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

目前工作的目的是了解Gbeta5/R7-RGS/R7BP复合体在人类神经内分泌系统中的细胞生物学。在一个相关的项目(DK043304-14)中，我们试图鉴定和表征Gbeta5复合体的功能，这些功能在昆虫和哺乳动物的神经系统之间进化上是保守的。在该项目中，我们使用了一种昆虫模型系统，在该系统中，果蝇Gbeta5在果蝇神经系统中转基因过度表达。两个独立的Gbeta5转基因果蝇品系的表型在缺乏转基因或缺乏从转基因启动子表达Gbeta5所必需的“驱动”的对照品系中并不明显。利用这些转基因果蝇，根据观察到的表型，使用实时定量聚合酶链式反应来估计相关转录本的水平。某些果蝇转录本在Gbeta5转基因果蝇品系中上调，但在对照品系中没有上调。在PC12细胞中的同源哺乳动物转录物上也观察到了对其中一些苍蝇转录物的影响，其中小鼠Gbeta5通过稳定的转染法诱导过表达。免疫印迹研究表明，Gbeta5过表达也上调了相应的蛋白产物。在这个项目中，我们目前的工作检测了a)Gbeta5/R7-RGS/R7BP复合体是否在神经内分泌细胞和组织中表达，以及b)Gbeta5在PC12和SH-SY5Y细胞中过度表达的表型是否提供了对其在垂体和胰岛可能的自然(或内源性)功能的有效洞察。使用的模型系统包括垂体和胰岛β细胞来源的细胞系，以及野生型和Gbeta5基因敲除小鼠。

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.