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Fig. 4 | Genome Biology

Fig. 4

From: Pooled extracellular receptor-ligand interaction screening using CRISPR activation

Fig. 4

Genome-wide CRISPRa selections identified known and novel ligands for adhesion GPCRs. Transformed gene enrichment P values plotted against a rank-ordered gene list for CRISPRa enrichment screens with cells selected using adhesion GPCR recombinant binding probes for ADGRL1, ADGRL3, and ADGRA2 (a), and ADGRB1 (b). A single screen was performed for each adhesion GPCR bait. c A highly-avid fluorescently labeled ADGRB1 binding probe stained cells transfected with cDNAs encoding full-length RTN4R, RTN4RL1, and RTN4RL2 (blue lines) but not mock-transfected cells compared to a control ADGRL1 binding probe (orange line), or streptavidin-phycoerythrin alone (red line). A representative of four independent experiments is shown. d The ectodomains of ADGRB1 and RTN4R family members directly interact. The extracellular regions of the named receptors were expressed as soluble biotinylated bait proteins, captured in individual wells of a streptavidin-coated plate and probed for interactions with pentameric beta-lactamase-tagged prey proteins using AVEXIS. Binding is quantified by absorbance at 485 nm of a hydrolysis product of the colorimetric beta-lactamase substrate, nitrocefin. Bars represent blank-subtracted mean ± s.d; n = 3. CD97-CD55 interaction was used as a positive control; negative control bait was the CD55 ectodomain. e The RTN-family binding interface on ADGRB1 is composed of the N-terminal three TSR domains. Schematic of the RTN-family and ADGRB1 proteins showing their domain organization. Binding of RTN4R and RTN4RL1 preys to fragments of ADGRB1 encompassing the full-length ectodomain (FL), thrombospondin repeats 1–3 (TSR1–3), TSRs 1–5, or the hormone receptor motif and GAIN domain (HRM+GAIN). Bars represent mean ± s.d.; n = 3

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