A cell surface interaction network of neural leucine-rich repeat receptors
© Söllner and Wright; licensee BioMed Central Ltd. 2009
Received: 9 June 2009
Accepted: 18 September 2009
Published: 18 September 2009
The vast number of precise intercellular connections within vertebrate nervous systems is only partly explained by the comparatively few known extracellular guidance cues. Large families of neural orphan receptor proteins have been identified and are likely to contribute to these recognition processes but due to the technical difficulty in identifying novel extracellular interactions of membrane-embedded proteins, their ligands remain unknown.
To identify novel neural recognition signals, we performed a large systematic protein interaction screen using an assay capable of detecting low affinity extracellular protein interactions between the ectodomains of 150 zebrafish receptor proteins containing leucine-rich-repeat and/or immunoglobulin superfamily domains. We screened 7,592 interactions to construct a network of 34 cell surface receptor-ligand pairs that included orphan receptor subfamilies such as the Lrrtms, Lrrns and Elfns but also novel ligands for known receptors such as Robos and Unc5b. A quantitative biochemical analysis of a subnetwork involving the Unc5b and three Flrt receptors revealed a surprising quantitative variation in receptor binding strengths. Paired spatiotemporal gene expression patterns revealed dynamic neural receptor recognition maps within the developing nervous system, providing biological support for the network and revealing likely functions.
This integrated interaction and expression network provides a rich source of novel neural recognition pathways and highlights the importance of quantitative systematic extracellular protein interaction screens to mechanistically explain neural wiring patterns.
Identifying the vast number of precise intercellular connections that ultimately account for higher cognitive functions in vertebrate nervous systems, and explaining how they develop, remains one of the main challenges facing neuroscience . Receptor proteins displayed on the surface of neurons are known to relay extracellular recognition events to elicit appropriate cellular responses such as axon guidance, neuron migration and synapse formation, but in comparison to the complex cellular networks that they regulate, relatively few extracellular recognition receptor interactions have been identified [2, 3]. Comparative genome analysis and large-scale gene expression studies, however, reveal that vertebrates contain large families of neurally expressed receptor proteins that are expanded relative to invertebrates . These genes are likely to account for the increased complexity of vertebrate nervous systems and two major families are the leucine-rich repeat (LRR) and extracellular immunoglobulin superfamily (IgSF). The neuronal roles of some proteins containing IgSF domains have been well documented (see  for a review) but the functions of LRR family members are less well characterized.
Despite this involvement in neurological diseases, very little is known about their function and especially their extracellular binding partners. Indeed, of the approximately 20 paralogous subfamilies of membrane-tethered vertebrate LRR-domain-containing receptors , extracellular binding partners have been identified for just five: the Lingo, Lrrc4, Flrt, Amigo and NgR subfamilies. One explanation for this disparity is that membrane-embedded receptor proteins are experimentally intractable: they are generally of low abundance and their amphipathic nature makes them difficult to solubilise since they usually contain both large hydrophilic glycans and at least one hydrophobic transmembrane region. Interactions between receptor proteins are also characterised by extremely low interaction strengths, often having half-lives of fractions of a second when measured in their monomeric state . The fleeting nature of these interactions is necessary to permit facile independent motility of migrating cells or growth cones when many receptor proteins arrayed on apposing cell membranes interact. These properties, however, make identifying novel extracellular recognition events mediated through cell surface proteins technically challenging.
The aim of this study was to identify novel receptor interactions that are involved in neural cellular recognition events, focussing in particular on the LRR and also IgSF receptor families. Furthermore, by identifying when and where each gene of an interacting pair is expressed during neural development, we could construct dynamic maps of the neural intercellular recognition program. Using a recombinant protein library of 150 neural receptor ectodomains and a highly stringent interaction assay suitable to detect low affinity extracellular interactions, we identified extracellular binding partners for orphan receptor families - such as the Lrrtms, Lrrns and Elfns - and novel partners for well-characterised receptors, including Unc5b. Paired spatiotemporal gene expression patterns of all genes within the network revealed when and where these interactions might occur during neural development. This neuroreceptor interaction network with integrated gene expression data provides a useful resource to mechanistically explain how complex cellular neural networks develop.
A protein interaction network of leucine-rich repeat neuroreceptors
To identify extracellular receptor interactions involved in neural recognition processes, we initially focused on the zebrafish LRR family since they represent a large group of receptor proteins expressed in the nervous system, many of which are 'orphan' receptors. We first identified members of this family by performing a comprehensive bioinformatics search of the zebrafish genome. Orthologues for each of the known type I membrane-tethered and glycophosphatidylinositol-linked mammalian subfamilies  were identified and at least one representative was successfully cloned by RT-PCR, with the one exception of the Lrig subfamily (Figure 1a; see Additional data file 4 for a comprehensive list). In total, ectodomain expression constructs were made for 53 genes, which accounts for the vast majority (approximately 80%) of this class of LRR neuroreceptors in the zebrafish genome. To identify novel interactions, we used the AVEXIS (for AVidity-based EXtracellular Interaction Screen) assay developed in our laboratory, which is able to detect very low affinity extracellular interactions (t1/2 ≤ 0.1 s) and can be scaled to screen thousands of binding events with a very low false positive rate . This assay requires that each ectodomain is expressed as a monomeric biotinylated bait as well as a multimerized, enzyme-tagged prey (Additional data file 1). In total, 49 baits and 52 preys were expressed at sufficient levels and were then normalized prior to screening . The biotinylated monomers were arrayed onto streptavidin-coated microtitre plates, and binary interactions identified by probing these arrays with the prey ectodomains. A primary screen between the LRR receptors of 49 × 52 = 2,548 interaction tests was performed and all positive interactions were then re-tested in both bait-prey orientations in a validation screen using fresh protein preparations. Seventeen interactions between 12 proteins were identified and classified into two confidence categories (Figure 1b; see Materials and methods for full details).
LRR neuroreceptors have binding partners within the IgSF
Since the IgSF is a well documented receptor family for LRR domains [26, 27], we next systematically screened the LRR proteins against a large library of 97 bait ectodomains belonging to the zebrafish IgSF (see Additional data file 5 for a comprehensive list). In total, 52 × 97 = 5,044 interactions were screened and positive interactions were subsequently retested using independent protein preparations in both bait-prey orientations. A further 17 interactions involving nine IgSF proteins were added to our neuroreceptor interaction network and similarly placed into two confidence categories (Figure 1c).
All interactions within the LRR-IgSF network except one  were previously unknown. The systematic nature of the screen revealed novel extracellular interactions for well described axon guidance receptors. For example, we identified novel LRR-domain-containing transmembrane ligands for the receptors Robo2 and 3, which we have shown bind to zebrafish Slit proteins (see Materials and methods) demonstrating that they were functionally active. Robo2 interacted with Lrrc24 and Lrrtm1, and Robo3 with Elfn1, suggesting that the Robo receptors are able to respond to local membrane-tethered signals in addition to secreted ligands such as Slit. Similarly, Unc5b, a known receptor for Netrin [29, 30], interacted with three out of the four Flrt-family homologs  (Figure 2). Other IgSF-LRR receptor interactions were found for the Lrrtm1 protein, which interacted with three out of four fibroblast growth factor receptor homologs in the screen (Fgfr4, Fgfrl1a and Fgfrl1b), and novel binding partners for both the axon guidance receptor Boc, and the myelin-associated glycoprotein Mag.
Interaction strengths between related neuroreceptors quantitatively vary
Paired receptor gene expression patterns reveal dynamic cellular neural recognition maps
The binding network of IgSF and LRR receptors (Figure 2) is a static representation of possible extracellular protein interactions and does not reflect the spatial and temporal ordering of recognition events used in the developing nervous system. To reveal when and where these binding events might occur, we determined the expression patterns of all the receptor genes within the network at four stages of zebrafish embryonic development (Additional data file 7 and see Materials and methods for details of an online database of paired stage and orientation-matched images) using mostly two-color fluorescent in situ hybridization to directly compare the expression of each gene encoding an interacting receptor pair within the same embryo.
This study represents the first step towards mapping an extracellular interaction network between neural receptor proteins, a resource that will be necessary to understand the intercellular recognition processes that ultimately underlie brain development and function. The importance of understanding these processes is becoming increasingly apparent as neurological disorders are more frequently being viewed as a product of abnormal brain development . Significantly, we have described here binding partners for three orphan LRR receptor subfamilies, including the Lrrtms, which have been implicated in neurological diseases, including schizophrenia. While the LRR and IgSF are both large families of neurally expressed receptors, there are several other families of cell surface proteins that contribute to neural recognition processes. A comprehensive extracellular network of interactions within the developing nervous system will require the addition of these protein families to our protein library. Crucially, however, we have shown that the systematic screening approach using the AVEXIS method has the scalability and sensitivity to detect transient interactions that are not generally detected by other high throughput protein binding assays. Beyond identifying extracellular binding partners for orphan receptor families, this systematic unbiased method can identify additional binding partners for receptors that already have identified ligands.
Currently, our protein library contains approximately 80% of the zebrafish neural LRR receptors, providing a high density coverage for this family of receptors, which are known to be important for synaptic target selection . We have shown that LRR receptor proteins are able to form both homophilic and heterophilic interactions within the family but also interact with receptors from the IgSF. Despite this large scale approach, we did not identify binding partners for all LRR subfamilies; indeed, both the Slitrk and Lrrc3 subfamilies still have no documented extracellular binding partner. LRR receptors are also known to bind other protein families such as the Netrin-G  and tumor necrosis factor-receptor family  and the future inclusion of these receptor families into our interaction screens is likely to reveal further binding partners for these subfamilies.
The AVEXIS assay was developed and implemented at a high stringency threshold to effectively eliminate false positives so as to produce high-quality datasets . Using this stringency, approximately 0.5% of unique interactions screened - calculated using just one bait-prey orientation - are positive. Although difficult to directly compare due to the ascertainment biases inherent in selecting proteins restricted to a particular subcellular localization (such as the plasma membrane) or screening within protein families previously demonstrated to interact, this interaction frequency lies between large-scale binary yeast-two-hybrid assays (approximately 0.01%)  and the LUMIER assay (approximately 8%) . The paucity of zebrafish protein interaction data makes a false negative rate difficult to assess, but by using the closest mammalian orthologue, the main class of false negatives comprised homophilic interactions. This is most likely due to prey-prey associations , although it should be noted that AVEXIS is able to detect some homophilic interactions and further work is required to determine the biochemical and/or structural reasons for this difference. A complementary scalable assay dedicated to identifying homophilic receptor interactions has been developed and could be used to specifically detect this class of interactions . AVEXIS may also not be generally suitable to detect interactions between ectodomains that interact in cis to form co-receptor complexes since no interaction between NgR1 and Lingo1 ectodomains was detected . During the preparation of this manuscript, an independent study reported the Flrt3-Unc5b interaction in Xenopus and demonstrated its role in cell adhesion processes during early embryogenesis .
The systematic nature of our screening approach revealed that many receptors have multiple binding partners with compatible expression patterns, raising the possibility of binding competition at the cell surface. While parameters such as abundance, local clustering and accessibility will also influence binding in vivo, the intrinsic binding affinity of a ligand for its receptor is important for resolving and measuring these effects. The finding that the three Flrt paralogs have different binding affinities for the Unc5b receptor, spanning at least an order of magnitude, was surprising and is likely to influence their ability to initiate signaling in vivo. Quantitative measurements of adhesion receptors in the immune system have shown that solution interaction strengths weaker than approximately 50 μM are unlikely to be high enough to support spontaneous interactions at physiological surface densities, highlighting the functional relevance of these measurements .
We have initially focused on two large families of neural receptor proteins - LRR and IgSF - as a starting point to begin a systematic approach to identify all extracellular recognition events required in the development of the vertebrate nervous system. In principle, this approach could be applied to other receptor families and secreted ligands. We anticipate that these networks of recognition receptors interpreted in the context of their corresponding gene expression patterns will provide a valuable new resource for neurobiology and will stimulate further research into the functional role of these interactions.
Materials and methods
Zebrafish were maintained on a 14/10 hour light/dark cycle at 28.5°C according to UK Home Office and local institutional regulations, and staged according to Kimmel . Embryos used for in situ hybridization were the progeny of a WIK/alb outcross; alb/alb embryos were used where endogenous pigment obscured staining signals.
Gene cloning and ectodomain library construction
The entire predicted extracellular and transmembrane regions of cell surface LRR-domain-containing genes were amplified by RT-PCR from mixed-stage zebrafish cDNA using oligonucleotides designed from automated gene predictions of the zebrafish genome . PCR products were either cloned or used as further templates to amplify predicted ectodomains, which were ligated into a mammalian expression vector based on pTT3 . The protein library was produced as previously described .
Interactions were identified using the AVEXIS procedure as described . Each plate contained both negative and positive controls as shown in Additional data file 1. Negative controls were the plate prey presented to the baits rat Cd4d3+4 (well H7), Cd200R (H8) and Cd200 (H9). Positive controls were Cd200R prey and Cd200 bait (H10) and the Cd200 bait diluted 1:10 (H11) and 1:100 (H12). Fifty-two LRR prey proteins were systematically screened against 49 LRR and 97 IgSF ectodomain bait proteins derived from membrane-bound receptors . The vast majority of the LRRs and 28 of the IgSF proteins (indicated in Additional data file 5) were initially screened in both bait-prey orientations. Protein pairs that showed positive interactions in the first-pass screen were re-expressed and systematically re-screened in the same matrix-style manner as both baits and preys in an independent validation screen. Interactions that were positive in the first screen and could be detected in a reciprocal fashion were considered as high confidence interactions. Other interactions, such as those that were dependent upon the bait-prey orientation, were regarded as lower confidence interactions. Full details of the screening results are shown in Additional data file 6 and the protein interactions from this publication have been submitted to the International Molecular Exchange Consortium (IMEx)  through IntAct (pmid: 17145710) and assigned the identifier IM-11659. Expected interactions, including those between the zebrafish Robo and Slit orthologs, were detected in subsequent and ongoing interaction screens showing that the recombinant proteins are functionally active and full details are available at IntAct: Robo1-Slit1b, 2, 3 (EBI-2268920, EBI-2269164, EBI-2269173), Robo2-Slit2 (EBI-2269141) and Robo3-Slit1b, 2 (EBI-2269026, EBI-2268001).
Fluorescent bead binding
The extracellular regions of rat Cd200, Lrrn1, Vasn, Robo2, Lrrtm1, Unc5b and Flrt3 used in the AVEXIS screening were cloned into a pTT3-based expression vector to produce a chimeric construct that contained the transmembrane domains of the rat Cd200R and the green fluorescent protein in the cytoplasmic region. HEK293E cells were transfected with these constructs, harvested 2 to 3 days later, washed three times in phosphate-buffered saline/1% bovine serum albumin, vortexed and approximately 5 × 105 cells aliquoted into each well of a flat-bottomed 96-well microtitre plate. Interactions were then detected using a modified version of the fluorescent bead binding experiments described in . Cells were then presented to biotinylated bait proteins immobilised around streptavidin-coated Nile Red fluorescent 0.4 to 0.6 μm microbeads (Spherotech Inc., Lake Forest, IL, USA) at a ratio of approximately 120 beads per cell. After incubating for an hour on ice the cells and beads were resuspended in 250 μl of phosphate-buffered saline/1% bovine serum albumin and analyzed for binding events using a BD LSR II flow cytometer and the data were analysed using FlowJo v7.5.3 software (Tree Star, Inc., Ashland, OR, USA).
Protein purification and BIAcore analysis
Protein purification and BIAcore analysis were performed as described . Briefly, the ectodomain of Unc5b was produced in mammalian cells as a Cd4d3+4-6His-tagged protein and purified on a 1 ml His-Trap column (GE Healthcare, Amersham, Bucks, UK). Protein aggregates, which are known to influence kinetic experiments, were removed by gel filtration using a 125 ml Superose6 column prior to BIAcore analysis. The indicated amounts of the Flrt-Cd4d3+4-bio baits were immobilized onto a streptavidin-coated sensor chip and approximate molar equivalents of Cd4d3+4-bio were used as a reference. All binding studies were performed in HBS-EP buffer (GE Healthcare, Amersham, Bucks, UK) at zebrafish physiological temperature (28°C). Flow rates of 100 μl min-1 were used for kinetic studies to minimize rebinding effects and data were collected at the maximum rate of 10 Hz. Equilibrium dissociation and both on and off rate constants were calculated using the appropriate fitting model in the BIAevaluation software.
Fluorescent two-color wholemount in situ hybridizations were essentially carried out as described . RNA probes were prepared from a template amplified from the protein expression constructs encoding the entire ectodomain fragments. To facilitate comparison, single color images of the gene expression patterns at several stages of development were stage and orientation-matched and are presented in an online database at . Expression data are also publicly available at .
Fluorescently labeled zebrafish embryos were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and images were captured either on a Leica SP5 confocal microscope or a Zeiss Axioplan2 compound microscope fitted with a Volocity OptiGrid structured light device (Improvision, Coventry, UK).
Additional data files
The following additional data are available with the online version of this paper: a figure showing an outline of the AVEXIS procedure (Additional data file 1); a figure showing validation of interactions using a fluorescent bead-based assay (Additional data file 2); a figure showing that interacting neuroreceptors display both complementary and overlapping expression patterns in the developing brain (Additional data file 3); a table listing the zebrafish LRR genes cloned and used to produce recombinant ectodomains (Additional data file 4); a table listing the 97 zebrafish IgSF ectodomain baits (Additional data file 5); a table classifying the neuroreceptor interactions using AVEXIS (Additional data file 6); a table listing the spatiotemporal expression of each gene within the interaction network (Additional data file 7).
avidity-based extracellular interaction screen
We thank Bernard and Christine Thisse for high throughput in situ analysis; Jim Stalker for the online database; Madushi Wanaguru for help with bead binding experiments; and Seth Grant, Elisabeth Busch-Nentwich and members of the laboratory for comments on the manuscript. Our work was supported by the Wellcome Trust (grant number 077108/Z/05/Z) and both a Marie Curie and Sanger postdoctoral fellowships to CS. None of the funding bodies had any influence in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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