Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.
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DOI：
10.1371/journal.pbio.1000172
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发表时间：
2009-08
期刊：
影响因子：
9.8
通讯作者：
Lohse MJ
中科院分区：
文献类型：
--
作者：
Calebiro D;Nikolaev VO;Gagliani MC;de Filippis T;Dees C;Tacchetti C;Persani L;Lohse MJ
对天然细胞中g蛋白偶联受体（GPCR）信号传导的实时监测表明，促甲状腺激素受体在内化后仍保持活性，挑战了当前的GPCR信号传导模型。g蛋白偶联受体（gpcr）通常被认为是从细胞表面向环AMP （cAMP）等第二信使发出信号，并在反复或长时间的刺激下被内化。一旦内化，它们应该停止向第二信使发送信号，但可能触发非经典信号，如丝裂原活化蛋白激酶（MAPK）激活。在这里，我们表明GPCR在内化后继续以持续的方式刺激cAMP的产生。我们培育了cAMP荧光传感器普遍表达的转基因小鼠，并研究了cAMP对促甲状腺激素（TSH）在从这些小鼠分离的天然3-D甲状腺滤泡中的反应。TSH刺激引起TSH受体内化进入前高尔基腔室，与g蛋白αs亚基和腺苷酸环化酶III密切相关。受体与TSH一起内化，产生了不同于细胞表面受体触发的下游细胞反应。这些数据表明，GPCR信号传导的经典范式可能需要修订，因为它们表明GPCR的cAMP信号传导可能发生在细胞表面和细胞内，但对细胞的影响不同。细胞通过细胞表面受体蛋白的活动对许多环境信号作出反应，细胞表面受体蛋白感知这些信号并将信息传递给细胞内的信号分子。g蛋白偶联受体（gpcr）是真核生物中最大的质膜受体家族。它们将细胞外刺激提供的信息转化为细胞内的第二信使，如环AMP （cAMP）。在长时间的刺激后，它们被内化在细胞内，这一事件迄今被认为终止了第二信使的产生。虽然GPCR信号传导的许多关键步骤已经被详细地了解，但确切地说，信号传导和终止实际上是如何在时间和空间（即，在亚细胞区室或微域）中发生的，仍然在很大程度上没有被探索。为了观察活细胞中的GPCR信号，我们培养了表达荧光传感器的小鼠，该传感器可以在显微镜下监测细胞内cAMP的水平。我们利用该系统直接在天然甲状腺滤泡中研究促甲状腺激素（TSH）受体发出的信号。我们的研究结果表明，TSH受体在激活后迅速内化，但继续刺激细胞内cAMP的产生，这种持续的cAMP的产生显然是下游成分局部激活所必需的。这些数据通过提示存在先前未被识别的cAMP生成细胞内位点以及由于细胞内GPCR信号传导而导致的差异信号传导结果，挑战了GPCR-cAMP通路的当前模型。这些细胞内位点可能提供了专门的信号平台，从而有助于cAMP产生的时空调节和GPCR家族中的信号特异性。
Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling. G-protein–coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH) in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein αs-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell. Cells respond to many environmental cues through the activity of cell surface receptor proteins, which sense these cues and convey that information to signaling molecules inside the cell. G-protein–coupled receptors (GPCRs) form the largest eukaryotic family of plasma membrane receptors. They convert the information provided by extracellular stimuli into intracellular second messengers, like cyclic AMP (cAMP). After prolonged stimulation, they are internalized inside cells, an event that to date has been thought to terminate the production of second messengers. Though many of the key steps of GPCR signaling are known in detail, precisely how signaling and termination actually occur in time and space (i.e., in subcellular compartments or microdomains) is still largely unexplored. To observe GPCR signaling in living cells, we generated mice expressing a fluorescent sensor that allows monitoring the intracellular levels of cAMP with a microscope. We utilized this system to study, directly in native thyroid follicles, the signal sent by the receptor for thyroid-stimulating hormone (TSH). Our findings indicate that TSH receptors are internalized rapidly after activation but continue to stimulate cAMP production inside cells and that this sustained, cAMP production is apparently required for localized activation of downstream components. These data challenge the current model of the GPCR-cAMP pathway by suggesting the existence of previously unrecognized intracellular site(s) for cAMP generation and of differential signaling outcomes as a result of intracellular GPCR signaling. Such intracellular site(s) may provide specialized signaling platforms, thus contributing to the spatiotemporal regulation of cAMP production and to signaling specificity within the GPCR family.
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