Abstract：
The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of β-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the β-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine β-arrestin recruitment. The ligand-dependent variance in β-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the β-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-β-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-β-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained β-arrestin binding: the V2 vasopressin receptor and a mutant β2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in β-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.
摘要：
G蛋白偶联受体的不同活性构象的稳定被认为是偏倚和平衡激动剂的不同功效的基础。这里,分析血管紧张素II1型受体（AT1R）激动剂对信号转导的激活表明，β-抑制蛋白结合的程度和动力学表现出明显的配体依赖性差异，当受体内化被抑制时丢失。当AT1R内吞被阻止时，即使β-抑制蛋白途径的弱部分激动剂也可以作为完全或接近完全的激动剂，提示受体构象并不完全决定β抑制蛋白募集.β-抑制蛋白易位的配体依赖性变异在核内体比在质膜大得多，表明β-抑制蛋白途径中的配体功效是时空确定的。实验研究和数学模型证明了多种因素如何同时影响激动剂对内体受体-β-抑制蛋白结合的影响，从而确定了功能选择性的程度。配体解离速度和G卵白活性特别强，内化依赖性对受体-β-抑制蛋白相互作用的影响。我们还表明，胞吞作用可调节其他两种具有持续β-抑制素结合的受体的激动剂功效：V2加压素受体和突变的β2-肾上腺素能受体。在没有内吞作用的情况下，β-arrestin2结合的激动剂依赖性变异显著减少.我们的结果表明，内吞作用决定了GPCR信号传导中的时空偏差，并且可以帮助开发更有效的，功能选择性化合物。
