(Received for publication, July 12, 1995; and in revised form, November 8, 1995)
From the
Neuroligin 1 is a neuronal cell surface protein that binds to a
subset of neurexins, polymorphic cell surface proteins that are also
localized on neurons (Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich,
B., Missler, M., Moomaw, C., and Südhof, T. C.
(1995) Cell 81, 435-443). We now describe two novel
neuroligins called neuroligins 2 and 3 that are similar in structure
and sequence to neuroligin 1. All neuroligins contain an N-terminal
hydrophobic sequence with the characteristics of a cleaved signal
peptide followed by a large esterase homology domain, a highly
conserved single transmembrane region, and a short cytoplasmic domain.
The three neuroligins are alternatively spliced at the same position
and are expressed at high levels only in brain. Binding studies
demonstrate that all three neuroligins bind to -neurexins both as
native brain proteins and as recombinant proteins. Tight binding of the
three neuroligins to
-neurexins is observed only for
-neurexins lacking an insert in splice site 4. Thus, neuroligins
constitute a multigene family of brain-specific proteins with distinct
isoforms that may have overlapping functions in mediating recognition
processes between neurons.
Neurexins are neuronal cell surface proteins that exhibit a high degree of diversity (Ushkaryov et al., 1992, 1994; Ushkaryov and Südhof, 1993). Three genes for neurexins have been described, each of which is transcribed from two promoters. This results in six principal neurexin transcripts that are subject to extensive alternative splicing, generating a family of thousands of differentially expressed proteins (Ullrich et al., 1995).
The diversity of the neurexins led to the hypothesis that different neurexin isoforms may have distinct binding activities and mediate recognition events between neurons. This hypothesis predicts that alternative splicing regulates the ligand interactions of neurexins and that neurons express neurexin ligands that are specific for certain splice variants of neurexins. The interactions between these ligands and the neurexins could mediate recognition events between neurons. The description of neuroligin 1 provided the first support for this hypothesis (Ichtchenko et al., 1995).
Neuroligin 1 was
purified by affinity chromatography on immobilized neurexin 1;
cloning revealed that it constitutes a cell surface protein with a
single transmembrane region and an extracellular domain homologous to
esterases such as acetylcholinesterase. However, neuroligin 1 lacks an
active site serine, suggesting that it is not catalytically active.
Interestingly, neuroligin 1 binds tightly only to
-neurexins but
not to
-neurexins, and only to those
-neurexins that lack an
insert in splice site 4 (Ichtchenko et al., 1995). Together,
these data suggested a model whereby neuroligin 1 and
-neurexins
lacking an insert in splice site 4 mediate specific interactions
between neurons. This model is supported by the recent discovery of
gliotactin in Drosophila. Gliotactin also contains an esterase
homology domain and a single transmembrane region and is distantly
related to neuroligin 1 (Auld et al., 1995). Gliotactin is
expressed transiently in glia cells during development and is essential
for the formation of the peripheral blood-nerve barrier, suggesting a
function in a transient cell-cell recognition event.
We now describe two new forms of neuroligin called neuroligins 2 and 3 and have systematically investigated the alternative splicing of all neuroligins. All three neuroligins are similar in structure and expressed at high levels only in brain. Furthermore, all neuroligins are alternatively spliced at the same position but with different insert patterns. Binding studies revealed that they exhibit comparable binding properties of neurexins. Thus, similar to neurexins, neuroligins form a multigene family of brain proteins that may collaborate with neurexins in mediating cell-cell interactions between neurons.
PCRs were performed with degenerate primers corresponding to a sequence from neuroligin 1, and the products were sequenced (Ichtchenko et al., 1995). The sequences obtained suggested the presence of new neuroligins in addition to neuroligin 1 and were used to isolate multiple overlapping cDNA clones. The cDNA clones encoded two new forms of neuroligin called neuroligins 2 and 3. Full-length sequences for neuroligins 2 and 3 were assembled from multiple overlapping cDNA clones (Fig. 1).
Figure 1: Structures and alternative splicing of the neuroligins. The sequences of neuroligins 1, 2, and 3 are shown in single-letter amino acid code aligned for maximal homology and are numbered on the right. Residues shared by at least two of the three neuroligins are shown on a blue background. The putative signal sequences are shown on a gray background, and the transmembrane regions on a black background. Signal sequence cleavage sites shown are predicted based on signal sequence cleavage site characteristics (von Heijne, 1986) and have only been confirmed for neuroligin 2 by sequencing (see text). Alternatively spliced sequences as determined from their absence or presence in multiple cDNA clones are shown on a green background. Note that the alternatively spliced sequence of neuroligin 3 contains two components that are similar to the sequence in either neuroligin 1 or 2; the two components can be inserted independently.
Alignment of the translated amino acid sequences of the three neuroligins revealed that they are highly homologous. Overall, the three neuroligins share 52% identical residues. In pairwise comparisons, the neuroligins are almost equally similar to each other (neuroligin 1 and 2, 59% identity; 1 and 3, 66% identity; and 2 and 3, 59% identity; Fig. 1). The sequence similarity between the neuroligins is distributed over the whole protein, with the extracellular domains (55% identity) and the transmembrane regions (91% identity) being more conserved than the cytoplasmic sequences (31% identity). As is often observed in gene families, sequence stretches of almost 100% identity alternate with clusters of divergence. The most conserved sequences between neuroligins were observed in the esterase homology domain (see below), and the most divergent sequences in the segment linking the esterase homology domain to the transmembrane region and in parts of the cytoplasmic domains (Fig. 1).
All neuroligins contain an
N-terminal hydrophobic sequence suggestive of a signal peptide.
Analysis of these sequences for signal sequence cleavage sites
indicated preferred cleavage sites that are shown in Fig. 1. In
order to confirm these sites directly, we purified neuroligins 1, 2,
and 3 from rat brain as a mixture by affinity chromatography on
immobilized neurexin 1 (see below) and attempted to obtain
N-terminal sequences. Only a single unequivocal sequence was obtained (XXGGGPGGGAP in single-letter amino acid code) that
corresponds to the N terminus of neuroligin 2. This sequence shows that
the N terminus of neuroligin 2 contains a cleaved sequence. The
N-terminal sequence starts one residue after the N terminus suggested
by the computer analysis of signal peptide cleavage sites (Fig. 1). However, it seems likely that the predicted cleavage
site at residue 14 is correct and that the N-terminal glutamine
resulting from the signal sequence cleavage is removed after cleavage
because a cleavage site corresponding to residue 15 would be atypical
(von Heijne, 1986). The N termini of the other two neuroligins appear
to be blocked and could not be sequenced.
Sequencing of multiple independent cDNA clones for neuroligins 2 and 3 demonstrated that at a single identical position, the cDNAs either contained or lacked a sequence with an intact reading frame, suggesting that this sequence is alternatively spliced (Fig. 1). This suggestion was supported by the previously observed alternative splicing of neuroligin 1 at the same position (Ichtchenko et al., 1995). Interestingly, neuroligins 1 and 2 each contains a single type of alternatively spliced insert that is not homologous between neuroligins 1 and 2. By contrast, neuroligin 3 had two types of alternatively spliced inserts, one of which is homologous to that of neuroligin 2, whereas the second contains a sequence homologous to the alternatively spliced neuroligin 1 insert in addition to the sequence homologous to the neuroligin 2-type insert (Fig. 1).
The three variants of alternative splicing observed for neuroligin 3 with their segmental homology to the alternatively spliced sequences in neuroligins 1 and 2 suggested the possibility that neuroligins 1 and 2 have additional splice variants. Furthermore, one might expect that neuroligin 3 mRNAs should also occur with alternatively spliced inserts consisting only of the neuroligin 1-similar insert but not the neuroligin 2-similar insert. To systematically investigate these possibilities and potential additional events of alternative splicing, we performed PCRs on rat brain cDNA using oligonucleotides that amplify the complete coding region of the three neuroligins and produced cDNA libraries for each neuroligin. More than 45 independent cDNA clones of each of the three neuroligins were isolated and mapped, and some clones were sequenced and used for expression and neurexin binding studies (see below). These experiments confirmed the alternative splicing pattern deduced from the cDNA clones isolated by library screening but failed to discover additional splice variants, suggesting that the most common splice variants had in fact been identified.
Data bank searches revealed that similar to neuroligin 1, neuroligins 2 and 3 are also homologous to esterase domain proteins (Fig. 2). The esterase domain starts immediately after the signal sequence. The active site serine of esterases is changed to a glycine in all neuroligins, suggesting that the neuroligins are not catalytically active. The esterase domains of the neuroligins are much more homologous to each other than they are to other member of the esterase gene family, indicating that they form a separate gene family (Table 1). Six cysteines form three intramolecular disulfide bonds in acetylcholinesterase (Sussman et al., 1991). The four cysteines forming the two N-terminal disulfide bonds are conserved precisely in the neuroligins, suggesting that the corresponding disulfide bonds are also conserved. The two cysteines of acetylcholinesterase that form the C-terminal disulfide bond are shifted to different positions in the neuroligins, indicating differences between neuroligins and acetylcholinesterase in the C-terminal disulfide bonds. In addition, neuroligins contain a conserved cysteine residue between the first and second disulfide loop. This cysteine does not appear to be used for intermolecular disulfide bonds since neuroligins have very similar migrations on reducing and nonreducing SDS-polyacrylamide gels (data not shown).
Figure 2: Homology of the extracellular domains of neuroligins to esterase domain proteins. The sequences of neuroligins 1, 2, and 3 (Nl 1, Nl 2, and Nl 3) are aligned with those of rat bile-activated lysophospholipase (Bal; Han et al., 1987), Torpedo acetylcholinesterase (Ace; Schumacher et al., 1986; Schumacher, 1988), rat carboxylesterase (Car; Robbi et al., 1990), rat thyroglobulin (Thy; Di Lauro et al., 1985), Drosophila neurotactin (Neu; Hortsch et al., 1990), and G. candidum lipase (Lip; Nagao et al., 1993) in the region of homology between these proteins. Residues present in at least 50% of the sequences are boxed on a blue background. Cysteine residues are shown on a red background to emphasize the presence of conserved cysteine residues in the different protein families involved in disulfide bonding. The serine, glutamate, and histidine residues that form the active sites of the catalytically active esterases are shown on a yellow background. The alternatively spliced sequences in the neuroligins (Fig. 1) have been deleted. Sequences are numbered on the right.
To investigate the tissue distribution of expression of different neuroligins, RNA blots were performed. Hybridization of RNAs from adult rats revealed a strongly positive signal for all three neuroligins that could only be detected in brain (Fig. 3; Ichtchenko et al., 1995). This indicates that, in adults, neuroligins are expressed at high levels only in the nervous system.
Figure 3: Tissue distribution of expression of neuroligins 2 and 3. Blots containing total RNA from the indicated rat tissues was hybridized with uniformly labeled cDNA probes from neuroligins 2 (NL2) and 3 (NL3). As shown on the bottom, RNA loading was controlled by rehybridizing blots with a combination of glyceraldehyde-3-phosphate dehydrogenase and cyclophilin cDNA probes. Numbers on the left indicate positions of molecular weight markers.
Neuroligin 1 was purified and
cloned based on its binding to -neurexins. To determine if
neuroligins 2 or 3 also bind to neurexins, we raised anti-peptide
antibodies against each neuroligin. The three neuroligins were
expressed by transfection in COS cells, and the specificity of the
antibodies was examined using proteins from the transfected COS cells
and from total rat brain homogenates. The results demonstrated that an
antibody against the C terminus of neuroligin 1 reacted with all
neuroligins, presumably because of their high degree of homology to
each other in this region (top panel, Fig. 4). All
other peptide antibodies, however, were specific for their respective
neuroligin (lower three panels, Fig. 4). In rat brain
only, the antibody specific for neuroligin 1 and the antibody against
all neuroligins but not the neuroligin 2 and 3 antibodies detected a
band at the sensitivity of the immunoblot shown, suggesting that
neuroligin 1 is more abundant than neuroligins 2 or 3.
Figure 4: Expression of neuroligins in COS cells and studies of antibody specificity. Total brain homogenates (lane 1) and COS cells transfected with neuroligin 1, 2, and 3 expression vectors (lanes 2-4) were analyzed by SDS-PAGE and immunoblotting using an antibody against the C terminus of neuroligin 1 that reacts with all three neuroligins (anti-NL1/2/3) and with antibodies specific for each of the three neuroligins (anti-NL1, -NL2, and -NL3). Note that at this level of analysis, only neuroligin 1 can be detected in total brain, presumably because the other neuroligins are present at much lower levels.
The
antibodies were used to study the binding properties of neuroligins.
For this purpose, we expressed -neurexins as IgG-fusion proteins
in COS cells and purified them from the medium as described (Ushkaryov et al., 1994). The purified
-neurexin IgG-fusion proteins
immobilized on protein A-Sepharose were then used as an affinity matrix
to isolate binding proteins. As shown in Fig. 5, elution of a
neurexin 1
affinity matrix loaded with rat brain proteins with
increasing salt concentrations in the presence of Ca
resulted in the release of a fraction of the bound neuroligins.
However, the majority of neuroligins remained attached to the matrix
until Ca
was removed by application of EGTA and EDTA.
This result suggests that all three neuroligins bind to neurexin 1
in a Ca
-dependent manner. The
Ca
-dependent binding of neuroligins 2 and 3 to
neurexin 1
was confirmed using recombinant neuroligins expressed
as full-length proteins, showing that it is a direct interaction (data
not shown).
Figure 5:
Ca-dependent binding of
neuroligins 1, 2, and 3 from brain to immobilized neurexin 1
-IgG.
Solubilized membrane proteins from rat brain were loaded in the
presence of Ca
onto a column of neurexin 1
-IgG
immobilized via protein A-Sepharose. The column was washed and eluted
first with a 0.1 to 1.0 M NaCl gradient in
Ca
-containing buffer, and then with 25 mM EGTA and 25 mM EDTA in the same buffer as indicated.
Fractions were analyzed by SDS-PAGE and immunoblotting with the
neuroligin antibodies whose specificity was studied in Fig. 4.
Note that most neuroligins bound to the immobilized neurexin 1
even in the presence of 1 M NaCl and could be eluted only by
withdrawal of Ca
.
One of the most striking aspects of the interaction of
neurexins with neuroligin 1 is that only -neurexins and only those
-neurexins lacking an insert in splice site 4 bind neuroligin 1
(Ichtchenko et al., 1995). To determine if neuroligins 2 and 3
exhibit similar properties, we tested the binding of brain neuroligins
to neurexins 1
, 2
, and 3
lacking inserts in splice site
4, and to neurexins 1
and 2
containing inserts in splice site
4 (Fig. 6). All neuroligins bound preferentially to
-neurexins lacking an insert, and all bound to all three
neurexins. However, slight differences in binding were observed,
especially for neurexin 2
which bound much better to neuroligin 2
than to the other two neuroligins (lane 5, Fig. 6).
Together, these results extend the previous description of a splice
site specific interaction of neuroligin 1 with
-neurexins to the
other two neuroligins and suggest the possibility that different
neurexins and neuroligins may have distinct affinities.
Figure 6:
Specificity of binding of neuroligins 1,
2, and 3 from brain to different neurexins. Control IgG-fusion protein (lane 1) or IgG-fusion proteins of neurexins 1, 2
,
and 3
without or with a splice site insert in the G-domain repeat (lanes 2-6; for neurexin 3
only the variant lacking
an insert was analyzed) were used as affinity matrices for protein
purification from total rat brain homogenate. Eluates were analyzed by
SDS-PAGE and immunoblotting with the indicated antibodies whose
specificity was defined in Fig. 4. The 60-kDa band detected with
the neuroligin 2 antibody is an artifactual reactivity with a
contaminant. Note the distinct elution patterns for different
neuroligins from the neurexin 2
column. Numbers on the left indicate positions of molecular weight
markers.
As described
above, neuroligins are also alternatively spliced similar to neurexins,
although much less extensively. In order to determine if the
alternative splicing of neuroligins had an effect on their interactions
with neurexins, we transfected neuroligin 3 with and without an
alternatively spliced insert into COS cells and examined its
interaction with neurexin 1. No effect of alternative splicing was
observed, suggesting that the alternative splicing of neuroligins does
not regulate its interaction with neurexins (data not shown).
The current study describes the structures, alternative
splicing, tissue distribution, and expression of two new neuroligins,
neuroligins 2 and 3. Neuroligins are expressed at high levels only in
brain where in situ hybridization localized them primarily to
neurons. ()Structurally, neuroligins resemble cell surface
receptors and are exposed on the surface of transfected cells. They are
composed of five domains: an N-terminal cleaved signal sequence, a
large extracellular domain homologous to esterases, a linker domain
between the transmembrane region and the esterase homology domain that
may be O-glycosylated, a single transmembrane region, and a
cytoplasmic tail. Sequence comparisons place the neuroligins into the
large family of esterase homology domain proteins that includes
thyroglobulin, acetylcholinesterase, and gliotactin. However, the
neuroligins are only distantly related to these proteins and form a
unique subset of this protein family ( Fig. 2and Table 1).
Functionally, neuroligins bind tightly to the extracellular domains of
-neurexins in a Ca
-dependent manner. This
interaction depends on the alternative splicing of the
-neurexins
since only those
-neurexins that lack an insert in splice site 4
bind tightly to neuroligins. Together, these data suggest that
neuroligins are neuronal cell surface proteins that interact with a
specific subset of
-neurexins.
Similar to neurexins, neuroligins are alternatively spliced, although to a much lesser extent. All neuroligins are alternatively spliced at the same position in the esterase homology domain; the position of alternative splicing maps to a loop in the crystal structure of acetylcholinesterase (Sussman et al., 1991) and to a position where Drosophila acetylcholinesterase contains an insert compared with other esterases (Fournier et al., 1988). In neuroligins 1 and 2, alternative splicing exists in the presence or absence of a single sequence that shares no homology between neuroligins 1 and 2. By contrast, neuroligin 3 has two types of alternatively spliced inserts. The first is homologous to that observed in neuroligin 1; the second contains in addition to the first insert sequence a sequence homologous to the alternatively spliced neuroligin 2 sequence. Extensive screening failed to reveal similar double inserts for the other neuroligins. The neuroligin 1-type alternatively spliced insert contains two cysteines, suggesting that it may form a disulfide-bonded ring, whereas the neuroligin 2-type insert has no cysteines (Fig. 1). Binding studies with recombinant neuroligins showed that the alternative splicing of neuroligins has no effect on their interaction with neurexins; however, this finding does not exclude the possibility that the alternative splicing may have other functional consequences.
Our experiments establish that neuroligins form a gene family encoding a series of alternative spliced proteins. The data provide further support for the notion that the interaction between neurexins and neuroligins may be important in neuronal cell-cell interactions. With three genes, the number of neuroligin genes mirrors that of neurexins and adds to the growing list of neuronal proteins that are expressed in a large number of isoforms. The question arises if this serves to impart distinct functional properties onto subsets of neurons that express certain combinations of neurexins and/or neuroligins, or if this is an evolutionary accident of random gene duplications. It is striking that in spite of their similarity, the different neurexins and neuroligins are on the average only 60% identical and exhibit major structural differences, suggesting an appreciable evolutionary distance and functional diversification. Although the different neuroligins have similar binding activities for neurexins, it is possible that they have different functions either due to distinct affinities for different neurexins (which could not be detected in our assays) or to interactions with other proteins. Future experiments including studies using knockout mice lacking one or several neuroligins or neurexins should help to clarify these issues.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U41662 [GenBank]and U41663[GenBank].