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。 许多精神和神经疾病的典型特征是特定神经递质的不平衡。 此外，许多作用于神经系统的滥用和治疗药物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。 待检验的一般假设是，单体GTdR ac 1对分泌反应性至关重要，因为它协调分泌颗粒可用性、引发和SNARE蛋白相互作用的关键事件。 分子，生物化学和膜片钳技术的组合将被用来确定网站的Rac 1行动内的功能可分离的阶段的分泌周期，并阐明效应途径，通过它直接对分泌反应的影响。研究将在牛嗜铬细胞上进行，这是一种广泛研究的生理相关神经内分泌细胞模型。 具体目标是：1）阐明Rac 1在调节Ca 2+依赖性胞吐中的生理作用，并明确其对Ca 2+敏感性、募集和易释放分泌颗粒库再填充的影响。2）表征Rac 1在响应分泌刺激时的激活特性。我们还将确定IQGAP 1（一种结合Ca 2 +/钙调蛋白和F-肌动蛋白的Rac效应蛋白）是否提供Rac 1的Ca 2+依赖性调节。3）为了确定主要Rac 1效应途径（即p21活化激酶（PAK））对Rac 1对分泌反应性的作用的贡献。 此外，我们将建立p35/细胞周期蛋白依赖性激酶5的作用，一个相互作用的蛋白激酶，调节PAK的激活，对Rac 1和PAK 1的分泌反应的调节，和4）确定Rac 1的作用对磷脂酰肌醇4-磷酸5-激酶（PIP 5 K）的活性在休息和刺激嗜铬细胞。 PIP 5 K是神经内分泌系统中启动分泌颗粒的重要辅因子，并直接与Rac 1相互作用。 这项工作提出试图提供一个更好的理解胞质和膜界定的信号通路，发挥重要的监管影响钙依赖性分泌反应。

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.