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Trans-induced cis interaction in the tripartite NGL-1,netrin-G1 and LAR adhesion complex promotesdevelopment of excitatory synapses
Yoo Sung Song1,2, Hye-Jin Lee2,3, Pavel Prosselkov4, Shigeyoshi Itohara4 and Eunjoon Kim2,3,*1Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Korea2Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon, 305-701, Korea3Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea4Laboratory for Behavioral Genetics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan
*Author for correspondence ([email protected])
Accepted 31 July 2013Journal of Cell Science 126, 4926–4938� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.129718
SummaryThe initial contact between axons and dendrites at early neuronal synapses is mediated by surface adhesion molecules and is thought to
induce synaptic maturation through the recruitment of additional synaptic proteins. The initiation of synaptic maturation should betightly regulated to ensure that synaptic maturation occurs selectively at subcellular sites of axo-dendritic adhesion. However, theunderlying mechanism is poorly understood. Here, we report that the initial trans-synaptic adhesion mediated by presynaptic netrin-G1
and postsynaptic NGL-1 (netrin-G1 ligand-1) induces a cis interaction between netrin-G1 and the receptor protein tyrosine phosphataseLAR (leukocyte antigen-related), and that this promotes presynaptic differentiation. We propose that trans-synaptic adhesions at earlyneuronal synapses trigger recruitment of neighboring adhesion molecules in a cis manner in order to couple initial axo-dendritic
adhesion with synaptic differentiation.
Key words: Synapse adhesion molecule, Netrin-G1, NGL-1, LAR
IntroductionSynaptic cell adhesion molecules (CAMs) are thought to regulate
various stages of synapse formation including the initial contact
between axons and dendrites, the formation and stabilization of
early synapses and their differentiation into mature synapses
(Dalva et al., 2007; Han and Kim, 2008; Sudhof, 2008; Brose,
2009; Shen and Scheiffele, 2010; Williams et al., 2010; Siddiqui
and Craig, 2011; Yuzaki, 2011; Ko, 2012). Recently, a large
number of synaptic adhesion molecules have been identified,
examples of which include neuroligins, neurexins, SynCAMs,
LRRTMs, NGLs, SALMs, LAR-PTPs, TrkC, Cblns-GluRd,
IL1RAPL1, IL1RAcP and Slitrks (Biederer et al., 2002; Lin
et al., 2003; Kim et al., 2006; Ko et al., 2006; Wang et al., 2006;
Sudhof, 2008; de Wit et al., 2009; Ko et al., 2009; Linhoff et al.,
2009; Woo et al., 2009b; Kwon et al., 2010; Matsuda et al., 2010;
Siddiqui et al., 2010; Uemura et al., 2010; Takahashi et al., 2011;
Valnegri et al., 2011; Yoshida et al., 2011; Takahashi et al., 2012;
Yoshida et al., 2012; Yim et al., 2013). However, it is unclear
how the trans-synaptic adhesion complexes are coupled to the
recruitment of additional synaptic proteins for synaptic maturation.
In one candidate mechanism for synaptic maturation, the
cytoplasmic regions of pre- and postsynaptic adhesion molecules
interact with multi-domain scaffolding proteins, which further
interact with and recruit other synaptic proteins (Han and Kim,
2008). In addition, synaptic adhesion molecules might interact
with adjacent membrane proteins (e.g. receptors and adhesion
molecules) in a cis manner, resulting in their recruitment and
stabilization at early synapses. Perhaps a more important question
would be whether the initial trans-synaptic interaction is coupled
to the recruitment of adjacent membrane and cytoplasmic
proteins in a regulated manner, allowing synaptic maturation to
occur only at sites of early synaptic adhesion. Such regulated
interactions could provide additional mechanisms of regulation
and flexibility for synaptic assembly and disassembly.
The netrin-G proteins, netrin-G1 and netrin-G2 (also known as
laminet-1 and laminet-2), were originally identified as novel
glycosylphosphatidyl inositol (GPI)-anchored adhesion molecules
that are expressed in distinct populations of neurons (Nakashiba
et al., 2000; Nakashiba et al., 2002; Yin et al., 2002). Netrin-G1
and netrin-G2 are structurally similar to conventional netrins but
do not interact with known netrin receptors, such as UNC-5H and
DCC/UNC-40 (Nakashiba et al., 2000; Nakashiba et al., 2002),
suggesting that they have novel ligands. Indeed, an early study
identified NGL-1 as a specific ligand of netrin-G1 (Lin et al.,
2003), whereas another study identified NGL-2 as a specific ligand
of netrin-G2 (Kim et al., 2006). More recently, NGL-3, which is an
orphan receptor, was found to interact with the LAR family of
receptor protein tyrosine phosphatases (PTPs), which contains
LAR, PTPd and PTPs (Woo et al., 2009b; Woo et al., 2009a;
Kwon et al., 2010).
Functionally, netrin-G proteins were suggested to regulate
neurite outgrowth and patterning of neuronal connections
(Nakashiba et al., 2000; Nakashiba et al., 2002; Yin et al., 2002;
Lin et al., 2003). More recently, however, a study using transgenic
mice lacking netrin-G1 or netrin-G2 expression demonstrated that
these molecules do not affect axonal path-finding but rather
4926 Research Article
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critically regulate the clustering of their postsynaptic partners (i.e.NGL-1 and NGL-2, see below) at specific segments of dendrites,which probably contributes to axonal cue-induced subdendriticdifferentiation (Nishimura-Akiyoshi et al., 2007).
NGL proteins and their ligands (netrin-G1, netrin-G2 and LAR-PTPs) have also been implicated in synapse formation. For
example, NGL proteins expressed in heterologous cells inducepresynaptic differentiation upon contacting axons of co-culturedneurons, suggesting that NGL-dependent trans-synaptic
interactions regulate synapse development (Kim et al., 2006;Woo et al., 2009a; Kwon et al., 2010). In line with this, deletion ofNgl2 in mice or acute knockdown of NGL-2 by electroporation
causes reductions in synaptic transmission and dendritic spinedensity at Schaffer collateral synapses in the CA1 region of thehippocampus in a synaptic-input-specific manner (DeNardo et al.,2012).
Notably, NGL-3 displays a presynapse-inducing activity muchgreater than those of NGL-1 and NGL-2 (Woo et al., 2009a). A
possible explanation for this difference might be that, whereasNGL-3 and LAR directly stimulate synapse formation in abidirectional manner, perhaps the major role for the interactions
between NGL-1 and NGL-2 with netrin-G1 or netrin-G2 mightbe to determine the initial sites of axo-dendritic contact, andcouple these events with subsequent maturation processes by
recruiting, for instance, neighboring adhesion molecules withdirect and stronger synaptogenic activities. In line with this,netrin-G1 and netrin-G2 are GPI-anchored proteins (Nakashibaet al., 2000; Nakashiba et al., 2002; Yin et al., 2002), and have
been suggested to have co-receptors.
Here, we show that the binding of NGL-1 to presynaptic netrin-
G1 induces a cis interaction between netrin-G1 and LAR, and thatthis promotes presynaptic differentiation. These results identifyLAR as a co-receptor of netrin-G1, reveal a novel mode of synapse
formation involving trans-induced cis interaction and suggest amechanism for adhesion-site-specific synapse maturation.
ResultsNGLs and their ligands do not interact in cis underbasal conditionsPresynaptic netrin-G1, netrin-G2 and LAR trans-synaptically
interact with NGL-1, NGL-2 and NGL-3, respectively (Lin et al.,2003; Kim et al., 2006; Woo et al., 2009a). These interactionssuggest that NGLs interact with each other on the postsynaptic
surface in a cis manner, and similarly, that netrin-G1, netrin-G2and LAR interact in cis with each other on the presynapticsurface.
We first explored this possibility in heterologous cells bydoubly expressing netrin-G1 or netrin-G2 with LAR. Clusteringof surface HA-netrin-G1 or HA-netrin-G2 by preclustered HA
antibodies had no effect on Myc-LAR, which exhibited a diffusedistribution on the cell surface (Fig. 1A,B). Similarly, Myc-antibody-induced clustering of Myc-LAR had no effect on HA-
netrin-G1 or HA-netrin-G2. By contrast, HA-neuroligin-1 andneurexin-1b-CFP were coclustered by neuroligin-1 clustering(Fig. 1A,B), consistent with their previously reported cis
interaction (Taniguchi et al., 2007). In coimmunoprecipitationexperiments, netrin-G1 or netrin-G2 and LAR did not form acomplex in heterologous cells (Fig. 1C), further suggesting that
they do not engage in a cis interaction. With respect toNGL, primary clustering of Myc-NGL-1 or Myc-NGL-2by preclustered Myc antibodies did not induce coclustering of
HA-NGL-3 (Fig. 1D). These results suggest that neither NGLsnor their ligands (netrin-G1, netrin-G2 and LAR) display cis
interactions under basal conditions.
NGL-1 binding to netrin-G1 causes netrin-G1 to interact incis with LAR in heterologous cells
We next tested whether ligand binding could induce cisinteractions between NGLs or their ligands. NGL-1 and netrin-
G1 were singly expressed in two different groups of HEK293Tcells; when these cells were mixed, the expressed proteinsconcentrated at the cell–cell interface, indicative of their trans-
cellular adhesion (Fig. 2A,B). Importantly, when HEK293T cellsdoubly expressing netrin-G1 and LAR were mixed with NGL-1-expressing cells, NGL-1, netrin-G1 and LAR all concentrated at
the cell–cell interface (Fig. 2A,B), suggesting that the transinteraction between NGL-1 and netrin-G1 caused netrin-G1 tointeract in cis with LAR. By contrast, NGL-2 failed to induce
coclustering of netrin-G2 with LAR (Fig. 2A,B). In controlexperiments, NGL-1 did not induce coclustering of netrin-G2 andLAR, and similarly, NGL-2 failed to induce coclustering ofnetrin-G1 and LAR. In addition, NGL-1 did not cocluster with
LAR in the absence netrin-G1 coexpression. Here, we used aphosphatase-dead form of LAR (LAR-FLAG-C1522S) in ordernot to make transfected cells unhealthy, which can occur through
tyrosine dephosphorylation of target proteins. This, however,suggests that the NGL-1-induced cis interaction between netrin-G1 and LAR does not require the phosphatase activity of LAR.
Biochemically, soluble NGL-1-Fc fusion proteins incubatedwith HEK293T cells doubly expressing netrin-G1 and LARcaused coprecipitation of NGL-1-Fc with LAR only in the
presence of netrin-G1 (Fig. 2C). By contrast, NGL-2-Fc failed tocoprecipitate with LAR even in the presence of netrin-G2(Fig. 2C). In a control experiment, NGL-1-Fc bound to netrin-G1
but not to LAR (Fig. 2D). These results suggest that netrin-G1binds its ligand (NGL-1), and thereafter interacts with LAR in acis manner. We next tested whether the binding of NGL-3 toLAR could cause LAR to cocluster with netrin-G1 or netrin-G2
in the reverse orientation. In HEK293T cells expressing LAR andnetrin-G1 or netrin-G2, NGL-3-Fc successfully clustered LAR onthe cell surface but failed to induce coclustering of LAR with
netrin-G1 or netrin-G2 (Fig. 2E). In addition, soluble LAR-Fcsuccessfully clustered NGL-3 on the cell surface but failed toinduce coclustering of NGL-3 with NGL-1 or NGL-2 (Fig. 2E).
These results suggest that NGL-1, but not NGL-2 or NGL-3, iscapable of inducing its specific receptor (netrin-G1) to interact incis with LAR.
NGL-1 binding to netrin-G1 on the axonal surface inducescoclustering of netrin-G1 with LAR
As NGL-1 and netrin-G1 are predominantly detected in dendritesand axons, respectively (Kim et al., 2006; Nishimura-Akiyoshiet al., 2007), we tested whether the NGL-1-binding-induced
coclustering of netrin-G1 and LAR occurs in axons. Whencultured hippocampal neurons transfected with netrin-G1 [8–9days in vitro (DIV)] were incubated with preclustered NGL-1-Fc
proteins, they specifically bound to netrin-G1 clusters on MAP2-negative axons (Fig. 3A). By contrast, control Fc proteins failedto cause netrin-G1 clustering on axons. We then doubly
expressed netrin-G1 and LAR in hippocampal neurons andincubated these neurons with NGL-1-Fc. Following NGL-1-Fctreatment, we observed coclustering of netrin-G1 with LAR at
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sites of NGL-1 binding (Fig. 3B,C). In control neurons treated
with Fc alone, LAR did not cocluster with netrin-G1 in axons and
mainly appeared in the soma and dendrites. It seems that LAR
proteins in axons are present in amounts smaller than those in
the cell body and proximal dendrites and/or are diffusely
distributed in axons, making it difficult to detect unless they
are clustered.
We next tested whether NGL-1 can induce coclustering of
endogenous netrin-G1 with LAR. Incubation of cultured neurons
with NGL-1-Fc induced coclustering of endogenous netrin-G1
Fig. 1. NGL proteins or their trans-synaptic ligands do not interact in cis under basal conditions. (A) Netrin-G1 and netrin-G2 do not interact in cis with
LAR on the cell surface. HEK293T cells doubly expressing HA-netrin-G1 or HA-netrin-G2 and Myc-LAR, or HA-neuroligin-1 and neurexin-1b-CFP, were
incubated with preclustered HA or Myc antibodies followed by immunostaining. (B) Quantification of the results in A. Clustering index indicates the average
intensity ratio of secondarily clustered molecules in the region of primary clustering versus non-clustering. For example, the first bar refers to the average intensity
ratio of Myc-LAR in the HA-netrin-G1 area versus non-HA area. Mean 6 s.e.m., n515, ***P,0.001, ANOVA. (C) Netrin-G1 and netrin-G2 do not form
coimmunoprecipitate complexes with LAR in heterologous cells. HEK293T cells doubly expressing Myc-netrin-G1 and LAR-FLAG-C1522S (phosphatase-dead),
or Myc-netrin-G2 and LAR-FLAG-C1522S were immunoprecipitated by FLAG antibodies, and immunoblotted with Myc antibodies. (D) NGL-1 and NGL-2 do
not interact in cis with NGL-3 on the cell surface. HEK293T cells doubly expressing Myc-NGL-1 and HA-NGL-3, or Myc-NGL-2 and HA-NGL-3, were
incubated with preclustered Myc antibodies followed by immunostaining. Scale bars: 10 mm.
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Fig. 2. NGL-1 binding to netrin-G1 induces netrin-G1 to interact in cis with LAR. (A) Surface NGL-1 in one cell induces trans-cellular coclustering of
netrin-G1 and LAR in another cell at the cell–cell interface, forming a zipper-like, protein-enriched line. A group of HEK293T cells expressing NGL-1-EGFP
were co-cultured with another group of cells doubly expressing Myc-netrin-G1 and LAR-FLAG-C1522S for 24 hours, followed by triple staining of the
proteins. Additional combinations of transfection include NGL-2-EGFP with Myc-netrin-G2 and LAR-FLAG-C1522S; NGL-1-EGFP with Myc-netrin-G2 and
LAR-FLAG-C1522S; NGL-2-EGFP with Myc-netrin-G1 and LAR-FLAG-C1522S; NGL-1-EGFP with Myc (Myc only) and LAR-FLAG-C1522S.
(B) Quantification of the results in (A). Clustering index indicates the average intensity ratio of LAR in the area of cell–cell interface versus non-interface area.
Mean 6 s.e.m., n515, ***P,0.001, ANOVA. (C) The binding of NGL-1 to netrin-G1 causes NGL-1 to coprecipitate with LAR. Soluble NGL-1-Fc or NGL-2-
Fc proteins were incubated with HEK293T cells doubly expressing Myc-netrin-G1 or Myc-netrin-G2 and LAR-FLAG-C1522S for 2 hours, followed by
precipitation of NGL-1-Fc and immunoblotting of netrin-G1 or netrin-G2 and LAR. (D) A control experiment showing that NGL-1 does not crossreact with
LAR, whereas it binds and clusters netrin-G1. HEK293T cells expressing Myc-LAR or Myc-netrin-G1 (positive control) were incubated with preclustered
soluble NGL-1-Fc for 2 hours, followed by immunostaining. (E) The interaction between NGL-3 and LAR does not cause NGL-3 to interact in cis with NGL-1
or NGL-12, or LAR to interact in cis with netrin-G1 or netrin-G2. Preclustered soluble NGL-3-Fc proteins were incubated with HEK293T cells expressing
LAR and netrin-G1 or netrin-G2. Alternatively, preclustered soluble LAR-Fc proteins were incubated with cells expressing NGL-3 and NGL-1 or NGL-2.
Scale bars: 10 mm.
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Fig. 3. See next page for legend.
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with endogenous LAR at sites of NGL-1-Fc binding (Fig. 3D).
The specificity of netrin-G1 and LAR antibodies could be
supported by the strong and punctate signals of endogenous
netrin-G1 and LAR induced by the incubation of cultured
neurons with their specific ligands NGL-1 and NGL-3,
respectively (supplementary material Fig. S1A,B). These results
suggest that the binding of NGL-1 to netrin-G1 on axonal
surfaces causes netrin-G1 to interact in cis with LAR.
LAR is required for NGL-1-dependent presynaptic
differentiation
NGL proteins have the capacity to induce presynaptic
differentiation in contacting axons (Kim et al., 2006; Woo et al.,
2009b). Incubation of Myc-netrin-G1-expressing neurons with
NGL-1-Fc caused coclustering of Myc-netrin-G1 with synapsin I
(a presynaptic protein; Fig. 4A,B) and VGlut1 (an excitatory
presynaptic vesicle protein; supplementary material Fig. S2A,B).
In addition, NGL-1-Fc induced uptake of synaptotagmin I
luminal domain antibodies at sites of NGL-1-Fc and Myc-
netrin-G1 coclustering (supplementary material Fig. S2C,D),
suggesting that NGL-1-Fc can induce recycling of presynaptic
vesicles. In control experiments, Fc alone did not cause synapsin
I coclustering at sites of netrin-G1 clusters (Fig. 4,A,B).
To test the possibility that LAR mediates NGL-1-induced
synapsin I clustering, we performed knockdown experiments in
cultured neurons using a previously reported shRNA for LAR (sh-
LAR) and a mismatch control (sh-LAR*) (Fig. 4C) (Mander et al.,
2005). In neurons transfected with sh-LAR (DIV 8–11 or 9–12),
NGL-1-Fc induced lower levels of synapsin I coclustering
compared with those in control neurons expressing an empty
shRNA vector (sh-vec) or sh-LAR*, as indicated by the intensity
ratios of synapsin I and netrin-G1. In rescue experiments, neurons
cotransfected with knockdown-resistant LAR-FLAG (LAR-
FLAGres) (Fig. 4C) and sh-LAR showed synapsin I coclustering
comparable to that in control neurons transfected with
sh-vec (Fig. 4D,E). Interestingly, neurons cotransfected with
phosphatase-dead LAR-FLAG (LAR-FLAG-C1522Sres) and sh-
LAR showed synapsin I coclustering comparable to that observed
in neurons transfected with wild-type LAR-FLAGres (Fig. 4D,E).
These results suggest that LAR is required for NGL-1-dependent
presynaptic differentiation, and that the phosphatase activity of
LAR is not required for this induction.
Domains mediating the cis interaction between netrin-G1and LAR
Netrin-G1 contains one VI domain (also known as the lamininglobular domain) and three V domains (V1–V3; laminin epidermal
growth factor-like or LE-like repeats). The extracellular region ofLAR contains three immunoglobulin domains (Ig) and eightfibronectin III (FNIII) repeats. To identify the domains responsible
for mediating the cis interaction between netrin-G1 and LAR, wegenerated deletion variants of these proteins (Fig. 5A), andperformed binding assays in which HEK293T cells expressingNGL-1 were mixed with HEK293T cells doubly expressing netrin-
G1 and LAR.
Unlike wild-type netrin-G1, a mutant netrin-G1 lacking thedomain V region (Myc-netrin-G1 DV) failed to cocluster withLAR upon NGL-1 binding (Fig. 5B,C). Myc-netrin-G1 DV
displayed normal binding to NGL-1 (Fig. 5B), as alsoconfirmed by NGL-1-Fc binding (Fig. 5D), consistent with thereported involvement of the N-terminal VI (lamin globular)
domain in NGL-1 binding (Seiradake et al., 2011). These resultssuggest that the domain V region of netrin-G1 is important for thecis interaction with LAR. The LAR deletion variants lacking the
eight FN domains (LAR-DFN) and the three Ig domains (LAR-DIg) both failed to interact with NGL-1-bound netrin-G1(Fig. 5E,F), suggesting that the FN and Ig domains in LAR areboth important for the interaction with NGL-1-bound netrin-G1.
Alternative splicing in netrin-G1 regulates NGL-1 andLAR binding
Netrin-G1 displays extensive alternative splicing, mainly in thedomain V region (Nakashiba et al., 2000; Nakashiba et al., 2002;
Yin et al., 2002; Aoki-Suzuki et al., 2005; Meerabux et al., 2005;Eastwood and Harrison, 2008). In the fetal human brain, netrin-G1c and netrin-G1d are most abundant (Meerabux et al., 2005)
(Fig. 6A). We used netrin-G1c in the above-describedexperiments. Netrin-G1m is the longest isoform and contains allthree V domains and a short unknown domain (Ukd). We first usedsoluble NGL-1-Fc binding assays to test whether these splice
variants display different levels of NGL-1 binding. Netrin-G1m onthe cell surface showed reduced levels of NGL-1 binding relativeto that in netrin-G1c and netrin-G1d (Fig. 6B,C), suggesting that
alternative splicing of netrin-G1 regulates NGL-1 binding. Netrin-G1m showed reduced cis interactions with LAR upon NGL-1binding in a mixed HEK cell assay (Fig. 6D,E). The extents of the
reductions were similar to those for NGL-1 binding to netrin-G1,suggesting that this reduction in cis interaction is largelyattributable to reduced NGL-1 binding. We also tested whether
neuronally expressed netrin-G1m display altered cis interactionswith LAR upon NGL-1 binding. Netrin-G1m displayed reducedcis interactions with LAR upon NGL-1-Fc binding, compared withnetrin-G1c and netrin-G1d (Fig. 6F,G). Together, these results
suggest that alternative splicing in netrin-G1 regulates its transinteraction with NGL-1, and these changes might subsequentlyaffect the cis interaction between netrin-G1 and LAR.
NGL-1 binding-defective netrin-G1 fails to interact in ciswith LAR
To obtain further evidence supporting the notion that NGL-1binding to netrin-G1 drives the interaction of netrin-G1 with
LAR, we generated a netrin-G1 point mutant (Y86T in thedomain VI or laminin globular domain) that we predicted wouldlose NGL-1 binding based on the crystal structure of netrin-G1
Fig. 3. NGL-1 binding to netrin-G1 on the axonal surface causes
coclustering of netrin-G1 with LAR. (A) NGL-1-Fc binds to netrin-G1
clusters on MAP2-negative axons. Cultured rat hippocampal neurons at 8–9
DIV were transfected with Myc-netrin-G1 for 3 days, and incubated with
preclustered NGL-1-Fc for 2 hours, followed by staining for Fc, Myc and
MAP2. (B) NGL-1-Fc binding to netrin-G1 induces coclustering of netrin-G1
with LAR. Cultured rat hippocampal neurons at 8–9 DIV were cotransfected
with Myc-netrin-G1 and LAR-FLAG-C1522S for 3 days, and incubated with
preclustered NGL-1-Fc for 2 hours, followed by staining for Fc, Myc and
FLAG. (C) Quantification of the results in B. Integrated intensities of LAR
clusters were normalized to those of netrin-G1. Mean 6 s.e.m., n515,
***P,0.001, ANOVA. (D) NGL-1-Fc binding to endogenous netrin-G1
induces coclustering of netrin-G1 with endogenous LAR. Cultured rat
hippocampal neurons at 11–12 DIV were incubated with preclustered NGL-1-
Fc for 2 hours, followed by staining for Fc, netrin-G1 and LAR.
Quantification of the results could not be performed because endogenous
netrin-G1 signals in Fc alone panels were almost undetectable unless they are
clustered by NGL-1-Fc. Scale bars: 10 mm.
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Fig. 4. See next page for legend.
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(Seiradake et al., 2011). This mutant indeed failed to interact with
soluble NGL-1-Fc (Fig. 7A). In cultured neurons expressing
netrin-G1-Y86T, NGL-1-Fc did not induce coclustering of netrin-
G1-Y86T with LAR (Fig. 7B,C). In this experiment, we used
both wild-type neurons and netrin-G1-deficient neurons obtained
from previously reported knockout mice (Nishimura-Akiyoshi
et al., 2007) to minimize the effect of endogenous netrin-G1
proteins. The results were qualitatively similar in the two cell
types (wild type and knockout), suggesting that the binding of
NGL-1 to netrin-G1 drives the cis interaction of netrin-G1 with
LAR.
DiscussionIn the present study, we found that the trans-synaptic adhesion
between postsynaptic NGL-1 and presynaptic netrin-G1 triggers a
cis interaction of netrin-G1 with LAR on the presynaptic surface.
This induced cis interaction is important for presynaptic
differentiation, suggesting that this is a case in which a
postsynaptic adhesion molecule plays an instructive role in
driving contact-dependent and regulated presynaptic differentiation.
The suggested cis interaction between netrin-G1 and LAR is
supported by similarities in their expression patterns. For
instance, mRNA encoding netrin-G1 is first detected in the
hippocampus from postnatal day 3 (P3), becomes abundant in the
CA1 region until P7 and is then abundant in the dentate gyrus
(DG) after P14 (Eastwood and Harrison, 2008). In the cortex,
netrin-G1 mRNA is detected on the piriform cortex on P0 and is
highly expressed in layers II and III of the entorhinal cortex
throughout the adult stage (Nakashiba et al., 2002; Nishimura-
Akiyoshi et al., 2007). Similarly, mRNA encoding LAR is
detected in the hippocampus on P0, is abundant in the CA1 and
CA3 regions at P7, and mainly exists in the DG after P14. In
cortical layers, LAR mRNA begins to be detected from P0 and
becomes specific to layer II and layer III from P14 (Honkaniemi
et al., 1998; Kwon et al., 2010).
This study extends the known roles of synaptic organizers. Pre-
and postsynaptic organizers are currently thought to couple initial
axo-dendritic synaptic adhesions with the recruitment of
cytosolic proteins at the pre- and postsynaptic sides, thereby
promoting synaptic maturation. Here, we suggest a novel
mechanism that neighboring adhesion molecules could also be
recruited by synaptic organizers. In the present study, we identify
LAR as a novel co-receptor for netrin-G1. Netrin-G1 is a GPI-anchored protein that lacks the cytoplasmic region, making itunlikely to interact with cytoplasmic proteins during synaptic
development. Thus, netrin-G1 has been expected to interact witha co-receptor that contains a cytoplasmic region.
LAR contains two cytoplasmic tyrosine phosphatase domains.The membrane proximal phosphatase domain (D1) is
catalytically active, whereas the membrane distal domain (D2)is inactive but can interact with various cytosolic proteins (Namet al., 1999; Blanchetot et al., 2002). An important binding
partner of the D2 domain is the multi-domain adaptor liprin-a(Pulido et al., 1995), which is further connected to keypresynaptic proteins, including RIM, ELKS/ERC and CASK(Schoch et al., 2002; Ko et al., 2003; Olsen et al., 2005). In
addition, studies using C. elegans, Drosophila and mammaliancells have indicated that LAR and liprin-a crucially regulate pre-and postsynaptic development (Wyszynski et al., 2002; Dunah
et al., 2005; Patel et al., 2006; Spangler and Hoogenraad, 2007;Jin and Garner, 2008).
Our results suggest that the tyrosine phosphatase activity ofLAR is not required for NGL-1-dependent coclustering of netrin-
G1 with synapsin I. Although further details remain to beexplored, it should be noted that LAR directly and trans-synaptically interacts with NGL-3, and that NGL-3 induces
presynaptic differentiation more strongly than NGL-1 in mixedculture assays (Woo et al., 2009b). In addition, LAR directly andtrans-synaptically interacts with IL1RAcP, a postsynaptic
adhesion molecule, in addition to NGL-3 (Yoshida et al.,2012). Whether LAR-dependent presynaptic differentiationrequires phosphatase activity thus remains to be studied under
the context of both cis and trans interactions.
Our study reveals that the netrin-G1–NGL-1 complex has abidirectional function in synaptic development. Netrin-G1 andnetrin-G2 are expressed in distinct subsets of neurons and
distribute mainly to axons (Nakashiba et al., 2000; Nakashibaet al., 2002; Yin et al., 2002), suggesting that netrin-G proteinsdifferentially regulate axonal functions in a pathway-specific
manner. Consistent with this notion, netrin-G1 and netrin-G2 arerequired for surface clustering of NGL-1 and NGL-2,respectively, in subdendritic segments of target neurons(Nishimura-Akiyoshi et al., 2007). These previous results
suggest that this is a case of an axonal factor (netrin-G1 ornetrin-G2) playing an instructive role in driving postsynapticdifferentiation. However, it was unclear whether NGL-1 and
NGL-2 play any instructive roles in presynaptic differentiation.Here, we demonstrate that NGL-1 regulates presynapticdifferentiation through netrin-G1 and LAR, which suggests a
novel role for a postsynaptic factor (NGL-1) in drivingpresynaptic differentiation, and is reminiscent of the reportedenhancement of presynaptic release by postsynaptic neuroligin-1
and PSD-95 through trans-synaptic adhesion (Futai et al., 2007).
An important aspect of the cis interaction between netrin-G1and LAR is that it is ‘induced’ by NGL-1 binding. Reported cisinteractions between neuronal adhesion molecules have been
shown to regulate neurite outgrowth and synaptic development,such as those of SynCAM–SynCAM for synaptic development(Fogel et al., 2011). Examples include the interactions of FLRT–
FGF for neurite outgrowth (Wheldon et al., 2010), ephirinA5–Eph3 for growth cone navigation and axon targeting (Marquardtet al., 2005; Carvalho et al., 2006) and NB-3–L1(CHL1) for
Fig. 4. LAR is required for NGL-1-dependent presynaptic
differentiation. (A) NGL-1-Fc binding induces coclustering of netrin-G1
with synapsin I. Cultured hippocampal neurons transfected with Myc-netrin-
G1 (DIV 8–11 or 9–12) were incubated with NGL-1-Fc for 2 hours, followed
by staining for Fc, Myc and synapsin I. (B) Quantification of the results in A.
Integrated intensities of synapsin I clusters were normalized to those of netrin-
G1. Mean 6 s.e.m., n515, ***P,0.001, Unpaired t-test. (C) Knockdown of
LAR expression by shRNA (sh-LAR) in heterologous cells. Controls were an
empty vector (sh-vec), a mismatch control sh-LAR (sh-LAR*) and
knockdown-resistant LAR rescue constructs (LAR-FLAG res; wild-type and
phosphatase-dead C1522S). Intensities of LAR were normalized to those in
the control lane (sh-vec). n56, **P,0.01, ANOVA. (D) Reduced NGL-1-Fc-
induced coclustering of netrin-G1 with synapsin I in LAR-knockdown
neurons and rescue of this effect by knockdown-resistant LAR (LAR-
FLAGres and LAR-FLAG-C1552Sres). Cultured hippocampal neurons
transfected with Myc-netrin-G1 and sh-LAR, sh-vec or sh-LAR*, or sh-LAR
and LAR-FLAGres or LAR-FLAG-C1522Sres (DIV 8–11 or 9–12) were
incubated with NGL-1-Fc for 2 hours, followed by staining for Fc, Myc and
synapsin I. (E) Quantification of the results in D. n515, **P,0.01, ANOVA.
Scale bars: 10 mm.
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Fig. 5. Domains that mediate the cis interaction between netrin-G1 and LAR. (A) Deletion variants of netrin-G1 and NGL-1. (B) Netrin-G1
lacking the domain V region (DV) fails to interact in cis with LAR upon NGL-1 binding. A group of HEK293T cells expressing NGL-1-EGFP were mixed
with another group of HEK293 cells doubly expressing Myc-netrin-G1 (wild-type or DV) and LAR for 1 day, followed by triple staining.
(C) Quantification of the results in (B). Clustering index indicates the average intensity ratio of LAR in the area of cell–cell interface versus non-interface
area. Mean 6 s.e.m., n515 **P,0.01, Unpaired t-test. (D) Myc-netrin-G1 DV displays normal binding to NGL-1-Fc. HEK293T cells expressing
Myc-netrin-G1 (wild type or DV) were incubated with preclustered NGL-1-Fc proteins. (E) LAR mutants lacking the FN or Ig domains fail to interact in
cis with NGL-bound netrin-G1. A group of HEK293T cells expressing NGL-1-EGFP and another doubly expressing Myc-netrin-G1 and LAR
(wild type and DFN/DIg mutant in pDisplay vector) were mixed cultured for 1 day, followed by triple staining. (F) Quantification of the results in E.
Clustering index indicates the average intensity ratio of LAR in the area of cell–cell interface versus non-interface area. n515, ***P,0.001, ANOVA.
Scale bars: 10 mm.
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Fig. 6. Alternative splicing in netrin-G1 regulates its interaction with NGL-1 and LAR. (A) Splice variants of netrin-G1 used in this study. (B) Netrin-G1m
show reduced levels of NGL-1 binding. HEK293T cells transfected with netrin-G1 splice variants were incubated with NGL-1-Fc (2 hours, non-permeabilized
live cells). (C) Quantification of the results in (B). Mean 6 s.e.m., n515, *P,0.01, ANOVA. (D) Netrin-G1m show reduced levels of cis interaction with
LAR in heterologous cells. One group of HEK293T cells expressing NGL-1-EGFP and another group doubly expressing Myc-netrin-G1 splice variants and
LAR-C1522S were mixed cultured for 1 day, followed by triple staining. (E) Quantification of the results in D. Clustering index indicates the average intensity
ratio of LAR in the area of cell–cell interface versus non-interface area. n515, **P,0.01, ANOVA. (F) Netrin-G1m show reduced levels of cis interaction with
LAR in neurons. Cultured hippocampal neurons doubly expressing Myc-netrin-G1 splice variants and LAR-FLAG-C1522S (DIV 8–11 or 9–12) were incubated
with NGL-1-Fc for 2 hours, followed by triple staining. (G) Quantification of the results in F. n515, **P,0.01, ANOVA. Scale bars: 10 mm.
Cis interaction of netrin-G1 and LAR 4935
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apical dendrite orientation (Ye et al., 2008). However, none of
these cis interactions are known to be induced by trans adhesions.
The NGL-1-induced netrin-G1-LAR cis interaction might
involve a conformational change. In the immune system, T
cells recognize specific antigens presented on antigen-presenting
cells (APCs) and form immunological synapses. Certain T cell
receptors (TCRs) undergo conformational changes upon binding
of a major histocompatibility complex/antigen peptide (MHCp)
ligand, leading to the exposure of a specific epitope on TCRs
(Risueno et al., 2005). Similarly, the ligand binding of integrins
causes a conformational change in which a bent and low-affinity
conformation is converted, by separation of the a- and b-
subunits, into an extended state that has a high affinity for
extracellular matrixes (Carman and Springer, 2003).
The regulated and stepwise cis interactions of NGL-1, netrin-G1
and LAR might have several advantages. First, this would ensure that
LAR, a receptor tyrosine phosphatase that could critically regulate
local phospho-protein environments, is clustered only at the sites of
axo-dendritic adhesion. Stepwise clustering might create additional
sites of regulation for synaptic development. In addition, it could
facilitate synaptic disassembly. For example, removal of trans-
synaptic ligands by proteolytic cleavage, as recently reported for
neuroligin-1 (Peixoto et al., 2012; Suzuki et al., 2012) and NGL-3
(Lee et al., 2013), might rapidly reduce the affinity of cis interactions
and trigger the disassembly of adhesion protein complexes.
Netrin-G1 has been associated with Rett syndrome, autism spectrum
disorders, schizophrenia, and bipolar disorder (Borg et al., 2005;
Eastwood and Harrison, 2008; O’Roak et al., 2012). Many aspects of
these disorders involve abnormalities in brain development. Therefore,
the critical role of netrin-G1 in mediating NGL-1-dependent LAR
clustering and presynaptic differentiation, as identified in this study,
might contribute to the development of these disorders.
There are several unanswered questions. Here, we mainly
investigated the NGL-1-dependent cis interaction of netrin-G1
with LAR. The LAR family contains two additional members,
PTPd and PTPs, which are known to regulate presynaptic
differentiation by trans-synaptically interacting with diversepostsynaptic adhesion molecules including NGL-3, TrkC,
Slitrks, IL1RAPL1 and IL1RAcP (Kwon et al., 2010;
Takahashi et al., 2011; Valnegri et al., 2011; Yoshida et al.,
2011; Takahashi et al., 2012; Yoshida et al., 2012; Yim et al.,
2013). Therefore, PTPd and PTPs could also regulate NGL-1-dependent presynaptic differentiation in a cis manner.
Another question is whether trans-induced cis interactionswould also be observed in other synaptic adhesion molecules such
as neurexins. Although this possibility remains to be explored, it is
conceivable that the mechanisms that we proposed in this study
cooperate with neurexins for presynaptic differentiation. Known
interactions of presynaptic proteins predict that LARs andneurexins cooperate, as we suggested previously (Woo et al.,
2009a). Specifically, LAR directly interacts with liprin-a (Pulido
et al., 1995) and neurexins indirectly interact with liprin-a through
CASK (Hata et al., 1996; Olsen et al., 2005). Given that liprin-aindirectly associates with synaptic vesicles through presynapticproteins including RIM and ELKS/ERC (Schoch et al., 2002; Ko
et al., 2003), LAR and neurexins might converge onto liprin-a for
cooperative presynaptic differentiation.
In conclusion, our study proposes a novel principle for synapse
formation: trans-synaptic adhesions induce cis interactions with
neighboring adhesion molecules to promote synaptic development.
In addition, our study suggests LAR as a co-receptor of netrin-G1
and proposes a novel role for synaptic organizers in recruitingneighboring membrane proteins.
Materials and MethodsDNA constructs and antibodies
A Myc epitope was inserted into the site between amino acid (aa) residues 43 and44 in human netrin-G1c (BC030220), and between residues 32 and 33 in mousenetrin-G2a (AB052336) in pEGFP-N1. For rat netrin-G1d and mouse netrin-G1m
Fig. 7. NGL-1 binding-defective netrin-G1 fails
to interact in cis with LAR. (A) Netrin-G1-Y86T
fails to interact with NGL-1. HEK293T cells
expressing Myc-netrin-G1 (WT and Y86T) were
incubated with NGL-1-Fc, followed by
immunostaining. (B) Netrin-G1-Y86T fails to
cocluster with LAR upon NGL-1 binding.
Cultured hippocampal neurons expressing Myc-
netrin-G1 and LAR-FLAG-C1522S or neurons
from netrin-G1-deficient mouse brain
coexpressing Myc-netrin-G1 (wild type or Y86T)
and LAR-FLAG-C1522S were incubated with
NGL-1-Fc for 2 hours followed by triple staining.
(C) Quantification of the results in B. Mean 6
s.e.m., n515, **P,0.01, ANOVA. Scale bars:
10 mm.
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(Meerabux et al., 2005), constructs were subcloned into pEGFP-N1 with N-terminalMyc tagging (EGFP not fused with the protein). Full-length human NGL-1(NM020929, aa 1–641) and mouse NGL-2 (DQ177325, aa 1–652) were subclonedinto pEGFP-N1. For Fc tagging, full-length ectodomains of NGL-1 (aa 1–484) andNGL-2 (aa 1–483) were subcloned into modified pEGFP-N1, where EGFP wasreplaced with human Fc. Human LAR (Y008115, aa 1–1881) C1522S (phosphatasedead) was subcloned into pEGFP-N1 with C-terminal FLAG tagging. Deletionvariants of LAR in pDisplay (LAR-DIg and LAR-DFn) have been described (Kwonet al., 2010). sh-RNA resistant LAR rescue constructs (LAR-FLAGres) wereobtained by introducing silent mutations using QuikChange kit; nucleotides 5461–5479 were replaced by 59-GGCCTGCACAGCTATACAG-39. For deletion of Vdomains in netrin-G1 (DV), aa 297–793 were deleted. For LAR deletion constructs,full-length LAR-Ecto (aa 17–1163), LAR-Ig1–3 (aa 35–295) and LAR-FN1–8 (aa309–1078) were subcloned into pDisplay. Polyclonal EGFP antibodies (#1997) weregenerated against H6-EGFP (aa 1–240) in guinea pigs. Synapsin I (Millipore), HA(Santa Cruz Biotechnology), FLAG (Sigma), Myc (Santa Cruz Biotechnology),VGlut1 (SYSY), synaptotagmin I luminal domain (SYSY) and LAR (NeuroMab)antibodies were purchased. Netrin-G1 antibody has been described (Nakashiba et al.,2002) we performed preclearing by repeatedly incubating netrin-G1 KO brain sliceswith the antibodies to increase the specificity.
HEK293T cell adhesion assays
HEK293T cells were cultured on 60 mm dishes and incubated for 24 hours after DNAtransfection. One dish was transfected with NGLs, and the other dish was doublytransfected with netrin-G1 or netrin-G2 and LAR. HEK293T cells from the two disheswere dissociated, transferred to 18 mm cover glasses and incubated for 24 hours.
Neuron transfection and immunocytochemistry
Primary hippocampal neurons were prepared from embryonic day 18 rats orpostnatal day 0–1 knockout mice. Neurons were transfected using a calcium-phosphate-based mammalian transfection kit (Clontech). Neurons were transfectedwith DNA constructs at 8–9 DIV and maintained until 11–12 DIV. Solublepreclustered NGL-1-Fc proteins were incubated with neurons at 11–12 DIV for2 hours. Neurons were fixed with 4% paraformaldehyde, 4% sucrose,permeabilized with 0.2% Triton X-100 in phosphate-buffered saline, andincubated with primary antibodies, followed by secondary Cy3-, Cy5- or FITC-conjugated antibodies (Jackson ImmunoResearch). For netrin-G1 and LARendostaining, neurons were fixed and permeabilized with methanol for10 minutes at 220 C. Synaptotagmin I luminal domain uptake assay wasperformed as previously described (Kraszewski et al., 1995). Briefly, culturedneurons were incubated with synaptotagmin I luminal domain antibodies (1:10) inKrebs-Ringer HEPES solution for 1 minute.
Image acquisition and quantification
Z-stacked images were randomly captured by confocal microscopy (LSM510,Zeiss) and were analyzed using MetaMorph image analysis software (UniversalImaging). For quantification of preclustered antibody and cell–cell interfacesignals on HEK cell surfaces, average intensities of the clusters from ,15 cellswere analyzed. For quantification of synapsin I clusters induced by netrin-G1clustering, captured neuronal images were thresholded, and the integratedintensities of synapsin I clusters were normalized to those of netrin-G1. Valuesdisplayed indicate mean 6 s.e.m. and statistical significance was determined byone-way ANOVA (Tukey’s test).
Author contributionsY.S. and E.K. designed the experiments, analyzed results and wrotethe manuscript; Y.S., H.J and P.P. performed experiments; E.K. andS.I. jointly supervised Y.S.
FundingThis study was supported by the Institute for Basic Science (IBS),and, in part, by the Funding Program for World-Leading InnovativeR&D on Science and Technology from the Japanese Society for thePromotion of Science and an Intramural Research Grant from theRIKEN Brain Science Institute.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.129718/-/DC1
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