RAMP2 Influences Glucagon Receptor Pharmacology via Trafficking and Signaling.
RAMP2 Influences Glucagon Receptor Pharmacology via Trafficking and Signaling.
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DOI：
10.1210/en.2016-1755
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发表时间：
2017-08-01
期刊：
影响因子：
4.8
通讯作者：
Bloom SR
中科院分区：
文献类型：
--
作者：
Cegla J;Jones BJ;Gardiner JV;Hodson DJ;Marjot T;McGlone ER;Tan TM;Bloom SR
内源性饱腹感激素为减肥药提供了一个有吸引力的靶点。高血糖素通过减少食物摄入量和增加能量消耗来减肥。为了进一步了解胰升糖素及其相关配体激活胰升糖素受体(GCGR)的细胞机制，我们研究了GCGR与受体活性修饰蛋白(RAMP)2的相互作用。我们采用竞争结合实验、细胞表面酶联免疫吸附实验、G-αS和G-αQ通路及β-arrestin募集的功能分析和小干扰RNA敲除相结合的方法来检测RAMP2对GCGR的影响。测试的配体是高血糖素、类胰高血糖素肽-1(GLP-1)、氧合酶调节蛋白和内部开发的GLP-1/高血糖素共激动剂类似物G(X)。用共聚焦显微镜观察RAMP2是否影响GCGR的亚细胞分布。在这里，我们证明了RAMP2和GCGR的共同表达导致GCGR的细胞表面表达减少。共聚焦显微镜证实了这一点，表明RAMP2与GCGR共定位，并导致显著的GCGR细胞重新分布。此外，RAMP2的存在影响了通过GαS和GαQ途径的信号传递，以及β-arrestin的招募。这项工作表明，RAMP2可能会改变GCGR的激动剂活性和运输，这可能与产生具有选择性激动剂活性的新的多肽类似物有关。采用竞争结合、功能分析评估G-α、S和GQ途径、β-arrestin募集和siRNA敲除相结合的方法检测RAMP2对GCGR的影响。
Endogenous satiety hormones provide an attractive target for obesity drugs. Glucagon causes weight loss by reducing food intake and increasing energy expenditure. To further understand the cellular mechanisms by which glucagon and related ligands activate the glucagon receptor (GCGR), we investigated the interaction of the GCGR with receptor activity modifying protein (RAMP)2, a member of the family of receptor activity modifying proteins. We used a combination of competition binding experiments, cell surface enzyme-linked immunosorbent assay, functional assays assessing the Gαs and Gαq pathways and β-arrestin recruitment, and small interfering RNA knockdown to examine the effect of RAMP2 on the GCGR. Ligands tested were glucagon; glucagonlike peptide-1 (GLP-1); oxyntomodulin; and analog G(X), a GLP-1/glucagon coagonist developed in-house. Confocal microscopy was used to assess whether RAMP2 affects the subcellular distribution of GCGR. Here we demonstrate that coexpression of RAMP2 and the GCGR results in reduced cell surface expression of the GCGR. This was confirmed by confocal microscopy, which demonstrated that RAMP2 colocalizes with the GCGR and causes significant GCGR cellular redistribution. Furthermore, the presence of RAMP2 influences signaling through the Gαs and Gαq pathways, as well as recruitment of β-arrestin. This work suggests that RAMP2 may modify the agonist activity and trafficking of the GCGR, with potential relevance to production of new peptide analogs with selective agonist activities. A combination of competition binding, functional assays assessing the Gαs and Gq pathways and β-arrestin recruitment, and siRNA knockdown was used to examine the effect of RAMP2 on the GCGR.
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影响因子：
4.8
作者：
Hilairet, S;Foord, SM;Bouvier, M
通讯作者：
Bouvier, M
DOI：
10.1038/nrd2760
发表时间：
2009-01
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.8
作者：
Bomberger, JM;Spielman, WS;Parameswaran, N
通讯作者：
Parameswaran, N
影响因子：
4.8
作者：
Kuwasako, K;Shimekake, Y;Sakata, T
通讯作者：
Sakata, T
影响因子：
--
作者：
GEARY, N;KISSILEFF, HR;HINTON, V
通讯作者：
HINTON, V