Neurochemical Basis of Opiate Addiction

阿片成瘾的神经化学基础

基本信息

批准号：
7634516
负责人：
HORACE LOH
金额：
$ 52.95万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-01-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7634516
关键词：
ADRBK1 gene Accounting Acute Address Adenoviruses Adenylate Cyclase Agonist Amino Acid Receptors Amino Acids Analgesics Antibodies Arrestins Attenuated Binding Biochemical Cell Line Cell model Cells Chronic Complex Consensus Cyclic AMP Data Dependence Development Dominant-Negative Mutation Dose Embryo Etorphine Event Exhibits Fibroblasts Funding G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GRK GTP-Binding Proteins Grant Hippocampus (Brain)Immunofluorescence Immunologic In Vitro Injection of therapeutic agent Knockout Mice Ligand Binding Mass Spectrum Analysis Methadone Modification Molecular Morphine Morphine Dependence Mouse Protein Mus National Institute of Drug Abuse Neurons Opiate Addiction Opiates Opioid Opioid Analgesics Opioid Receptor Peptides Pharmaceutical Preparations Phospho-Specific Antibodies Phosphorylation Phosphorylation Site Phosphotransferases Protein Binding Protein Kinase Protein Kinase Inhibitors Proteins Receptor Activation Receptor Signaling Recruitment Activity Reporting Research Personnel Rhodopsin Role Signal Transduction Signaling Molecule Small Interfering RNA Tetracycline Control Transgenic Mice Withdrawal arrestin 2 base beta-arrestin cellular targeting desensitization in vivo inhibitor/antagonist kinase inhibitor knock-down member mutant neurochemistry programs protein kinase inhibitor receptor receptor coupling response restoration scaffold src-Family Kinases

项目摘要

DESCRIPTION (provided by applicant): This is a competitive renewal application of NIDA grant DA000564-34. Since opioid receptors have been identified to be members of the rhodopsin subfamily of GPCRs, the general mechanism for GPCR desensitization has been applied to account for in vivo opiate tolerance. In this mechanism, opioid receptors are phosphorylated by GRKs in the presence of agonists and beta-arrestin is recruited to the receptor vicinity resulting in the blunting of the signals. Such mechanism is supported by studies with beta-arrestin knockout mice in which morphine tolerance was attenuated. However, a direct correlation between receptor phosphorylation and desensitization could not be demonstrated. Nevertheless, it remains our central hypothesis that covalent modification of opioid receptor and the signaling complex, or receptosome, during chronic agonist treatment is the key for the eventual manifestation of opiate tolerance and dependence. Therefore, in the proposed studies, we will continue our on-going studies to investigate the role of MOR phosphorylation on morphine tolerance response. We will determine the protein kinases involved in the phosphorylation of the consensus motif TXXXPS within the receptor. We will re-determine the agonist-dependent MOR phosphorylation sites by mass spectrometry analyses of purified receptor. We will validate our observations in clonal cell models with primary neuronal cultures. We will investigate the role of receptor phosphorylation in morphine tolerance by the use of GRK null mice together with inhibitors of protein kinases involved in MOR phosphorylation. The role of receptor phosphorylation in morphine tolerance will also be investigated by restoration of morphine acute and chronic responses in MOR null mice with adenovirus carrying the wild type and phosphorylation receptor mutants. In addition to receptor desensitization, our preliminary data also suggested that Src activation during chronic morphine treatment could be the basis for adenylyl cyclase (AC) superactivation. Since beta-arrestin has been implicated in Src activation, receptor phosphorylation could be the trigger for such event. Thus, we will examine the mechanism for and the role of beta-arrestin in Src activation. We will investigate the consequence of Src activation on AC superactivation, and will identify the cellular targets of Src with mass spectrometry analysis. The role of Src activation in the in vivo morphine dependence and withdrawal responses will be established by the use of Src inhibitor PP2 and inducible siRNA approach. All these studies will allow us to elucidate the molecular components involved in opiate tolerance and dependence.

描述（由申请人提供）：这是 NIDA 拨款 DA000564-34 的竞争性续展申请。由于阿片受体已被鉴定为 GPCR 视紫红质亚家族的成员，因此 GPCR 脱敏的一般机制已应用于解释体内阿片类药物耐受性。在这种机制中，阿片受体在激动剂存在下被 GRK 磷酸化，β-抑制蛋白被募集到受体附近，导致信号减弱。这种机制得到了β-抑制蛋白敲除小鼠的研究的支持，其中吗啡耐受性减弱。然而，无法证明受体磷酸化和脱敏之间的直接相关性。尽管如此，我们的中心假设仍然是，在长期激动剂治疗过程中，阿片受体和信号复合物或受体体的共价修饰是阿片耐受和依赖性最终表现的关键。因此，在拟议的研究中，我们将继续正在进行的研究，以调查 MOR 磷酸化对吗啡耐受反应的作用。我们将确定参与受体内共有基序 TXXXPS 磷酸化的蛋白激酶。我们将通过纯化受体的质谱分析来重新确定激动剂依赖性 MOR 磷酸化位点。我们将用原代神经元培养物验证我们在克隆细胞模型中的观察结果。我们将通过使用 GRK 缺失小鼠以及参与 MOR 磷酸化的蛋白激酶抑制剂来研究受体磷酸化在吗啡耐受中的作用。受体磷酸化在吗啡耐受中的作用也将通过在携带野生型和磷酸化受体突变体的腺病毒的MOR缺失小鼠中恢复吗啡急性和慢性反应来研究。除了受体脱敏之外，我们的初步数据还表明，长期吗啡治疗期间的 Src 激活可能是腺苷酸环化酶 (AC) 超激活的基础。由于 β-arrestin 与 Src 激活有关，因此受体磷酸化可能是此类事件的触发因素。因此，我们将研究 β-arrestin 在 Src 激活中的机制和作用。我们将研究 Src 激活对 AC 超激活的影响，并通过质谱分析确定 Src 的细胞靶标。 Src 激活在体内吗啡依赖性和戒断反应中的作用将通过使用 Src 抑制剂 PP2 和诱导型 siRNA 方法来确定。所有这些研究将使我们能够阐明与阿片类药物耐受性和依赖性有关的分子成分。