Role of Racl in Regulated Exocytosis

神经元之间的信息传递和神经系统的正常功能依赖于突触处神经递质的调节释放。一些精神和神经疾病的典型特征是特定神经递质的失衡。此外，许多作用于神经系统的滥用和治疗性药物都是在改变突触传递的水平上起作用的。这项拟议研究的长期目标是了解调节神经递质或神经激素释放的分子机制，因为它最终可能导致改进临床治疗和改进药物设计。小的RAS样GTP结合蛋白是许多细胞过程的关键调节因子，包括形态发生、细胞骨架动力学、膜运输、转化和蛋白激酶级联反应。需要检验的一般假设是，单体GTPase rac1对分泌反应是必不可少的，因为它协调对分泌颗粒可获得性、启动和SNARE蛋白相互作用至关重要的事件。结合分子、生化和膜片钳技术将被用来确定在分泌周期的功能可分离的阶段中rac1的作用位置，并阐明其直接影响分泌反应的效应途径。这些研究将在牛嗜铬细胞上进行，牛嗜铬细胞是一种被广泛研究的、与生理相关的神经内分泌细胞模型。其具体目的是：1)阐明rac1在调节钙依赖的胞吐作用中的生理作用，并特异性地确定对易释放的分泌颗粒池的钙敏感性、募集和再充盈的影响；2)表征rac1对分泌刺激的反应而激活的特性。我们还将确定IQGAP1，一个结合了钙/钙调蛋白和F-肌动蛋白的RAC效应蛋白，是否提供对rac1的钙依赖调节；3)确定主要的rac1效应通路，即p21激活的激酶(PAK)，在rac1对分泌反应性的影响中所起的作用。此外，我们还将确定p35/细胞周期蛋白依赖性激酶5(一种调节PAK激活的相互作用的蛋白激酶)在rac1和PAK1调节分泌反应性中的作用，以及4)确定rac1在静息和刺激的嗜铬细胞中对磷脂酰肌醇4-磷酸5-激酶(PIP5K)活性的作用。PIP5K是神经内分泌系统分泌颗粒启动的重要辅助因子，直接与rac1相互作用。这项工作试图提供对胞浆和膜分隔的信号通路的更多了解，这些信号通路对钙依赖的分泌反应产生重要的调节影响。

Information transfer between neurons and the normal functioning of the nervous system is dependent upon the regulated release of neurotransmitter at synapses. A number of psychiatric and neurological conditions are typified by an imbalance of particular neurotransmitters. In addition, many abused and therapeutic drugs that act on the nervous system act at the level of altering synaptic transmission. The long-term objective of the proposed research is to understand molecular mechanisms which regulate neurotransmitter or neurohormone release, as it may ultimately lead to enhanced clinical treatments as well as improved drug design. Small Ras-like GTP binding proteins are key regulators of many cellular processes including morphogenesis, cytoskeletal dynamics, membrane trafficking, transformation, and protein kinase cascades. The general hypothesis to be tested is that the monomeric GTPase Rac1 is essential to secretory responsiveness in that it coordinates events critical for secretory granule availability, priming and SNARE protein interactions. A combination of molecular, biochemical and patch-clamp techniques will be used to determine the sites of Rac1 action within functionally separable stages of the secretory cycle, and to elucidate effector pathways through which it directly exerts effects on secretory responsiveness. The investigations will be performed on bovine chromaffin cells, which present an extensively studied, physiologically relevant, neuroendocrine cell model. The specific aims are: 1) To elucidate the physiological role of Rac1 on regulation of Ca2+- dependent exocytosis and to specifically determine effects on Ca2+ sensitivity, recruitment, and refilling of the readily releasable secretory granule pool, 2) To characterize the properties of Rac1 activation in response to secretory stimuli. We will also determine if IQGAP1, a Rac effector protein that binds Ca2+/calmodulin and F-actin, provides Ca2+-dependent regulation of Rac1, 3) To determine the contribution of the predominant Rac1 effector pathway, i.e. the p21 activated kinases (PAK), to Rac1 effects on secretory responsiveness. In addition, we will establish the role of p35/cyclin dependent kinase 5, an interacting protein kinase that regulates PAK activation, on Rac1 and PAK1 regulation of secretory responsiveness, and 4) To determine the role of Rac1 on phosphatidylinositol 4-phosphate 5- kinase (PIP5K) activity in resting and stimulated chromaffin cells. PIP5K is an essential co-factor for priming of secretory granules in neuroendocrine systems and directly interacts with Rac1. The work proposed attempts to provide a greater understanding of cytosolic and membrane delimited signaling pathways that exert an important regulatory influence on Ca2+- dependent secretory responsiveness.