Analysis of the Functional Roles of a Novel G-alpha Nucleotide Cycle

新型 G-α 核苷酸循环的功能作用分析

• 批准号: 8555896
• 负责人: JOHN H KEHRL
• 金额: $ 42.41万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS 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/8555896
• 关键词: Alleles; Animal Model; B-Lymphocytes; Back; Binding; Biochemical; Biosensor; Bone Marrow; CD19 gene; Caenorhabditis elegans; Cell Cycle; Cell Nucleus; Cell Size; Cell division; Cells; Centrosome; Cholinesterase Inhibitors; Chromosomes, Human, Pair 7; Collaborations; Complex; Cytokinesis; Defect; Development; Dissociation; Drosophila genus; Dynein ATPase; Embryo; Ensure; Excision; Fluorescence; Fluorescence Resonance Energy Transfer; G Beta Gamma; GTP-Binding Protein Regulators; GTP-Binding Protein alpha Subunits, Gs; GTPase-Activating Proteins; Generations; Genetic; Gi-alpha protein; Glues; Guanine Nucleotide Dissociation Inhibitors; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Hematopoietic; Heterotrimeric GTP-Binding Proteins; Human; Human G(i) Alpha Proteins; Image; Immune; Immunologic Deficiency Syndromes; In Vitro; Length; Lymphocyte; Malignant Neoplasms; Mammalian Cell; Mammals; Measures; Metaphase; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic Spindle Apparatus; Mitotic spindle; Mus; Normal Cell; Normal tissue morphology; Nuclear; Nucleotides; Phenotype; Phosphatidylinositols; Phospholipids; Phosphorylation; Process; Production; Protein Isoforms; Proteins; RGS Domain; RGS Proteins; RGS3 gene; Recruitment Activity; Regulation; Resistance; Role; Sampling; Screening for cancer; Signal Transduction; Site; Spleen; T-Lymphocyte; Time; Ubiquitination; Universities; Vps34 Phosphatidylinositol 3 Kinase; Wisconsin; cell cortex; cell type; chromosome movement; daughter cell; genetic regulatory protein; inhibitor/antagonist; macrophage; mutant; novel; protein complex; protein expression; protein function; receptor; segregation; tumor

These studies have focused on the role of Gi-proteins and their regulators in mitosis and cytokinesis. In model organisms such as Caenorhabditis elegans and Drosophila receptor-independent heterotrimeric G protein function is vital for the orientation of mitotic spindle, generation of microtubule pulling force, aster-induced cytokinesis, and centration of the nucleus-centrosome complex. This new paradigm is now being extended to mammalian cells. We and others have shown that Gi proteins and their regulators such as AGS3, LGN, and RGS14 localize in centrosomes, at the mitotic cell cortex, and at the midbody region. At these sites AGS3, LGN, and RGS14 likely bind Gi alpha proteins and function similar to G beta/gamma subunits. We have shown a role for a non-GPCR activator of Gi protein termed Ric-8A in human cell division. Ric-8A expression occurs in most human cells and at high levels in lymphocytes. We have evidence that Ric-8A is important for recruiting a signaling complex to the metaphase cell cortex consisting of NuMA, LGN, dynein, p150 glued, and Gi alpha1. Interference with the localization of this complex caused defects in mitotic spindle orientation and normal cell division. In collaboration with Zhen Huang at the University of Wisconsin we have begun studies to examine mice in which Ric-8A has been conditionally deleted from B or T lymphocytes. The non-conditional disruption of Ric-8A causes embryonic lethality. The Ric-8A LoxP mice have been re-derived and crossed to CD19-CRE. Since CD19 and Ric-8A are both located on chromosome 7, mice with only one interrupted allele of Ric-8A in B cells can be characterized. The initial analysis of these mice has revealed a reduction in the numbers of transitional B cells compared to control mice. To examine mice in which both alleles of Ric-8A are subject to deletion in B cells and other hematopoietic cell types we have obtained Vav-1 CRE mice and crossed them with the Ric-8A LoxP mice. We have verified that Ric-8A is deleted in the spleen cells of these mice and in bone marrow derived macrophages. Prelimary results indicate that these mice suffer from a severe immunodeficiency. We have shown that Ric-8A protein expression, phosphorylation and ubiquitination vary during the cell cycle, reaching their maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis Ric-8A co-localized with the phosphatidylinositol 3-kinase (PI3-K) Vps34 at the midbody along with G alphai and LGN, where these proteins functioned to regulate the production of PtdIns(3)P, a phospholipid needed for proper cytokinesis. Because cytokinesis defects often accompany human cancers we screened primary human tumors for Ric-8A expression levels. We found that they were significantly altered in the tumor samples compared to normal tissues. In C. elegans RGS7 functions in early cell divisions and RGS7 mutants show hyper-asymmetric movementof mitotic spindles. Among the mammalian RGS proteins, RGS3 most closely resembles C. elegans RGS7. We have shown that one isoform of RGS termedPDZ-RGS3 functions to regulate microtubule dynamics and cytokinesis. Two independent mouse lines each with a targeted disruption of Rgs3 have been identified, however, one line is an embryonic lethal while the other is viable. Extensive back-crossing of the two lines onto a C57/Bl6 background has not resolved the differences between the two lines. We are now beginning to immune phenotype the non-lethal Rgs3-/-mice.

这些研究集中在Gi蛋白及其调节剂在有丝分裂和胞质分裂中的作用。在模式生物如秀丽隐杆线虫和果蝇中，受体独立的异源三聚体G蛋白功能对于有丝分裂纺锤体的定向、微管拉力的产生、紫菀诱导的胞质分裂和核-中心体复合物的集中是至关重要的。这种新的模式现在正在扩展到哺乳动物细胞。我们和其他人已经表明，Gi蛋白及其调节因子，如AGS 3，LGN和RGS 14定位在中心体，在有丝分裂细胞皮质，并在中间体区域。在这些位点，AGS 3、LGN和RGS 14可能结合Gi α蛋白，并且功能类似于G β/γ亚基。我们已经显示了Gi蛋白的非GPCR激活剂Ric-8A在人类细胞分裂中的作用。Ric-8A在大多数人类细胞中表达，在淋巴细胞中表达水平较高。我们有证据表明，Ric-8A是重要的招募信号复合物的中期细胞皮质组成的NuMA，LGN，动力蛋白，p150胶合，和Gi α 1。干扰该复合物的定位引起有丝分裂纺锤体方向和正常细胞分裂的缺陷。 我们与威斯康星州大学的Zhen Huang合作，开始研究从B或T淋巴细胞中有条件地删除Ric-8A的小鼠。Ric-8A的无条件破坏导致胚胎死亡。Ric-8A LoxP小鼠已重新衍生并与CD 19-CRE杂交。 由于CD 19和Ric-8A均位于7号染色体上，因此可以表征在B细胞中仅具有一个Ric-8A中断等位基因的小鼠。 对这些小鼠的初步分析显示，与对照小鼠相比，过渡性B细胞的数量减少。 为了检查其中Ric-8A的两个等位基因在B细胞和其它造血细胞类型中均缺失的小鼠，我们获得了Vav-1 CRE小鼠，并将它们与Ric-8A LoxP小鼠杂交。 我们已经证实Ric-8A在这些小鼠的脾细胞和骨髓衍生的巨噬细胞中缺失。 实验结果表明，这些小鼠患有严重的免疫缺陷。 我们已经表明，Ric-8A蛋白表达，磷酸化和泛素化在细胞周期中变化，在有丝分裂时达到最高水平。为了通过FLIM（荧光寿命成像显微镜）测量Ric-8A的构象变化而创建的FRET生物传感器显示Ric-8A在有丝分裂期间处于闭合状态，特别是在胞质分裂期间。降低Ric-8A表达延迟分裂细胞的分裂时间，这与细胞间桥长度增加和多核化相关。 在胞质分裂期间，Ric-8A与磷脂酰肌醇3-激酶（PI 3-K）Vps 34共定位于中间体沿着有G α和LGN，在那里这些蛋白质起调节PtdIns（3）P的作用，PtdIns（3）P是一种适当胞质分裂所需的磷脂。 因为胞质分裂缺陷通常伴随人类癌症，所以我们筛选了Ric-8A表达水平的原发性人类肿瘤。 我们发现，与正常组织相比，它们在肿瘤样本中发生了显着变化。 In C.线虫RGS 7在早期细胞分裂中起作用，RGS 7突变体显示有丝分裂纺锤体的超不对称运动。 在哺乳动物的RGS蛋白中，RGS 3与C.优雅的RGS 7。我们已经证明RGS的一种亚型PDZ-RGS 3具有调节微管动力学和胞质分裂的功能。 已经鉴定了两个独立的小鼠品系，每个品系具有Rgs 3的靶向破坏，然而，一个品系是胚胎致死的，而另一个品系是存活的。将两个品系广泛回交到C57/B16背景上没有解决两个品系之间的差异。 我们现在开始对非致死性Rgs 3-/-小鼠进行免疫表型。