Determinants of phospholipase C-Beta regulation

肌醇磷脂水解的刺激是细胞内对广泛的细胞外化学信号信使激活跨膜受体的初始反应。 肌醇磷脂水解是由大多数细胞外信号分子通过G蛋白亚基激活磷脂酶C-β（PLC-β）酶刺激的。 肌醇磷脂水解的进一步调节（例如抑制）可由替代因素介导，包括共价修饰和与膜结合底物的接近。 该项目的长期目标是通过激酶、脂质结合结构域和非G蛋白分子间相互作用准确、严格地描述PLC-β 3同工酶的调节。 将在受控良好的体外试验中定量磷酸化介导的PLC-β 3水解活性抑制的动力学。将评估PLC-β 3分子内推定的脂质结合结构域的磷脂和膜结合亲和力，以进一步将结构与PLC-β活性的功能和调节相关联。 此外，将鉴定PLC-β 3与除G蛋白或激酶以外的推定相互作用蛋白的关联，并在体内和体外定量这些分子间相互作用的功能。 这些研究的目的是在分子水平上详细描述PLC-β 3活性的多种调节手段，这些手段超出了充分表征的G蛋白活化途径，将有助于更好地理解信号转导的基本机制。 PLC-β酶调节的详细功能图可能有助于设计选择性干预病理生理PLC-β介导过程的新型治疗剂;与PLC-β活化相关的激素和神经递质反应包括平滑肌收缩、血小板聚集、激素分泌、平滑肌肥大和增生、神经元活化和恶性细胞增殖。

Stimulation of inositol phospholipid hydrolysis is the initial intracellular response to transmembrane receptor activation by a wide array of extracellular chemical signaling messengers. Inositol phospholipid hydrolysis is stimulated by most extracellular signaling molecules through G protein subunits which activate phospholipase C-beta (PLC-beta) enzymes. Further regulation (e.g. inhibition) of inositol phospholipid hydrolysis may be mediated by alternative factors including covalent modification and proximity to membrane bound substrate. This project's long-term goal is an accurate, rigorous description of PLC-beta3 isoenzyme regulation by kinases, lipid binding domains, and non-G protein intermolecular interactions. The kinetics of phosphorylation-mediated inhibition of PLC-beta3 hydrolytic activity will be quantitated in well-controlled in vitro assays. Phospholipid and membrane binding affinities of putative lipid binding domains within the PLC-beta3 molecule will be assessed to further associate structure with function and regulation of PLC- beta activity. Additionally, the association of PLC-beta3 with putative interacting proteins, other than G proteins or kinases, will be identified and the function of these intermolecular interactions quantitated both in vivo and in vitro. These studies, which aim to detail at a molecular level multiple means of regulation of PLC-beta3 activity beyond the well-characterized G protein activation pathway, will contribute to a greater understanding of the basic mechanisms of signal transduction. A detailed, functional map of PLC-beta enzyme regulation may contribute to the design of novel therapeutic agents that intervene selectively into pathophysiological PLC-beta-mediated processes; hormone and neurotransmitter responses associated with PLC-beta activation include, among many others, smooth muscle contraction, platelet aggregation, hormone secretion, smooth muscle hypertrophy and hyperplasia, neuronal activation, and malignant cell proliferation.