The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics
The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics
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DOI：
10.1039/b823243a
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发表时间：
2009-01-01
影响因子：
--
通讯作者：
Zhang, Jin
中科院分区：
文献类型：
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作者：
DiPilato, Lisa M.;Zhang, Jin
最近，膜筏和小泡因其作为信号平台的作用而受到广泛关注，特别是由于它们参与许多疾病的发病机制，包括艾滋病毒以及神经和心血管疾病。β -肾上腺素能受体（β - ar）是g蛋白偶联受体（gpcr）大家族的一员，通过无处不在的第二信使cAMP转导细胞外信息，由于该途径的多个组分被这些膜微域区隔，因此β - ar介导的信号传导一直是大量研究的焦点。然而，这些膜筏如何在细胞环境中表现和调节信号动力学尚不清楚。在这里，我们描述了一种基于单细胞实时荧光成像的活细胞实验，通过改进的基于fret的cAMP生物传感器，来监测第二信使动力学的筏调节。用甲基- β -环糊精（M β - CD）去除胆固醇后，HEK-293细胞中β (2)- ar介导的cAMP积累增强并延长，表明膜筏完整性有助于形成β - ar信号传导。单细胞成像与分离研究并行表明，动力学的增强和变化是由受体介导的，并与受体的再分布有关。最后，胆固醇消耗的效果是受体类型特异性的，因为M β - CD治疗在刺激筏排除的前列腺素E受体时没有显示出相同的效果。这项研究强调了研究膜筏的活细胞实时成像分析的潜力，包括高灵敏度和时空分辨率，以更好地了解健康和患病状态下膜微结构域的细微差别。
Recently, membrane rafts and caveolae have received much attention for their role as signaling platforms, particularly due to their involvement in the pathogenesis of a number of diseases, including HIV as well as neurological and cardiovascular conditions. Signaling mediated by the beta-adrenergic receptor (beta-AR), a member of the large family of G-protein coupled receptors (GPCRs) that transduce extracellular messages via the ubiquitous second messenger, cAMP, has been a focus of raft studies since multiple components of the pathway are compartmentalized by these membrane microdomains. However, how these membrane rafts behave and regulate signaling dynamics in a cellular context is poorly understood. Here, we describe a live-cell assay based on single-cell, real-time fluorescence imaging, via an improved FRET-based cAMP biosensor, to monitor raft regulation of second messenger dynamics. Upon cholesterol depletion with methyl-beta-cyclodextrin (M beta CD), beta(2)-AR-mediated cAMP accumulation was enhanced and prolonged in HEK-293 cells, demonstrating that membrane raft integrity helps shape beta-AR signaling. Single-cell imaging in parallel with fractionation studies reveal that the enhancement and change of dynamics are mediated by the receptor and correlated with its redistribution. Finally, the effect of cholesterol depletion is receptor-type specific as M beta CD treatment did not show the same effect when the raft-excluded prostaglandin E receptor was stimulated. This study highlights the potential of a live-cell, real-time imaging assay for studying membrane rafts, including high sensitivity and spatiotemporal resolution, to achieve a better understanding of the nuances of membrane microdomains in both healthy and diseased states.