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 GABA 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 GABA 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.

The role of 3’UTR-protein complexes in the regulation of protein multifunctionality and subcellular localization

“…Likewise, we also predicted a 3'UTR-protein complex formed with the nascent protein RHAMM/HMMR, a regulator of the stability of the mitotic spindle in normal cells 8,10 that acts as an extracellular CD44ligand promoting cell motility and invasion in cancer cells 10 , IGF2BP2, involved in mRNA storage and transport, and the Dynactin subunit 1 (DCTN1) as intermediate, a protein present at the "spindle" as well as at the "membrane" and notably involved in the transport of organelles and vesicles by tethering the dynein cargo to the microtubule 39 . We can thus hypothesize that the 3'UTR-RHAMM complex plays a role in the molecular mechanism allowing the extracellular localization of RHAMM in tumor cells.…”

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B

“…Since that the binding assay results suggested that the G6b-B ligand was primarily composed of HS glycans, we opted to confirm and extend these finding by using a genome-scale cell-based CRISPR knockout (KO) screening approach, identifying all genes required for the synthesis and cell surface display of the G6b-B ligand (Sharma, Bartholdson, Couch, Yusa, & Wright, 2018). We observed that a fluorescently labelled highly-avid recombinant G6b-B binding reagent robustly stained several human cell lines providing the basis for a cellular genetic screen (Figure 3A).…”

“…We observed that a fluorescently labelled highly-avid recombinant G6b-B binding reagent robustly stained several human cell lines providing the basis for a cellular genetic screen (Figure 3A). A genome-wide mutant cell library was generated by transducing Cas9-expressing HEK293 cells with a library of lentiviruses each encoding a single gRNA from a pool of 90,709 individual gRNAs targeting 18,009 human genes (Sharma et al, 2018). Transduced cells that had lost the ability to bind to the recombinant protein were isolated using fluorescent-activated cell sorting and genes required for cell surface binding of G6b-B were identified by comparing the relative abundance of gRNAs in the sorted versus unsorted control population (Li et al, 2014).…”

“…A total of 600,000 cells were collected from which genomic DNA was extracted, gRNA sequences amplified by PCR and their abundances determined by next generation sequencing. The enrichment of gRNA sequences targeting specific genes in the sorted versus unsorted populations were quantified from the sequence data using the MAGeCK software (Li et al, 2014) as previously described (Sharma et al, 2018).…”

Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms

“…However, by carefully designing the screening parameters, for example by generating marker lines specific to the biological process of interest, other facets of cellular signalling processes such as differentiation, motility, proliferation, metabolism and immunity can also be studied with this tool (refer to Table 1 of [ 14 ]). We have recently been working on devising a genome-wide KO screen that can be used to identify low-affinity interactions mediated by the receptors present on the surface of the cells [ 67 ] (unpublished). The field of identification of low-affinity receptor-ligand interactions has been challenging owing to the transient nature of such interactions, which make them difficult to detect in many existing biochemical methods.…”