(Received for publication, July 18, 1995; and in revised form, September 13, 1995)
From the
The cell adhesion molecule L1 has been implicated in mediating cell-cell adhesion and in promoting neurite outgrowth. The extracellular region of L1 contains six immunoglobulin (Ig)-like domains in the amino-terminal region, followed by five fibronectin type III-like repeats. L1 is capable of undergoing homophilic binding as well as heterophilic interactions. To map the homophilic binding domain in L1, three glutathione S-transferase (GST) fusion proteins (GST-Ig1-2-3, GST-Ig4-5-6, and GST-Fn) were prepared and coupled to Covaspheres and their homophilic binding activity was determined using the Covasphere-to-substratum binding assay. Only GST-Ig1-2-3 was capable of homophilic binding. Next, His-tagged recombinant Ig-domain proteins (His-Ig1-2, His-Ig1, and His-Ig2) were expressed and subjected to similar assays. Only His-Ig1-2 and His-Ig2 were capable of homophilic interactions. Binding of His-Ig2-conjugated Covaspheres to substrate-coated His-Ig2 was inhibited by anti-Ig1-2-3 Fab and soluble His-Ig2. These results indicate that the L1 homophilic binding site resides within Ig2. To examine effects of these L1 recombinant proteins on neurite outgrowth, neural retinal cells were cultured on different substrate-coated fusion proteins. Both GST-Ig1-2-3 and His-Ig2 were potent inducers of neurite extension. These results thus indicate that the L1 Ig-like domain 2 alone is sufficient to mediate L1-L1 interaction and promote neurite outgrowth from retinal cells.
Intercellular adhesion plays an important role during neural
development, when specific synaptic connections are established
primarily by extension of axons along restricted
pathways(1, 2) . The molecular basis of these
processes involves cell adhesion molecules and diffusible factors. In
recent years, an increasing number of cell adhesion molecules have been
found associated with neuronal cells. These cell adhesion molecules
have been categorized according to their structure into three major
groups: the cadherins(3) , the integrins (4) , and
proteins of the immunoglobulin (Ig) ()superfamily(5, 6) .
L1 was first described as a 200-kDa transmembrane glycoprotein in the central nervous system, and it belongs to the Ig superfamily(7, 8) . L1 consists of six C2-type Ig-like domains in the amino-terminal region, followed by five fibronectin type III-like repeats, a transmembrane domain, and a cytoplasmic domain(8) . NILE in rat, NgCAM, G4, and 8D9 in chicken are the species homologues of mouse L1(9, 10, 11) . L1 cDNAs have been cloned from mouse(8) , rat(12, 13) , and human(14, 15) . The L1 gene in human has been mapped to chromosome Xq28(16, 17) . An association has been made between mutations in the L1 gene and the X-linked hydrocephalus phenotype(18, 19, 20) .
L1 can undergo homophilic binding as well as heterophilic interactions with several other cell adhesion molecules, such as NCAM(21, 22) , TAG-1/axonin-1(23, 24) , F3/F11(25) , glia(26, 27) , and the extracellular matrix protein laminin(28) . Some of these heterophilic interactions are known to modulate L1 functions. For instance, NCAM has been shown to undergo cis-interactions with L1, which in turn facilitates L1-L1 homophilic binding(21) . Neurocan, in contrast, binds to L1 and inhibit neuronal adhesion and neurite extension promoted by the L1 substrate(29) .
L1 has been implicated in a wide range of neuronal cell differentiation. Substrate-coated L1 is a potent inducer of neurite outgrowth from a number of primary neurons(14, 30, 31) . Axonal growth involves both adhesion and the transmission of extracellular signals into the interior of a growth cone to activate intracellular events (32, 33) . L1 appears to play an important role in this signal transduction process(34, 35) . A L1-Fc chimeric protein has been reported to induce protein tyrosine phosphorylation in neuronal cells (36) as well as promote neurite outgrowth(37) , suggesting that the clustering of L1 molecules may trigger the signaling pathway leading to neurite extension.
It is evident that the formation of adhesion complexes via L1 homophilic binding may serve as an initiation point for many important signaling events. However, very little is known about the homophilic binding site of L1 and the mechanism of its interaction. In this report, experiments were carried out to investigate the relationship between L1 homophilic binding and its neuritogenic activity. We found that the second Ig-like domain of L1 was capable of binding to cell membrane-associated L1, as well as undergoing homophilic binding by itself. In addition, the Ig-like domain 2 of L1 was capable of promoting neurite outgrowth from retinal neurons, suggesting an intimate relationship between L1 homophilic binding and L1-mediated neurite outgrowth.
The unbound protein was removed by washing
with water, and the coverslips were blocked with 1% BSA in
-minimal essential medium at room temperature for 30 min. These
coverslips were then transferred to 24-well Linbro plates, and retinal
cells suspended in N2 medium were seeded on top of them. Rat L1 protein
was either adsorbed on to a nitrocellulose substrate according to the
method of Lagenaur and Lemmon (42) or on to a
poly-L-lysine substrate. In inhibition studies, Fab or
recombinant proteins were used to precoat either the substratum or the
retinal cells at final concentrations of 250 and 40 µg/ml,
respectively. Neurite extension was allowed to proceed for 16-18
h. Retinal cells were fixed for 20 min in 3.7% formaldehyde in PBS by
gradually replacing the culture medium with the fixative. After three
washes with PBS, coverslips were mounted in vinol, containing
1,4-diazabicyclo(2,2,2)octane and p-phenelenediamine, to
retard photobleaching. Samples were examined by epifluorescence
microscopy. Retinal neurons bearing neurites were recorded, and
100 neurites were measured in each experiment.
Figure 1: Construction and expression of GST fusion proteins. A, schematic drawings of GST fusion proteins: GST-Ig1-2-3, GST-Ig4-5-6, and GST-Fn. The restriction enzyme sites NarI and BamHI were used in the construction of GST-Ig1-2-3. The PCR product extending from the BamHI site at nucleotide position 1114 to the end of Ig-like domain 6, at L1 nucleotide position 1848, was used to construct GST-Ig4-5-6. The PCR fragment from nucleotide position 1849 to 3344 was used to construct GST-Fn. All the fragments were subcloned into pGEX-3T vector, and the GST protein was fused to the amino terminus of the recombinant proteins. B, gel profiles of purified GST fusion proteins. Protein samples were separated on 10% gels and stained with Coomassie Brilliant Blue. Lane a, GST-Ig1-2-3; lane b, GST-Ig4-5-6; lane c, GST-Fn.
To obtain L1 domain-specific antibodies, rabbits were immunized with the purified fusion proteins. The antisera were absorbed against acetone powder to remove antibodies that recognized bacterial protein and the GST moiety of these fusion proteins. The IgG fraction was isolated from each antiserum to obtain L1 domain-specific antibodies. Western blots were carried out using these purified IgG to ensure that they did not cross-react with the other two fusion proteins (data not shown).
Figure 2: Epifluorescence micrographs showing the binding of the GST-Ig1-2-3-conjugated Covaspheres to substrate-coated fusion proteins. Round spots on Petri dishes were coated with anti-Ig1-2-3 IgG (a), GST-Ig1-2-3 (b), GST (c), or GST-Ig4-5-6 (d). GST-Ig1-2-3-conjugated Covaspheres were allowed to adhere to the substratum for 30 min. After washing five times with PBS, the bound Covaspheres were observed by epifluorescence microscopy.
The relative percentages of Covaspheres
bound per unit area were estimated by normalizing the results to the
level of Covasphere binding on the IgG-coated substrate (Fig. 3). The amount of GST-Ig1-2-3-conjugated Covaspheres bound
to the GST-Ig1-2-3 substrate was 25-fold higher than that attached
on the GST substrate. In contrast, binding of GST-Ig4-5-6-conjugated
Covaspheres to the GST-Ig4-5-6 substrate was at the background level (Fig. 3). In the case of GST-Fn-conjugated Covaspheres, a higher
background level of binding to the GST substrate was observed, but
there was no significant difference between the level of binding to the
GST-Fn substrate and the level of binding to GST (Fig. 3).
Furthermore, GST-Ig1-2-3-conjugated Covaspheres did not attach to
substrate-coated GST-Ig4-5-6 or GST-Fn (data not shown). It was evident
that the Ig-like domains 1, 2, and 3 did not interact with other
extracellular segments of L1. These results thus demonstrate that the
L1 homophilic binding site resides within the first three Ig-like
domains, and that the last three Ig-like domains and the fibronectin
domains may not be directly involved in L1 homophilic interactions.
Figure 3: Binding of fusion protein-conjugated Covaspheres to different substrates. Round spots on Petri dishes were coated with different proteins. Fusion protein-conjugated Covaspheres were allowed to adhere to the substrate for 30 min. Proteins used as substrate were: domain-specific IgG (black bars), GST (stippled bars), GST-Ig1-2-3 (hatched bar), GST-Ig4-5-6 (white bar), and Gst-Fn (cross-hatched bar). The number of Covaspheres attached per unit area was estimated, and the results were normalized to the amount of Covaspheres bound to the substratum coated with domain-specific IgG. Data represent the mean ± S.D. (n = 6-9).
The dose effect of substratum-associated GST-Ig1-2-3 on Covasphere binding was also examined. When equal amounts of Covaspheres were loaded onto substrates coated with different concentrations of GST-Ig1-2-3, Covasphere attachment was found to be dose-dependent and maximal binding was achieved when the substratum was coated with 1 µM GST-Ig1-2-3 (Fig. 4A). When binding was carried out on a GST-coated substratum, no significant binding was observed up to a concentration of 5 µM.
Figure 4:
Binding specificity of
GST-Ig1-2-3-conjugated Covaspheres to GST-Ig1-2-3 substrate. A, dose effect of GST-Ig1-2-3 fusion protein used to coat the
substratum on Covasphere binding. Spots on Petri dish were coated with
different concentrations of either GST-Ig1-2-3 () or GST (
).
GST-Ig1-2-3-conjugated Covaspheres were allowed to adhere to coated
spots. The relative amounts of Covaspheres attached were estimated
relative to the amount bound to anti-Ig1-2-3 IgG. B,
inhibition of the attachment of GST-Ig1-2-3-conjugated Covaspheres to
substrate-coated GST-Ig1-2-3 by domain-specific antibodies.
GST-Ig1-2-3-conjugated Covaspheres were mixed with different
concentrations of anti-Ig1-2-3 Fab (
), anti-Ig4-5-6 Fab (
),
or goat-anti-mouse-IgG Fab (
) before being placed on
GST-Ig1-2-3-coated spots. The relative amounts of Covaspheres bound per
unit area were estimated. C, inhibition of the binding of
GST-Ig1-2-3-conjugated Covaspheres by soluble GST-Ig1-2-3. Binding of
Covaspheres to substrate-coated GST-Ig1-2-3 was carried out in the
presence of different concentrations of GST-Ig1-2-3 (
),
GST-Ig4-5-6 (
), or GST (
). Data represent the mean
± S.D. (n =
6-9).
To demonstrate
the specificity of Covasphere binding, competition experiments were
carried out using either anti-Ig1-2-3 Fab or soluble GST-Ig1-2-3.
Anti-Ig1-2-3 Fab blocked the binding of GST-Ig1-2-3-conjugated
Covaspheres to substrate-coated GST-Ig1-2-3 in a dose-dependent manner (Fig. 4B). Fifty percent inhibition was achieved at
35 nM anti-Ig1-2-3 Fab. However, the effects of
anti-Ig4-5-6 Fab was negligible up to a concentration of 5
µM. The attachment of GST-Ig1-2-3-conjugated Covaspheres
to substrate-coated GST-Ig1-2-3 was also inhibited by soluble
GST-Ig1-2-3 (Fig. 4C). The inhibition was
dose-dependent, and 50% inhibition was achieved at
80 nM soluble GST-Ig1-2-3. In contrast, the attachment of GST-Ig1-2-3
Covaspheres to GST-Ig1-2-3 substrate was not affected by GST or
GST-Ig4-5-6, up to a concentration of 3 µM.
Figure 5: Construction and expression of His-tagged domain proteins. A, schematic diagrams of the three recombinant domain proteins. PCR fragments containing the coding sequences for Ig1-2, Ig1, and Ig2 were generated and subcloned into the pQE-8 expression vector. B, gel profiles of the recombinant proteins. Proteins samples were separated on 12% SDS-polyacrylamide gels under reducing conditions and then stained with Coomassie Brilliant Blue. Lane a, His-Ig1-2; lane b, His-Ig1; lane c, His-Ig2.
The fusion proteins were used to coat Petri dishes and then assayed
for their ability to bind GST-Ig1-2-3-conjugated Covaspheres. A large
number of GST-Ig1-2-3-conjugated Covaspheres attached to the His-Ig1-2
substrate, suggesting that the third Ig-like domain of L1 is not needed
for homophilic interactions. When recombinant proteins containing a
single Ig-like domain were tested, Covaspheres attached to the His-Ig2
substrate, but not to the His-Ig1 substrate (Fig. 6A),
suggesting that it is Ig2, and not Ig1, that is directly involved in
L1-L1 binding. Consistent with this observation, His-Ig2 was able to
function as a competitor to displace GST-Ig1-2-3-conjugated Covaspheres
in the attachment assay. Only residual binding (5%) was observed
when binding was carried out in 10 µM soluble His-Ig2 (Fig. 6A). In contrast, a relative level of 70% binding
was retained when the same concentration of His-Ig1 was included in the
assay.
Figure 6: Binding of fusion protein-conjugated Covaspheres to substrate-coated domain proteins. In A, Petri dishes were coated with different recombinant proteins: GST-Ig1-2-3, His-Ig1-2, His-Ig1, His-Ig2, or GST at a concentration of 3 µM. GST-Ig1-2-3-conjugated Covaspheres were allowed to adhere to the substrate for 30 min. In inhibition studies, Covaspheres were mixed with 10 µM His-Ig2 (stippled bar) or His-Ig1 (hatched bar) before placing on substrate coated GST-Ig1-2-3. The relative amounts of Covaspheres bound were estimated relative to the amount of Covaspheres bound to substrate-coated anti-Ig1-2-3 antibodies. In B, His-Ig2-conjugated Covaspheres were assayed for their ability to attach to substrate-coated His-tagged recombinant proteins. Values were normalized to the amount of Covaspheres bound to the His-Ig2 substrate. Data represent the mean ± S.D. (n = 9).
To determine whether Ig2 can bind to Ig2, His-Ig2-conjugated Covaspheres were assayed for their ability to attach to substrate-coated His-Ig2. Binding of Covaspheres was observed on the His-Ig2 substrate, but not on GST (Fig. 6B). Positive results were also obtained when these Covaspheres were deposited on substratum coated with either GST-Ig1-2-3 or His-Ig1-2. However, His-Ig2-conjugated Covaspheres did not bind to substrate-coated His-Ig1. These results are consistent with the notion that L1-L1 binding is mediated by homophilic interactions between the second Ig-like domains of two apposing L1 molecules.
Whether His-Ig2 was able to interact with L1 molecules expressed by neural retinal cells was also examined. Retinal cells were isolated from day 6 chick embryos and cultured on coverslips. His-Ig2-conjugated Covaspheres were deposited on top of these cells, and the number of cells showing positive Covasphere binding was estimated. About 40% of retinal cells were decorated with His-Ig2-conjugated Covaspheres, whereas binding of His-Ig1-conjugated Covaspheres to these cells was at the background level (Table 1). To determine whether His-Ig2 was interacting with L1 molecules on the surface of retinal cells, cells were first incubated with either soluble His-Ig2 or anti-Ig1-2-3 Fab. After removal of the excess protein, His-Ig2-conjugated Covaspheres were placed on the precoated cells. Precoating the cells with either soluble His-Ig2 or anti-Ig1-2-3 Fab blocked the binding of Covaspheres to retinal cells, and the inhibition was dose-dependent. The data thus indicate that His-Ig2 was binding to L1 molecules on the surface of retinal cells.
Figure 7: Epifluorescence micrographs of neurites extended by retinal cells. Neural retinal cells were isolated from E5 chick embryos and labeled with DiI. Retinal cells were cultured for 18 h on different fusion protein-coated substrates: GST-Ig1-2-3 (a), GST-Ig4-5-6 (b), and GST (c). Bar, 10 µm.
Quantitative analysis showed that the majority of neurites (>80%)
extending from retinal cells cultured on GST-Ig4-5-6, GST-Fn, or GST
were <25 µm, with mean neurite lengths ranging between 15 and 20
µm. (Fig. 8). In contrast, retinal cells cultured on top of
GST-Ig1-2-3 sent out much longer neurites, with a wider range of size
distribution (Fig. 8A). Approximately 90% of them were
25 µm. As a positive control, retinal cells were cultured on
rat L1-coated substratum. The patterns of neurite length distribution
for GST-Ig1-2-3 and intact L1 were almost identical (Fig. 8A), and their mean neurite lengths were 42.3 and
47.4 µm, respectively (Fig. 8B). The data indicated
that GST-Ig1-2-3 retained most of the neuritogenic activity of native
L1. In comparison to substratum coated with GST where cells yielded a
mean neurite length of 13 µm, retinal cells cultured on the
GST-Ig1-2-3 substrate extended neurites with a 3-fold increase in their
average length, whereas GST-Ig4-5-6 and GST-Fn did not lead to a
significant increase in neurite outgrowth over the GST control.
Figure 8:
Neurite outgrowth from retinal cells on
different coated substrata. Coverslips were first coated with 0.01%
poly-L-lysine, followed by one of the GST fusion proteins at 1
µM concentration. Purified rat L1 was used as the positive
control. Retinal cells were seeded on coverslips and cultured in N2
medium. Cultures were incubated at 37 °C for 18 h. Cells were fixed
with formaldehyde, and fluorescence images were recorded on VCR for
neurite length measurements. A, size distribution of neurites
extending from retinal cells cultured on different fusion protein
substrates (, rat L1;
, GST-Ig1-2-3;
, GST-Ig4-5-6;
, GST-Fn;
, GST). B, mean neurite lengths for
cells cultured on different protein substrates. Data represent the mean
± S.D. of three experiments.
Competition experiments were carried out using either soluble fusion protein or anti-Ig1-2-3 Fab. When retinal cells were cultured in the presence of soluble GST-Ig1-2-3, neurite outgrowth was reduced to the background level. The pattern of neurite length distribution was similar to that of cells cultured on GST (Fig. 9). Similar inhibitory effects were observed when cells were cultured in the presence of anti-Ig1-2-3 Fab. In both cases, the active L1 sites on retinal cells and substratum were blocked by the competitor. Nevertheless, the number of cells attached to the coverslip did not decrease, suggesting that the anchorage of cells to the substratum per se was not sufficient to promote neurite outgrowth and that neurite outgrowth was dependent on the cellular response to the L1 substrate.
Figure 9: Inhibitory effect of GST fusion proteins and anti-domain Fab on neurite outgrowth. A, retinal cells were cultured on GST-Ig1-2-3-coated substratum (solid bar) or on GST (hatched bar) in the absence of competitors. B, the GST-Ig1-2-3 substrate was preincubated with competitors (a, goat anti-mouse Fab (5 µM); b, anti-Ig1-2-3 Fab (5 µM)) for 15 min at room temperature and then washed, before retinal cells were deposited on the coverslip. C, competitors (c, goat-anti-mouse Fab (5 µM); d, anti-Ig1-2-3 Fab (5 µM); c, GST (40 µg/ml); d, GST-Ig1-2-3 (40 µg/ml)) were added to retinal cells prior to seeding on the protein substrate.
Figure 10: Inhibition of neurite outgrowth by recombinant domain proteins. Retinal cells were cultured on top of GST-Ig1-2-3-coated coverslips in the presence of 1 µM His-Ig1-2, His-Ig1, or His-Ig2. Data represent the mean ± S.D. of three experiments.
It is therefore likely that the Ig-like domain 2 of L1 harbors both homophilic binding and neuritogenic activities. To test whether the second Ig-like domain of L1 was capable of promoting neurite outgrowth, retinal cells were cultured on top of substrate-coated His-Ig2 (Fig. 11). Relatively long neurites were extended by retinal cells cultured on top of the His-Ig2 substrate, with a mean neurite length of 52 µm (Fig. 11B). Similar results were obtained when cells were cultured on substrate-coated His-Ig1-2. Their length distribution patterns were similar to that of cells cultured on GST-Ig1-2-3 (Fig. 11A). In contrast, only short neurites were found on the His-Ig1 substrate, which yielded a mean neurite length of 18.5 µm. These results thus indicate that the second Ig-like domain alone is sufficient to promote neurite outgrowth from retinal neurons.
Figure 11:
Neurite outgrowth on domain
protein-coated substratum. Nitrocellulose-coated coverslips were coated
with 1 µM recombinant proteins. Retinal cells were
deposited on them and cultured for 18 h. Cells were fixed, and the
neurite lengths were measured. A, size distribution of
neurites extending from retinal cells cultured on different protein
substrates: GST (), GST-Ig1-2-3 (
), His-Ig1 (
),
His-Ig2 (
), His-Ig1-2 (
). B, Mean neurite lengths
of retinal cells. Data represent the mean ± S.D. of 3
experiments.
Homophilic interactions between L1 molecules not only result in cell-cell adhesion, but also elicit neurite outgrowth(22, 27) . In this report, we first focused on mapping the L1 homophilic binding site. Fusion proteins containing different segments derived from the extracellular region of L1 were assayed for homophilic binding activity. Only fusion proteins that contained Ig-like domain 2 were capable of undergoing homophilic interactions, suggesting that the homophilic binding site of L1 resides within its Ig-like domain 2. Since these fusion proteins do not interact with each other, it becomes evident that Ig-like domain 2 interacts directly with Ig-like domain 2 on an apposing L1 molecule. The initial interactions centered at Ig-like domain 2 may lead to subsequent interactions at other secondary sites on L1, further stabilizing the homophilic binding reaction. It is of interest to note that Ig-like domain 2 has the greatest interspecies homology among the extracellular domains of L1(14) . This probably accounts for the ability of L1 to mediate homophilic interactions among several vertebrate species(14) .
Several cell adhesion molecules in
the Ig superfamily are also known to mediate cell-cell adhesion by a
homophilic binding mechanism. The classical example is
NCAM(43, 44, 45, 46) , and its
homophilic binding site has been mapped to a decapeptide sequence
(KYSFNYDGSE) within its third Ig-like domain(47) . This
sequence corresponds to the C` -strand and the C`-E loop of the Ig
fold(40, 47) . The charged residues as well as the
aromatic side-chains appear to play a crucial role in NCAM homophilic
binding(40) . The NCAM homophilic binding sequence is unique to
Ig-like domain 3, and it probably interacts isologously with the same
sequence on NCAM molecule present on apposing cells(39) . A
similar strategy of binding is used by the cell adhesion molecule gp80
in Dictyostelium
discoideum(48, 49, 50, 51) .
gp80 is a primitive member of the Ig superfamily of recognition
molecules(51, 52) , and it mediates cell-cell adhesion
in a Ca
-independent manner. The homophilic binding
site has been mapped to an octapeptide sequence (YKLNVNDS), which is
also predicted to adopt a
-strand conformation followed by the
beginning of a
-turn structure(53) . As in NCAM, both the
amino-terminal Tyr residue and the two internal charged residues are
vital to the homophilic binding activity of gp80. Furthermore, the
homophilic binding site of gp80 is capable of undergoing isologous
interaction with the same sequence in an anti-parallel
manner(53) .
The exact location of the homophilic binding
site within Ig-like domain 2 of L1 is not yet known. However, two point
mutations within this domain have been implicated in X-linked
hydrocephalus and mental retardation. One of the mutations resulted in
the replacement of Arg-184 with Gln, while the other mutation
substitutes Gln for His-210 (20) . Both mutations may affect
the folding of the Ig-like domain 2, resulting in the abolition or
reduction in the affinity of L1 homophilic interactions. It is of
interest to note that Arg-184 lies within a region corresponding to the
predicted C` -strand of the Ig fold(14) , suggesting that
Arg-184 and its flanking sequences may participate in L1 homophilic
binding in a manner similar to the C`
-strand in the third Ig-like
domain of NCAM.
Whereas L1 and NCAM undergo homophilic binding via interactions between two identical domains, the carcinoembryonic antigen (CEA), which is also a member of the Ig superfamily, adopts a heterologous binding mechanism. This involves the reciprocal interactions between the amino-terminal Ig-like domain of one molecule and an internal Ig-like domain of the apposing molecule(54) . Since the Ig superfamily consists of a great variety of recognition molecules, it is conceivable that different mechanisms may be utilized in the adhesive processes mediated by different molecules. It remains to be determined whether the two mechanisms utilized by L1/NCAM and CEA are widely adopted by other members of the Ig superfamily.
In addition to being able to undergo homophilic binding, the Ig-like domain 2 of L1 is a potent inducer of neurite outgrowth from neural retinal cells. Our results showed that the Ig-like domains 4-6 and all five fibronectin type III repeats failed to promote neurite outgrowth from retinal cells. In contrast, Appel et al.(55) reported that L1 fusion proteins containing Ig-like domains 1-2, 3-4, 5-6, or fibronectin type III repeats 1-2 were all capable of promoting neurite outgrowth from small cerebellar neurons. Interestingly, a more recent study on NgCAM, a chicken homolog of L1, showed that only the fourth and fifth fibronectin-like domains of NgCAM were required for stimulating neurite outgrowth from dorsal root ganglia cells(56) . It is possible that, depending on the relative levels of endogenous L1 and L1 receptors, different types of primary neurons may respond differently to these external peptide substrates. These apparently conflicting results may also reflect the complexities involved in neurite outgrowth.
It should be pointed out that the second Ig-like domain alone is sufficient to stimulate neurite outgrowth. Since the potency of His-Ig2 in our neurite outgrowth assay was comparable to that of intact L1, the other structural domains of L1 do not seem to be required in the initial step of activating the neurite outgrowth pathway. Our results suggest that an intimate relationship exists between L1 homophilic binding and L1-induced neurite outgrowth. Similar observations have been made when retinal cells were cultured on top of a monolayer of NCAM-expressing L cell transfectants. Here retinal neurons extend much longer neurites than those cultured on control cells(38, 57) . However, mutations in the NCAM homophilic binding site abrogates the ability of NCAM to stimulate neurite outgrowth(38) .
Lemmon et al.(27) have shown that L1 stimulated neurite outgrowth via a
homophilic binding mechanism. It is therefore conceivable that the
substrate-coated His-Ig2 may interact with the Ig-like domain 2 of L1
on cells, which in turn generates neurite outgrowth signals either by
inducing conformational changes in the L1 molecule or by altering L1
interactions with neighboring membrane and cytoplasmic components.
Direct association between the cytoplasmic domain of L1 and ankyrin has
been reported(58) . The cytoplasmic domain of L1 has also been
found to associate with both protein kinase C and non-protein kinase C
kinase activities and it can be phosphorylated (59, 60) . Both tyrosine phosphorylation and
Ca influx have been found to be key steps in the
signaling pathways initiated by cell adhesion
molecules(30, 35, 57) .
A recent study (37) showing that L1 clustering by a soluble bivalent L1-Fc chimeric proteins leads to an increase in neurite outgrowth is consistent with the notion that L1-L1 homophilic interaction serves as the first step in the signaling cascade that leads to neurite extension. It is of interest to note that NCAM behaves somewhat differently in this respect. We have previously found that a synthetic peptide which contains the NCAM homophilic binding site within a 21-amino acid sequence is a potent inducer of neurite outgrowth from retinal cells(38) . This suggests that while NCAM homophilic binding is required, clustering of NCAM molecules may not be essential.
The signaling cascade involved in L1-dependent neurite outgrowth is
a subject of considerable debate. Several recent reports implicate an
essential role for the FGF receptor in neurite outgrowth induced by
several cell adhesion molecules, including L1, NCAM, and
N-cadherin(36, 37) . A similar neurite outgrowth
response can be elicited by treatment with basic FGF(61) ,
suggesting that L1-L1 binding may lead to the activation of FGF
receptor. Since both the L1-Fc chimera and basic FGF induce increases
in tyrosine phosphorylation on a common set of neuronal
proteins(36) , an identical pathway that involves the
activation of FGF receptor has been postulated for all cell adhesion
molecule-dependent neurite outgrowth(36, 61) . On the
other hand, studies using src or fyn knock-out mice
have indicated that pp60 is an essential
component of the intracellular signaling pathway in L1-mediated neurite
outgrowth (62) . Whereas the L1 response is dependent on the
nonreceptor tyrosine kinase pp60
,
NCAM-stimulated neurite outgrowth is dependent on p59
since neuronal cells derived from fyn-minus mice fail to
respond to NCAM(63) . These results argue for the involvement
of distinctly different components in the early steps of signaling
pathways induced by L1 and NCAM. However, the nature of the association
between either L1 and src or NCAM and fyn is still
not known. Similarly, there is no direct evidence demonstrating
physical interactions between L1 and FGF receptor. Future experiments
to address this and related issues will be required to resolve the
discrepancy between these models.
L1 is a multidomain molecule and is known to undergo heterophilic interactions with other molecules such as NCAM(21, 22, 64) , TAG-1/axonin-1(23, 24) , F3/F11(25) , and brain proteoglycans(29, 65) . Interactions with these membrane and extracellular components may have important roles at specific stages of brain development. In addition to the mutations detected in Ig-like domain 2, several other mutations in L1 have been reported to co-segregate with X-linked hydrocephalus(18, 19, 66) . Some mutations have been found to affect the expression of L1; others may have deleterious effects on L1 interactions with its ligands. Further investigation of the role of L1 in brain development will depend on the identification of its homophilic and heterophilic binding sites as well as the elucidation of their mechanisms of interaction. These studies should help us better understand how the hydrocephalus-related mutations affect L1 functions and provide new insights in the cause of X-linked hydrocephalus and mental retardation.