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Methods

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

¹ Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom; ² Pfam Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom

Extracellular protein–protein interactions are essential for both intercellular communication and cohesion within multicellular organisms. Approximately a fifth of human genes encode membrane-tethered or secreted proteins, but they are largely absent from recent large-scale protein interaction datasets, making current interaction networks biased and incomplete. This discrepancy is due to the unsuitability of popular high-throughput methods to detect extracellular interactions because of the biochemical intractability of membrane proteins and their interactions. For example, cell surface proteins contain insoluble hydrophobic transmembrane regions, and their extracellular interactions are often highly transient, having half-lives of less than a second. To detect transient extracellular interactions on a large scale, we developed AVEXIS (avidity-based extracellular interaction screen), a high-throughput assay that overcomes these technical issues and can detect very transient interactions (half-lives 0.1 sec) with a low false-positive rate. We used it to systematically screen for receptor–ligand pairs within the zebrafish immunoglobulin superfamily and identified novel ligands for both well-known and orphan receptors. Genes encoding receptor–ligand pairs were often clustered phylogenetically and expressed in the same or adjacent tissues, immediately implying their involvement in similar biological processes. Using AVEXIS, we have determined the first systematic low–affinity extracellular protein interaction network, supported by independent biological data. This technique will now allow large-scale extracellular protein interaction mapping in a broad range of experimental contexts.

E-mail gw2{at}sanger.ac.uk; fax 44-1223-496802.

[Supplemental material is available online at www.genome.org. The sequences of all the genes cloned for this study have been submitted to GenBank, and their accession numbers are listed in Supplemental Table S1. All protein–protein interaction data have been submitted to the IntAct database under accession nos. EBI-1578837 and EBI-1578841 for AVEXIS and SPR validation data, respectively.]

Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.7187808.

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