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

本研究的目的是了解Gbeta 5/R7-RGS/R7 BP复合物在人体中的信号传导功能。 为此，我们首先试图确定和表征的功能的Gbeta 5复杂的进化保守的昆虫和哺乳动物的神经系统之间。 我们实验室目前的工作是使用果蝇、培养的PC 12和SH-SY 5 Y细胞以及初级啮齿类动物脑神经元作为模型系统来研究这种共享功能。 我们正在继续使用一种昆虫模型系统，其中果蝇Gbeta 5在果蝇神经系统中转基因过表达。 两个独立的G β 5转基因果蝇品系具有在缺乏转基因或缺乏从转基因启动子表达G β 5所必需的“驱动”的对照品系中不明显的表型。使用这些转基因果蝇，并根据观察到的表型，定量实时PCR被用来估计相关转录本的水平。某些果蝇转录本在Gbeta 5转基因果蝇品系中上调，但在对照果蝇品系中未上调。 在PC 12细胞中的同源哺乳动物转录物上也观察到对这些果蝇转录物中的一些的影响，其中小鼠G β 5通过稳定转染诱导过表达。 免疫印迹研究表明，相应的蛋白质产物也上调了Gbeta 5过表达。 目前正在研究观察到的变化的分子机制，以及Gbeta 5功能丧失的影响（以观察表型是否与过表达模型相反），例如在Gbeta 5敲除小鼠的神经元和大脑中。

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.