Role of Lipid Rafts in Adenylyl Cyclase Sensitization

描述(申请人提供)：阿片类药物依赖在这些药物的滥用中起着主要作用，是临床上关注的问题。如果我们要改善疼痛的治疗，治疗和/或预防阿片类药物滥用，重要的是要了解依赖的潜在机制。急性时，类阿片激活Gai/o蛋白以抑制腺酰环化酶，但在慢性治疗后，腺酰环化酶激活的代偿性增加可在细胞水平上表现为依赖性，称为敏化。本项目的目的是在细胞水平上更好地了解依赖发生的机制，特别是腺酰环化酶的代偿性变化是如何通过与其他信号蛋白在质膜微域(称为脂筏)的共同定位来调节的。MU-阿片受体存在于脂筏中，被致敏的腺酰环化酶亚型往往定位于脂筏中，而一些具有致敏作用的信号蛋白能够重新定位到脂筏内或脂筏外。为了确定脂筏在u-阿片介导的腺酰环化酶敏化的发生和表达中的作用，我们将：1)检测u-阿片信号成分在脂筏和非脂筏组分中的分布，并建立检测其功能的方法。2)从经Mu-阿片激动剂处理的细胞中制备富含脂筏和非脂筏的质膜组分，比较腺酰环化酶的增敏作用，并确定增敏变化是否与信号蛋白在脂筏内或外的重新定位有关。3)确定脂筏是否在不同受体和不同细胞类型的腺苷环化酶敏化过程中发挥必要的作用。在长期接触阿片类药物，如吗啡或海洛因后，神经元细胞膜上的信号蛋白发生变化，如腺苷环化酶。这些补偿性变化导致在阿片类药物移除时停用。为了更好地治疗和预防阿片依赖，目标是了解导致这些变化的机制，特别是与调节信号蛋白在细胞膜微域内外的位置有关的脂筏。

DESCRIPTION (provided by applicant): Opioid dependence plays a major role in abuse of these drugs and is a concern in the clinic. It is important to understand mechanisms underlying dependence if we are to improve the treatment of pain and to treat and/or prevent opioid abuse. Acutely, mu- opioids activate Gai/o proteins to inhibit adenylyl cyclase, but following chronic treatment dependence can be demonstrated at the cellular level by the compensatory increase in adenylyl cyclase activation, referred to as sensitization. The objective of this project is to better understand the mechanisms of the development of dependence at the cellular level, particularly how the compensatory changes in adenylyl cyclase are modulated by co-localization with other signaling proteins in plasma membrane microdomains referred to as lipid rafts. Mu-opioid receptors are found in lipid rafts and the adenylyl cyclase isoforms which are sensitized tend to be localized in lipid rafts, while some signaling proteins which have a role in sensitization are able to relocalize into or out of lipid rafts. To determine the role of lipid rafts in the development and expression of mu-opioid mediated adenylyl cyclase sensitization, we will: 1) Examine the distribution of mu-opioid signaling components in lipid rafts and non-raft fractions and develop assays to measure their function. 2) Prepare lipid-raft enriched and non-raft plasma membrane fractions from cells treated with mu-opioid agonist to compare sensitization of adenylyl cyclase and determine if changes in sensitization are related to relocalization of signaling proteins into or out of lipid rafts. 3) Determine if lipid rafts play a necessary role in adenylyl cyclase sensitization across different receptors and in different cell types. Changes occur in signaling proteins, such as adenylyl cyclase, in membranes of neuronal cells following long term exposure to opioid drugs, such as morphine or heroin. These compensatory changes result in withdrawal on removal of the opioid. To better treat and prevent opioid dependence, the goal is to understand the mechanisms causing these changes, particularly in relation to the location of modulatory signaling proteins in or out of cell membrane microdomains referred to as lipid rafts.