G PROTEIN SIGNALING IN CELL DIFFERENTIATION

细胞分化中的 G 蛋白信号传导

• 批准号: 6349113
• 负责人: Ifeanyi J Arinze
• 金额: $ 11.77万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349113
• 关键词: G protein; affinity chromatography; biological signal transduction; cell differentiation; cell line; confocal scanning microscopy; genetic transcription; immunofluorescence technique; pertussis toxin; phosphorylation; western blottings

The overall objective of this project is to study the involvement of the G- protein Gialpha2, and related G-protein subunits, in cellular differentiation, using the hematopoietic cell lines K562 and U937 as experimental paradigms. Differentiation will be induced in these cells with one or more of the following cell-differentiation-inducing agents, sodium butyrate, retinoic acid and phorbol myristic acetate, so as to achieve different cell phenotypes. The project will accomplish four specific objectives. Firstly, using pertussis toxin and antisense methods to inactive Gialpha2, the hypothesis will be tested that cell differentiation in K562 cells (measured by hemoglobin expression) requires Gialpha2. Secondly, the idea that transcriptional mechanisms underlie the previously reported robust changes in Gialpha2 levels will be verified and confirmed. Thirdly and fourthly, it will be established whether the mechanism of the participation of Gialpha2, and related G-protein subunits, in cell differentiation involves their phosphorylation and/or translocation to the cell nucleus. The G-protein subunits will be detected by immunoblotting with affinity-purified antibodies. Detection of their translocation to the nucleus will also be accomplished by using immunofluorescence and confocal microscopy. Conventional wisdom of how G proteins mediate signal transduction has not traditionally considered their translocation to the cell's nuclear compartment as a potential mode of action. Therefore, this project has the potential to uncover innovative roles of G-protein signaling molecules which do not involve their traditional role of transmembrane effector activation immediately resulting in the formation of second messenger(s).

该项目的总体目的是使用造血细胞系K562和U937作为实验范式研究G蛋白gialpha2和相关G蛋白亚基的参与。在这些细胞中，将通过以下一个或多个细胞差异诱导的剂，丁酸钠，视黄酸和凤凰肉芽杆菌醋酸酯诱导分化，以实现不同的细胞表型。该项目将实现四个特定目标。首先，将百日咳毒素和反义方法用于无活性的gialpha2，将检验假设K562细胞中的细胞分化（通过血红蛋白表达测量）需要gialpha2。其次，将验证和确认转录机制是先前报道的GIALPHA2水平的强大变化的想法。第三和第四，将确定gialpha2参与的机制以及相关的G蛋白亚基在细胞分化中是否涉及它们的磷酸化和/或转移到细胞核。通过用亲和纯化抗体进行免疫印迹来检测G蛋白亚基。通过使用免疫荧光和共聚焦显微镜来检测其转移到细胞核的易位。传统上，传统上，传统的智慧介导信号转导，传统上并未将其转移到细胞的核室是一种潜在的作用方式。因此，该项目有可能发现G蛋白信号分子的创新作用，而G蛋白信号分子不涉及跨膜效应子激活的传统作用，从而立即形成了第二信使。