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Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.
EMBO Reports ( IF 7.7 ) Pub Date : 2020-09-14 , DOI: 10.15252/embr.202050437
Thore Böttke 1 , Stefan Ernicke 1 , Robert Serfling 1 , Christian Ihling 2 , Edyta Burda 3 , Vsevolod V Gurevich 4 , Andrea Sinz 2 , Irene Coin 1
Affiliation  

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.

中文翻译:

探索GPCR-arrestin与遗传编码的交联剂的界面。

β-arrestin(βarr1和βarr2)是G蛋白偶联受体(GPCR)信号的普遍调节因子。现有数据表明,β-arrestins以不同方式对接至不同的受体。然而,GPCR-arrestin复合物的结构表征是具有挑战性的,需要替代方法来研究GPCR-arrestin复合物。在这里,我们从指环作为抑制蛋白与GPCR相互作用的主要部位开始,我们将非规范氨基酸进行光和化学交联,将其遗传结合到βarr1和βarr2中,并探索了与GPCR形成稳定或瞬时复合物的结合拓扑结构。抑制蛋白:血管加压素受体2(视紫红质样),促肾上腺皮质激素释放因子受体1和甲状旁腺激素受体1(均为促胰液素样)。我们显示,每个受体在抑制蛋白上都留下独特的足迹,而两个β-抑制蛋白产生非常相似的交联模式。此外,我们表明该方法允许定义相对于GPCR的抑制蛋白的方向。最后,我们提供了在细胞中形成抑制蛋白低聚物的直接证据。
更新日期:2020-11-06
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