The activation of heterotrimeric G proteins through G-protein-coupled receptors (GPCRs) is a ubiquitous signaling mechanism in eukaryotic biology. The three principal molecular components of this cascade are the GPCR, Gα subunit, and Gβγ subunit. Measurement of interactions between these components and their downstream effectors in live cells is paramount to understanding how cells fine-tune their physiology in response to many external stimuli. Multicolor fluorescence fluctuation spectroscopy (FFS) approaches allow the sensitive detection of heteromeric interactions by using spectrally distinct fluorophores to label biomolecules of interest. We considered three imaging FFS approaches to measuring molecular interactions from the signals produced by a spectrally resolved confocal microscopy: raster spectral image correlation spectroscopy (RSICS), spectral spatial cumulant analysis, and native resolution spatial cumulant analysis. We characterized these approaches using simulation and experiments on heteromers with known stoichiometries. We found that RSICS had the best sensitivity for measuring heteromeric interactions and employed it to measure G protein complexes. As measured by RSICS, interactions between the G protein subunits Gαi and Gβγ were sensitive to the stimulation of two GPCRs, the D2 dopamine receptor and the α-2A adrenergic receptor. Interactions between GPCRs and G proteins were not detectable above background, supporting a collisional model of GPCR/G protein interactions in contrast to a preassembly model where strong interactions would be present. These data are uniquely available by this FFS framework, which is appropriate for not only multiplexed measurements of G protein biology but any dynamic protein complexes in the cell.

中文翻译：

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通过光谱分辨成像荧光波动光谱测量 G 蛋白活化


通过 G 蛋白偶联受体 （GPCR） 激活异源三聚体 G 蛋白是真核生物学中普遍存在的信号转导机制。该级联反应的三个主要分子组分是 GPCR、Gα 亚基和 Gβγ 亚基。测量这些成分与其在活细胞中的下游效应子之间的相互作用对于了解细胞如何响应许多外部刺激来微调其生理机能至关重要。多色荧光波动光谱 （FFS） 方法通过使用光谱不同的荧光基团来标记感兴趣的生物分子，从而灵敏地检测异聚相互作用。我们考虑了三种成像 FFS 方法来测量光谱分辨共聚焦显微镜产生的信号中的分子相互作用：光栅光谱图像相关光谱 （RSICS）、光谱空间累积量分析和自然分辨率空间累积量分析。我们使用对具有已知化学计量的异源体的模拟和实验来表征这些方法。我们发现 RSICS 在测量异聚相互作用方面具有最佳灵敏度，并将其用于测量 G 蛋白复合物。通过 RSICS 测量，G 蛋白亚基 Gαi 和 Gβγ 之间的相互作用对两种 GPCR （D2 多巴胺受体和 α-2A 肾上腺素能受体）的刺激敏感。GPCR 和 G 蛋白之间的相互作用在背景之上无法检测到，这与存在强相互作用的预组装模型相反，支持 GPCR/G 蛋白相互作用的碰撞模型。这些数据通过该 FFS 框架是唯一可用的，它不仅适用于 G 蛋白生物学的多重测量，也适用于细胞中的任何动态蛋白质复合物。