G-Protein Signaling in Cell Differentiation

本项目的总体目标是研究异源三聚体G蛋白亚基GI(Alpha2)在细胞分化中的作用。造血细胞系K562和U937将作为实验范例。用丁酸钠和佛波酯诱导这些细胞分化，以获得不同的终末表型。该项目将实现三个具体目标。首先，将使用百日咳毒素和反义方法灭活GI(Alpha2)来检验K562细胞分化(通过血红蛋白表达来衡量)需要Gi(Alpha2)的假设。其次，转录机制负责胃肠道(α2)水平变化的观点将得到验证和证实。第三，将评估Gi(Alpha2)或相关G蛋白亚基在分化过程中移位到细胞核的可能性。G蛋白亚基将通过亲和纯化的抗体进行免疫印迹检测。核移位将通过细胞分离方法以及免疫荧光和共聚焦显微镜进行分析。关于G蛋白如何介导信号转导的传统智慧，传统上并不认为它们转位到核间室是一种潜在的作用方式。因此，这个项目有可能揭示Gα亚基的新角色，这些亚基不涉及它们在形成部位附近激活跨膜效应器的传统角色。该项目还将为少数民族学生提供培训，他们在科学界的代表性历来不足。

The overall objective of this project is to study the involvement of the heterotrimeric G protein subunit, Gi(alpha2), in cellular differentiation. The hematopoietic cell lines K562 and U937 will be used as experimental paradigms. Differentiation will be induced in these cells with sodium butyrate and phorbol myristic acetate to achieve different terminal phenotypes. The project will accomplish three specific objectives. First, pertussis toxin and antisense methods to inactivate Gi(alpha2) will be used to test the hypothesis that differentiation of K562 cells (measured by hemoglobin expression) requires Gi(alpha2). Second, the idea that transcriptional mechanisms are responsible for changes in Gi(alpha2) levels will be verified and confirmed. Third, the probability that Gi(alpha2) or related G protein subunits may be translocated to the nucleus during differentiation will be assessed. G protein subunits will be detected by immunoblotting with affinity-purified antibodies. Nuclear translocation will be assayed by cell fractionation methods as well as by immunofluorescence and confocal microscopy. Conventional wisdom of how G proteins mediate signal transduction has not traditionally considered their translocation to the nuclear compartment as a potential mode of action. Therefore, this project has the potential of revealing novel roles for G alpha subunits that do not involve their traditional role in activating transmembrane effectors near their site of formation. The project will also provide training of students of ethnic minority origin, who are historically under-represented in the scientific community.