Jain Abhinav R, McGraw Claire, Robinson Anne S
Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA 70118, USA.
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Biomedicines. 2020 Dec 13;8(12):603. doi: 10.3390/biomedicines8120603.
Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (AR and AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for AR. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with AR C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling. To further study the G protein coupling, we used this humanized yeast platform to explore coupling to human-yeast G-protein chimeras in a cellular context. Although the C-terminus was essential for Gα protein-associated signaling, chimeras of AR with a C-terminus of AR coupled to the AR-specific Gα (i.e., Gαi1 versus Gαs). This surprising result suggests that the C-terminus is important in the signaling strength, but not specificity, of the Gα protein interaction. This result has further implications in drug discovery, both in enabling the experimental use of chimeras for ligand design, and in the cautious interpretation of structure-based drug design using truncated receptors.
最近为确定腺苷受体（AR和AR）的高分辨率晶体结构所做的努力，利用了对天然受体的修饰，以促进受体结晶和结构测定。一种常见的修饰是截短无结构的C末端，迄今为止获得的所有腺苷受体晶体结构都采用了这种方法。已证明这种截短受体的配体结合与AR的全长受体相似。然而，C末端已被确定为蛋白质-蛋白质相互作用的位点，这可能对这些重要药物靶点的生理功能至关重要。我们发现，与转染到哺乳动物细胞（HEK-293）中的全长受体构建体相比，具有AR C末端截短的变体缺乏与cAMP相关的信号传导。此外，我们表明，在人源化酵母系统中，使用基于酵母MAPK反应荧光测定法，全长C末端的缺失会影响下游信号传导，尽管全长受体显示出类似天然的G蛋白偶联。为了进一步研究G蛋白偶联，我们利用这个人源化酵母平台在细胞环境中探索与人类-酵母G蛋白嵌合体的偶联。尽管C末端对于与Gα蛋白相关的信号传导至关重要，但具有AR C末端的AR嵌合体与AR特异性Gα（即Gαi1与Gαs）偶联。这一惊人结果表明，C末端在Gα蛋白相互作用的信号强度而非特异性方面很重要。这一结果在药物发现方面有进一步的意义，既有助于将嵌合体用于配体设计的实验，也有助于谨慎解读使用截短受体的基于结构的药物设计。
