Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning

In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor (ß2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for ß2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors. Overall design: The overall design of this experiment is to test the functional consequences of every amino acid substitution to the beta-2 adrenergic receptor (B2AR). This occurs in two stages that occurs roughly as follows (for more details, see Figure 1 of our bioRxiv preprint ). First, we use DNA microarray synthesis to generate a library of mutants at every position in the protein (think alanine scan, cysteine scan, aspartic acid scan, ...). We then clone these mutants into a plasmid backbone that's tagged with a DNA barcode. We use NGS to associate each variant with its corresponding barcode (this corresponds to the the Map_* and Eric_* fastq's that are eventually processed into the NextSeq_MiSeq.map.csv.gz). Second, the mapped library is integrated into human cell lines such that each cell only receives a single B2AR mutant - barcode pair. Activation of the receptor by agonist incubation results in transcription of the mutant's barcode, which we can detect with RNA-seq (the second set of fastq's drug-*).

在人体中，813种G蛋白偶联受体（G protein-coupled receptors, GPCRs）负责转导各类化学刺激以改变细胞状态，同时也是最大的一类药物靶点。其多样的结构构象与多种信号转导模式，使得解析其结构与功能颇具挑战。本研究开发了一套基于G蛋白信号转导条形码转录报告系统的平台，可在人类细胞系中对GPCR变体大型文库进行表征分析。本研究针对β₂肾上腺素能受体（beta-2 adrenergic receptor, ß2AR），在4种不同浓度的激动剂异丙肾上腺素处理下，测试了其7828种理论单氨基酸替换中的7800种。我们鉴定出了对β₂AR信号转导至关重要的氨基酸残基、人群中可能导致功能丧失的突变位点，以及可调节基础活性的残基。通过无监督学习，我们解析出了对信号转导关键的残基，涵盖所有主要结构基序与分子相互作用界面。我们还发现了一个此前未被表征的结构锁扣，该结构横跨前两个细胞外环，在A类GPCR中高度保守，且在受体的失活与激活状态下均具有构象刚性。更广泛而言，本研究将深度突变扫描与工程化转录报告基因相结合，建立了一种可推广的方法，用于探究各类药物受体的药物基因组学、结构与功能特征。
实验整体设计：本实验的核心设计为测试β₂肾上腺素能受体（B2AR）的所有单氨基酸替换所产生的功能效应。实验大致分为两个阶段进行（详细内容参见我们的bioRxiv预印本图1）：第一阶段，我们通过DNA微阵列合成技术，构建该蛋白所有位点的突变体文库（可类比丙氨酸扫描、半胱氨酸扫描、天冬氨酸扫描等策略）；随后将这些突变体克隆至带有DNA条形码标记的质粒骨架中，并利用下一代测序（Next-Generation Sequencing, NGS）将每个变体与其对应的条形码进行关联（对应最终被处理为NextSeq_MiSeq.map.csv.gz的Map_*与Eric_*格式fastq文件）。第二阶段，将完成映射的文库整合至人类细胞系中，确保每个细胞仅携带一组β₂AR突变体-条形码配对；通过激动剂孵育激活受体后，突变体对应的条形码会被转录，我们可通过RNA测序（RNA-seq）检测到该信号（对应第二组fastq格式文件drug-*）。