Mechanism of G Protein Beta5/ R7-RGS Protein/ R7BP Complex Signal Transduction

本研究的目的是了解Gbeta5/R7-RGS/R7BP复合物在人类中的信号功能。为此，我们首先试图识别和表征Gbeta5复合物的功能，该复合物在昆虫和哺乳动物神经系统之间具有进化保守性。我们实验室目前的工作是利用黑腹果蝇、培养的PC12和SH-SY5Y细胞以及啮齿动物的初级脑神经元作为模型系统来研究这些共享功能。我们正在继续使用昆虫模型系统，其中果蝇Gbeta5在果蝇神经系统中转基因过表达。两个独立的Gbeta5转基因蝇系具有一种表型，这种表型在缺乏转基因或缺乏来自转基因启动子的Gbeta5表达所必需的“驱动”的控制系中不明显。利用这些转基因果蝇，并根据观察到的表型，使用实时荧光定量PCR来估计相关转录本的水平。在Gbeta5转基因蝇系中，某些转录本上调，而在对照蝇系中没有上调。通过稳定转染诱导小鼠Gbeta5在PC12细胞中过表达，也观察到对某些蝇类转录本的影响。免疫印迹研究表明，Gbeta5过表达也上调了相应的蛋白产物。目前正在研究观察到的变化的分子机制，以及Gbeta5基因敲除小鼠的神经元和大脑中Gbeta5功能丧失（看看表型是否与过表达模型相反）的影响。

The purpose of the current work is to understand the signaling function of the Gbeta5/R7-RGS/R7BP complex in humans. To this end we are first trying to identify and characterize the functions of the Gbeta5 complex that are evolutionarily conserved between the insect and mammalian nervous systems. Current work in our laboratory examines such shared functions using Drosophila melanogaster, cultured PC12 and SH-SY5Y cells, and primary rodent brain neurons as model systems. We are continuing to use an insect model system in which Drosophila Gbeta5 is transgenically over-expressed in the fly nervous system. Two independent Gbeta5 transgenic fly lines have a phenotype that is not evident in control lines lacking the transgene or lacking the "driver" that is necessary for Gbeta5 expression from the transgenic promoter. Using these transgenic flies, and based on the observed phenotype, quantitative real-time PCR was used to estimate the levels of relevant transcripts. Certain fly transcripts were up-regulated in the Gbeta5 transgenic, but not control, fly lines. The effects on some of these fly transcripts were also observed on the homologous mammalian transcripts in PC12 cells in which mouse Gbeta5 was inducibly over-expressed by stable transfection. Immunoblot studies showed that the corresponding protein products were also up-regulated by Gbeta5 overexpression. The molecular mechanism of the observed changes, and the effect of Gbeta5 loss-of-function (to see if the phenotype is opposite to the over-expression models) such as in neurons and brain of Gbeta5 knockout mice, are currently being examined.