The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways
The melanin-concentrating hormone receptor couples to multiple G proteins to activate diverse intracellular signaling pathways
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DOI：
10.1210/en.141.12.4524
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发表时间：
2000-12-01
期刊：
影响因子：
4.8
通讯作者：
Graziano, MP
中科院分区：
文献类型：
--
作者：
Hawes, BE;Kil, E;Graziano, MP
黑色素浓缩激素受体(MCH)最近被鉴定为孤儿G蛋白偶联受体SLC-1。在这项研究中，使用一个表达MCH受体的CHO细胞系(K-d=1.3 nM；结合容量，3.6pmol/mg蛋白)来评估MCH受体与G(I)、G(O)和G(Q)蛋白的偶联能力。结果表明，MCH以百日咳毒素(PTX)敏感的方式抑制Forsklin刺激的CHO-MCHR细胞内cAMP的产生(EC50=100 pM)，表明MCH受体与G蛋白Gi亚家族的一个或多个成员偶联。此外，CH还能刺激肌醇磷脂代谢(EC50=50 nM)和细胞内游离钙水平(EC50=10 nM)的增加。PTX可部分抑制MCH刺激的肌醇磷酸生成和细胞内游离钙的增加(分别为60%和40%)，表明这些信号通路中至少有两个组分。其中一个组分对PIX敏感，因此通过G(I)/G(O)蛋白介导。一种独特的G蛋白偶联(可能是G(Q)型)介导了PTX不敏感成分。为了区分G(I)偶联和G(O)偶联，检测了MCH刺激的丝裂原活化蛋白(MAP)激酶活性。G(I)和G(O)使用不同的信号通路来调节CHO细胞中MAP激酶的激活。蛋白激酶C(PKC)活性在依赖于G(O)的MAP激酶信号通路中是必不可少的，但在依赖于G(I)的MAP激酶信号通路中不是必需的。MCH刺激的MAPK活性通过抑制PKC活性或细胞内PKC的耗竭而降低(50%)，但不会被消除，这表明MCH刺激的MAPK活性是通过G(I)和G(O)依赖的信号机制介导的。这项研究的结果首次清楚地证明了MCH受体与多个G蛋白偶联，介导了几种不同的细胞内信号通路。
The receptor for melanin-concentrating hormone (MCH) was recently identified as the orphan G protein-coupled receptor SLC-1. In this study, a CHO cell line expressing the MCH receptor(K-d = 1.3 nM; binding capacity, 3.6 pmol/mg protein) is used to assess the ability of the MCH receptor to couple to G(i), G(o), and G(q) proteins. The results demonstrate that MCH inhibits forskolin-stimulated cAMP production in a pertussis toxin- (PTX)-sensitive manner in CHO-MCHR cells (EC50 = 100 pM), indicating that the MCH receptor couples to one or more members of the Gi subfamily of G proteins. in addition, CH stimulates increases in phosphoinositide metabolism (EC50 = 50 nM) and in intracellular free Ca2+ levels (EC50 = 10 nM). MCH-stimulated inositol phosphate production and increases in intracellular free Ca2+ are partially inhibited (60% and 40%, respectively) by PTX pretreatment, demonstrating that there are at least two components of each of these signaling pathways. One component is PIX sensitive and therefore mediated through a G(i)/G(o) protein. A distinct G protein-coupled (probably G(q) type) mediates the PTX-insensitive component. To distinguish G(i) vs. G(o) coupling, MCH-stimulated mitogen-activated protein (MAP) kinase activity was examined. G(i) and G(o) use separate signaling pathways to mediate MAP kinase activation in CHO cells. Protein kinase C (PKC) activity is essential in the G(o)-dependent MAP kinase signaling pathway, but is not required in the G(i)-dependent MAP kinase signaling pathway. MCH stimulated MAP kinase activity is decreased (50%), but not abolished, by inhibition of PKC activity or depletion of cellular PKC, indicating that MCH-stimulated MAP kinase activity is mediated through both G(i)- and G(o)-dependent signaling mechanisms. The results of this study are the first to clearly demonstrate that the MCH receptor couples to multiple G proteins to mediate several diverse intracellular signaling pathways.