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中神经递质的调节释放。许多精神疾病和神经疾病的典型特征是特定神经递质失衡。此外，许多滥用和治疗药物作用于神经系统，在改变突触传递的水平上起作用。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，因为它可能最终导致增强临床治疗以及改进药物设计。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。需要验证的一般假设是，单体GTPase Rac1对分泌反应性至关重要，因为它协调分泌颗粒可用性、启动和SNARE蛋白相互作用的关键事件。分子、生化和膜片钳技术的结合将用于确定Rac1在分泌周期的功能可分离阶段的作用位点，并阐明其直接影响分泌反应的效应途径。研究将在牛染色质细胞上进行，这是一个广泛研究的，生理学相关的神经内分泌细胞模型。具体目的是：1)阐明Rac1在Ca2+依赖性胞吐调节中的生理作用，并特异性地确定对Ca2+敏感性、招募和易释放分泌颗粒池的再填充的影响；2)表征Rac1在分泌刺激下的激活特性。我们还将确定IQGAP1，一种结合Ca2+/钙调蛋白和f -肌动蛋白的Rac效应蛋白，是否提供Ca2+依赖性的Rac1, 3)。以确定主要的Rac1效应途径，即p21活化激酶（PAK），对Rac1对分泌反应性的影响的贡献。此外，我们将确定p35/cyclin依赖性激酶5（一种调节PAK激活的相互作用蛋白激酶）在Rac1和PAK1分泌反应性调节中的作用。4)确定Rac1在静息和刺激的染色质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因子，与Rac1直接相互作用。这项工作提出，试图提供细胞质和膜划定的信号通路，发挥Ca2+依赖性分泌反应的重要调节影响的更大的理解。

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.