Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany.
Institute of Pharmacy, Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany.
EMBO Rep. 2020 Nov 5;21(11):e50437. doi: 10.15252/embr.202050437. Epub 2020 Sep 14.
β-arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that β-arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β-arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.
β-arrestins（βarr1 和 βarr2）是广泛存在的 G 蛋白偶联受体（GPCR）信号转导调节剂。现有数据表明，β-arrestins 以不同的方式与不同的受体结合。然而，GPCR- arrestin 复合物的结构特征难以确定，因此需要替代方法来研究 GPCR-arrestin 复合物。在这里，我们从手指环作为 arrestins 与 GPCR 相互作用的主要位点出发，将非天然氨基酸引入到 βarr1 和 βarr2 中，用于光交联和化学交联，并探索与 arrestins 形成稳定或瞬时复合物的 GPCR 的结合拓扑结构：血管加压素受体 2（视紫红质样）、促肾上腺皮质素释放因子受体 1 和甲状旁腺激素受体 1（均为分泌素样）。我们表明，每种受体在 arrestins 上留下独特的足迹，而两种β-arrestins 产生非常相似的交联模式。此外，我们表明该方法允许定义 arrestin 相对于 GPCR 的取向。最后，我们提供了细胞中 arrestin 寡聚体形成的直接证据。
