RT Journal
A1 Sharma, Sumana
A1 Bartholdson, S. Josefin
A1 Couch, Amalie C.M.
A1 Yusa, Kosuke
A1 Wright, Gavin J.
T1 Genome-scale identification of cellular pathways required for cell surface recognition
JF Genome Research 
JO Genome Research 
YR 2018 
FD September 01 
VO 28 
IS 9 
SP 1372 
OP 1382 
DO 10.1101/gr.231183.117 
UL http://genome.cshlp.org/content/28/9/1372.abstract 
AB Interactions mediated by cell surface receptors initiate important instructive signaling cues but can be difficult to detect in biochemical assays because they are often highly transient and membrane-embedded receptors are difficult to solubilize in their native conformation. Here, we address these biochemical challenges by using a genome-scale, cell-based genetic screening approach using CRISPR gene knockout technology to identify cellular pathways required for specific cell surface recognition events. By using high-affinity monoclonal antibodies and low-affinity ligands, we determined the necessary screening parameters, including the importance of establishing binding contributions from the glycocalyx, that permitted the unequivocal identification of genes encoding directly interacting membrane-embedded receptors with high statistical confidence. Importantly, we show that this genome-wide screening approach additionally identified receptor-specific pathways that are required for functional display of receptors on the cell surface that included chaperones, enzymes that add post-translational modifications, trafficking proteins, and transcription factors. Finally, we demonstrate the utility of the approach by identifying IGF2R (insulin like growth factor 2 receptor) as a binding partner for the R2 subunit of GABAB receptors. We show that this interaction is direct and is critically dependent on mannose-6-phosphate, providing a mechanism for the internalization and regulation of GABAB receptor signaling. We conclude that this single approach can reveal both the molecular nature and the genetic pathways required for functional cell surface display of receptors recognized by antibodies, secreted proteins, and membrane-embedded ligands without the need to make any prior assumptions regarding their biochemical properties.