The Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.
The Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA; The Department of Tumor Biology, University of Miami Sylvester Comprehensive Cancer Center, Miami, Florida, USA; The Institute for Data Science Computing, University of Miami, Coral Gables, Florida, USA.
J Biol Chem. 2021 Jan-Jun;296:100167. doi: 10.1074/jbc.RA120.016352. Epub 2020 Dec 13.
Of the 800 G protein-coupled receptors (GPCRs) in humans, only three (GPR4, GPR65, and GPR68) regulate signaling in acidified microenvironments by sensing protons (H). How these receptors have uniquely obtained this ability is unknown. Here, we show these receptors evolved the capability to sense H signals by acquiring buried acidic residues. Using our informatics platform pHinder, we identified a triad of buried acidic residues shared by all three receptors, a feature distinct from all other human GPCRs. Phylogenetic analysis shows the triad emerged in GPR65, the immediate ancestor of GPR4 and GPR68. To understand the evolutionary and mechanistic importance of these triad residues, we developed deep variant profiling, a yeast-based technology that utilizes high-throughput CRISPR to build and profile large libraries of GPCR variants. Using deep variant profiling and GPCR assays in HEK293 cells, we assessed the pH-sensing contributions of each triad residue in all three receptors. As predicted by our calculations, most triad mutations had profound effects consistent with direct regulation of receptor pH sensing. In addition, we found that an allosteric modulator of many class A GPCRs, Na, synergistically regulated pH sensing by maintaining the pK values of triad residues within the physiologically relevant pH range. As such, we show that all three receptors function as coincidence detectors of H and Na. Taken together, these findings elucidate the molecular evolution and long-sought mechanism of GPR4, GPR65, and GPR68 pH sensing and provide pH-insensitive variants that should be valuable for assessing the therapeutic potential and (patho)physiological importance of GPCR pH sensing.
在人类的 800 个 G 蛋白偶联受体（GPCR）中，只有三个（GPR4、GPR65 和 GPR68）通过感应质子（H）来调节酸化微环境中的信号转导。这些受体如何独特地获得这种能力尚不清楚。在这里，我们表明这些受体通过获得埋藏的酸性残基获得了感应 H 信号的能力。使用我们的信息学平台 pHinder，我们鉴定了这三个受体共有的一组埋藏的酸性残基，这一特征与所有其他人类 GPCR 不同。系统发育分析表明，该三联体出现在 GPR65 中，GPR65 是 GPR4 和 GPR68 的直接祖先。为了了解这些三联体残基的进化和机制重要性，我们开发了深度变体分析，这是一种基于酵母的技术，利用高通量 CRISPR 构建和分析大量 GPCR 变体文库。使用深度变体分析和 HEK293 细胞中的 GPCR 测定，我们评估了所有三个受体中每个三联体残基对 pH 感应的贡献。正如我们的计算预测的那样，大多数三联体突变具有深远的影响，与受体 pH 感应的直接调节一致。此外，我们发现许多 A 类 GPCR 的别构调节剂 Na+通过维持三联体残基的 pK 值在生理相关的 pH 范围内，协同调节 pH 感应。因此，我们表明所有三个受体都作为 H 和 Na+的符合探测器起作用。总之，这些发现阐明了 GPR4、GPR65 和 GPR68 pH 感应的分子进化和长期寻求的机制，并提供了 pH 不敏感的变体，这些变体应该对评估 GPCR pH 感应的治疗潜力和（病理）生理重要性具有重要价值。
