 1 integrin antibody but not
the L1 antibody significantly reduced migration when used alone.
Differences in age of mice, antibodies, labeling, and culture
conditions may have contributed to the smaller inhibition of migration
produced by L1 antibodies in our experiment compared with Lindner et
al. (1983). The results support a potential role for L1 and 1
integrins in coordinate regulation of radial migration of cerebellar
granule cells.
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Figure 11.
Effect of L1 and 1 integrin antibodies on
migration of newly generated BrdU-labeled neurons in cerebellar slice
cultures. BrdU-labeled cell distribution in postnatal day 4 mouse
cerebellar slices after 0 hr in culture (A),
after 21 hr in culture (B), after 21 hr in
culture with nonimmune IgG (C), after 21 hr in
culture with L1 polyclonal antibody 6096 (D),
after 21 hr in culture with 1 integrin monoclonal antibody CD29
(E), and after 21 hr in culture with both L1
antibody 6096 and 1 integrin antibody CD29
(F). At 0 hr, BrdU-labeled cells were restricted
primarily to the EGL (A). In the next 21 hr,
newly generated BrdU-positive neurons migrated into the IGL under
control conditions (B) or in the presence of
nonimmune IgG (C). In the presence of both L1 and
1 integrin antibodies (F), migration was
impeded, and neurons did not migrate as far as neurons in the presence
of only L1 antibody (D) or 1 integrin antibody
(E). EGL, External granular layer;
ML, molecular layer; IGL, internal
granular layer. The borders of the Purkinje cell layer and ML/IGL were
indistinct and thus were not delineated in this figure. Scale bar
(shown in F for
A-F), 50 µm.
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DISCUSSION |
This study describes a novel function for L1 as a potentiator of
neuronal cell migration toward extracellular matrix proteins through
1 integrins by a MAP kinase signaling pathway dependent on
receptor-mediated endocytosis. L1 potentiated haptotactic migration through the fibronectin-specific integrin 51 in HEK293 cells but
appeared capable of activating other integrin subclasses, because
migration of B35 neuroblastoma cells to laminin and vitronectin was
also stimulated by L1. Potentiated migration depended on determinants of L1 endocytosis: dynamin I, c-Src, and the AP2/clathrin binding sequence (RSLE) in the neuronal form of L1. Determinants within the
cytoplasmic domain of L1 that are mutated in the CRASH syndrome and the
RGD sequence in the extracellular Ig6 domain of L1 were essential for
potentiating cell migration. Surprisingly, clustering of cell surface
L1 caused enhanced 1 integrin accumulation into endocytic vesicles
in addition to L1 internalization. Results were consistent with a model
in which L1 potentiated neuronal cell migration toward extracellular
matrix proteins through L1 and 1 integrin endocytosis leading to MAP
kinase activation. Function-blocking L1 and 1 integrin antibodies
cooperatively retarded the migration of granule neurons in early
postnatal mouse cerebellar slices, underscoring the potential for a
functional L1 integrin interaction in neuronal migration in
vivo.
Several lines of evidence support the interpretation that potentiation
of integrin-mediated haptotactic migration by L1 derived from its
ability to activate MAP kinase through endocytosis of L1. (1) Dominant
negative dynamin I (K44A), which suppresses L1 endocytosis and MAP
kinase activation (Schaefer et al., 1999; Schmid et al., 2000),
inhibited L1-potentiated migration to fibronectin. (2) The L1 RSLE
sequence, which mediates clathrin-dependent rapid L1 endocytosis
(Schaefer et al., 1999; Long et al., 2001), was required for
L1-potentiated migration as well as MAP kinase activation. Because the
ERK inhibitor PD98059 does not block L1 internalization, MAP kinase
activation occurs subsequent to L1 endocytosis (Schmid et al., 2000).
Because the L1(-RSLE) form recycles more slowly than L1(+RSLE), it
remains to be determined whether this form would stimulate
migration and MAP kinase activity at longer times than were evaluated
in our assays. (3) Inhibition of c-Src kinase by PP2 or the
kinase-inactive Src (K295M) mutant blocked L1-potentiated migration.
c-Src is required for dynamin-mediated internalization of L1 (Schmid et
al., 2000) and other receptors (Carpenter, 2000). Interestingly, c-Src
can phosphorylate tyrosine residues in components of the endocytic
machinery, because 2 adrenergic stimulation causes c-Src-induced
tyrosine phosphorylation of dynamin (Ahn et al., 1999; Luttrell et al.,
1999), and epidermal growth factor stimulation induces c-Src-induced
tyrosine phosphorylation and membrane recruitment of clathrin (Wilde et
al., 1999).
L1-potentiated migration and MAP kinase activation may also depend on
its ability to stimulate the internalization of 1 integrins as
observed in this study. One model consistent with all of the evidence
is that transient association of L1 with 1 integrin through the L1
RGD sequence induces endocytosis of both L1 and 1 integrins, leading
to activation of MAP kinase through 1 integrin signaling to
potentiate migration toward extracellular matrix proteins. In accord
with this model, mutation of the L1 RGD sequence coordinately abolished
its ability to stimulate the accumulation of 1 integrin into
endocytic vesicles and to activate MAP kinase. Furthermore, the time of
maximal ERK phosphorylation induced by L1 (10 min) (Schmid et al.,
2000) closely followed the time of maximal 1 integrin accumulation
into endocytic vesicles (5 min). 1 integrins have been shown to
internalize with similar kinetics into early endosomes in non-neuronal
cells by a dynamin-dependent pathway (Ng et al., 1999). The possibility
that L1 signals to MAP kinase through 1 integrins is consistent with
the common intermediates shared by L1 and early integrin signaling
pathways (Src, PI3 kinase, Rac1, Vav-2, ERK; see introductory remarks). Indeed PI3 kinase was shown here to be required for haptotactic migration of B35 cells to fibronectin. PI3 kinase may contribute to
migration through its ability to influence integrin endocytosis or
recycling (Siddhanta et al., 1998; Ng et al., 1999), because its
product PI-3,4,5 phosphate can bind the plextrin homology domain of
dynamin I and stimulate dynamin GTPase activity in vitro (Barylko et al., 1998); however, its exact function in haptotactic migration has not been determined.
L1 also potentiated migration of B35 cells toward laminin, which can
bind in a non-RGD manner to certain integrins such as 61. An
interesting possibility is that interactions between L1 and an RGD
binding integrin might stimulate migration by inside-out signaling to
increase the affinity of non-RGD binding integrins such as 61 for
extracellular matrix. Such cross-talk among integrin subclasses to
increase integrin affinity is established (Simon et al., 1997; Blystone
et al., 1999) and can be mediated by signaling from Ig-like cell
adhesion molecules such as PECAM (Chiba et al., 1999). Alternatively,
L1 may regulate non-RGD-binding integrins through its ability to
interact with integrins through its third fibronectin III domain
(Silletti et al., 2000) or through interaction with other cell
surface molecules such as the tetraspan cell surface molecule CD9,
which can enhance laminin-induced cell migration and neurite growth
through 61 integrin (Schmidt et al., 1996).
Current and previous findings do not preclude a mechanism in which
internalization of only L1 and not 1 integrins is the critical
factor in MAP kinase activation. Nonetheless 1 integrins are clearly
required for L1-potentiated migration as shown by antibody perturbation
experiments, but they could perform a function other than signaling to
MAP kinase. For example, integrin endocytosis at the rear of cells may
facilitate detachment from the substrate necessary for forward
migration, and together with integrin recycling to the leading edge,
endocytosis may help establish an adhesive gradient needed for
directional cell motility (Bretscher, 1996; Lauffenburger and
Horwitz, 1996; Condic and Letourneau, 1997).
There is increasing evidence that signaling to MAP kinase can occur
from endosomal compartments (Ceresa and Schmid, 2000). Elevated levels
of internalized L1 or 1 integrins could prolong signaling from
endocytic compartments or place receptor complexes in an appropriate
cellular location to interact with downstream signaling molecules
necessary for stimulating motility. The substrates of MAP kinase
important for L1-potentiated haptotactic cell migration have not been
identified but might include cytoplasmic targets such as myosin light
chain kinase, the phosphorylation of which by ERK1,2 has been shown to
be required for collagen-dependent FG carcinoma cell motility
(Klemke et al., 1997), or nuclear transcription factors modulating
expression of genes important for cell motility such as those encoding
cytoskeletal proteins.
The RGD sequence in the L1 Ig6 domain was necessary for potentiating
haptotactic migration to fibronectin through 51 integrin, for
stimulating 1 integrin accumulation in endocytic vesicles, and for
MAP kinase activation. The L1 RGD sequence may directly interact with
RGD-binding integrins either in cis or trans, or it may simply stabilize a conformation necessary for the signaling properties of L1. However, structural modeling predicts that the L1 RGD
sequence is located at the molecular surface within a turn, where
it would be available for binding to integrins (Drescher et al., 1996).
Because we did not observe colocalization of L1(+RSLE) and 1
integrins in internalized vesicles and did not detect a stable
association between these proteins by co-immunoprecipitation from
transfected HEK293 cells or cerebellar extracts, such an association
within the plasma membrane might be transient or low in affinity. A
cis-interaction of the L1 and integrins 51, v3, and 91 mediating adhesion in M21 melanoma cells that requires two
dibasic sequences in the third fibronectin III domain of L1 but could
also involve the RGD sequence has been described previously (Silletti
et al., 2000). The third fibronectin III domain was shown to be
critical for neurite outgrowth by cerebellar neurons in
vitro (Stallcup, 2000). A potential cis-interaction
between L1 and integrins was implicated previously in neurite growth of sensory neurons (Felsenfeld et al., 1994) and PC12 cells (Yip and Siu,
2001). Through its RGD sequence, L1 has also been shown to interact
functionally, although not physically, with several RGD-binding
integrins (51, v1, 41, v3, and IIb3) to mediate adhesion and neurite outgrowth on L1 substrates (Moos et al.,
1988; Ruppert et al., 1995; Ebeling et al., 1996; Montgomery et al.,
1996; Felding-Habermann et al., 1997; Yip et al., 1998). In addition to
enhancing 1 integrin internalization, potential binding of the
L1-RGD sequence to integrins might have the effect of increasing
integrin affinity for ligands, inasmuch as low concentrations of RGD
peptides superactivate integrin affinity for RGD-containing ligands
(Legler et al., 2001).
While our studies were in progress, it was reported that ectodomain
cleavage of L1 by ADAMs (A Disintegrin and Metalloproteinase) can promote migration of transfected CHO cells through v5
integrin independent of the L1 cytoplasmic domain (Mechtersheimer et
al., 2001). Our experiments do not rule out an involvement of
metalloprotease cleavage of L1 in the mechanism of haptotoactic
migration. However, in our studies with B35 neuroblastoma cells and
HEK293 cells, the cytoplasmic domain of L1 plays an essential role in
stimulating cell migration by providing linkage to AP2/clathrin
necessary for signaling and perhaps also to the actin cytoskeleton
through ankyrin as shown by impairment of potentiated migration by
particular CRASH mutations in the L1 cytoplasmic domain.
Three mutations in the intracellular domain of L1 (S1194L, S1224L
TOP
ABSTRACT
INTRODUCTION
