(Received for publication, January 25, 1995; and in revised form, May 23, 1995)
From the
Drosophila neuroglian is a transmembrane glycoprotein that has strong structural and sequence homology to the vertebrate L1 gene family of cell adhesion molecules (Bieber, A. J., Snow, P. M., Hortsch, M., Patel, N. H., Jacobs, J. R., Traquina, Z. R., Schilling, J., and Goodman, C. S.(1989) Cell 59, 447-460). Two different neuroglian protein forms that are generated by a differential splicing process are expressed in a tissue-specific fashion by embryonic and larval cells (Hortsch, M., Bieber, A. J., Patel, N. H., and Goodman, C. S. (1990) Neuron 4, 697-709). The two neuroglian polypeptides differ only in their cytoplasmic domains. Both of these neuroglian species, when transfected into and expressed in Drosophila S2 cells, induce the calcium-independent, homophilic aggregation of transformed cells. A third artificial neuroglian protein form was constructed by substituting the neuroglian transmembrane segment and cytoplasmic domains with the glycosyl phosphatidylinositol attachment signal of the Drosophila fasciclin I protein. This cDNA construct generates a glycosyl phosphatidylinositol-anchored form of neuroglian, which retains the ability to induce homophilic cell aggregation when expressed in S2 cells, and was able to interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that neuroglian mediates a calcium-independent, homophilic cell adhesion activity and that neither cytoplasmic neuroglian domains nor a direct interaction with cytoskeletal elements is essential for this property.
A growing number of putative neural cell adhesion molecules
(CAMs) ()with immunoglobulin and fibronectin type III
protein domains are currently being identified and characterized from a
wide range of different species. Among the best studied examples are
representatives of the NCAM and the L1 CAM families. These molecules
have been implicated in many different developmental and cellular
processes including neurite outgrowth, axonal fasciculation, neuronal
cell migration, muscle formation and innervation, neuron-Schwann cell
interactions(1, 2, 3, 4, 5) .
Drosophila neuroglian is a member of the L1 CAM gene family (6) . It encodes a transmembrane polypeptide with six
immunoglobulin and five fibronectin type III protein domains and has
strong sequence homology to the mouse L1 glycoprotein. Differential
splicing of the primary neuroglian transcript in Drosophila embryos results in the production of two polypeptide species
differing only in their cytoplasmic domains(7) . The larger of
the neuroglian protein forms (designated neuroglian, in
accordance with its apparent molecular mass in kDa) is restricted to a
subset of neuronal cells in the peripheral and central nervous system,
whereas the smaller neuroglian polypeptide (neuroglian
)
is also expressed by many non-neuronal cell types and tissues. Multiple
interchangeable cytoplasmic protein domains have also been reported for
a number of other immunoglobulin-type CAMs including
NCAM(8, 9) , L1(10, 11) , and
myelin-associated glycoprotein (MAG)(12, 13) . This
diversity of cytoplasmic domains, and therefore protein forms, could
result in differential interactions with cytoskeletal elements,
different involvement in cellular second messenger systems, or
different degrees of intracellular regulation of the extracellular
adhesive properties of these molecules.
The importance of the
cytoplasmic domain for interactions with the cellular cytoskeleton and
ligand binding and specificity has been studied in detail for some
members of the integrin and cadherin CAM families. Deletions of the
cytoplasmic domain of the lymphocyte function associated antigen-1
(LFA-1) integrin -subunit do not interfere with cell
surface expression or heterodimer assembly, but abolish binding to its
intercellular adhesion molecule-1 ligand and disrupt the association of
the LFA-1 integrin with the actin cytoskeleton(14) . L-CAM and
E-cadherin both depend on the integrity of their cytoplasmic protein
domains for their calcium-dependent homophilic adhesive
function(15, 16) .
Some adhesion molecules
belonging to the immunoglobulin gene superfamily have been functionally
tested for their requirement for an intact cytoplasmic domain. One
example of functional differences between alternate cytoplasmic domains
is seen for the major protein forms of NCAM. In comparison to the
smaller transmembrane NCAM form (NCAM 140) the largest NCAM protein
form (NCAM 180) is less able to support neurite outgrowth from rat
cerebellar neurons when expressed in 3T3 fibroblast cells(17) .
The NCAM 180 form also interacts with the cellular spectrin-actin
cytoskeleton, in contrast to the other two major NCAM species, which do
not(18) . Besides the two major transmembrane forms of NCAM, a
naturally occurring, GPI-anchored form has also been
described(9, 19) . Lesley et al.(20) reported that the CD44 receptor loses its ability to
bind hyaluronic acid after deletion of its cytoplasmic domain. Smaller
deletions in the CD44 cytoplasmic protein domain that abolish CD44
binding to the cytoskeleton adapter molecule ankyrin have similar
effects on its adhesive ability(21) . Similarly, PECAM-1
molecules with a complete deletion of the cytoplasmic domain do not
support cell-cell adhesion of transformed mouse L-cells(22) .
Interestingly, DeLisser and co-workers (22) found that partial
deletions of the PECAM-1 cytoplasmic domain result in a basic change of
adhesion mechanism from a calcium-dependent, heterophilic mode to a
calcium-independent, homophilic mode. Carboxyl-terminal deletions of
the myelin P cytoplasmic protein domain abolish
P
-mediated homophilic adhesion of transformed
cells(23) .
These and other studies strongly support the
notion that the adhesive function of many different types of CAMs often
depend on, or is regulated by, the cytoplasmic portions of these
membrane molecules. By expressing different protein forms of Drosophila neuroglian in Drosophila S2 cells, we have
analyzed the adhesive properties of this invertebrate member of the L1
CAM gene family. S2 cells have been used as host cells for the
functional characterization of a number of Drosophila and
vertebrate CAMs(24, 25, 26) . The experiments
reported here show that both of the naturally occurring forms of
neuroglian act as homophilic CAMs. Furthermore, the substitution of the
neuroglian transmembrane segment and the cytoplasmic domains, with the
GPI anchor signal of the Drosophila CAM fasciclin I, produces
a novel neuroglian polypeptide (neuroglian) that is
anchored in the plasma membrane by a GPI moiety and is still able to
induce homophilic cell aggregation of transformed S2 cells. These
findings indicate that neither of the two neuroglian cytoplasmic
domains is required for neuroglian-dependent homophilic cell adhesion.
A GPI-anchored form of neuroglian was
constructed as outlined in Fig.1. A 3.4-kb KpnI/AvaII cDNA fragment, encoding the amino-terminal
1121 amino acid residues of the 1138-amino acid neuroglian
extracellular domain, was isolated from a neuroglian cDNA. The Drosophila fasciclin I GPI anchor signal (28) was
derived by PCR from a fasciclin I cDNA in Bluescript
SK. In the PCR the universal sequencing primer was
used along with a 23-mer oligonucleotide
(5`-GATCAACGTATACGGACCCGATG-3`) that was identical to a small part of
the coding strand of the fasciclin I cDNA in all but 2 nucleotide
residues (introducing an AvaII site underlined in the
sequence). PCR products were digested with AvaII and SalI, and an 1.1-kb DNA fragment was isolated from an agarose
gel. This fasciclin I cDNA-derived PCR product encodes the
carboxyl-terminal 53 amino acids of the Drosophila fasciclin I
open reading frame and most of the 3`-untranslated region. Both the
3.4-kb neuroglian cDNA fragment and the 1.1-kb fasciclin I PCR product
were subcloned into KpnI/SalI-cut pRmHa3 vector DNA.
Figure 1:
Construction of a Drosophila neuroglian cDNA containing the GPI anchor signal of Drosophila fasciclin I. A 3.4-kb KpnI/AvaII Drosophila neuroglian cDNA fragment, encoding the first
amino-terminal 1121 amino acid residues (comprising the signal sequence
and almost the entire extracellular domain), and a 1.1-kb AvaII/BamHI PCR product, encoding the 53
carboxyl-terminal amino acid residues and the 3`-untranslated region of Drosophila fasciclin I, were ligated together into KpnI/BamHI-cut pRmHa3 vector DNA. An in-frame AvaII site was introduced into the fasciclin I cDNA by the 5`
PCR primer. The resulting expression vector, pRmHa3-nrg,
drives the expression of a hybrid protein consisting of almost the
entire neuroglian extracellular domain bound to the membrane by a GPI
anchor.
S2
cells were transfected with DNA-calcium phosphate coprecipitates using
the pPC4 plasmid to confer -amanitin resistance as the selectable
marker(31) . Individual subclones were expanded and assayed by
Western blotting for high level expression of neuroglian protein. These
cell lines are designated S2:pRmHa3-nrg
,
S2:pRmHa3-nrg
, and S2:pRmHa3-nrg
,
respectively. Prior to assay, protein expression was induced by the
addition of 0.7 mM CuSO
to the culture medium for
1-2 days.
In adhesion assays which
required that two different cell lines be mixed to determine the extent
of the reaction between their expressed surface molecules, one cell
line was marked by labeling with the vital fluorescent membrane dye DiI
. Cells were stained by adding 1 µl of a 2.5 mg/ml solution of DiI
(in ethanol) for each ml of culture medium, and the cells were allowed
to incorporate the dye for 2-12 h before washing the cells three
times in culture medium to remove excess dye. Labeled cells were then
mixed with unlabeled cells of the appropriate type, and the cells were
induced together and assayed for aggregation. For all combinations of
cell lines that were tested, several assays with different ratios of
the two different cell types were examined, but the total cell
concentration in all assays was 1 10
cells/ml.
Cell aggregates were visualized and photographed on a Zeiss Axiophot microscope with Nomarski interference optics or on a Nikon Diaphot with phase contrast optics. DiI epifluorescence was visualized on a Zeiss Axiophot microscope using a rhodamine optical filter set.
Figure 6:
Quantitative analysis of
neuroglian-induced cell aggregation. Cell aggregation was quantified as
the loss of single cells over time. At t = 0 h, each
cell line was diluted to 1 10
cells/ml and induced
with 0.7 mM CuSO
. Throughout the experiment the
cultures were agitated constantly on a rotary shaker at 100 rpm. At the
selected time points, aliquots of cells were removed and mounted on
microscope slides for quantitation using an automatic object analysis
program. The residual cells were used for the assessment of neuroglian
expression by Western blot analysis.
,
S2:pRmHa3-nrg
;
, S2:pRmHa3-nrg
;
, S2:pRmHa3-nrg
;
, S2
cells.
Prior to PI-PLC
treatment of S2 cells, transformed cells were induced at a
concentration of 0.5 10
cells/ml for 2 days with
0.7 mM CuSO
and then gently dissociated into a
single cell suspension by pipetting. A small aliquot of cells was
incubated for 2 h at 37 °C with 50 units/ml PI-PLC. An equal volume
of Schneider's medium containing 12.5% fetal calf serum was
added, and cells were incubated at room temperature in a 24-well tissue
culture plate. Cell aggregation assays were performed as described
previously.
The three neuroglian
expression constructs (pRmHa3-nrg,
pRmHa3-nrg
, and pRmHa3-nrg
) were introduced
into S2 cells by DNA-calcium phosphate coprecipitation and the
expressed proteins were analyzed as described below and under
``Experimental Procedures.''
Figure 2:
Different protein forms of neuroglian are
present in membrane preparations from embryonic and transformed S2
cells. Western blots of different membrane preparations were stained
with various anti-Drosophila neuroglian antibodies. About
3-20 µg of membrane proteins were loaded per lane. Lanes1, a crude membrane preparation from 20-23-h-old Drosophila embryos; lanes2, from
untransformed S2 cells; lanes3, from
S2:pRmHa3-nrg cells; lanes4, from
S2:pRmHa3-nrg
cells; lanes5, from
S2:pRmHa3-nrg
cells. S2 cells were induced for 2 days
with 0.7 mM CuSO
before a crude membrane
preparation was isolated. PanelA was incubated with
the mouse anti-neuroglian mAb 3F4, which binds to the extracellular
domain of neuroglian and recognizes all three neuroglian protein forms. PanelB was incubated with a rat polyclonal antiserum
that recognizes the carboxyl-terminal amino acid residues specific for
the neuroglian
form. PanelC was
incubated with the mouse anti-neuroglian mAb BP-104, which is specific
for the neuroglian
protein
form.
Figure 4:
The neuroglian protein form
is anchored in the S2 cell membrane by a glycosyl phosphatidylinositol
membrane anchor. Membrane preparations isolated from transformed,
induced S2 cells (S2:pRmHa3-nrg
, lanes
1-4; pRmHa3-nrg
, lanes5 and 6) were incubated for 2 h in the presence (lanes
3-6) or absence (lanes1 and 2)
of PI-PLC (20 units/ml) and subsequently subjected to an extraction at
pH 10 in order to separate soluble (S) from integral membrane (P) proteins. Whereas the neuroglian
protein is
partially released from the membranes by PI-PLC, the phospholipase has
no effect on the membrane association of the neuroglian
protein.
A significant portion of the neuroglian
protein in embryonic membranes and the membrane preparations from S2
cells expressing the two transmembrane neuroglian forms appeared to be
proteolytically processed to a smaller polypeptide species with an
apparent molecular mass of about 150 kDa (Fig.2A,
indicated by the arrow). Since this degradation product was
absent in membranes expressing neuroglian and was not
detected by antibodies against either of the neuroglian cytoplasmic
domains, we assume that the processing site must be very close to the
transmembrane segment, probably on the extracellular aspect of the
membrane. Most of this neuroglian degradation product can be released
from the membrane by an alkaline pH wash and is therefore not firmly
inserted into the lipid bilayer (data not shown). Interestingly, a
similar proteolytic processing has been reported for the mouse L1
glycoprotein(36) . This degradation process could constitute an
endogenous mechanism to regulate cell adhesion by the proteolytic
inactivation of neuroglian protein. Although the preparation of
membranes was performed in the presence of a panel of various protease
inhibitors, we cannot exclude at the present time that the observed
neuroglian degradation might have also occurred during the isolation of
the membrane fractions.
The covalently attached GPI anchor of the
hybrid neuroglian form should provide a strong membrane association,
and accordingly, no immunoreactive neuroglian protein was found in cell
free tissue culture medium of induced S2:pRmHa3-nrg cells (Fig.3A, lane1). The cells from an
equivalent volume of tissue culture medium contained large amounts of
neuroglian
protein (Fig.3A, lane2). Protein fractionation of membrane preparations at an
alkaline pH has been routinely used for the separation of integral
membrane proteins from those not embedded in the lipid
bilayer(37) , and, like the transmembrane forms of neuroglian,
the neuroglian
protein was not released from the membrane
by an exposure to an alkaline pH of 10 (Fig.3, B and C), demonstrating that the protein is securely associated with
the membrane.
Figure 3:
The
neuroglian form is not secreted into the tissue culture
medium and behaves like an integral membrane protein.
S2:pRmHa3-nrg
cells were induced for 2 days with 0.7
mM CuSO
and 20 µl of the tissue culture medium
were loaded onto the 7.5% SDS-polyacrylamide electrophoresis gel shown
in panelA, lane1. Induced cells
from an equivalent volume were loaded in lane2. PanelsB and C show pH 10 extraction
experiments; lanes1 contain the starting material
before pH 10 extraction, soluble and peripheral membrane proteins are
in the soluble fractions (lanes2), and integral
membrane proteins are found in the pellet fractions (lanes3). PanelB shows the experiment using
S2 cell membranes containing the neuroglian
form and panelC the experiment with S2 cell membranes
containing the neuroglian
form. PanelsA and B are Western blots stained with the mouse
anti-neuroglian mAb 3F4, and panelC is a Western
blot using the mouse mAb BP-104. Neuroglian
is not
released from the membrane by pH10 treatment and therefore behaves like
an integral membrane protein.
The membrane association of GPI-anchored proteins can
be severed by digestion with bacterial PI-PLC. This enzyme catalyzes
the hydrolysis and release of a diacylglycerol group from the GPI
moiety. Treatment of S2 cell membranes containing neuroglian with PI-PLC led to a loss of membrane association of this
neuroglian form during exposure to pH 10 (Fig.4). As expected
PI-PLC incubation of membranes containing the transmembrane
neuroglian
polypeptide had no effect on its membrane
attachment (Fig.4, lanes5 and 6).
The 53 carboxyl-terminal amino acid residues of the fasciclin I
polypeptide are therefore sufficient to direct the attachment of a GPI
membrane anchor to the neuroglian hybrid protein.
Figure 5:
Expression of neuroglian on the surface of
S2 cells causes the cells to aggregate. Cells were plated at 1
10
cells/ml in 60-mm Petri plates and induced with 0.7
mM CuSO
for 24 h. After induction, the cells were
gently dissociated by repeated pipetting and cell concentrations were
readjusted to 1
10
cells/ml. The cells were then
shaken at 100 rpm on a rotating platform, at room temperature, for
10-12 h prior to examination. PanelA,
untransformed S2 cells; panelB,
S2:pRmHa3-nrg
; panelC,
S2:pRmHa3-nrg
; panelD,
S2:pRmHa3-nrg
. Untransformed S2 cells show no ability to
form aggregates (A), whereas cells that express any of the
three neuroglian forms show significant aggregation (B-D). Scalebar, 200
µm.
Untransformed S2 cells (Fig.5A and 6) or S2 cells transformed with neuroglian cDNAs in the antisense orientation do not aggregate. Furthermore, the neuroglian-induced cell aggregation is calcium-independent (data not shown).
Our initial attempts to quantify cell aggregation involved
preinduction with CuSO, followed by dissociation of
aggregates and assessment of reaggregation over the next 2-4 h.
This approach was complicated by the fact that after 24 h of induction
many of the neuroglian-expressing cells are so tightly bound that they
could not be dissociated quantitatively without destroying the cells.
For this reason we chose to begin the quantification experiments at the
point of induction and to assess aggregation at time points over 3 days
of induction. Therefore, data for these experiments represent a
combination of the kinetics of protein induction and the kinetics of
cell aggregation.
None of the cell lines showed any tendency to aggregate prior to induction. Within 12 h after induction, cell lines expressing any of the three neuroglian forms show significant levels of aggregation as compared to untransfected S2 cells (Fig.6). At this time point, expression levels of the various neuroglian proteins as measured by Western blot analysis, were less than 20% of the eventual maximal levels (data not shown). Maximal levels of protein expression for all three neuroglian forms were attained by 36-48 h after induction (data not shown) with maximal levels of cell aggregation occurring at 48-60 h.
At maximal aggregation the
nrg- and nrg
-expressing S2 cell lines
attained greater than 80% of cells incorporated into multicellular
aggregates. Antibody staining demonstrated that the unincorporated
cells were generally cells that no longer expressed high levels of the
protein. Both the nrg
and nrg
cell lines
tended to form large aggregates, as shown in Fig.5.
The
nrg cells also showed significant aggregation as compared
to untransfected S2 cells but less than that observed with the other
neuroglian forms. Despite efforts to isolate and compare cell lines
that express similar levels of the different neuroglian forms, we have
been unable to isolate a line that consistently retains high levels of
expression of the 167-kDa form of neuroglian. At maximal induction, the
nrg
line expressed about 50% of the protein expressed by
the nrg
and nrg
lines (data not shown).
This lower level of neuroglian protein expression may account for the
generally lower level of total aggregation in the quantitation
experiments and also for the smaller size of the nrg
aggregates (Fig.5). As with the nrg
and
nrg
lines, the cells that did not participate in cellular
aggregation tended to be cells that had lost the ability to express
high levels of neuroglian protein. The experiments shown in Fig.5and Fig. 6demonstrate that all three neuroglian
protein forms induce a robust aggregation response upon cell surface
expression in S2 cells.
The preincubation of induced
S2:pRmHa3-nrg cells with PI-PLC completely abolished the
formation of cell aggregates (Fig.7), but PI-PLC treatment had
no influence on the aggregation of S2:pRmHa3-nrg
cells.
A strong membrane attachment is therefore required for
neuroglian-induced cell aggregation.
Figure 7:
PI-PLC treatment abolishes
neuroglian induced cell aggregation. Induced
S2:pRmHa3-nrg
(panelsA and B)
or S2:pRmHa3-nrg
cells (panels C and D)
were incubated in the presence (panelsA and C) or in the absence (panelsB and D) of 50 units/ml PI-PLC. Cells were incubated with PI-PLC for
2 h at 37 °C, dispersed into single cells, and then allowed to
aggregate on a shaking platform at room temperature. A representative
field of each cell population is shown. Removal of neuroglian
from the cell surface abolishes the ability of
S2:pRmHa3-nrg
cells to aggregate. Scalebar, 200 µm.
Figure 8:
Neuroglian-expressing cells do not
interact with untransformed S2 cells. PanelsA and B, untransformed S2 cells were labeled with DiI, and the
labeled cells were then mixed with an equal number of unlabeled
S2:pRmHa3-nrg cells. The cells were induced together and
assayed for aggregation as described. PanelA shows
all cells under Nomarski optics, and panelB shows
the same field of view with only the labeled S2 cells visualized using
rhodamine epifluorescence. PanelsC and D,
an experiment similar to that described for panelsA and B, except that S2:pRmHa3-nrg
cells were
used as both the labeled and unlabeled cell types.
Neuroglian-expressing cells form aggregates only with other
neuroglian-expressing cells and do not interact with untransformed S2
cells, demonstrating that neuroglian is not binding to a heterophilic
receptor that is constitutively expressed by S2 cells. Scalebar, 200 µm.
Figure 9:
The three neuroglian protein forms
interact with each other. Cell lines expressing different forms of the
neuroglian protein were mixed to determine the extent of the
interaction between the different forms. In each experiment a cell line
expressing one form of neuroglian was marked by labeling with DiI, and
the labeled cells were then mixed with an equal number of unlabeled
cells expressing a different form of neuroglian. The cells were induced
together and assayed for aggregation as described. PanelsA and B, C and D, and E and F represent the same fields of view showing all cells
under Nomarski optics (A, C, and E) or only
the labeled cell type visualized using rhodamine epifluorescence (B, D, and F). PanelsA and B show pRmHa3-nrg mixed with
DiI-labeled pRmHa3-nrg
. PanelsC and D show pRmHa3-nrg
mixed with DiI-labeled
pRmHa3-nrg
. PanelsE and F show pRmHa3-nrg
mixed with DiI labeled
pRmHa3-nrg
. Each neuroglian-expressing cell line formed
mixed aggregates with all other neuroglian-expressing lines regardless
of the specific form of the neuroglian protein, demonstrating that the
specificity of neuroglian-mediated homophilic adhesion is not affected
by the cytoplasmic domains. Scalebar, 200
µm.
The structural and sequence analysis of Drosophila neuroglian indicates a strong homology to other members of the L1-CAM gene family(6) . The data presented here confirm that this similarity also extends to some functional aspects. Like its vertebrate ``siblings'' L1, Nr-CAM, NILE, and Ng-CAM(4, 38, 39, 40) , Drosophila neuroglian functions as a calcium-independent, homophilic CAM.
Two different neuroglian protein forms are generated in vivo by alternative splicing(7) . The two neuroglian polypeptides differ only in their cytoplasmic domains and are expressed in a tissue-specific fashion during development. When transfected into and expressed in Drosophila S2 cells, both of these neuroglian species mediate homophilic cell adhesion. A third artificial form of neuroglian, in which the transmembrane segment and cytoplasmic domains are replaced with a GPI moiety, retains this ability to induce cell adhesion and can interact with both of the two naturally occurring neuroglian polypeptides. These results demonstrate that the neuroglian cytoplasmic domains are not essential for neuroglian-mediated homophilic cell adhesion activity.
Members from
all major groups of substrate and cell adhesion molecules have been
found to be associated with different components of the cellular
cytoskeleton. This includes several members of the immunoglobulin
domain superfamily. The 180-kDa form of NCAM and members of the L1-CAM
family either bind either directly or via an ankyrin linker to the
cellular, submembranous actin-spectrin
network(18, 41, 42) . We recently obtained
experimental evidence that both alternative cytoplasmic domains are
capable of interacting with the membrane cytoskeleton protein ankyrin. ()Such interactions of CAMs with cytoskeletal elements might
be of great functional importance for the recruitment of CAMs or
cytoskeletal components to regions of cell-cell contact, for the
regulation of cell migration and cell shape, for the mediation of
CAM-induced signaling events, or for the regulation of adhesive
specificity and strength. From the data presented here, it appears that
the binding of neuroglian protein to elements of the cytoskeleton is
not required for its homophilic recognition ability. However, it is
still possible that such interactions might have a modulating effect on
extracellular neuroglian-mediated homophilic binding.
Besides these homophilic contacts, some L1-type CAMs are also engaged in heterophilic recognition events(38, 40, 43) . Mouse L1-CAM has been shown to interact with NCAM(44) , and chicken Ng-CAM binds to axonin-1/TAG-1 as well as to neurocan, a brain chondroitin sulfate proteoglycan(45, 46, 47) . Both chicken Ng-CAM as well as Nr-CAM/Bravo, both members of the L1 family, interact with the immunoglobulin domain F11 molecule(43, 48) . The heterophilic recognition properties of CAMs appear to be more sensitive to deletions in their cytoplasmic domains than their homophilic. In the case of PECAM-1, partial deletions of its cytoplasmic domain inactivates its heterophilic binding to heparin but uncovers a calcium-independent homophilic interaction(22) . However, in contrast to neuroglian the total deletion of its cytoplasmic domain renders the PECAM-1 molecule completely adhesion-incompetent. It would be of interest to determine whether hetero- and homophilic adhesion processes of other CAMs are regulated differently by their cytoplasmic domains. Since no potential heterophilic binding partner for Drosophila neuroglian has been identified so far, we are currently unable to address this aspect for the neuroglian molecule.