* Department of Pharmacology, New York University Medical Center, New York 10016; SUGEN, Inc., Redwood City,
California 94063-4720; and § Becton Dickinson Research Center, Becton Dickinson and Company, P.O. Box 12016, Research
Triangle Park, North Carolina 27709-2016
Receptor protein tyrosine phosphatase (RPTP
) is expressed as soluble and receptor forms
with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the
C domain (
C) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate,
CFS was less effective in supporting cell adhesion, but
it was a more effective promoter of neurite outgrowth
than
CF.
S had no effect by itself, but it potentiated
neurite growth when mixed with
CF. Neurite outgrowth induced by
CFS was inhibited by antibodies
against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound
CFS. NIH3T3 cells transfected to express
CFS on their surfaces
induced neuronal differentiation in culture. These results suggest that binding of glial RPTP
to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.
Abetter understanding of molecular mechanisms of
cell-cell interactions in the nervous system is
emerging from studies of neural cell adhesion molecules (CAMs)1 and other receptors that transmit signals
across the plasma membrane to control cell behavior (Edelman and Crossin, 1991 Phosphorylation of proteins inside cells on tyrosine is an
important mechanism for mediating transmembrane signaling that is regulated by the balanced actions of protein
tyrosine kinases and protein tyrosine phosphatases (Schlessinger and Ullrich, 1992 We have previously identified contactin as a neuronal
receptor for the C domain of RPTP To gain further insight into the function of the individual isoforms of RPTP Cells
COS7 cells, 293 cells, and an NIH-3T3 subline, 2.2 cells, which do not express endogenous EGF receptors (Honegger et al., 1987 Proteins and Antibodies
Chick Ng-CAM and N-CAM were purified from membrane fractions of
embryonic day 14-16 chick embryo brains using immunoaffinity chromatography columns as described (Grumet and Edelman, 1988 Human Fc Fusion Proteins
Recombinant human Fc fusion proteins of the extracellular regions of
RPTP Neurite Outgrowth Assay
Neurite outgrowth assays were performed using 35-mm petri dishes coated
with different protein substrates (Friedlander et al., 1994
Immunofluorescence
Tectal neurons that adhered and extended processes on substrates coated
with Protein Binding Assay
1 µl of various proteins (10 µg/ml in PBS) was incubated on 35-mm petri
dishes for 1 h. The dishes were washed with PBS and blocked with 1%
BSA for 1 h and then incubated with culture supernatant from COS7 cells
expressing RPTP DNA Constructs, Transfection, and
Co-precipitation Studies
Full length chick Nr-CAM cDNA was constructed from Nr-CAM or contactin cDNAs were transfected into COS7 cells using lipofectamine. After 72 h, the cells were lysed in extraction buffer (50 mM Hepes,
pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1.5 mM MgCl2,
10% glycerol, 20 mM PMSF, 10 µg/ml leupeptin, 0.1 M NaF, 10 mM sodium
tetraphosphates, and 0.6 mM sodium vanadate) and cleared for 30 min in
a microfuge. For the mixtures of the individual cell lysates, separate cultures of COS7 cells transfected to express Nr-CAM or contactin were extracted and then mixed in a 1:1 ratio. For experiments involving mixtures
of cells expressing either Nr-CAM or contactin, the cells were removed
from tissue culture dishes with 0.25% trypsin and 1 mM EDTA (GIBCO
BRL) 24 h after transfection and combined in new dishes at a 1:1 ratio. After incubation for an additional 48 h, the cultures were extracted as described. In all cases, protein was precipitated by protein A that had been
preincubated with antibodies against Nr-CAM or contactin, or with Fc fusion proteins.
Coprecipitation of Nr-CAM with contactin from detergent extracts of
E14 chick embryo brain membranes was performed using Pansorbin (Calbiochem Corp., La Jolla, CA; Zisch et al., 1995 Binding of various Fc fusion proteins to COS7 cells transiently transfected with contactin or Nr-CAM was measured by scintillation counting
after incubation with 125I-protein A (Peles et al., 1995 Neurite Outgrowth on Transfected Cell Monolayer
Monolayer cultures of To analyze functions of the extracellular region of RPTP
Table I.
Binding of Neurons to
Studies using Neurite Outgrowth Promoted by The striking observation that neurite outgrowth on The results implicate Nr-CAM as well as contactin in neurite outgrowth of chick tectal neurons induced by RPTP
Nr-CAM and Contactin Bind to Distinct
Domains in RPTP In view of the interactions of RPTP
Given that tectal neurite outgrowth on RPTP Table II.
Binding of ; Doherty and Walsh, 1994
). Neurons express various CAMs, including several members of
the Ig superfamily that also contain fibronectin type III repeats (Grumet, 1991
; Rathjen and Jessel, 1991
). Most proteins in this family such as Ng-CAM (Grumet, 1992
) or its
likely mammalian homolog L1 (Schachner et al., 1990
) are
expressed after neurons become postmitotic. Nr-CAM is
closely related to Ng-CAM, consisting of six Ig domains
and five fibronectin type III repeats, as well as a single
transmembrane region and a cytoplasmic domain (Grumet et al., 1991
; Kayyem et al., 1992
) that can bind to the cytoskeletal protein ankyrin (Davis and Bennett, 1994
). Despite structural similarities between these two proteins,
Ng-CAM/L1 is a much more potent promoter of neurite
growth than Nr-CAM (Grumet and Sakurai, 1996
), whereas
Nr-CAM appears to be important for axonal guidance in
regions such as the floor plate of the spinal cord (Stoeckli and Landmesser, 1995
). In contrast to these transmembrane CAMs, contactin/F11/F3, which contains six Ig domains and four fibronectin type III repeats, is anchored in
the plasma membrane by a glycophosphatidylinositol (GPI)
linkage (Ranscht, 1988
; Brummendorf et al., 1989
; Gennarini et al., 1989
). Although contactin lacks a cytoplasmic
domain, it has been implicated in transmembrane signaling in neurons (Pesheva et al., 1993
), most likely by associating with transmembrane and cytoplasmic proteins (Olive et al., 1995
; Zisch et al., 1995
).
). Receptor-like protein tyrosine
phosphatase
(RPTP
; Krueger and Saito, 1992
; Levy et
al., 1993
) is expressed primarily in the nervous system and
is synthesized by glial progenitors, radial glial cells, and astrocytes (Canoll et al., 1993
; Milev et al., 1994
; Engel et al.,
1996
; Meyer-Puttlitz et al., 1996
; Sakurai et al., 1996
). It consists of a carbonic anhydrase domain (C) in its NH2-terminal region, followed by a fibronectin type III repeat (F), a
long cysteine-free spacer region (S) in its extracellular region, and two phosphatase domains in its intracellular part (Barnea et al., 1994b
; Maurel et al., 1994
). There are three
splicing isoforms of RPTP
, one that is secreted and two
that are membrane bound, which differ by a sequence of
860-amino acid residues. Proteins corresponding to all
three forms have recently been detected in the developing
nervous system and in glioma cell lines (Sakurai et al.,
1996
). The secreted form, also named phosphacan (Rauch et al., 1991
; Maurel et al., 1994
), as well as the long receptor form, are chondroitin sulfate proteoglycans (Barnea et
al., 1994a
; Shitara et al., 1994
), while the short receptor
form is detected primarily without glycosaminoglycan
(Sakurai et al., 1996
). Receptor forms are expressed on
glial progenitors and radial glial cells, while the soluble
form is detected in developing fiber tracts associated with
more mature astroglia (Canoll et al., 1993
; Milev et al.,
1994
; Canoll et al., 1996
). The expression of receptor forms changes moderately during neural development,
while expression of the soluble form increases dramatically (Meyer-Puttlitz et al., 1995
; Sakurai et al., 1996
).
, raising the possibility that the interaction between contactin and RPTP
may
generate bidirectional signals between neurons and glia
(Peles et al., 1995
). All three isoforms of RPTP
contain the
C domain, suggesting that they all interact with contactin.
We also showed that the soluble proteoglycan form of
RPTP
, phosphacan, can bind to neurons and inhibits neurite outgrowth in culture (Milev et al., 1994
). This binding
was shown to be mediated, at least in part, by Ng-CAM
and N-CAM (Milev et al., 1994
). Thus, it is reasonable to
hypothesize that neuron-glia interactions mediated by the
different isoforms of RPTP
involve different CAMs and
receptors on neurons.
on cell interactions, we began a
functional analysis of the short receptor form, which is the
major receptor form expressed on astrocytes and glioma
cells (Canoll et al., 1996
; Sakurai et al., 1996
). Astrocytes
can support neurite outgrowth and modulate neuronal differentiation, and our data suggest that some of these activities may be mediated by specific interactions of RPTP
on astrocytes with contactin and Nr-CAM expressed on neurons.
Materials and Methods
), were maintained in DME medium (Biowhittaker, Inc., Walkersville, MD) with 10%
FCS (GIBCO BRL, Gaithersburg, MD). The
CFS/EK chimera construct, which has the extracellular region of RPTP
short form fused with
the intracellular region of the EGF receptor kinase, was retrovirally infected into 2.2 cells, and stable transformants were established by G418
selection (Peles et al., 1995
). For transient expression of
CFS/EK, COS7
cells were transfected with the cDNA using lipofectamine reagent (GIBCO
BRL) and used for analysis 72 h after transfection. IMR32 cells, a human
neuroblastoma cell line, were cultured as described (Peles et al., 1995
).
Primary chick tectal neurons were prepared from embryonic day 9 chick embryos, and rat cerebellar cells and cortical cells were prepared from
postnatal 2-4-d-old rats (Grumet and Edelman, 1988
).
); Nr-CAM
was purified from the same extract using the anti-Nr-CAM monoclonal
antibody, C3B23A7 (generously provided by Dr. Jeffrey Denburg, University of Iowa, Iowa City, IA; Denburg et al., 1995
). Rat L1 was purified
from membrane fractions of postnatal 7 d rat brains (Friedlander et al.,
1994
). Polyclonal antibodies against N-CAM, Ng-CAM (Grumet and
Edelman, 1988
), Nr-CAM (Grumet et al., 1991
), and their Fab
fragments
were prepared as described. Purified human contactin and anti-human
contactin polyclonal antibody were prepared as described (Reid et al.,
1994
). Fab
fragments of rabbit antibodies and monoclonal antibodies
against chick contactin were generously provided by Dr. Fritz Rathjen
(Max-Delbrueck-Centrum for Molecular Medicine, Berlin-Buch, Germany; Rathjen et al., 1987
).
were expressed in either COS7 cells (for transient transfection) or
293 cells (for stable) and purified from culture supernatants using protein
A beads (Zymed Labs., Inc., S. San Francisco, CA).
C (amino acids from
1 to 313),
CF (amino acids from 1 to 415), and
F (amino acids from 301 to 414) fusion proteins were prepared as described (Peles et al., 1995
).
cDNAs encoding
CFS (amino acids from 1 to 630),
FS (amino acids
from 301 to 630), and
S (amino acids from 415 to 630) were synthesized
by PCR and ligated with cDNA of human Fc portion in pCDM8 vector in
frame. Nucleotide sequences were confirmed by sequencing. Protein concentrations were determined by Western blotting using anti-human Fc antibody (Jackson ImmunoResearch Labs., Inc., West Grove, PA) with kit-Fc
fusion protein as a control. Human contactin-Fc fusion protein was prepared from culture supernatant of COS7 cells transiently transfected with
human contactin-Fc cDNA using lipofectamine reagent (Peles et al.,
1995
).
). Chick primary
tectal neurons (5 × 104 cells) or IMR32 cells (2.5 × 104 cells) were incubated for 48 h in DME/F12 medium (Biowhittaker, Inc.) supplemented
with ITS+ (Collaborative Research Inc., Lexington, MA) and fixed with
Hanks/balanced salt solution (GIBCO BRL) /2% paraformaldehyde/3%
sucrose. Treatment with phospho-inositol specific phospholipase C (PIPLC; generous gift of Dr. J. Salzer, New York University Medical Center,
New York) was performed as described (Peles et al., 1995
). When antibodies were used, they were added to cultures 3 h after seeding the neurons.
After fixation, pictures were taken using a microscope (Diaphot; Nikon
Inc., Melville, NY), and neurite lengths were measured on the pictures.
Neurites that were more than one cell diameter were measured, and only the longest neurite for each cell was considered. More than 100 cells for
each substrate were quantified. Data were analyzed with Microsoft Excel,
and the percentage of neurons with neurites longer than a given length
was plotted in Figs. 3, 4, and 8.
Fig. 3.
Inhibition by anti-contactin antibodies of neurite outgrowth induced by CFS. Primary chick tectal cells were plated
on dishes coated with
CFS fusion protein (80 µg/ml) and cultured for 48 h in the presence of Fab
fragments (500 µg/ml) of
nonimmune (A) or anti-contactin polyclonal antibody (B). The
plates were fixed and photographed. Quantification of lengths of
tectal cell neurites on
CFS fusion protein (C) in the presence of
100 µg/ml Fab
fragments of nonimmune and anti-contactin polyclonal antibodies, or no added Fab
. The percentage of neurons
with neurites longer than a given length in µm was determined as
described in Materials and Methods. The average neurite lengths
without Fab
were 67 ± 8 µm (n = 100), with normal rabbit 73 ± 7 µm (n = 57), and with anti-contactin 16 ± 2 µm (n = 82). This
level of anti-contactin produced maximal inhibition insofar as
higher levels of anti-contactin did not yield greater levels of inhibition; the average lengths at 300 and 500 µg/ml were 22 ± 2 (n = 99) and 25 ± 3 µm (n = 119), respectively. Bar, 100 µm.
[View Larger Versions of these Images (97 + 94 + 16K GIF file)]
Fig. 4.
Inhibition by anti-
Nr-CAM antibodies of neurite extension induced by
CFS. Primary chick tectal
cells were plated on dishes coated with
CFS fusion protein (80 µg/ml) and cultured
for 48 h in the presence of
Fab
fragments (500 µg/ml)
of nonimmune (A) or anti-
Nr-CAM polyclonal antibody (B). The plates were
fixed and photographed.
Quantification of lengths of
tectal cell neurites on
CFS
fusion protein (C) and on
Ng-CAM (D) in the presence
of Fab
fragments. The average neurite lengths on
CFS
(C) treated with Fab
were
59 ± 4 µm (n = 193) for normal rabbit, 55 ± 2 µm (n = 302) for anti-Ng-CAM, and 16 ± 3 µm (n = 93) for anti-NrCAM. The average neurite
lengths on Ng-CAM (D)
treated with Fab
were 83 ± 5 µm (n = 198) for normal
rabbit, 34 ± 2 µm (n = 118) for anti-Ng-CAM, and 80 ± 5 µm (n = 187) for anti-Nr-CAM. The percentage of neurons with neurites
longer than a given length in micrometers was determined as described in Materials and Methods. The results of one representative experiment are shown, and similar results were obtained in five different experiments. Anti-Nr-CAM polyclonal antibody, inhibited neurite outgrowth on Nr-CAM substrate completely at the concentration of 500 µg/ml (data not shown). Bar, 100 µm.
[View Larger Versions of these Images (108 + 106 + 15 + 16K GIF file)]
Fig. 8.
S induces neurite growth when combined with
CF.
Primary chick tectal cells (5 × 104 cells) were plated on purified
CFS,
CF,
S (40 µg/ml), or on combinations (40 µg/ml final concentration) of the latter two as noted in the right panels of the figure. After 48 h, cultures were fixed and photographed. The graph
shows quantification of the lengths of neurites on
CF and
CFS
and on mixtures of
CF +
S at different ratios. Neurite lengths
were measured and analyzed as described in Materials and Methods. The average neurite lengths were:
CFS, 73 ± 7 µm (n = 109);
CF, 26 ± 2 µm (n = 148); 1X[CF] + 3X[S], 67 ± 6 µm (n = 161); 2X[CF] + 2X[S], 39 ± 3 µm (n = 153); and 3X[CF] + 1X[S], 27 ± 2 µm (n = 192). Representative data are shown, and
similar results were obtained in three experiments. Bar, 100 µm.
[View Larger Versions of these Images (152 + 19K GIF file)]
CFS, or with 10 µg/ml polylysine followed by 40 µg/ml laminin
(Becton Dickinson, Bedford, MA), were fixed as described above, treated
with PBS/100 mM glycine, and blocked with PBS/2% goat serum. Double
staining was performed using 30 µg/ml monoclonal anti-F11 (Rathjen et
al., 1987
) and 1:100 dilution of polyclonal anti-Nr-CAM antiserum (Grumet et al., 1991
) for 1 h, followed by treatment for 1 h with 1:250 dilutions
of lissamine-labeled anti-mouse Ig and fluorescein labeled anti-rabbit Ig
(Jackson ImmunoResearch Labs., Inc.). After washing, fluorescence for
lissamine and fluorescein was visualized in a microscope (Diaphot; Nikon Inc.) and recorded with appropriate filters using a Sony CCD camera interfaced with a frame grabber (AG-5; Scion Corp., Frederick, MD) in a
PowerMacintosh 7100 computer.
fusion proteins (0.5-1 µg/ml) for 1 h. After washing the
dishes with PBS for 5 min, the proteins were fixed by treatment with 3%
paraformaldehyde/PBS. To visualize the bound fusion protein, incubations were performed with biotinylated anti-human Fc followed by avidin
conjugated with alkaline phosphatase and the alkaline phosphatase substrate, according to the manufacturer's specifications (Vector Labs, Inc.,
Burlingame, CA). All incubations were performed at room temperature.
Reaction products are seen as blue/black deposits on the petri dish and
were scanned using Silverscan III, analyzed with Adobe photoshop and
NIH Image 1.54 software. Anti-human Fc antibody was spotted and detected as a control substrate.
gt 11 clones
(Mauro et al., 1992
) using Bluescript II KS (Stratagene, La Jolla, CA) as a
cloning vector; the cDNA was inserted into pCMP1 using BamHI and
EcoRV. This cDNA encodes an isoform of Nr-CAM lacking amino acids,
inserted after the second Ig domain and half of the fifth fibronectin type
III repeat as a result of alternative RNA splicing (Grumet et al., 1991
).
Human contactin cDNA was cloned into pCMP1 (Peles et al., 1995
).
); binding of Fc fusion proteins (5 ml of ~1 µg/ml) was performed with 100 µl of a 50% suspension
of Pansorbin in PBS, and the complexes were washed with HNTG buffer
(50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol). The immunoprecipitates were resolved on SDS-PAGE, immunoblotted with antibodies, and analyzed with the ECL detection system
(NEN Dupont, Wilmington, DE; Peles et al., 1995
).
).
CFS/EK transfectant and parental 2.2 cells were
grown to confluence in DME medium with 10% FCS on 2.5-mm wells of
24-well slides (Cel-line Assoc., Inc., The Sea Ranch, CA) coated with
polylysine and fibronectin (Friedlander et al., 1989
). After overnight culture, each well was gently washed with DME medium/F12/ITS+, and dissociated primary tectal neurons (1,000 cells/10 µl) were added. After 24 h,
the slides were fixed, and the neurons were incubated with anti-Ng-CAM
polyclonal antibodies followed by biotinylated anti-rabbit IgG (Vector
Labs, Inc.) and avidin-rhodamine (Molecular Probes, Inc., Eugene, OR)
and visualized by fluorescence microscopy. Neurite outgrowth was measured on photographs and analyzed as described.
Results
CFS Protein Induces Neurite Outgrowth and
Neuronal Differentiation
,
we made several recombinant Fc fusion proteins containing different subdomains of the extracellular region of
RPTP
(Fig. 1). We showed previously that the carbonic
anhydrase domain (
C) of RPTP
alone or together with
the fibronectin type III repeat (
CF) induces adhesion
and neurite growth of primary chick neurons and IMR32 neuroblastoma cells (Peles et al., 1995
). Similarly, a fusion protein called
CFS, containing most of the extracellular
region of the short receptor form of RPTP
including the
S region (Fig. 1), also promoted adhesion of chick primary
neurons. The number of neurons that adhered to
CFS
was, however, lower than those that adhered to
CF substrates (Table I). Remarkably, the average length of neurites on
CFS (73 ± 7 µm; n = 110) was much longer than
those on
C and
CF (26 ± 2 µm; n = 149) and resembled
the morphology of neurites induced on Ng-CAM, which
on average were longer (118 ± 7 µm; n = 165; Fig. 2). In
contrast, neurons extended short thick processes on
C
(Peles et al., 1995
) and
CF fusion proteins, as well as on
anti-Ng-CAM antibodies (average length of 48 ± 2 µm;
n = 110) and on Nr-CAM (Grumet and Sakurai, 1996
; Fig. 2). Fc fusion proteins with
F and
S (see Fig. 8) did
not promote neuronal adhesion or neurite growth, and an
Fc fusion protein of the extracellular region of MCK10
containing the discoidin I domain (Alves et al., 1995
) supported neuronal adhesion but not neurite growth (data not
shown), indicating that the Fc region itself does not significantly affect neuronal adhesion and neurite growth.
Fig. 1.
Fusion proteins representing extracellular region of
RPTP. (A) Schematic representations of the short form of
RPTP
and different subdomains used to construct fusion proteins with human IgG-Fc. The short receptor form (the deletion
variant) consists of an NH2-terminal carbonic anhydrase domain
(C), fibronectin type III repeat (F), a spacer region (S), and cytoplasmic protein tyrosine phosphatase domains (PTP). Vertical
bar represents the transmembrane region (TM). (B) Expression
of the chimeric IgG molecules in COS cells. Various RPTP
fusion proteins containing different combinations of C, F, and S regions as illustrated in A, were purified, separated on an SDS gel,
and immunoblotted with antibodies against human IgG. The diffuse higher molecular weight components in
FS and
S probably represent proteoglycans insofar as they were not observed after chondroitinase treatment (Sakurai et al., 1996
), indicating that
the short form of RPTP
is a "part time" proteoglycan. Molecular weight makers are shown in kD.
[View Larger Versions of these Images (20 + 61K GIF file)]
CFS and
CF
Fig. 2.
Extracellular regions of RPTP promote neuronal adhesion and neurite outgrowth. Primary chick tectal cells were plated
on purified
CFS or
CF fusion proteins (80 µg/ml), Ng-CAM
(20 µg/ml), and anti-Ng-CAM monoclonal antibody 4B9 (100 µg/ml
of IgG) and incubated for 48 h. After fixation with paraformaldehyde, photographs were taken. In each case the average length of
neurites was measured (see text). Bar, 100 µm.
[View Larger Version of this Image (127K GIF file)]
C and
CF indicated that the carbonic
anhydrase domain of RPTP
binds to the GPI-linked neuronal cell recognition molecule contactin (Peles et al.,
1995
). We have confirmed that
CFS also binds to contactin and induces neurite growth (Figs. 2 and 3). As shown
previously on
C-coated substrates (Peles et al., 1995
),
when chick tectal neurons were pretreated with PI-PLC, neuronal adhesion and neurite outgrowth on
CFS was reduced dramatically (data not shown), indicating that these
activities involve GPI-linked molecules on the neuronal
cell surface. To verify the involvement of the GPI-linked
protein contactin, we tested the effect of antibodies on
neurite growth after allowing chick tectal neurons to adhere to substrates coated with
CFS. Neurite growth was
inhibited by Fab
fragments of polyclonal antibodies against
chick contactin (Fig. 3).
CFS also promoted adhesion of
human neuroblastoma IMR32 cells which express contactin, and anti-human contactin antibodies inhibited neuronal
adhesion and neurite growth on substrates coated with
C
(Peles et al., 1995
) and
CFS (data not shown). These results indicate the involvement of contactin in neuronal adhesion and neurite growth induced by
CFS.
CFS Is Inhibited by
Anti-Nr-CAM Antibodies
CFS
differs from that seen on
C and
CF provided a clue that
molecules other than contactin may also be involved in
neurite outgrowth on RPTP
. Indeed, we showed previously that Ng-CAM and N-CAM (Milev et al., 1994
) as
well as Nr-CAM (Milev et al., 1996
) bind to phosphacan, the soluble form of RPTP
. Interestingly, Ng-CAM and
Nr-CAM can also bind to contactin (Brummendorf et al.,
1993
; Morales et al., 1993
). Therefore, we investigated
whether any of these CAMs are also involved in the promotion of neurite outgrowth by RPTP
. Whereas polyclonal
antibodies against Ng-CAM and N-CAM had no effect on
neurite outgrowth on
CFS substrates, anti-Nr-CAM antibodies inhibited it (Fig. 4). Quantitation of neurite outgrowth from tectal cells on
CFS substrates revealed
strong inhibition by anti-Nr-CAM antibodies, whereas the
effects of anti-N-CAM (data not shown) and anti-NgCAM antibodies were negligible (Fig. 4 C). In control experiments, anti-Ng-CAM antibodies inhibited neurite outgrowth on Ng-CAM, as expected, whereas anti-Nr-CAM
antibodies did not (Fig. 4 D).
.
Consistent with this idea, we found that the majority of
neurons that adhered to and extended neurites on
CFS
expressed both Nr-CAM and contactin (Fig. 5). Most tectal neurons were stained positively for Ng-CAM, while
only a few of these cells were Nr-CAM and contactin positive when cultured on substrates coated with laminin and
polylysine (data not shown). These results suggest that the
majority of tectal neurons is not stained with antibodies
against Nr-CAM and contactin, and a subpopulation of
tectal neurons that adhere selectively to
CFS are NrCAM and contactin positive.
Fig. 5.
Coexpression of
Nr-CAM and contactin on
tectal neurons. Primary chick
tectal cells were isolated on
dishes coated with CFS fusion protein, as described in
the legend to Fig. 3, and
stained by immunofluorescence with monoclonal antiF11 (
-con) and polyclonal anti-Nr-CAM antibodies (
Nr) as described in Materials
and Methods. Note that the
majority of cells that bound
to
CFS were stained by
both antibodies to contactin
and Nr-CAM.
[View Larger Version of this Image (86K GIF file)]
/phosphacan with NgCAM, N-CAM, and Nr-CAM, it was of interest to define
the binding properties of its different extracellular domains.
We showed previously that the C domain of RPTP
contains a binding site for contactin (Peles et al., 1995
). A
solid phase protein binding assay was used to analyze
binding of various combinations of extracellular domains of RPTP
as Fc fusion proteins (Fig. 6).
C as well as
CF
bound to contactin but not to Ng-CAM, N-CAM, NrCAM, fibronectin, or laminin.
CFS binding was detected
to contactin, Nr-CAM, Ng-CAM, and N-CAM, confirming
our previous findings that RPTP
binds to these CAMs.
Moreover,
S (containing only the S domain) alone or
in combination with the fibronectin type III repeat of
RPTP
(i.e.,
FS) also bound to Nr-CAM, Ng-CAM, and
N-CAM but not to contactin, laminin, or fibronectin. These
adhesion molecules did not bind
F, which consists of only
the fibronectin type III repeat. These data suggest that different domains of RPTP
interact with different CAMs;
the C domain binds to contactin while the S domain interacts with Nr-CAM, Ng-CAM, and N-CAM.
Fig. 6.
Binding of domains in RPTP to various adhesion molecules. Proteins (10 µg/ml) were coated on dishes and incubated
with different RPTP
fusion proteins (0.5-1 µg/ml). Binding of
the Fc fusion proteins was detected by biotinylated anti-human
Fc antibody, followed by streptavidin-alkaline phosphatase, and
visualized by NBT/BCIP alkaline phosphatase substrates. Only
fusion proteins containing the S domain bound to Ng-CAM (Ng),
Nr-CAM (Nr), and N-CAM (N), while fusion proteins containing
the C domain bound to contactin (Con). None of the fusion proteins tested bound to laminin (Lm) or fibronectin (Fn). Similar
results were obtained in three independent experiments.
[View Larger Version of this Image (52K GIF file)]
CFS Binds to a Complex of Contactin with Nr-CAM
involves
both contactin and Nr-CAM, it was of interest to study interactions among these proteins. To analyze the interaction of RPTP
with contactin and Nr-CAM when these
CAMs are expressed on the cell surface, we transfected
COS7 cells with plasmids encoding these proteins alone or
in combination. The Fc fusion proteins
CF and
CFS
both bound to cells expressing contactin (Table II). No significant change in binding was detected when Nr-CAM
was coexpressed with contactin. In control experiments,
CF, an Fc fusion protein containing C and F domains of
RPTP
(Barnea et al., 1993
), exhibited no binding. Negligible binding was also observed with the
FS fusion protein (Table II), and with
F and
S, containing only the F
and S domains of RPTP
, respectively (data not shown).
The results indicate that the C domain of RPTP
is important for binding to contactin when expressed on COS7
cells in the absence or presence of Nr-CAM. Although the
S domain bound to purified Nr-CAM in the solid phase assay (Fig. 6), no interaction was detected when Nr-CAM
was expressed on cells, suggesting that binding of RPTP
to cells expressing contactin and Nr-CAM is mediated primarily through contactin and not through Nr-CAM.
CF and
CFS Fc-Fusion Proteins to
Cells Expressing Contactin and Nr-CAM
Previous studies using immobilized forms of Nr-CAM
showed that it binds to contactin/F11 (Morales et al., 1993).
Using solid phase protein binding assays, we confirmed
that a contactin Fc chimera binds to Nr-CAM (Fig. 7 A).
Ng-CAM has also been found to bind to contactin (Brummendorf et al., 1993
), however, we found that the contactin fusion protein bound to Nr-CAM but not to Ng-CAM, L1, and N-CAM.
To determine whether contactin interacts with Nr-CAM
in nervous system tissue during development, we analyzed
these proteins in membrane extracts prepared from embryonic day 14 chick brains following precipitation with Fc
fusion proteins. Immunoblotting with specific antibodies
showed that Nr-CAM and contactin coprecipitated with
CF but not with
CF used as a control (Fig 7 B). Insofar as
CF binds to contactin but not directly to Nr-CAM,
these results suggest that Nr-CAM can bind to contactin.
To distinguish whether contactin can interact with NrCAM laterally on the same cell or between apposing cells,
we used the transient transfection strategy described above
(Table II). When COS7 cells were double transfected with
cDNAs encoding for contactin and Nr-CAM, both proteins were expressed (Fig. 7 C). Immunoprecipitates of
cell extracts with antibodies against contactin were found to contain Nr-CAM, as detected by immunoblotting with
anti-Nr-CAM antibodies. When Fc fusion proteins were
linked to protein A beads, Nr-CAM coprecipitated with
CF and
CFS Fc fusion proteins but not with the
CF Fc
fusion protein. These results confirm an interaction between Nr-CAM and contactin and suggest that a complex
of these proteins in cells binds specifically to extracellular
regions of RPTP
.
The interaction between Nr-CAM and contactin was
only detected when both proteins were expressed in the
same cells and not when mixtures were prepared from
cells transfected with each protein individually. When extracts of COS7 cells individually transfected to express either Nr-CAM or contactin were mixed and analyzed by
immunoprecipitation and immunoblotting, Nr-CAM did
not coprecipitate with anti-contactin antibodies or with
any of the Fc fusion proteins (Fig. 7 C). In contrast, contactin coprecipitated with CF and
CFS but not with
CF Fc fusion protein (data not shown), as expected from
its ability to bind to the C domain of RPTP
(Peles et al., 1995
). In addition, we could not detect an interaction between Nr-CAM and contactin following coculture for 48 h
of mixtures of COS7 cells that had been transfected individually to express either Nr-CAM or contactin (Fig. 7 D).
The observation that Nr-CAM coprecipitated specifically
with contactin only from extracts of doubly transfected
cells suggests that Nr-CAM and contactin interact laterally
in the plane of the plasma membrane to form a complex.
Neurite Outgrowth Promoted by RPTP
Involves the S Region
Although CF bound to contactin and could coprecipitate
Nr-CAM as a complex (Fig. 7),
CFS was more potent
than
CF in promoting neurite growth (Fig. 2), suggesting
the importance of the S region in neurite growth. This idea
was tested directly using an Fc fusion protein with the S region.
S by itself was a very poor substrate for neuronal
adhesion, and when cells adhered to substrates coated with
S, they did not extend processes (Fig. 8). Interestingly, when combined with
CF,
S potentiated neurite
growth in a dose-dependent manner as determined by
measurements of neurite lengths (Fig. 8). The potentiation
of neurite growth by
S occurred specifically in combination with the CF domains of RPTP
and not with other adhesive substrates including laminin, fibronectin, and antibodies against Ng-CAM. For example, the S domain was
found to be slightly inhibitory for neurite outgrowth on
substrates coated with 4B9 monoclonal antibody to NgCAM reducing the average outgrowth from 48 ± 2 µm (n = 149) to 40 ± 6 µm (n = 106). The ability of the S region to
potentiate neurite extension was only observed in combination with the C domain.
Fibroblasts Expressing CFS Promote Neurite
Outgrowth and Neuronal Differentiation
Given that extracellular regions of the short form of RPTP
promote neurite outgrowth when presented as substrates,
it was of interest to test their effects as cell surface proteins. For this purpose, we expressed on NIH3T3 cells a
chimeric receptor protein consisting of extracellular domains of RPTP
and intracellular regions of EGF receptor kinase called
CFS/EK. Initial experiments using transiently transfected cells indicated that
CFS/EK promoted neurite growth. Therefore, stable transfectants were isolated and their expression of the chimeric protein was confirmed by immunoblotting (data not shown). Chick tectal
neurons cultured on monolayers of stable
CFS/EK transfectants for 24 h had on average longer neurites than neurons found on monolayers of the parental cells (Fig. 9).
Moreover, on the
CFS/EK transfectant, some cells developed very long neurites that were >300 µm in length, while such highly differentiated tectal neurons were never
seen on the parental cells. Similar results were observed
using COS7 cells stably transfected with
CFS/EK. Fab
fragments of antibodies against Nr-CAM inhibited neurite
growth on
CFS/EK transfectants, indicating a role for
Nr-CAM in neurite growth for these tectal cells (data not
shown). The cell bodies of these differentiated neurons
were large and oval shaped (Fig. 9, A-C), but we could not
definitively identify these cells because molecular markers for distinguishing tectal neurons are not available. In contrast to the tectal neurons, cerebellar neurons did not extend long neurites on the
CFS/EK transfectant (data not
shown), suggesting that different types of neurons respond
differently to the short form of RPTP
, which is the major
receptor form expressed on astrocytes (Sakurai et al., 1996
).
A major conclusion of this study is that the extracellular
region of the short receptor form of RPTP (
CFS) is a
potent promoter of neurite growth acting through the neuronal CAMs, contactin and Nr-CAM. These CAMs are
expressed on overlapping subsets of developing axons, and
the combined observations have led us to hypothesize that
they form a complex in a subset of neurons by interacting
laterally in the same plasma membrane (Fig. 10).
CFS can bind to the contactin-Nr-CAM complex, and this interaction is critically dependent on the binding of contactin to the C domain. In addition, maximal neurite growth
induced by RPTP
involves the S domain.
CAMs were initially characterized as ligands or counter
receptors on apposing cells that mediate cell adhesion by
homophilic and heterophilic mechanisms (Edelman, 1984).
While many CAMs can support cell adhesion and induce
neurite growth, adhesiveness is a poor predictor of neurite
growth (Lemmon et al., 1992
). This has stimulated the hypothesis that in addition to adhesion per se, specific signaling events are involved in CAM-mediated neurite growth
(Doherty and Walsh, 1994
; Doherty et al., 1995
). Accordingly, there may be two different modes of neurite growth:
one that is permissive, being adhesion dependent, and the
other that involves specific signaling. Ng-CAM/L1 may be
particularly complicated because it can act both as a ligand
to promote neurite growth (Lagenaur and Lemmon, 1987
;
Fig. 2) and as a neuronal receptor that mediates signaling
(Schachner, 1993
). As a substrate, the anti-Ng-CAM antibody appears to act in an adhesion-dependent manner and
supports only limited process extension. We found striking morphological similarities between the robust responses
of neurons to
CFS and Ng-CAM and the weaker responses to
CF and anti-Ng-CAM antibody (Fig. 2). These
results suggest that
CF mediates initial adhesion-dependent processes while
CFS provides additional signals that
promote neurite growth and that the S region may be important for the signaling.
Contactin has been implicated as a neuronal receptor
that mediates repulsion by a mechanism involving binding
of tenascin-R (Pesheva et al., 1993), and RPTP
functions
as a ligand for contactin that promotes neurite outgrowth.
Given that contactin is not a transmembrane protein, its
ability to modulate neurite growth may be adhesion dependent, but it probably also involves other receptors that
can transmit signals to the cytoplasm. Therefore, modulation of neurite outgrowth via contactin in particular, and possibly by CAMs in general, is likely to involve a hierarchy of lateral interactions with other CAMs and/or receptors in the neuronal membrane. Similarly, axonin-1 is another GPI-linked protein that may interact laterally with
Ng-CAM to regulate axonal growth in certain neurons
(Rader et al., 1996
), and both of these CAMs can bind to
RPTP
/phosphacan (Milev et al., 1994
, 1996), suggesting that other complexes of neuronal CAMs may also interact
with RPTP
.
The promotion of neurite growth by glial RPTP requires interactions with contactin and Nr-CAM, which
probably form a complex on the responding neuron (Fig.
10). This activity is dependent on binding of the C domain
of RPTP
to contactin (Peles et al., 1995
). The involvement
of the S domain was suggested by comparing
CF with
CFS (Fig. 2) and was demonstrated in mixing experiments where it was able to potentiate neurite growth by
CF (Fig. 8). Therefore, we propose that the extracellular
region of RPTP
has at least two distinct domains that are
important for neurite growth: the C domain that mediates
adhesion-dependent events via contactin and the S domain that is necessary but not sufficient for extension of
long neurites, probably by transmembrane signaling via
Nr-CAM and possibly other CAMs and/or receptors.
Whereas the binding studies using purified proteins indicated an interaction of the S domain of RPTP
with NrCAM, this may be a weak interaction insofar as it was not
detected when Nr-CAM was expressed on cells. The action of the S domain may involve intramolecular interactions with other extracellular domains in RPTP
that modulate its binding affinity for Nr-CAM. These other domains
(i.e., C and F) are not simply acting to promote adhesion
because other adhesion molecules could not substitute for
their ability to potentiate neurite growth by
S. It is possible that the CF domains interact with the S region of
RPTP
and modulate its conformation and its ability to
promote neurite growth. Inasmuch as
CF bound neurons
more robustly than
CFS did (Table II), it is unlikely that
the S region is important for binding, per se. An excess of
S over
CF was required for maximal neurite growth in
the mixing experiments (Fig. 8), possibly because of its low
affinity of binding to Nr-CAM alone or in complexes with
other membrane proteins such as contactin. In any case, it
appears that simultaneous binding of C and S domains to
complexes of neuronal receptors may be important for
promoting neurite growth.
Both Ng-CAM and Nr-CAM can bind to contactin
(Brummendorf et al., 1993; Morales et al., 1993
), but only
contactin and Nr-CAM have been implicated in neurite
growth promoted by the short form of RPTP
. These results combined with the coprecipitation studies suggest
that a lateral interaction between Nr-CAM and contactin (Fig. 10) in certain cells, such as a subset of tectal neurons, may be particularly important for neuronal differentiation
and neurite growth that is promoted by RPTP
. Significant levels of contactin, Nr-CAM, as well as RPTP
/phosphacan are found during neural development, but it has
been difficult to compare their patterns of expression in a
single species because of limitations with the available antibodies. The available data suggest that RPTP
/phosphacan (Milev et al., 1994
; Meyer-Puttlitz et al., 1996
), contactin/
F11/F3 (Ranscht, 1988
; Faivre-Sarrailh et al., 1992
), and
Nr-CAM/Bravo (Grumet et al., 1991
; Krushel et al., 1993
;
Denburg et al., 1995
) have overlapping distributions in
vertebrate fiber tracts in the retina, optic nerve, spinal
cord, and tectum (Lustig, M., and M. Grumet, unpublished
observations). Therefore, it is of interest to determine
whether changes in the expression and interactions between RPTP
and various combinations of neuronal CAMs during development modulate the behavior of neurons and glia locally.
Receptor complexes containing contactin and Nr-CAM
may be able to relay specific transmembrane signals or recruit additional proteins for neurite outgrowth (Fig. 10).
The FGF receptor has been implicated in neurite growth
involving transmembrane CAMs such as Ng-CAM/L1
(Doherty and Walsh, 1994), and Nr-CAM has a peptide
sequence between its third and forth Ig domains that is similar to that found in Ng-CAM/L1 (Grumet et al., 1991
;
Williams et al., 1994
). It is possible that Nr-CAM, as a
complex with contactin, mediates signaling via the FGF
receptor. Alternatively, a recently identified contactinassociated transmembrane protein of 190 kD that coprecipitates with RPTP
(Peles et al., 1995
, 1997
) may be involved in transducing neurite growth signals generated by RPTP
. Nr-CAM might also act in signaling by linking to
the cytoskeleton via ankyrin (Davis and Bennett, 1994
;
Dubreuil et al., 1996
).
The short form of RPTP induced neurite outgrowth
both as a cell surface molecule (
CFS/EK) and a substrate
bound to plastic (
CFS), suggesting that this activity of
RPTP
is biologically relevant. Previous studies have
shown that astrocytes and Schwann cells support neurite
outgrowth in culture by mechanisms involving integrins,
cadherins, N-CAM, and Ng-CAM/L1 (Bixby et al., 1988
; Tomaselli et al., 1988
). The ability of RPTP
to interact
with neural CAMs in the Ig family (Milev et al., 1994
, 1996)
suggests that it might induce regulatory signals in neurons.
In addition, binding of contactin and Nr-CAM to RPTP
may generate signals in glia via this receptor, but, as yet,
candidate substrates in glia for the tyrosine phosphatase
have not been detected. Recently, it has been proposed
that binding of extracellular regions of RPTPµ to its ligand
does not induce any change in phosphatase activity.
Rather, ligand binding may regulate the localization of
phosphatases on the cell surface (Gebbink et al., 1995
).
Similarly, binding to neural CAMs and extracellular matrix molecules might induce changes in localization of
RPTP
on glial cells that modulate local signaling by clustering of phosphatases. While receptor forms of RPTP
expressed on glia may mediate communication between neurons and glia, the secreted form has the potential for
blocking these activities (Fig. 10). Interestingly, the level
of expression of the secreted form increases dramatically
during development (Meyer-Puttlitz et al., 1995
; Sakurai
et al., 1996
)
Remarkably, a subset of neurons bearing long neurites
with oval-shaped cell bodies were observed when tectal
cells were incubated on CFS transfectants (Fig. 9). The
characteristics of these cells are similar to those seen for
the chick tectobulbar neurons which extend efferent fibers
from the tectum and have large oval-shaped cell bodies
(Kroger and Schwarz, 1990
). Bundles of axons in the tectobulbar tracts grow through the tectum without contacting axons growing in from the retina. Perhaps, glial cells in
the tectum expressing RPTP
provide cues for neuronal
differentiation, and axonal growth. Using other chick neurons such as cerebellar cells, we did not detect dramatic effects on
CFS, but we did observe that cortical neurons
from P2 rat brain extended long neurites on
CFS (Sakurai, T., and M. Grumet, unpublished observations). While
this manuscript was in preparation, it was reported
(Maeda and Noda, 1996
) that protein moieties in the NH2terminal half of phosphacan can promote differentiation
of certain cortical neurons which is consistent with our observations.
RPTP/phosphacan is also found in certain locations
during development that axons avoid such as the roof
plate, and phosphacan has been found to inhibit neurite
growth in culture (Milev et al., 1994
; Maeda and Noda,
1996
). In addition to the C, F, and S domains, phosphacan
and the long receptor form of RPTP
contain an 860-
amino acid domain that has been postulated to function as
an inhibitor of neurite growth (Grumet et al., 1996
). It is interesting that the roof plate of the spinal cord is deficient in adhesion molecules in general while the fiber tracts
have a rich assortment of CAMs (see references in Brummendorf and Rathjen, 1995
). Therefore, it is likely that
RPTP
/phosphacan differentially affects neurite growth
depending on the relative abundance of the different
forms of the molecule. Responses may also vary depending on the receptors available in particular neurons as well
as on extracellular binding proteins such as tenascin (Grumet et al., 1994
), which may modulate interactions of
RPTP
with other proteins. Additional studies are needed
to determine whether contactin and Nr-CAM also act as
neuronal receptors mediating inhibition of neurite growth
by phosphacan.
In conclusion, RPTP has two distinct domains that
bind to different neuronal receptors, including contactin
and Nr-CAM, that can form a complex in the plasma membrane. These CAMs probably act in the form of complexes
possibly including other neuronal receptors to mediate
glial signals that control neurite growth.
Received for publication 19 July 1996 and in revised form 25 October 1996.
Please address all correspondence to Martin Grumet, Department of Pharmacology, New York University Medical Center, New York, NY 10016. Tel.: (212) 263-7126; Fax.: (212) 263-7133; E-mail: grumem01{at}mcrcr6.med.nyu.eduWe thank Drs. Fritz Rathjen and Jeffrey Denburg for their generous gifts of antibodies, Dr. James Salzer for PI-PLC, and Drs. David R. Friedlander and Irit Lax for helpful discussions and for critically reading the manuscript.
This work was supported by grants from the National Institutes of Health (NS21629 and NS33921). M. Lustig was supported in part by a National Institutes of Health MD-Ph.D. training grant.
C, carbonic anhydrase domain;
CAM, cell adhesion molecule;
F, fibronectin type III repeat;
GPI, glycosylphosphatidylinositol;
PI-PLC, phospho-inositol specific phospholipase C;
RPTP, receptor-like protein tyrosine phosphatase
;
S, spacer region.