A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for “orphan” receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets

A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for “orphan” receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets

Dscam-mediated cell recognition regulates neural circuit formation

The Dscam family of immunoglobulin cell surface proteins mediates recognition events between neurons that play an essential role in the establishment of neural circuits. The Drosophila Dscam1 locus encodes tens of thousands of cell surface proteins via alternative splicing. These isoforms exhibit exquisite isoforms-specific binding in vitro that mediates homophilic repulsion in vivo. These properties provide the molecular basis for self-avoidance, an essential developmental mechanism that allows axonal and dendritic processes to uniformly cover their synaptic fields. In a mechanistically similar fashion, homophilic repulsion mediated by, Drosophila Dscam2 prevents processes from the same class of cells from occupying overlapping synaptic fields through a process called tiling. Genetic studies in the mouse visual system support the view that vertebrate DSCAM also promotes both self-avoidance and tiling. By contrast, DSCAM and DSCAM-L promote layer-specific targeting in the chick visual system, presumably through promoting homophilic adhesion. The fly and mouse studies underscore the importance of homophilic repulsion in regulating neural circuit assembly, whereas the chick studies suggest that DSCAM proteins may mediate a variety of different recognition events during wiring in a context-dependent fashion

Data from: An extracellular biochemical screen reveals that FLRTs and Unc5s mediate neuronal subtype recognition in the retina

In the inner plexiform layer of the mouse retina, ~70 neuronal subtypes form a stereotyped circuit that underlies visual processing. During development, subtypes organize into an intricate laminar structure. This organization is choreographed by extracellular interactions that mediate cell recognition events. To identify recognition proteins involved in lamination, we utilized microarray data from 13 subtypes to identify differentially-expressed cell surface and secreted proteins. Using these candidates, we performed a biochemical screen and identified ~50 previously-unknown receptor-ligand pairs. We tested the response of retinal neurons to several candidates and found that both members of one interaction pair, FLRT2-Unc5C, induce repulsion; each in a different neuronal subtype(s). Consistent with a repulsive role in mediating lamination, we observed a complementary expression pattern of FLRT2 and Unc5C in vivo. We identified that Starburst amacrine cells express FLRT2 and are repelled by Unc5C. These data support a repulsive mechanism for laminar restriction of Starburst amacrines

Mouse retina extracellular receptor-ligand biochemical screen

Biochemical screen for extracellular receptor-ligand interactions in the developing mouse retina. The extracellular domain (ECD) region of 126 cell surface or secreted proteins were tested for pairwise binding in a matrix. ECDs along the x-axis were C-terminally fused to the Fc region of human IgG1. ECDs along the y-axis were C-terminally fused to alkaline phosphatase (AP). Proteins were tested for binding using a high-throughput, ELISA-based binding assay. In this assay, ECD-AP receptor protein is captured on an ELISA plate using an anti-AP antibody. Binding of ECD-Fc ligand protein to the ECD-AP receptor protein is detected by inclusion of an anti-Fc antibody conjugated to HRP. HRP activity is measured using O.D. 650 nm one hour following addition of substrate. Raw data values are reported