Construction of a Large Extracellular Protein Interaction Network and Its Resolution by Spatiotemporal Expression Profiling

Abstract

Extracellular interactions involving both secreted and membrane-tethered receptor proteins are essential to initiate signaling pathways that orchestrate cellular behaviors within biological systems. Because of the biochemical properties of these proteins and their interactions, identifying novel extracellular interactions remains experimentally challenging. To address this, we have recently developed an assay, AVEXIS (avidity-based extracellular interaction screen) to detect low affinity extracellular interactions on a large scale and have begun to construct interaction networks between zebrafish receptors belonging to the immunoglobulin and leucine-rich repeat protein families to identify novel signaling pathways important for early development. Here, we expanded our zebrafish protein library to include other domain families and many more secreted proteins and performed our largest screen to date totaling 16,544 potential unique interactions. We report 111 interactions of which 96 are novel and include the first documented extracellular ligands for 15 proteins. By including 77 interactions from previous screens, we assembled an expanded network of 188 extracellular interactions between 92 proteins and used it to show that secreted proteins have twice as many interaction partners as membrane-tethered receptors and that the connectivity of the extracellular network behaves as a power law. To try to understand the functional role of these interactions, we determined new expression patterns for 164 genes within our clone library by using whole embryo in situ hybridization at five key stages of zebrafish embryonic development. These expression data were integrated with the binding network to reveal where each interaction was likely to function within the embryo and were used to resolve the static interaction network into dynamic tissue- and stage-specific subnetworks within the developing zebrafish embryo. All these data were organized into a freely accessible on-line database called ARNIE (AVEXIS Receptor Network with Integrated Expression; www.sanger.ac.uk/arnie) and provide a valuable resource of new extracellular signaling interactions for developmental biology.

A Floor-Plate Extracellular Protein-Protein Interaction Screen Identifies Draxin as a Secreted Netrin-1 Antagonist

“…Seven of the detected interactions were already known for zebrafish proteins (Martin et al, 2010); a large fraction (29 out of 64) are orthologous interactions, meaning they had been previously described for proteins from other organisms. The final discovery screen interaction network ( Figure 2A; Figure S1) also contains 28 novel interactions, not reported previously.…”

“…Using the AVEXIS platform (Bushell et al, 2008), specifically designed to detect ePPIs, we screened within a library of extracellular domains (ECDs) from cell-surface and secreted (CSS) proteins expressed by FP cells or other cell types of the developing CNS, covering more than 30,000 potential binding events. Combining our data with results from previous AVEXIS screens (Bushell et al, 2008;Martin et al, 2010; Sö llner and Wright, 2009), we assembled an extracellular FP interaction network (FPnet) consisting of 47 interactions between 44 proteins. Among the identified interactions, we detected binding between the two secreted axon guidance molecules Netrin-1 Serafini et al, 1994Serafini et al, , 1996 and Draxin (Islam et al, 2009).…”

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

“…While other scalable methods have been developed to detect extracellular interactions [11][12][13][14][15] , one advantage of AVEXIS is that the experimental parameters such as the bait and prey activities have been quantified to detect even the weakest of interactions (t 1/2 ≤ 0.1 s) while retaining a low false positive rate 3 . In addition, the streamlined and robust sample preparation procedures have enabled this assay to be implemented on a much larger scale than other assays and we have recently described a systematic interaction screen totalling over ~16,500 potential interactions 16 .…”

“…The assay is not generally suitable for multipass membrane proteins such as ion transporters or pumps since they are unlikely to be correctly folded outside the context of a plasma membrane. We have applied this to a variety of different systems and have successfully expressed extracellular proteins from zebrafish 3,16,18 , human, mouse and P. falciparum for interaction screens.…”

“…The frequency at which interactions are detected depends upon the content of the protein library being screened. As a guide to the reader, a screen of >30,000 interactions within the zebrafish immunoglobulin superfamily resulted in 188 positives -a frequency of 0.6% 3,16,18 . A very rapid check that can be performed to verify any positive hits is to determine whether the interaction can be detected in both bait-prey orientations; that is, can the same interaction be detected irrespective of whether the binding proteins are present as either a bait or a prey.…”