Cell surface signalling laboratory

The Cell surface signalling laboratory is identifying new extracellular protein-protein interactions between membrane-embedded receptors that mediate cellular recognition processes. We are investigating how these interactions are involved in fundamental biological questions relating to vertebrate development and the pathology of cardiovascular and infectious diseases.

The team, headed by Gavin Wright, has developed a screening assay called AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes many of the technical problems associated with identifying interactions between membrane-embedded receptor proteins. Using this method, the team takes a unique large-scale approach to identify networks of extracellular protein interactions. The rationale is to compile recombinant protein libraries containing the full repertoire of receptor proteins displayed on the surface of known interacting cell types and screen them against each other to identify novel interactions. We have built libraries of receptor proteins expressed on neurons, and identified receptor interactions likely to be involved in neuronal connections. To understand the function of these interactions in a living animal we have used the zebrafish as a model organism to observe what happens when genes encoding interacting receptor-ligand pairs are disrupted. More recently, we have applied these technologies to tackle questions relating to human diseases such as cardiovascular disorders, and the recognition and invasion of human cells by pathogens.

Background

Kidney cells

Kidney cells [The Wellcome Trust Sanger Institute]
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How do the trillions of genetically identical cells within our bodies develop into the large number of specialised cell types at the appropriate time and place? Many of the instructive signals received by cells originate from their immediate environment: the cell surface. More specifically, signals are sent and received by membrane proteins: special proteins which bridge the membrane surrounding each cell and are able to convey extracellular binding events within the cell.

It is these proteins, and especially the extracellular interactions that they make with other proteins, that are the main interest of the laboratory.

Research

The laboratory aims to discover entirely new signalling pathways by identifying novel cell surface receptor-ligand pairs. Despite the known importance of these interactions in biology, relatively few cell surface proteins have known binding partners because they are biochemically difficult to manipulate.

Screening

Screening [The Wellcome Trust Sanger Institute]
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Biochemical difficulties:

  • the hydrophobic transmembrane region makes these proteins difficult to solubilise;
  • they contain complex and structurally important posttranslational modifications such as disulphide bonds and sugars;
  • the interactions made with other cell surface proteins are highly transient, having half-lives of 1 second or less;

We have developed a scalable screening assay that overcomes these technical limitations and can be used to screen for tens of thousands of potential interactions with a low false positive rate. It is based on detecting direct binding events between soluble recombinant proteins produced in mammalian cells; a typical screening plate is shown.

Zebrafish development.

Zebrafish development. [The Wellcome Trust Sanger Institute]
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To determine the in vivo functional significance of identified cell surface interactions, we use the popular model vertebrate, the zebrafish. Because zebrafish produce many externally developing translucent embryos, they are an excellent system to study the earliest events of vertebrate development.

Zebrafish also have many other experimental advantages, such as rapid gene expression pattern determination by wholemount in situ hybridisation and also the ability to specifically remove gene function.

Expression patterns of two genes.

Expression patterns of two genes. [The Wellcome Trust Sanger Institute]
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The expression patterns of two genes whose protein products physically interact are shown at 14 hours of zebrafish development. One gene is expressed in the developing somites (left) and its binding partner (right) is expressed at the boundaries of this expression, suggesting that they interact in vivo. The interaction networks that we have determined, together with gene expression patterns can be found here

We use the Institute's zebrafish genome sequencing project and zebrafish mutation resource to isolate heritable mutations in the genes that encode proteins for which we have identified interactions. By observing the phenotype of these mutants in the context of the gene expression patterns and biochemical data, we are usually able to understand the role of the interaction during vertebrate development.

One of the main difficulties in the design of novel disease-treating drugs is getting the drug across the largely impermeable membrane that surrounds each of our cells so that it can access its target within the cell. One way of solving this problem is to target the proteins which are found in the extracellular space so that soluble drugs can be delivered systemically. By identifying the function of new cell surface interactions, the research in the laboratory is providing novel therapeutic opportunities for which drugs can be designed.

A neuroreceptor network

The development of the vast number of precise neural connections within the vertebrate brain is perhaps one of the most complex examples of intercellular communication known. A molecular understanding of this process will require a comprehensive protein interaction network of extracellular receptors interpreted in the context of their developmental gene expression patterns. To begin to address this problem, we have used our screening assay described here to determine an interaction network of cell surface receptor-ligand pairs that are expressed in the developing nervous system belonging to the leucine-rich repeat (LRR, red nodes) and immunoglobulin superfamilies (IgSF, blue nodes). The network below was produced by a systematic protein interaction screen between the ectodomain fragments of 52 LRR and 98 IgSF receptors. For each receptor in the network, wholemount in situ hybridization was used to determine when and where the gene is expressed during development. Expression patterns for each receptor-ligand pair were then stage-matched and can be viewed by clicking on the lines connecting the genes. Domain diagrams for each interacting receptor are also shown with the expression patterns for reference. Clicking the nodes will reveal either ZFIN or Ensembl entries for the genes, if available. Some in situ data were provided by B. and C. Thisse (University of Virginia).

Selected Publications

  • A cell surface interaction network of neural leucine-rich repeat receptors.

    Söllner C and Wright GJ

    Genome biology 2009;10;9;R99

    PUBMED: 19765300; DOI: 10.1186/gb-2009-10-9-r99; PMC: 2768988

  • Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions.

    Wright GJ

    Molecular bioSystems 2009

    PUBMED: 19593473; DOI: 10.1039/B903580J

  • Large-scale screening for novel low-affinity extracellular protein interactions.

    Bushell KM, Söllner C, Schuster-Boeckler B, Bateman A and Wright GJ

    Genome research 2008;18;4;622-30

    PUBMED: 18296487; DOI: 10.1101/gr.7187808; PMC: 2279249

  • Setting the tempo in development: an investigation of the zebrafish somite clock mechanism.

    Giudicelli F, Ozbudak EM, Wright GJ and Lewis J

    PLoS biology 2007;5;6;e150

    PUBMED: 17535112; DOI: 10.1371/journal.pbio.0050150; PMC: 1877819

  • Delta proteins and MAGI proteins: an interaction of Notch ligands with intracellular scaffolding molecules and its significance for zebrafish development.

    Wright GJ, Leslie JD, Ariza-McNaughton L and Lewis J

    Development (Cambridge, England) 2004;131;22;5659-69

    PUBMED: 15509766; DOI: 10.1242/dev.01417

  • Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor.

    Foster-Cuevas M, Wright GJ, Puklavec MJ, Brown MH and Barclay AN

    Journal of virology 2004;78;14;7667-76

    PUBMED: 15220441; DOI: 10.1128/JVI.78.14.7667-7676.2004; PMC: 434103

  • Characterization of the CD200 receptor family in mice and humans and their interactions with CD200.

    Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, Song Y, Jenmalm M, Gorman D, McClanahan T, Liu MR, Brown MH, Sedgwick JD, Phillips JH and Barclay AN

    Journal of immunology (Baltimore, Md. : 1950) 2003;171;6;3034-46

    PUBMED: 12960329

  • Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta.

    Itoh M, Kim CH, Palardy G, Oda T, Jiang YJ, Maust D, Yeo SY, Lorick K, Wright GJ, Ariza-McNaughton L, Weissman AM, Lewis J, Chandrasekharappa SC and Chitnis AB

    Developmental cell 2003;4;1;67-82

    PUBMED: 12530964

  • Down-regulation of the macrophage lineage through interaction with OX2 (CD200).

    Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, Barclay AN and Sedgwick JD

    Science (New York, N.Y.) 2000;290;5497;1768-71

    PUBMED: 11099416

  • Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function.

    Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH and Barclay AN

    Immunity 2000;13;2;233-42

    PUBMED: 10981966