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蛋白连锁的信号级联反应。 作为对激活的G蛋白的响应，PLC-β同工酶加速了 磷脂酰肌醇-4，5-二磷酸(PtdIns(4，5)P2)的水解 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 导致细胞内钙释放，同时激活DAG 蛋白激酶C同工酶，这些事件最终控制着不同的 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(S) 同工酶受G蛋白、磷脂、钙等潜能的影响 在分子水平上，人们对调节剂知之甚少。但是，有了 最近提纯多毫克功能性PLC-β的能力 同工酶，我们现在处于有利的位置，可以清楚地定义模式 PLC-β调节。将开展一系列研究，以 优化均相和单分散PLC-β的生产 全酶和结构域片段。这些特化的蛋白质将 然后用于定量结合分析的双重方法和 了解PLC-β调控的结晶学。特别关注 将致力于描绘N-末端PH结构域的作用 PLC-β结合磷脂酰肌醇和可能的Gbetagamma亚基。 磷脂酰肌醇和Gbetagamma结合的定量测定 PLC-β的PH域将由表面等离子激元获得 共振测量和微量热法。由于不同PLC-测试版 同工酶表现出明显的G蛋白调节，结合数据 不同同工酶的等值PH结构域可以区分 同工酶特异的G蛋白激活。X射线结晶学也将 用于确定PLC-β结构域的原子分辨结构 或全长蛋白质。这些结构将提供关键信息 关于域间交互、相对域定向和特定 小分子的结合部位。总体而言，绑定的组合 数据和结构信息旨在提供原子- 用于理解并可能操纵 PLC-β同工酶对界面催化的调控这部作品 也将为更详细地理解 其他激活的Galpha亚基对PLC-β同工酶的调节 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.