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-betas) 通过 G 蛋白连接的信号级联反应。 PLC-β 同工酶响应激活的 G 蛋白，加速 磷脂酰肌醇 4,5 二磷酸 (PtdIns(4,5)P2) 水解为 第二信使 sn-1,2-二酰基甘油 (DAG) 和 D-肌醇- 1,4,5-三磷酸（Ins(1,4,5)P3）。 可溶性 Ins (1,4,5)P3 的生产 DAG 激活时导致细胞内钙的释放 蛋白激酶 C 同工酶，这些事件最终控制着多种 一系列细胞功能，包括增殖、激发、 分泌、收缩。 PLC-beta 的调节机制 G 蛋白、磷脂、Ca2 和其他潜在同工酶 人们对分子水平上的调节剂知之甚少。 然而，随着 最近能够纯化数毫克功能性 PLC-beta 同工酶，我们现在处于有利的位置来明确定义模式 PLC-beta 调节。 将开展一系列研究 优化同质和单分散 PLC-beta 的生产 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和 晶体学来了解 PLC-beta 调节。 特别注意 将描述 N 端 PH 结构域的作用 PLC-β 结合磷酸肌醇和可能的 Gbetagamma 亚基。 磷酸肌醇和 Gbetagamma 结合的定量测量 PLC-betas 的 PH 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-beta 同工酶表现出独特的 G 蛋白调节、结合数据 不同同工酶的等价 PH 结构域可能会界定不同的模式 同工酶特异性 G 蛋白激活。 X射线晶体学也将 用于确定 PLC-beta 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 关于域间相互作用、相对域方向和特定 小分子的结合位点。总体而言，结合 数据和结构信息旨在提供原子 理解并可能操纵的解决框架 PLC-β同工酶对界面催化的调节。 这部作品 也将为更详细地理解提供基础 其他活化的 Galpha 亚基对 PLC-β 同工酶的调节和 Gbetagamma 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.