Transmembrane Signaling Mechanisms

跨膜信号传导机制

• 批准号: 8553770
• 负责人: Lutz Birnbaumer
• 金额: $ 179.44万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 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/8553770
• 关键词: Adenylate Cyclase; Affect; Bacteria; Binding; Birth; Breeding; Calmodulin; Cattle; Cells; Collaborations; Crystallography; Data; Endotoxins; Extramural Activities; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genes; Genetic Epistasis; Go Alpha Subunit; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Inflammatory; Insecta; Knock-out; Learning; Ligation; London; Lymphocyte; Medical; Molecular; Mus; Mutation; N-terminal; Nature; Peptide Hydrolases; Pertussis Toxin; Phenotype; Play; Process; Property; Proteins; Puncture procedure; Recombinant DNA; Reporting; Research Personnel; Resistance; Retina; Rhodopsin; Role; Scientist; Sepsis; Signal Transduction; South Carolina; Stimulus; Structure; Structure of beta Cell of islet; Techniques; Time; Transducin; Universities; Work; alpha Subunit Transducin; base; cell type; cooking; dimer; dopaminergic neuron; insulin granule; insulin secretion; meetings; mutant; myristoylation; novel; programs; promoter; receptor; recombinase; tissue/cell culture

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.

在前几年，我们已经破坏了编码百日咳毒素敏感的G蛋白Gi2， Gi1， Gi3和Go的基因。还生成了Gi2和Go的条件敲除。涉及Gi2和Go的双敲除是致命的。我们希望从Gi2和Gi3的结合中学习，并通过在出生后的不同时间去除固定的基因来提高Go KO小鼠的存活率。已经建立了育种计划，在几个特定启动子下添加cre重组酶，以便在所有问题或特定细胞类型中去除基因，例如在多巴胺能神经元中去除Go或淋巴细胞中去除Gi3 KO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，