Transmembrane Signaling Mechanisms

在前几年，我们已经扰乱了编码百日咳毒素敏感G蛋白GI2、GI1、GI3和GO的基因。GI2和GO的条件基因敲除也被生成。涉及GI2和围棋的双重击倒是致命的。我们希望从GI2和GI3的结合中学习，并通过在出生后不同时间移除GO KO小鼠的成活基因来提高GO KO小鼠的存活率。已经建立了一些育种计划，在几个特定的启动子下添加cre重组酶，以便在所有问题或特定细胞类型中移除基因，例如在多巴胺能神经元中移除GO或在淋巴细胞中移除Gi3KO小鼠的GI。大多数表型研究都是与外部研究人员合作完成的， 编码Gsα亚单位的基因座(称为GNAS复合体基因座)是印记的，即它的转录只来自两个等位基因中的一个，使我们和小鼠的这些基因产物成为单倍体。该基因由5个转录单位组成，其中Gs-α为1个，其余为XXlas、Nespas、Nesp和1A(未转录的RNA)。对于Gs-α，这只出现在大约五个组织中，但对于其他转录本来说是普遍的。印迹是启动子不同甲基化的结果。这使我们对DNA甲基化产生了兴趣，并开发了一种高分辨率的方法来在基因组范围内调查DNA甲基化。在这种方法中，我们用甲基化敏感的限制性内切酶切割基因组DNA，用EcoP15I 25-NT标签从新创建的末端剪下，并用三种商业技术中的任何一种对这些标签进行大规模平行测序。这种方法现在可以用来探索组织之间的差异，以及发育、年龄和环境暴露对DNA甲基化的影响。我们现在终于可以评估血液传播的中性粒细胞的甲基化模式，并测试这样一种假设，即这种模式的变化可以用来报告环境暴露在骨髓造血室的干细胞壁龛中留下的足迹。需要收集的更多信息将有助于描述差异甲基化结构域或区域(DMRS)，以及甲基化状态是否可以告知等位基因排斥现象，而印记只是等位基因排斥现象的多种表现形式之一。 第二项研究集中在Gs-α突变体的特性上，这些特性可能会为受体激活Gs的分子机制提供信息。我们之前分析了Gs-αR265E突变体。今年我们重点研究了影响镁：GS-αT204A、T204Q和T204E结合的突变体。结合起来，他们揭示了赋予Gs-α激活腺酰环酶能力的构象变化(效应器激活功能)独立于导致其GTPase活性激活的构象变化(自动关闭功能)。我们试图使转导蛋白α亚基中的同源突变结晶，以更好地理解改变性质的原子基础，但这是不可能的，很可能是因为T204的镁结合功能的丧失放松了结构，产生了一个相当无序的分子。 最后，我们继续在刘燕顺博士(工作人员科学家)的指导下，研究视紫红质与其同源G蛋白转导蛋白的共结晶。视紫红质是从牛视网膜和稳定转基因的组织培养细胞中提取的。转导蛋白将使用重组DNA技术来制造，这种技术允许我们在细菌中表达阿尔法亚基，在昆虫细胞中表达β-伽马亚基。然后，α和β-伽马二聚体将被提纯并组装成转导蛋白(α-β-伽马三聚体)。 我们继续与校外科学家合作，分析G蛋白缺陷小鼠的表型。今年最值得注意的发现是，Go G蛋白是胰岛素分泌的负调节因子，它通过减少胰岛β细胞中容易释放的胰岛素颗粒来实现这一点。

In previous years we had disrupted the genes encoding the pertussis toxin sensitive G proteins Gi2, Gi1, Gi3 and Go. Conditional knockouts for Gi2 and Go were also generated. Double knockouts involving Gi2 and Go are lethal. We expect to learn from combining Gi2 with Gi3 and enhanced survival of Go KO mice by removing the floxed genes at various times after birth. Breeding programs have been set up to add cre recombinase under several specific promoters so as to remove the genes both generally in all issues or in specific cell types such as in dopaminergic neurons to remove Go or lymphocytes to remove Gi from Gi3 KO mice. Most phenotypic studies are done in collaboration with out side investigators, The locus encoding the Gs alpha subunit (termed GNAS complex locus is imprinted, i.e. its transcription proceeds from only one of the two alleles, making us and mice haploid for these gene products. The locus comprises five transcription units of which Gs-alpha is one and XXLas, Nespas, Nesp and 1A (a non-transcribed RNA) are the others. For Gs-alpha this occurs in only five tissues or so, but is generalized for the other transcripts. Imprinting is the result of differential methylation of promoters. This led us to become interested in DNA methylation and to the development of a high resolution method to survey DNA methylation at a genome wide scale. In this method we cut genomic DNA with methylation sensitive restriction enzymes, snip off with EcoP15I 25-nt tags from the newly created ends and subject these tags to massively parallel sequencing by any one of three commercially available technologies. The method is now ready to be used to explore differences between tissues, and effects of development, age and environmental exposures on DNA methylation. We are now, finally, in position to assess the methylation pattern of blood borne neutrophils and test the hypothesis that changes in this patterns can be used to report on environmental exposures leaving a footprint in the stem cell niche of the hematopoietic compartment of the bone marrow. Additional information to be gathered will contribute to the description of differentially methylated domains or regions (DMRs) and whether the methylation status can inform about allelic exclusion phenomena of which imprinting is but one of many forms in which allelic exclusion is manifested. A second study focused on properties of Gs-alpha mutants that may inform on the molecular mechanism by which receptors activate Gs. We previously analyzed the Gs-alpha R265E mutant. This year we concentrated on mutants that affect binding of Mg: Gs-alpha T204A, T204Q and T204E. Combined, they revealed that the conformational change that confers the ability of Gs-alpha to activate adenylyl cyclase (effector-activating function) is independent of the conformational change responsible for activation of its GTPase activity (auto-turnoff function). We attempted to crystallize the cognate mutation in a transducin alpha subunit to better understand the atomic basis of the changed properties, but this was not possible, very likely because the loss of the Mg binding function of T204 relaxes the structure creating a rather disorganized molecule. Finally, we are continuing under the guidance of Dr. Yanshun Liu (staff scientist) to work on the co-crystalization of rhodopsin with its cognate G protein transducin. Rhodopsin is both extracted from bovine retinas and and from stably transfected tissue culture cells. Transducin will be made using recombinant DNA techniques that allow us to express the alpha subunit in bacteria, and the beta-gamma subunit in insect cells. Alpha and beta-gamma dimers will then by purified and assembled into transducin (alpha-beta-gamma trimer). We continue collaborating with extramural scientists in the analysis of the phenotypes that arise in G protein deficient mice. The most notable finding this year has been the discovery that the Go G protein is a negative regulator of insulin secretion and does so by diminishing the readily releasable pool of insulin granules in the pancreatic beta cell.