G-Protein Signaling in Cell Differentiation

该项目的总体目标是研究异源三聚体G蛋白亚基Gi（α 2）在细胞分化中的参与。 造血细胞系K562和U937将用作实验范例。 将在这些细胞中用丁酸钠和佛波醇肉豆蔻乙酸酯诱导分化以实现不同的终末表型。 该项目将实现三个具体目标。 首先，将使用百日咳毒素和抗G1（α 2）的反义方法来检验K562细胞的分化（通过血红蛋白表达测量）需要G1（α 2）的假设。 其次，转录机制负责Gi（α 2）水平变化的想法将得到验证和确认。 第三，将评估Gi（α 2）或相关G蛋白亚基在分化期间可易位至细胞核的概率。 G蛋白亚基将通过用亲和纯化的抗体进行免疫印迹来检测。 将通过细胞分级分离方法以及免疫荧光和共聚焦显微镜检测核易位。 关于G蛋白如何介导信号转导的传统观点并没有传统地认为它们易位到核区室是一种潜在的作用模式。 因此，该项目有可能揭示G α亚基的新作用，这些亚基不涉及它们在其形成位点附近激活跨膜效应物的传统作用。 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.