Abstract：
Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with β-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in β-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated β-arrestin 1/2 recruitment for diverse ligands, and β-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased β-arrestin 1 recruitment but increased β-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected β-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in β-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive β-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in β-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in β-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on β-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without β-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and β-arrestins. Our study offers valuable information about ligand induced β-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.
摘要：
胰高血糖素样肽-1受体（GLP-1R）是2型糖尿病和肥胖症的主要药物靶标。配体引发的GLP-1R与G蛋白的相互作用已经得到了很好的研究，但不是β-抑制素1/2。因此,生物发光共振能量转移（BRET），使用诱变和操作模型来评估GLP-1R上85个细胞外表面残基在由三种代表性GLP-1R激动剂（GLP-1，exendin-4和胃泌酸调节素）触发的β-抑制素1/2募集中的作用。残基选择性调节不同配体的β-抑制蛋白1/2募集，并鉴定了β-抑制蛋白亚型。跨膜螺旋1（TM1）-胞外域（ECD）接头上残基K130-S136，L142和Y145的突变减少了β-抑制素1的募集，但增加了β-抑制素2的募集。其他胞外环（ECL）突变，包括P137A，Q211A,D222A和M303A选择性影响β-抑制素1募集,而D215A,L217A,Q221A,S223A,Y289A,S301A,F381A和I382A更多地参与配体的β-抑制蛋白2募集。与GLP-1和exendin-4相比，泌酸调节素更广泛地参与GLP-1R细胞外表面以驱动β-抑制素1/2募集；I147，W214和L218参与β-抑制素1募集，而L141、D215、L218、D293和F381在β-抑制素2募集胃泌酸调节素中尤为明显。此外,β-抑制蛋白1/2C结构域上的非保守残基有助于与GLP-1R的相互作用。在有或没有β-抑制素1/2过表达的配体刺激下，GLP-1R稳定表达的细胞系的进一步蛋白质组学分析证明了与同源配体和β-抑制素相关的常见和偏倚调节的蛋白质和途径。我们的研究提供了有关GLP-1R介导的配体诱导的β-抑制素募集以及随之而来的细胞内信号传导事件的有价值的信息。
