G PROTEIN COUPLED PLC B ISOZYMES

一大类神经递质和激素激活磷脂酶 C β-同工酶（PLC-β）通过G蛋白连接的信号级联。 响应于活化的G蛋白，PLC-β同工酶加速了细胞的增殖。 磷脂酰肌醇-4，5二磷酸（PtdIns（4，5）P2）水解成 第二信使sn-1，2-二酰基甘油（DAG）和D-肌肌醇- 1，4，5-三磷酸（Ins（1，4，5）P3）。 可溶性Ins（1，4，5）P3的生产 导致细胞内钙的释放，同时DAG激活 蛋白激酶C同工酶，这些事件最终控制了多种 一系列细胞功能，包括增殖，兴奋， 分泌和收缩。 PLC-β的调节机制 同工酶由G蛋白、磷脂、Ca 2+等电位 调节因子在分子水平上了解甚少。 但随着 最近纯化多毫克功能PLC-β的能力 同工酶，我们现在处于有利地位，明确定义模式 PLC-β调节。 将进行一系列研究， 优化生产均匀和单分散的PLC-β 全酶和结构域片段。 这些特征蛋白质将 然后用于定量结合分析的双重方法， 晶体学来理解PLC-β调节。 特别注意 将给出描绘的N-末端PH结构域的作用， PLC-β结合磷酸肌醇和可能的β γ亚基。 定量测量磷酸肌醇和γ-氨基丁酸与 PLC-β的PH结构域将从表面等离子体获得 共振测量和微量热法。 由于不同的PLC-β 同工酶表现出不同的G蛋白调节， 来自不同同工酶的等价PH结构域可以界定 同工酶特异性G蛋白激活。 X射线晶体学也将 用于确定PLC-β结构域的原子分辨率结构 或全长蛋白质。 这些结构将提供关键信息 域间的相互作用，相对域方向，以及特定的 小分子的结合位点。总的来说，结合约束力 数据和结构信息旨在提供原子- 解析框架，用于理解并可能操纵 通过PLC-β同工酶调节界面催化。 这项工作 也将为更详细地了解 通过其他活化的G α亚基调节PLC-β同工酶， γ-二聚体。

A large class of neurotransmitters and hormones activate phospholipase C beta-isozymes (PLC-betas) through G protein-linked signaling cascades. In response to activated G proteins, PLC-beta isozymes accelerate the hydrolysis of phosphatidylinositol-4,5 bisphosphate (PtdIns(4,5)P2) to the second messengers sn-1,2-diacylglycerol (DAG) and D-myo-inositol- 1,4,5-trisphosphate (Ins(1,4,5)P3). Production of soluble Ins (1,4,5)P3 leads to the release of intracellular calcium while DAG activates protein kinase C isozymes, and these events ultimately control a diverse array of cellular functions including proliferation, excitation, secretion, and contraction. The mechanism(s) of regulation of PLC-beta isozymes by G proteins, phospholipids, Ca2+, and other potential regulators is poorly understood at the molecular level. However, with the recent ability to purify multiple milligrams of functional PLC-beta isozymes, we are now in a favorable position to define clearly the modes of PLC-beta regulation. A series of studies will be carried out to optimize the production of homogeneous and monodisperse PLC-beta holoenzymes and domain fragments. These characterized proteins will then be utilized in a dual approach of quantitative binding analysis and crystallography to understand PLC-beta regulation. Particular attention will be given to delineating the role of the N-terminal PH domains of PLC-betas in binding phosphoinositides and possibly Gbetagamma subunits. Quantitative measurements of phosphoinositide and Gbetagamma binding to the PH domains of PLC-betas will be obtained from surface plasmon resonance measurements and microcalorimetry. Since different PLC-beta isozymes exhibit distinct G protein regulation, binding data for equivalent PH domains from different isozymes may delimit modes of isozyme-specific G protein activation. X-ray crystallography also will be used to determine atomic resolution structures of PLC-beta domains or full-length proteins. These structures will provide key information on interdomain interactions, relative domain orientations, and specific binding sites for small molecules. Overall, the combination of binding data and structural information is intended to provide an atomic- resolution framework for understanding and possibly manipulating the regulation of interfacial catalysis by PLC-beta isozymes. This work also will provide the foundation for understanding in greater detail the regulation of PLC-beta isozymes by other activated Galpha subunits and Gbetagamma dimers.