Neurexin–neuroligin signaling in synapse development Ann Marie Craig and Yunhee Kang Neurexins and neuroligins are emerging as central organizing and knockdown of neuroligins in culture led to the idea molecules for excitatory glutamatergic and inhibitory that these molecules control the balance of GABAergic GABAergic synapses in mammalian brain. They function as cell and glutamatergic inputs [4,5]. We focus our discussion adhesion molecules, bridging the synaptic cleft. Remarkably, here on studies from the past two years that address how each partner can trigger formation of a hemisynapse: alternative splicing in both neurexin and neuroligin tran- neuroligins trigger presynaptic differentiation and neurexins scripts regulates their function. We also discuss initial trigger postsynaptic differentiation. Recent protein interaction results from studies of knockout mice and disease-linked assays and cell culture studies indicate a selectivity of function mutations. conferred by alternative splicing in both partners. An insert at site 4 of b-neurexins selectively promotes GABAergic synaptic Structure of neurexins and neuroligins function, whereas an insert at site B of neuroligin 1 selectively There are three neurexin genes in mammals, each of promotes glutamatergic synaptic function. Initial knockdown which has both an upstream promoter that is used to and knockout studies indicate that neurexins and neuroligins generate the larger a-neurexins and a downstream pro- have an essential role in synaptic transmission, particularly at moter that is used to generate the b-neurexins (Figure 1) GABAergic synapses, but further studies are needed to assess [6]. Alternative splicing at five sites and N- and O-gly- the in vivo functions of these complex protein families. cosylation contribute additional diversity. By contrast, Drosophila melanogaster and Caenorhabditis elegans have Addresses only a single a-neurexin homolog. b-Neurexins contain Brain Research Centre, University of British Columbia, 2211 Wesbrook a single LNS domain (laminin, neurexin, sex-hormone- Mall, Vancouver V6T 2B5, Canada binding protein domain; also known as a Laminin G Corresponding author: Craig, Ann Marie ([email protected]) domain), whereas a-neurexins contain six LNS domains organized into modules with three EGF-like domains. The crystal structure of the neurexin 1b LNS domain Current Opinion in Neurobiology 2007, 17:43–52 revealed a remarkable conservation with the agrin LNS domain in the position of alternative splice sites [7]. This This review comes from a themed issue on Development structural similarity might reflect functional similarity. Edited by Ben Barres and Mu-Ming Poo Alternative splicing of agrin regulates its role as an essential synaptic organizer at the mammalian neuro- muscular junction [8]; as discussed later in this review, 0959-4388/$ – see front matter alternative splicing of neurexin regulates its role at # 2006 Elsevier Ltd. All rights reserved. synapses in the CNS. A recent structure–function study has confirmed that binding to neuroligins and synapto- DOI 10.1016/j.conb.2007.01.011 genic activity is mediated by same face of the neurexin 1b LNSdomainthatcontainsthesplicesites(Figure 1) [9]. The crystal structure of the second LNS domain of Introduction neurexin 1a revealed that this region containing the Neurexin 1a was first identified in 1992 [1] by affinity splice sites forms a highly variable surface surrounding chromatography of rat brain extract on a column of a coordinated Ca2+ ion [10]. Ca2+ binding occurred with a-latrotoxin (a component of black widow spider venom low affinity (Kd 400 mM) and was reduced to below that stimulates massive synaptic vesicle fusion). Over the detectable levels by addition of the eight-residue or subsequent 15 years, pioneering studies by Sudhof and fifteen-residue splice inserts. Thus, some of the Ca2+- colleagues have characterized neurexins and their bind- dependent interactions of neurexins might be influenced ing partners the neuroligins. The intense current interest by reductions in cleft Ca2+ concentrations following in these proteins was triggered by the finding of Scheif- synaptic activity (but note that interaction of neurexin fele et al.[2] that neuroligins presented on the surface of 1b with neuroligin 1 was half-maximal at Ca2+ concen- non-neuronal cells induce synaptic vesicle clustering and trations of 2 mM[11], which is well below the estimated formation of functional release sites in contacting gluta- range in the synaptic cleft). matergic axons. A complementary study by Graf et al. [3] then showed that neurexins presented alone to dendrites Neurexins have three known extracellular binding part- trigger postsynaptic differentiation, inducing clusters of ners in the mammalian brain: neuroligins, dystroglycan GABA postsynaptic components even more so than and neurexophilins. Neuroligins exhibit Ca2+-dependent glutamate postsynaptic components. Overexpression binding to a-neurexins and b-neurexins, dystroglycan www.sciencedirect.com Current Opinion in Neurobiology 2007, 17:43–52 44 Development Figure 1 Structure of neurexins and neuroligins. In humans, there are three neurexin genes and five neuroligin genes. Each neurexin gene uses an upstream promoter to generate the larger a-neurexins and a downstream promoter to generate the smaller b-neurexins. Thus, b-neurexins can be thought of as N-terminally truncated a-neurexins that have a short b-specific leader (bN). In a-neurexins, the LNS (laminin, neurexin, sex-hormone-binding protein) domains are organized with EGF (epidermal growth-factor)-like domains into three homologous modules, I–III. The position of each of five sites of alternative splicing (SS1–SS5) is indicated. Neuroligins contain an extracellular acetylcholinesterase (AChE)-homologous domain that contains one or two sites of alternative splicing (SSA, plus SSB in the case of neuroligin 1). Both neurexins and neuroligins contain a highly glycosylated region (CH) and a transmembrane domain (TM; not present in some splice variants of neurexin 3), and terminate in PDZ-domain-binding sites (PDZ BD). Shown between the neurexins and neuroligins are structures of AChE, a model for the AChE-homologous domain of neuroligins, and the neurexin 1b LNS domain [7]. The position of splice sites SS2–SS4 is shown on a single LNS domain for simplicity, although SS2 and SS3 actually occur in different LNS domains of a-neurexins. Note also that the left face of the neurexin LNS as shown here binds neuroligin [9] but the precise structure and interacting region of neuroligin has not been reported yet. The structure files E.C.3.1.1.7 (AChE) and d1c4ra (neurexin LNS) were downloaded from the Research Collaboratory for Structural Bioinformatics protein data bank (http://www.rcsb.org/pdb/home/home.do) and visualized using the program Visual Molecular Dynamics [66]. shows Ca2+-dependent binding preferentially to a-neur- homo-multimers through the AChE-homologous domain, exins, and neurexophilin binding is Ca2+-independent a structural association that is important for neuroligin and specific to a-neurexin. Neuroligins were first ident- function [15,16]. However, hetero-oligomerization versus ified using an affinity column of neurexin 1b and they are homo-oligomerization is a potential regulatory step that the most intensively studied neurexin binding partners. has not yet been explored. The AChE-homologous region There are five neuroligin genes in humans: NLGN1, of neuroligins contains alternative splice site A, and there NLGN2, NLGN3, NLGN4 and NLGN4Y [12]. The major is an additional splice site B within this region specifically extracellular domain of neuroligins is homologous to in neuroligin 1 (Figure 1). Thus, both neuroligins and acetylcholinesterase (AChE) but lacks cholinesterase neurexins exist in numerous isoforms that derive from activity and mediates binding to neurexins. Curiously, multiple genes and alternative splicing. Neuroligins and overexpression of AChE reduces levels of b-neurexins neurexins both have relatively short intracellular domains in vivo and in culture, and impairs genesis of glutama- that terminate in PDZ-domain-binding sites, which are tergic synapses in culture, indicating that there is cross- presumably important for linking them to other synaptic talk between the two proteins [13,14]. Neuroligins form proteins. Current Opinion in Neurobiology 2007, 17:43–52 www.sciencedirect.com Neurexin–neuroligin signaling in synapse development Craig and Kang 45 Synaptic localization and interacting partners GOPC and SPAR [29–31]. Thus, it came as a surprise when Because neurexin 1a can function as an a-latrotoxin two groups [3,32] independently found that neuroligin 2 receptor to mediate transmitter release [17], it is assumed concentrates not at glutamate postsynaptic sites but that neurexins at least partially localize to the presynaptic at GABA postsynaptic sites. This is in contrast to the terminus; this assumption has been confirmed by anti- glutamatergic postsynaptic localization of neuroligin 1 body labeling and localization of tagged recombinant (Figure 2)[33]. The mechanisms by which neuroligin 2 neurexins [1,3,16]. Ultrastructural localization using is inhibited from binding to glutamate-specific PDZ high-quality antibodies is still needed to define the pre- domain proteins such as PSD-95 in neurons, how it loca- cise localization of neurexins in relation to different lizes to GABAergic synapses and which proteins it binds to synapse types during development. At the cellular