Proximity Labeling to Identify β-Arrestin1 Binding Partners Downstream of Ligand-Activated G Protein-Coupled Receptors.
Proximity Labeling to Identify β-Arrestin1 Binding Partners Downstream of Ligand-Activated G Protein-Coupled Receptors.
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DOI：
10.3390/ijms24043285
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发表时间：
2023-02-07
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
β-抑制蛋白是多方面的衔接蛋白，其调节G蛋白偶联受体（GPCR）信号传导的各个方面。β-抑制蛋白被募集到质膜上激动剂活化和磷酸化的GPCR，从而防止G蛋白偶联，同时还靶向GPCR，通过网格蛋白包被的小凹进行内化。此外，β-arrestins可以激活各种效应分子，以发挥它们在GPCR信号传导中的作用;然而，它们相互作用的伴侣的全部程度仍然未知。为了发现潜在的新型β-arrestin相互作用伴侣，我们使用基于APEX的邻近标记结合亲和纯化和定量质谱。我们将APEX框内附加到β-arrestin 1（β arr 1-APEX）的C-末端，我们表明这不会影响其支持激动剂刺激的GPCR内化的能力。通过使用免疫共沉淀，我们发现β arr 1-APEX与已知的相互作用蛋白相互作用。此外，在激动剂刺激后，通过链霉亲和素亲和纯化和免疫印迹评估，β arr 1-APEX标记了已知的β arr 1相互作用伴侣。以类似的方式制备等分试样，并通过串联质量标签标记和高含量定量质谱法进行分析。发现几种蛋白质在GPCR刺激后丰度增加。生物化学实验证实了两个新的蛋白质与β-arrestin 1相互作用，我们预测这是新的配体刺激的β arr 1相互作用伴侣。我们的研究强调，基于β arr 1-APEX的邻近标记是识别参与GPCR信号转导的新参与者的有价值的方法。
β-arrestins are multifaceted adaptor proteins that regulate various aspects of G protein-coupled receptor (GPCR) signaling. β-arrestins are recruited to agonist-activated and phosphorylated GPCRs at the plasma membrane, thereby preventing G protein coupling, while also targeting GPCRs for internalization via clathrin-coated pits. In addition, β-arrestins can activate various effector molecules to prosecute their role in GPCR signaling; however, the full extent of their interacting partners remains unknown. To discover potentially novel β-arrestin interacting partners, we used APEX-based proximity labeling coupled with affinity purification and quantitative mass spectrometry. We appended APEX in-frame to the C-terminus of β-arrestin1 (βarr1-APEX), which we show does not impact its ability to support agonist-stimulated internalization of GPCRs. By using coimmunoprecipitation, we show that βarr1-APEX interacts with known interacting proteins. Furthermore, following agonist stimulation βarr1-APEX labeled known βarr1-interacting partners as assessed by streptavidin affinity purification and immunoblotting. Aliquots were prepared in a similar manner and analyzed by tandem mass tag labeling and high-content quantitative mass spectrometry. Several proteins were found to be increased in abundance following GPCR stimulation. Biochemical experiments confirmed two novel proteins that interact with β-arrestin1, which we predict are novel ligand-stimulated βarr1 interacting partners. Our study highlights that βarr1-APEX-based proximity labeling represents a valuable approach to identifying novel players involved in GPCR signaling.
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影响因子：
19
作者：
Bendris N;Schmid SL
通讯作者：
Schmid SL
DOI：
10.1074/jbc.m116.754887
发表时间：
2016-12-30
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Alvarez-Curto E;Inoue A;Jenkins L;Raihan SZ;Prihandoko R;Tobin AB;Milligan G
通讯作者：
Milligan G
影响因子：
14.8
作者：
Hung V;Udeshi ND;Lam SS;Loh KH;Cox KJ;Pedram K;Carr SA;Ting AY
通讯作者：
Ting AY
影响因子：
7.3
作者：
D'Agostino G;Artinger M;Locati M;Perez L;Legler DF;Bianchi ME;Rüegg C;Thelen M;Marchese A;Rocchi MBL;Cecchinato V;Uguccioni M
通讯作者：
Uguccioni M
影响因子：
7.3
作者：
Chandan, Naincy R.;Abraham, Saji;SenGupta, Shuvasree;Parent, Carole A.;Smrcka, Alan, V
通讯作者：
Smrcka, Alan, V