Mechanism of Drug-Induced Adenylyl Cyclase Super-Sensitization

描述（由申请人提供）：吸毒成瘾是一种影响美国数百万人的毁灭性疾病。这种行为的核心是影响单个神经元和神经元回路的复杂生化过程。一个主要的挑战是了解成瘾的分子和细胞基础以及相关的依赖、戒断、耐受性和致敏行为。许多滥用药物直接或间接作用于受体，进而激活Gi/Go类异三聚体G蛋白，当这些受体受到慢性刺激时，cAMP信号通路上调。对慢性胃肠道刺激的早期适应性反应是腺苷酸环化酶活性的增加，称为腺苷酸环化酶超敏化。多年来的研究已经提供了大量关于腺苷酸环化酶超敏化的分子基础的信息，并涉及许多腺苷酸环化酶的调节因子，包括Gs1、Gi1、23和蛋白激酶；然而，这种现象背后的主要机制在很大程度上仍然是未知的。我的实验室开发了一种基于细胞的腺苷酸环化酶超敏化模型系统，适用于中等高通量筛选方法。这项工作使我们能够开发并执行全基因组范围的siRNA筛选，以识别沉默时阻断腺苷酸环化酶超敏化的基因，这种超敏化是在慢性激素激活Gi通路时产生的。我们将研究在腺苷酸环化酶超敏化过程中发生的腺苷酸环化酶的磷酸化（目的1），研究TTK的作用，TTK是一种双特异性蛋白激酶蛋白，我们在筛选中发现TTK在腺苷酸环化酶超敏化的发展中所起的作用（目的2）。我们的siRNA筛选结果以及后续研究表明，蛋白质周转在腺苷酸环化酶超敏化的发展中起作用；提案的目的3检查泛素介导的蛋白质降解途径在腺苷酸环化酶超敏化中的作用。在目标4中，我们将描述泛素介导的蛋白质周转机制中已鉴定的AKAP靶点在腺苷酸环化酶超敏化发展中的作用。我们的全基因组siRNA筛选已经产生了丰富的数据集，这将为我们提供一个独特的机会来识别腺苷酸环化酶超敏化的信号通路的新成分。这将为药物成瘾的重要生物学过程提供洞见。

DESCRIPTION (provided by applicant): Drug addiction is a devastating disease affecting millions of people in the United States. At the core of this behavior are complex biochemical processes that affect individual neurons and neuronal circuits. A major challenge is to understand the molecular and cellular basis of addiction and associated behaviors of dependence, withdrawal, tolerance, and sensitization. Many abused drugs act directly or indirectly on receptors that in turn activate the Gi/Go class of heterotrimeric G proteins and upon chronic stimulation of these receptors, the cAMP signaling pathway is up-regulated. An early adaptive response to chronic Gi stimulation is an increase in the activity of adenylyl cyclase termed adenylyl cyclase super-sensitization. Studies over the years have provided a great deal of information regarding the molecular basis responsible for adenylyl cyclase super-sensitization and have implicated the involvement of many of the regulators of adenylyl cyclase, including Gs1, Gi1, 23, and protein kinases; however, the major mechanisms underlying this phenomenon is still largely unknown. My laboratory has developed a cell based model system for of adenylyl cyclase super-sensitization that is amenable to moderate high throughput screening approaches. This work has allowed us to develop and execute a genome wide siRNA screen to identify genes that when silenced, block the adenylyl cyclase super-sensitization that develops in response to chronic hormonal activation of a Gi pathway. We will examine the phosphorylation of adenylyl cyclase that occurs during adenylyl cyclase super- sensitization (aim 1), study the role of TTK, a dual-specificity protein kinase protein that we identified in our screen in the development of the super-sensitivity of the adenylyl cyclase enzyme (aim 2). Our results from the siRNA screen, as well as follow up studies implicate a role of protein turnover in the development of adenylyl cyclase super-sensitization; aim 3 of the proposal examines the role of the ubiquitin-mediated protein degradation pathway in adenylyl cyclase super-sensitization. In aim 4 we will characterize the role of an identified AKAP target of the ubiquitin-mediated protein turnover machinery in the development of adenylyl cyclase super-sensitization. Our genome-wide siRNA screen has produced a rich dataset that will provide us with a unique opportunity to identify novel components of the signaling pathways that underlie adenylyl cyclase super-sensitization. This will provide insights into the biologically important process of drug addiction.