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MECHANISM OF CALCIUM SPIKING IN SIGNAL TRANSDUCTION

信号转导中的钙尖峰机制

基本信息

批准号：
3384998
负责人：
LUBERT STRYER
金额：
$ 21.07万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-01 至 1994-08-31
项目状态：
已结题

项目摘要

Many cells exhibit calcium spikes (periodic transient increases in cytosolic CA2+) when stimulated by a neurotransmitter, hormone, or growth factor. The overall goal of this research is to elucidate the molecular mechanism of calcium spiking and delineate its role in signal transduction. The rat basophilic leukemic cell (RBL cell) will be studied as a model secretory cell, the rat hepatocyte as an integrator of diverse stimuli, and the PC12 cell as a model neuron. Calcium spiking will be monitored by photon-counting fluorescence microscopy of single cells containing an indicator such as fluo-3. Biochemical, fluorescence, and ultrastructural studies of intact cells, permeabilized cells, purified proteins, and reconstituted membrane assemblies will be carried out to answer: (1) How are calcium spikes generated? We will test a molecular model for calcium spiking that is based on four elements: cooperativity and positive feedback between inositol 1,4,5- trisphosphate (IP3) and cytosolic Ca2+, delayed deactivation by mitochondrial uptake of Ca2+, and reactivation by refilling of the endoplasmic reticulum Ca2+ store. Specific inhibitors will pinpoint the contributions of particular processes to spike generation. (2) How is calcium spiking modulated? The effects of calcium influx into the cell, intracellular pH, the level of phosphoinositides, and phosphorylation state of components of the spike generator will be determined. (3) How is spiking altered by the interplay of the phosphoinositide cascade with other signal transduction pathways? The modulatory actions of the cyclic AmP cascade, growth factors, voltage-sensitive calcium channels, and lithium ion will be investigated. (4) How do calcium spikes trigger effector events such as exocytosis and memory? The distribution of F- actin and myosin in stimulated RBL cells will be determined by fluorescence and immunoelectron microscopy to determine whether individual spikes induce discrete cytoskeletal rearrangements and to relate them to granule release. The Ca2+/calmodulin-dependent protein kinase of PC12 cells will be studied as a model memory protein. We will measure how calcium spikes switch this protein between different functional states as expressed by their degree of phosphorylation- autophosphorylation activity, and kinase activity for exogenous substrates such as tyrosine hydroxylase. A deeper understanding of calcium spiking is likely to reveal how digital logic is used within cells to process information and achieve timing control (as in circadian rhythms). Some neuropsychiatric disorders may arise from kinetic mismatches of components of the spike generator. Information about spiking should lead to a better understanding of the therapeutic action of lithium ion in manic-depressive disorders.

许多细胞表现出钙峰（周期性短暂增加，细胞溶质CA 2+），当受到神经递质、激素或生长因子本研究的总体目标是阐明钙峰化的分子机制及其在信号传导中的作用转导大鼠嗜碱性白血病细胞（RBL细胞）将被作为分泌细胞模型研究，大鼠肝细胞作为整合细胞以PC 12细胞为模型神经元。钙将通过光子计数荧光显微镜监测加标，含有指示剂（如fluo-3）的单细胞。生物化学，完整细胞的荧光和超微结构研究，透化细胞、纯化的蛋白质和重构的膜组件将被进行回答：（1）钙峰是如何产生的？我们将测试钙峰值的分子模型，该模型基于四个元素：肌醇1，4，5- 三磷酸盐（IP 3）和胞质Ca 2+，延迟失活线粒体摄取Ca 2+，并通过重新填充线粒体而重新激活。内质网钙库。特异性抑制剂可以精确定位特定过程对尖峰产生的贡献。 (2)怎么样调节钙峰值？钙离子流入细胞的影响，细胞内pH、磷酸肌醇水平和磷酸化将确定尖峰发生器的组件的状态。 (3)如何是由磷酸肌醇级联反应的相互作用，其他信号传导途径？的调节作用，环AmP级联，生长因子，电压敏感性钙通道，和锂离子将被研究。 (4)钙峰值是如何触发的效应事件，如胞吐和记忆？ F的分布- 在刺激的RBL细胞中的肌动蛋白和肌球蛋白将通过荧光和免疫电子显微镜来确定是否单个尖峰诱导离散的细胞骨架重排，与颗粒释放有关。 Ca 2 +/钙调素依赖蛋白将PC 12细胞的激酶作为模型记忆蛋白进行研究。我们将测量钙峰值如何在不同的蛋白质之间转换以磷酸化程度表示的功能状态- 自磷酸化活性和外源性的激酶活性底物如酪氨酸羟化酶。更深入地了解钙尖峰可能揭示数字逻辑是如何在细胞处理信息并实现定时控制（如昼夜节律节奏）。一些神经精神障碍可能是由运动性的尖峰发生器的组件不匹配。信息加标应能更好地理解治疗作用锂离子在躁狂抑郁症中的应用

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Calcium-myristoyl protein switch.

DOI：
10.1073/pnas.89.23.11569
发表时间：
1992-12
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
S. Zozulya;L. Stryer
通讯作者：
S. Zozulya;L. Stryer

Association of the beta isoform of protein kinase C with vimentin filaments.

蛋白激酶 C β 亚型与波形蛋白丝的关联。