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

G蛋白Beta5/ R7-RGS蛋白/ R7BP复合物信号转导机制

• 批准号: 7967421
• 负责人: WILLIAM F SIMONDS
• 金额: $ 36.46万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967421
• 关键词: Binding; Binding Proteins; Biological Models; Brain; Cell Nucleus; Cell membrane; Cells; Complex; Cytosol; Drosophila genus; Drosophila melanogaster; Enzymes; Family; GTP-Binding Protein Regulators; GTP-Binding Proteins; Goals; Guanine Nucleotides; Human; Immunoblotting; Insecta; Ion Channel; Knockout Mice; Laboratories; Lipids; Modeling; Modification; Molecular; Mus; Nervous system structure; Neurons; Nuclear; PC12 Cells; Pattern; Phenotype; Property; Protein Isoforms; Proteins; RGS Proteins; RNA Splicing; Research Project Grants; Retinal Photoreceptors; Rodent; Role; Signal Transduction; Stimulus; Structure; System; Time; Transcript; Transducers; Transfection; Transgenes; Transgenic Organisms; Work; base; extracellular; fly; genetic regulatory protein; loss of function; mammalian genome; overexpression; palmitoylation; promoter; protein complex; receptor; receptor coupling

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.

目前工作的目的是了解 Gbeta5/R7-RGS/R7BP 复合物在人类中的信号传导功能。 为此，我们首先尝试鉴定和表征昆虫和哺乳动物神经系统之间进化保守的 Gbeta5 复合物的功能。 我们实验室目前的工作使用果蝇、培养的 PC12 和 SH-SY5Y 细胞以及原代啮齿动物脑神经元作为模型系统来检查此类共享功能。 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度表达。 两个独立的Gbeta5转基因蝇系具有在缺乏转基因或缺乏转基因启动子表达Gbeta5所必需的“驱动器”的对照系中不明显的表型。使用这些转基因果蝇，并根据观察到的表型，使用定量实时 PCR 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，但在对照果蝇品系中则不然。 在 PC12 细胞中的同源哺乳动物转录本上也观察到了对其中一些果蝇转录本的影响，其中小鼠 Gbeta5 通过稳定转染诱导过表达。 免疫印迹研究表明，Gbeta5 过表达也上调了相应的蛋白质产物。 目前正在研究观察到的变化的分子机制，以及 Gbeta5 功能丧失（观察表型是否与过度表达模型相反）对 Gbeta5 敲除小鼠的神经元和大脑的影响。