Neuroendocrine Cell Biology of the G Protein Beta5/ R7-RGS Protein/ R7BP Complex

目前工作的目的是了解人类神经内分泌系统中Gbeta 5/R7-RGS/R7 BP复合物的细胞生物学。 在一个相关的项目（DK 043304 -14）中，我们试图识别和表征昆虫和哺乳动物神经系统之间进化保守的Gbeta 5复合物的功能。在该项目中，我们使用了一个昆虫模型系统，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。 在这个项目中，我们目前的工作检查a）是否，以及在细胞内的何处，G β 5/R7-RGS/R7 BP复合物在神经内分泌细胞和组织如垂体和胰岛中表达，和B）通过G β 5在PC 12和SH-SY 5 Y细胞中过表达观察到的表型是否提供了对其在垂体和胰岛中可能的天然（或内源性）功能的有效洞察。采用的模型系统包括垂体和胰岛β细胞衍生的细胞系，以及野生型和Gbeta 5敲除小鼠。

The purpose of the current work is to understand the cell biology of the Gbeta5/R7-RGS/R7BP complex in the human neuroendocrine system. In a related project (DK043304-14) we are trying to identify and characterize the functions of the Gbeta5 complex that are evolutionarily conserved between the insect and mammalian nervous systems. In that project we are using 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. In this project our current work examines a) whether, and where within the cell, the Gbeta5/R7-RGS/ R7BP complex is expressed in neuroendocrine cells and tissues such as the pituitary and pancreatic islets, and b) whether the phenotype observed by Gbeta5 over-expression in PC12 and SH-SY5Y cells provides valid insight into its possible natural (or endogenous) function in the pituitary and pancreatic islets. Model systems employed include pituitary- and pancreatic islet beta-cell-derived cell lines, as well as wild-type and Gbeta5 knockout mice.