Genome-scale identification of cellular pathways required for cell surface recognition.

Abstract:

: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.

journal_name

Genome Res

journal_title

Genome research

authors

Sharma S,Bartholdson SJ,Couch ACM,Yusa K,Wright GJ

doi

10.1101/gr.231183.117

subject

Has Abstract

pub_date

2018-09-01 00:00:00

pages

1372-1382

issue

9

eissn

1088-9051

issn

1549-5469

pii

gr.231183.117

journal_volume

28

pub_type

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