Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.
Stem Cell Genetics Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.
Genome Res. 2018 Sep;28(9):1372-1382. doi: 10.1101/gr.231183.117. Epub 2018 Jun 18.
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.
细胞表面受体介导的相互作用引发了重要的指导信号，但在生化分析中很难检测到，因为它们通常是高度瞬时的，并且膜嵌入受体难以在其天然构象中溶解。在这里，我们通过使用基于基因组规模的基于细胞的遗传筛选方法，使用 CRISPR 基因敲除技术来识别特定细胞表面识别事件所需的细胞途径，解决了这些生化挑战。通过使用高亲和力单克隆抗体和低亲和力配体，我们确定了必要的筛选参数，包括建立糖萼结合贡献的重要性，这使得能够明确识别编码与高统计置信度直接相互作用的膜嵌入受体的基因。重要的是，我们表明，这种全基因组筛选方法还鉴定了受体特异性途径，这些途径是功能性表达细胞表面受体所必需的，包括伴侣蛋白、添加翻译后修饰的酶、运输蛋白和转录因子。最后，我们通过鉴定 IGF2R（胰岛素样生长因子 2 受体）作为 GABA 受体 R2 亚基的结合伴侣来证明该方法的实用性。我们表明，这种相互作用是直接的，并且严重依赖于甘露糖-6-磷酸，为 GABA 受体信号转导的内化和调节提供了一种机制。我们得出结论，这种单一方法可以揭示通过抗体、分泌蛋白和膜嵌入配体识别的受体的分子性质和功能细胞表面显示所需的遗传途径，而无需对其生化特性做出任何预先假设。
