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）：在过去的5年中，PI能够利用当前的K05奖在其实验室中开发两个新的NIDA资助的项目，并能够在UCLA的Chris Evans博士的实验室中度过一个学期的假期，以探索斑马鱼作为替代模型，以研究替代模型，以研究副业依赖性和依赖性的分子机制。由于K05奖，由于他在明尼苏达大学的行政和教学承诺缓解了他的行政和教学承诺，因此之所以成为可能。因此，该K05奖励申请的目的仍然是一种机制，允许PI继续他成功地从明尼苏达大学夺走缺席的计划，并花费时间在其合作者实验室中，以追求新的或替代方法和技术，以研究耐受性和依赖性的分子机制。从PI实验室正在进行的项目中可以清楚地看出，阿片受体通过受体形成信号。在微域内用阿片受体的细胞蛋白脚手架脚手架极大地影响受体信号。通过募集不同的蛋白质与受体附近，例如β-arrestin，src，rgs和AGS，可以调节信号的大小和持续时间。 PI启动了研究，以鉴定可以调节阿片类药物活性的细胞蛋白。使用小鼠脑文库的酵母两杂化筛选，蛋白质候选物（例如FK506结合蛋白FKBP12）特异性地与MOR的羧基尾结构域相互作用可以调节激动剂诱导的细胞内Ca2+运动。但是，使用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进行结合的蛋白质的鉴定。因此，我们将继续进行持续的项目，以使用蛋白质组学方法和使用整个受体蛋白质的蛋白质组学方法和酵母两杂化筛选来确定阿片类受体的成分。将通过siRNA对这些蛋白质的过表达，或者通过siRNA敲低神经母细胞瘤N2A细胞中的这些蛋白质水平，以确定它们对两种受阿片受体调节的效应子的影响，即腺苷酸环化酶和细胞内CA2+稳态。这些蛋白质水平在富含表达内源性MOR的神经元的原发性海马培养物中的改变也将进行。可诱导的siRNA载体将被开发并用于涉及受体形成的蛋白质的时间敲低。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。