Transmembrane Signaling Mechanisms

在过去的几年中，我们已经破坏了编码百日咳毒素敏感G蛋白Gi 2，Gi 1，Gi 3和Go的基因。还产生了Gi 2和Go的条件性敲除。涉及Gi 2和Go的双击倒是致命的。 我们希望从Gi 2和Gi 3的组合中学习，并通过在出生后的不同时间去除floxed基因来提高Go KO小鼠的存活率。 已经建立了育种程序，以在几种特异性启动子下添加cre重组酶，从而通常在所有组织中或在特定细胞类型中去除基因，例如在多巴胺能神经元中去除Go或在淋巴细胞中去除Gi，以从Gi 3 KO小鼠中去除Gi。 大多数表型研究都是与外部研究人员合作完成的， 第二项研究集中在Gs-alpha突变体的特性上，这些突变体可能为受体激活Gs的分子机制提供信息。 我们之前分析了GS-α R265 E突变体。 今年我们集中研究了影响Mg结合的突变体：Gs-alpha T204 A、T204 Q和T204 E。 结合起来，他们揭示了赋予Gs-alpha激活腺苷酸环化酶（效应子激活功能）能力的构象变化独立于负责激活其GT3活性（自动关闭功能）的构象变化。 我们试图使转导素α亚基中的同源突变结晶，以更好地理解改变的性质的原子基础，但这是不可能的，很可能是因为T204的Mg结合功能的丧失使结构松弛，从而产生相当混乱的分子。 在过去的一年中，我们还建立了一种在细菌中合成G蛋白α亚基的技术，并通过共表达脱甲酰基酶（DEF），Met-氨基肽酶（MAP）和人N-末端肉豆蔻酰转移酶（NMT）来实现其翻译后肉豆蔻酰化。与NMT和MAP的共表达以前曾有报道，但在我们的手中没有修饰超过10%的蛋白质。DEF的添加导致90%豆蔻酰化的G蛋白α亚基。 我们将准备几个G α并使用它们来探索它们可能调节的新目标。 由于Gi 2 α KO小鼠的可用性而可能的一个重要且出乎意料的发现是缺乏Gi 2的小鼠对内毒素（LPS）和脓毒症（盲肠结扎和穿孔，CLP）过敏，这是与James A.库克在南卡罗来纳州医科大学。另一项尚未发表的研究表明，缺乏TRPC 6通道的小鼠对LSP和CLP具有抵抗力，这是可以理解的，因为TRPC 6通道调节Ca进入细胞，缺乏Ca使小鼠对需要Ca发挥作用的刺激更具抵抗力。 我们将进行上位性研究，其中我们将Gi 2 KO小鼠与TRPC 6 KO小鼠杂交，以确定这些作用的主导作用，并了解需要Gi 2保护免受LPS和CLP的机制。 我们在刘延顺博士（研究员）的指导下继续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染的组织培养细胞中提取的。转导蛋白将使用重组DNA技术，使我们能够在细菌中表达α亚基，在昆虫细胞中表达β-γ亚基。然后将α和β-γ二聚体纯化并组装成转导素（α-β-γ三聚体）。 在与LSB的Robert伦敦和Geoffrey Mueller的合作中，Yanshun Liu研究了钙调素（CaM）与Orai 1的CaM结合结构域（CaMBD）相互作用的性质。 晶体学和NMR数据都得到了导致的结论，即CaM与Orai 1的相互作用代表了一种新的结构，因为它具有2个CaMBD与单个CaM的化学计量比，从而保留了cAM的扩展形式。钙调素诱导的抑制奥赖依赖性钙进入细胞的模型发生在钙库耗尽时， 我们继续与校外科学家合作，分析G蛋白缺陷小鼠中出现的表型。 今年最值得注意的发现是发现Go G蛋白是胰岛素分泌的负调节剂，并且通过减少胰腺β细胞中易于释放的胰岛素颗粒池来实现，并且发现Gi 2在炎症过程中起作用。

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 outside investigators, 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. During this last year we also established a technique to synthesize G protein alpha subunits in bacteria and to effect their post-translational myristoylation by co-expression of a deformylase (DEF), a Met-amino peptidase (MAP)and a human N-terminal myristoyltransferase (NMT). Co-expression with NMT and MAP had been reported before but in our hands did not modify more than 10% of the protein. Addition of DEF led to a G protein alpha subunit that is 90% myristoylated. We will prepare several G alphas and use them to explore novel target they may regulate. One important and unexpected finding that was possible because of the availability of the Gi2 alpha KO mouse is that mice lacking Gi2 are hypersensitive to endotoxin (LPS) and sepsis (cecal ligation and puncture, CLP), which was carried out in collaboration with James A. Cook at Medical University of South Carolina. We do not know the basis for this result and do not understand it. Another study, as yet unpublished, has shown that mice lacking the TRPC6 channel are resistant to LSP and CLP, which is understandable because TRPC6 channels regulate Ca entry into cells and lack of Ca renders the mice more resistant to stimuli that require Ca to exert their effects. We will perform an epistasis study, in which we cross Gi2 KO mice with TRPC6 KO mice, to determine the dominant of these effects and learn about the mechanism requiring Gi2 that protects against LPS and CLP. 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). In collaboratin with Robert London and Geoffrey Mueller from LSB Yanshun Liu examined the nature of the interaction of Calmodulin (CaM) with a CaM binding domain (CaMBD) of Orai1. Both crystallography and NMR data were obtained leading to the coclusion that the interaction of CaM with Orai1 represents a novel structure as it has stoichiometryof 2 CaMBD with a single CaM such that the extended form of cAM is preserved. A modelof the CaM induced inhibition of Orai dependent Ca entry into cells occurring upon store depletion, 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 and the discovery that Gi2 plays a role in inflammatory processes.