Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, The Heart Centre, Copenhagen University Hospital, Rigshospitalet, and The Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2010 Nov 30;5(11):e14135. doi: 10.1371/journal.pone.0014135.
Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or β-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of β-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit β-arrestins. Since uncoupling of G proteins by increased ability to recruit β-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.
METHODOLOGY/PRINCIPAL FINDINGS: We studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49-3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to β-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit β-arrestin in response to Sar1-Ile4-Ile8 (SII) Ang II, a ligand solely activating the β-arrestin pathway.
CONCLUSIONS/SIGNIFICANCE: Our analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit β-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and β-arrestin recruitment, respectively.
七次跨膜受体（7TMRs）可以采用不同的活性构象，从而选择性地激活 G 蛋白或β-arrestin 依赖性信号通路。这为开发针对特定受体的选择性生物学效应的新型治疗药物提供了机会。已经进行了许多关于途径分离的研究，其中许多是关于血管紧张素 II 型 1 型受体（AT1R）的。已经表明，某些配体或突变促进了β-arrestin 的内化和/或募集，而不激活 G 蛋白。然而，潜在的分子机制在很大程度上仍未得到解决。例如，尚不清楚这种选择性 G 蛋白偶联的缺失是由于缺乏与 G 蛋白相互作用的能力，还是由于受体募集β-arrestin 的能力增强所致。由于通过增强募集β-arrestin 的能力而导致 G 蛋白解偶联可能导致与缺乏与 G 蛋白相互作用的能力不同的细胞或体内结果，因此必须区分这两种机制。
方法/主要发现：我们研究了先前发表的五个显示途径分离的 AT1R 突变体：D74N、DRY/AAY、Y292F、N298A 和 Y302F（Ballesteros-Weinstein 编号：2.50、3.49-3.51、7.43、7.49 和 7.53）。我们发现 D74N、DRY/AAY 和 N298A 突变体比 WT 更容易募集β-arrestin。相比之下，受体突变体 Y292F 和 Y302F 对 Sar1-Ile4-Ile8（SII）Ang II 的反应显示出募集β-arrestin 的能力受损，SII Ang II 是一种仅激活β-arrestin 途径的配体。
结论/意义：我们的分析表明，这些 AT1R 突变体诱导的潜在构象很可能代表了不同的解偶联 G 蛋白的主要机制，对于某些突变体，其原因可能是它们募集β-arrestin2 的能力增强。因此，这些发现对药物发现和 7TMR 生物学具有重要意义，并说明了分别揭示 G 蛋白偶联和β-arrestin 募集的确切分子决定因素的必要性。
