G Proteins and Opiate Receptor Functions

G 蛋白和阿片受体功能

基本信息

批准号：
7657315
负责人：
PING-YEE LAW
金额：
$ 12.91万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-07-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7657315
关键词：
5&apos Flanking Region Adenovirus Vector Adenoviruses Adenylate Cyclase Affect Agonist Amino Acid Sequence Arrestins Binding Brain Cell model Cells Chronic Complex Cyclic AMP Data Dependence Development Dimerization Dominant-Negative Mutation Drug effect disorder Educational process of instructing Elements Funding G-Protein-Coupled Receptors GTP-Binding Proteins Genes Goals Hippocampus (Brain)Homeostasis In Vitro Individual Laboratories LacZ Genes Laws Left Libraries Life Mass Spectrum Analysis Minnesota Modeling Molecular Movement Mus Mutation National Institute of Drug Abuse Neuroblastoma Neurons Opiates Opioid Opioid Receptor Peptide Sequence Determination Pertussis Toxin Phospholipase Plant Resins Potassium Channel Principal Investigator Progress Reports Proteins Proteomics Reagent Receptor Signaling Recruitment Activity Regulation Reporter Genes Role Ryanodine Scaffolding Protein Screening procedure Senior Scientist Award Signal Transduction Small Interfering RNA Structure Study models System Tacrolimus Binding Protein 1A Tacrolimus Binding Proteins Tail Technology Testing Time Transgenic Mice Universities Wheat Germ Agglutinins Yeasts Zebrafish beta-arrestin career dimer in vivo interest nano-electrospray programs promoter protein aminoacid sequence protein expression receptor receptor expression receptor function restoration scaffold success tandem mass spectrometry three dimensional structure vector voltage yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): During the last 5 years, PI was able to utilize the current K05 award to develop two new NIDA funded projects in his laboratory, and was able to spend a semester leave in Dr. Chris Evans' laboratory at UCLA to explore the use zebrafish as an alternative model for studying the molecular mechanism of opiate tolerance and dependence, a life-long career goal of PI. This was made possible because of the relief from his administrative and teaching commitments at University of Minnesota due to the K05 award. Therefore, the objective of this K05 award application remains to be a mechanism allowing PI to continue his successful program of taking periodic leaves of absence from University of Minnesota and spend time in his collaborator's laboratory in the pursuit of new or alternative approaches and technologies in studying the molecular mechanism of tolerance and dependence. It is clear from the on-going projects in PI's laboratory that opioid receptor signals via the formation of receptorsomes. The scaffolding of cellular proteins with opioid receptor within the microdomains greatly affect the receptor signaling. By recruiting different proteins to the receptor vicinity, e.g., beta-arrestin, Src, RGS and AGS, the magnitude and duration of signals could be modulated. PI has initiated studies to identify the cellular proteins that could modulate opioid receptor activities. Using yeast two-hybrid screens of mouse brain library, protein candidates, such as the FK506 binding protein FKBP12 that specifically interacts with the carboxyl tail domain of MOR could modulate the agonist-induced intracellular Ca2+ movement. However, the use of a specific receptor domain limits the identification of proteins that require multiple domains or tertiary receptor structure for binding. Thus we will continue our on-going projects to determine the components of opioid receptorsomes by the use of proteomic approaches and yeast two-hybrid screens using whole receptor protein. Over-expression of these proteins with adenoviruses, or the knockdown of these proteins levels in neuroblastoma N2A cells by siRNA will be carried out to determine their effects on two the effectors regulated by opioid receptors, i.e., adenylyl cyclase and intracellular Ca2+ homeostasis. The alteration of these proteins levels in primary hippocampal cultures enriched in neurons expressing endogenous MOR will be carried out also. The inducible siRNA vector will be developed and used in the temporal knockdown of the proteins involved in the receptorsomes formation. Genetic alteration of the proteins levels will be carried out in mice and other models to test the effects of these proteins in chronic opiate drug actions.

描述（由申请人提供）：在过去 5 年中，PI 能够利用当前的 K05 奖项在他的实验室开发两个新的 NIDA 资助项目，并能够在 UCLA Chris Evans 博士的实验室度过一个学期的假期探索使用斑马鱼作为研究阿片类药物耐受和依赖的分子机制的替代模型，这是PI的终生职业目标。这一切之所以成为可能，是因为他获得了 K05 奖，从而免除了他在明尼苏达大学的行政和教学工作。因此，本次 K05 奖项申请的目的仍然是建立一种机制，使 PI 能够继续他从明尼苏达大学定期休假的成功计划，并花时间在合作者的实验室中寻求新的或替代的研究方法和技术。耐受和依赖性的分子机制。从 PI 实验室正在进行的项目可以清楚地看出，阿片受体通过受体体的形成发出信号。微域内具有阿片受体的细胞蛋白支架极大地影响受体信号传导。通过将不同的蛋白质（例如 β-arrestin、Src、RGS 和 AGS）募集到受体附近，可以调节信号的强度和持续时间。 PI 已启动研究以确定可以调节阿片受体活性的细胞蛋白。使用小鼠脑文库的酵母双杂交筛选，候选蛋白质，例如与 MOR 羧基尾结构域特异性相互作用的 FK506 结合蛋白 FKBP12，可以调节激动剂诱导的细胞内 Ca2+ 运动。然而，特定受体结构域的使用限制了需要多个结构域或三级受体结构才能结合的蛋白质的鉴定。因此，我们将继续我们正在进行的项目，通过使用蛋白质组学方法和使用完整受体蛋白的酵母双杂交筛选来确定阿片受体体的成分。将用腺病毒过度表达这些蛋白质，或通过 siRNA 在神经母细胞瘤 N2A 细胞中敲低这些蛋白质水平，以确定它们对阿片受体调节的两个效应器（即腺苷酸环化酶和细胞内 Ca2+ 稳态）的影响。还将对富含表达内源性 MOR 的神经元的原代海马培养物中的这些蛋白质水平进行改变。诱导型 siRNA 载体将被开发并用于暂时敲低参与受体体形成的蛋白质。蛋白质水平的遗传改变将在小鼠和其他模型中进行，以测试这些蛋白质在慢性阿片类药物作用中的作用。