G Proteins and Opiate Receptor Functions

G 蛋白和阿片受体功能

• 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
• 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' 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

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奖在他的实验室开发两个新的NIDA资助项目，并能够在加州大学洛杉矶分校的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的终身职业目标。这是由于获得了K05奖，他从明尼苏达大学的行政和教学工作中解脱出来。因此，这项K05奖申请的目标仍然是建立一种机制，使PI能够继续他在明尼苏达大学的成功计划，定期休假，并花时间在他的合作者的实验室中追求新的或替代的方法和技术来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，阿片受体通过受体体的形成来传递信号。细胞蛋白微结构域内阿片受体的支架式结构极大地影响了受体的信号转导。通过在受体附近招募不同的蛋白质，如β -阻滞蛋白、Src、RGS和AGS，可以调节信号的大小和持续时间。PI已经开始研究确定可以调节阿片受体活性的细胞蛋白。利用小鼠脑文库的酵母双杂交筛选，候选蛋白，如FK506结合蛋白FKBP12，特异性地与MOR的羧基尾部结构域相互作用，可以调节激动剂诱导的细胞内Ca2+运动。然而，使用特定受体结构域限制了识别需要多个结构域或三级受体结构结合的蛋白质。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酵母双杂交筛选全受体蛋白来确定阿片受体体的成分。在神经母细胞瘤N2A细胞中，腺病毒过度表达这些蛋白，或通过siRNA敲低这些蛋白的水平，以确定它们对阿片受体调节的两种效应器的影响，即腺苷酸环化酶和细胞内Ca2+稳态。在表达内源性MOR的神经元富集的原代海马培养物中，这些蛋白水平的改变也将被进行。可诱导的siRNA载体将被开发并用于参与受体形成的蛋白质的时间敲低。蛋白质水平的基因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。