LRRTM2 Induces Excitatory Presynaptic Specializations in the Artificial Synapse-Formation Assay
We transfected COS cells with plasmids encoding only mVenus (control), or mVenus-fusion proteins of LRRTM2 or neuroligin-1, and co-cultured the transfected COS cells with cultured hippocampal neurons. After two days of co-culture, samples were fixed, immunolabeled for mVenus and synaptic markers, and analyzed by quantitative fluorescence microscopy ().
LRRTM2 Expression in COS cells and in Cultured Hippocampal Neurons Increases Excitatory Synapse Density
Immunostaining for synapsin confirmed that LRRTM2, similar to neuroligin-1, induced formation of stable presynaptic terminals onto transfected COS cells (Linhoff et al., 2009
). Analysis with antibodies to the vesicular glutamate- and GABA-transporters (VGLUT1 and VGAT, respectively) demonstrated that LRRTM2 only induced formation of excitatory, VGLUT1-containing synapses on transfected COS cells, whereas neuroligin-1 induced formation of both excitatory and inhibitory-synapses ( and S1A
). Neither LRRTM2 nor neuroligin-1 produced formation of synaptic specializations containing the postsynaptic marker PSD-95.
Neuronal Overexpression of LRRTM2 Increases Excitatory Synapse Numbers
We next examined whether the effect of LRRTM2 can also affect synapse density in neurons. We transfected cultured hippocampal neurons at 10 days in vitro (DIV10) with plasmids encoding only mVenus (control), or mVenus-fusion proteins of LRRTM2 or neuroligin-1, and fixed and immunostained the neurons at DIV14. Using image analysis, we quantified the density and signal intensity of immunoreactive synaptic puncta identified with antibodies for various pre- and postsynaptic markers and for mVenus (). LRRTM2 potently increased the density of excitatory, but not inhibitory synapses on transfected neurons, as measured with both pre- and postsynaptic marker proteins. In this assay, LRRTM2 thus again acted similar to neuroligin-1, but was more effective (). Moreover, LRRTM2 strongly increased the signal of presynaptic markers per punctum on the transfected neurons, also similar to neuroligin-1. However, different from neuroligin-1, LRRTM2 had no significant effect on the postsynaptic signal ().
Neurexins are Candidate Presynaptic Ligands for LRRTM2
To search for presynaptic ligands of postsynaptic LRRTM2, we produced recombinant LRRTM2 composed of the entire extracellular sequence of LRRTM2 fused to the Fc-domain of human IG (Ig-LRRTM2), analogous to previously generated neurexin fusion proteins (Ushkaryov et al., 1994
). As a control, we used IgC that is composed of the first 18 residues of mature neurexin-1α fused to the Fc-domain of human IG. Ig-fusion proteins were produced in transfected HEK293 cells, and purified on protein A-Sepharose (). We then performed affinity chromatography experiments with solubilized rat brains on the immobilized Ig-fusion proteins ().
Affinity Purification of Neurexins on Immobilized LRRTM2
Silver staining of SDS polyacrylamide gels loaded with the input, wash and eluate fractions from affinity chromatography experiments suggested that multiple bands are purified on immobilized LRRTM2. We performed mass spectrometry analyses of all proteins in the silver-stained gels larger than Ig-LRRTM2. Of 140 identified peptides, 31 peptides were derived from neurexins, with all three α-neurexins represented, and 18 peptides were from synaptotagmin-1, which binds to neurexins (; Hata et al., 1993
). Besides neurexins, no other cell-surface proteins were identified in the LRRTM2 affinity-purified fractions, suggesting that neurexins are the most abundant and/or the most tightly binding extracellular interaction partners of LRRTM2.
To confirm that LRRTM2 indeed pulls down neurexins present in detergent-solubilized membrane fractions, we used immunoblotting to analyze which proteins were captured by immobilized LRRTM2 (). We observed a high degree of enrichment of α- and β-neurexins in the LRRTM2-bound fraction, but not of any other cell-surface protein tested, confirming the mass spectroscopy results.
LRRTM2 Directly Binds α- and β-Neurexins in Cell Surface Labeling Assays
To test whether neurexins can directly bind to LRRTM2 on the surface of a cell, we bound various recombinant Ig-fusion proteins of neurexins to HEK293T cells that express full-length LRRTM2-mVenus. We fixed the cells without detergent, and measured surface-bound Ig-fusion proteins by indirect immunofluorescence (). As a negative control, we used IgC, and as a positive control, we employed cells transfected with two different neuroligin-1 splice variants. We found that both neurexin-1α and -1β avidly bound to LRRTM2 in this assay, but only when the neurexins lacked an insert in SS#4. In contrast (but as reported previously, see Boucard et al., 2005
), neurexin-1α containing or lacking an insert in SS#4 did not bind to neuroligin-1 containing an insert in splice sites A and B, but did bind to neuroligin-1 lacking an insert in splice sites A and B. Neurexin-1β, similar to neurexin-1α, also bound to LRRTM2 dependent on SS#4 of neurexin-1β, but was independent of either neurexin-1β or neuroligin-1 alternative splicing (). Thus, binding of neurexin-1α and -1β to LRRTM2 and to neuroligin-1 is differentially controlled by alternative splicing at SS#4 of neurexins, suggesting that neurexin-binding to LRRTM2 and neuroligins operates via distinct but related mechanisms. This conclusion is reinforced by the finding that binding of both neurexin ligands is reversed by EGTA, i.e., is Ca2+
Analysis of LRRTM2/Neurexin Interaction with Cell-Surface Binding and Cell-Adhesion Assays
LRRTM2-binding to Neurexins Mediates Cell Adhesion
To investigate whether binding of LRRTM2 to neurexins is a trans-interaction, i.e. capable of promoting intercellular cell-adhesion (as expected for an interaction between a postsynaptic cell-adhesion molecule with presynaptic neurexins), we examined the ability of surface-expressed LRRTM2 to mediate cell-adhesion with cells expressing neurexins. We transfected HEK293T cells with (i) mVenus or tdTomato alone; (ii) mVenus-fusion proteins of LRRTM2; (iii) mVenus-fusion protein of neuroligin-1, (iv) mCherry-fusion proteins of various neurexins; and (v) tdTomato co-transfected with untagged LRRTM2. One day after transfection, cells were dissociated, and mVenus-expressing and tdTomato- or mCherry-expressing cells were mixed. Cells were imaged immediately after mixing and after a 60 min incubation at room temperature with mild agitation, and free cells were counted at each time point to quantify cell adhesion ().
Since at least in some instances, leucine-rich repeat proteins mediate homophilic cell adhesion (e.g., see the role of connectin in Drosophila synapse formation; Nose et al., 1992
), we first examined whether LRRTM2 mediates homophilic cell adhesion by mixing red (td-Tomato) and green (mVenus) LRRTM2-expressing cells. However, we observed no cell-adhesion (Figure S3A
). We next examined whether LRRTM2-binding to neurexins can mediate cell adhesion. Indeed, cells expressing LRRTM2 formed large aggregates with cells expressing neurexin-1α or -1β, provided that the neurexins lacked an insert in SS#4 (Nrx1αSS4−
, but not Nrx1αSS4+
; ). All three β-neurexins bound with the same splice-site dependence (Figures S3B–S3C
). This splice-site dependence was different from that observed with neuroligin-1, where cells expressing neuroligin-1 lacking an insert in the neuroligin-splice sites A and B (NL1ΔAB
) formed aggregates with all neurexins tested, consistent with the binding data shown in .
However, similar to neuroligin-1, LRRTM2-mediated cell-adhesion to neurexin-containing cells was strictly dependent on Ca2+
-binding to neurexins. Binding was abolished by EGTA, and was blocked by a single amino-acid substitution in neurexin-2β that abolishes Ca2+
-binding to the neurexin LNS domain, and that also abolishes neuroligin-1 mediated cell adhesion (, S3B
, and S4
). No cell adhesion in negative controls was observed ( and Figure S4
). Taken together, these data demonstrate that LRRTM2 forms an intercellular junction via a trans-interaction with α- and β-neurexins in a manner that resembles that of neuroligin-1, but differs from neuroligin-1 in its distinct regulation by alternative splicing of the neurexins.
LRRTM2 and Neuroligin-1 Cannot Bind Simultaneously to Neurexin-1β
To corroborate the surface-binding and cell-adhesion assays with an independent approach, and to additionally test whether neurexins also bind to other LRRTM isoforms, we performed pull-down experiments using the immobilized Ig-fusion protein of neurexin-1β lacking an insert in SS#4 (IgNrx1αSS4−; ). We found that immobilized Ig-neurexin-1β effectively captured all LRRTM isoforms (LRRTM1–4) and neuroligin-1 expressed in HEK293 cells. However, Ig-neurexin-1β did not bind to NGL-3, and IgC used as a negative control was unable to bind to either LRRTMs or neuroligin-1.
Exclusive Binding of Neurexin-1β to either LRRTM2 or Neuroligin-1, and Estimation of the LRRTM2-Neurexin Interaction Affinity
We next reversed the assay, and employed the Ig-fusion protein of LRRTM2 described above (see ) to pull down neurexins expressed as mCherry fusion proteins in HEK293 cells. Strikingly, LRRTM2 pulled down only neurexins lacking an insert in SS#4, and exhibited no activity towards neuroligin-1, confirming the binding specificity observed in the cell-surface binding and cell-adhesion assays ().
Neurexin binding by both neuroligin-1 and LRRTM2 depends on Ca2+-binding to neurexins, and both binding reactions are regulated, although differentially, by SS#4 of neurexins. These observations indicate that despite their lack of homology, LRRTM2 and neuroligin-1 may bind to overlapping sites on the neurexin LNS domain. To test this hypothesis, we investigated whether LRRTM2 and neuroligin-1 simultaneously bind to neurexins. We produced recombinant Ig-LRRTM2, FLAG-tagged neuroligin-1 lacking an insert in splice sites A and B (FLAG-NL1ΔAB), and HA-tagged neurexin-1β lacking an insert in SS#4 (HA-Nr×1βSS4−). We then mixed these proteins in different combinations, and tested whether pulldowns of neuroligin-1 or LRRTM2 would capture not only neurexin-1β, but also LRRTM2 or neuroligin-1, respectively ().
Unequivocally, only neurexin-1β but not with LRRTM2 was brought down with neuroligin-1. Similarly, only neurexin-1β but not neuroligin-1 was brought down with LRRTM2 (). These data demonstrate that neuroligin-1 and LRRTM2 cannot simultaneously bind to neurexin-1β.
Finally, we estimated the binding affinity for the interaction of LRRTM2 and neurexin-1β. LRRTM2-mVenus expressing and control HEK293T cells were incubated with increasing amounts of Ig-neurexin-1β fusion protein lacking an insert in SS#4, and proteins bound to the cell surface were measured with an HRP-tagged secondary antibody. After subtraction of non-specific binding, we calculated a nanomolar Kd by Scatchard analysis (5.83 ± 1.47 nM; ). This result indicates that LRRTM2 binds to neurexins with high affinity, although it should be noted that the dimeric neurexin ligands used in these experiments produce an increased binding affinity.
Soluble Neurexin-1β Impairs the Synaptogenic Activity of LRRTM2
Since neurexins directly interact with LRRTM2 (–), the question arises whether this interaction is essential for the synaptogenic activity of LRRTM2 (). As a first test of this question, we examined whether Ig-neurexin-1β lacking or containing an insert in SS#4 (IgNrx1βSS4−
) can block the function of LRRTM2 in the artificial synapse formation assay (). In these experiments, we used IgC as a negative, and neuroligin-1 as a positive control, and restricted the co-culture time to 12 h to avoid cellular uptake and degradation of the Ig-fusion proteins (Chubykin et al., 2005
Soluble Ig-Neurexin Impairs Synaptogenic Activity of LRRTM2: Model of LRRTM2 Action
but not IgNrx1βSS4+
specifically reduced the synaptogenic activity of LRRTM2 (), suggesting that neurexins are indeed presynaptic receptors for LRRTM2 in the artificial synapse formation assay, similar to neuroligin (Ko et al., 2009
). Interestingly, the synaptogenic activity of neuroligin-1 was also specifically reduced by IgNrx1βSS4−
but not by IgNrx1βSS4+
, although neuroligin-1 (NL1 ΔAB
) binds to both neurexin isoforms in vitro (), possibly because of the differences in neuroligin-1 binding affinities of the two different neurexin splice variants (Boucard et al., 2005
), or because of the dependence of the artificial synapse-formation assay on α-neurexin binding by neuroligin-1 (Ko et al., 2009