Cellular Pharmacology of Kappa Opioid Receptor

Kappa 阿片受体的细胞药理学

• 批准号: 7232651
• 负责人: LEE-YUAN LIU-CHEN
• 金额: $ 32.11万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232651
• 关键词: 6-carboxyfluorescein; Absence of pain sensation; Accounting; Adenylate Cyclase; Affect; Agonist; Analgesics; Antipruritics; Binding; Biochemical; C-terminal; Calcium Channel; Cavia; Cell Line; Cell membrane; Cell surface; Cells; Cellular biology; Characteristics; Chinese Hamster Ovary Cell; Chronic; Classification Scheme; Dependence; Development; Didelphidae; Diuresis; Diuretics; Down-Regulation; Dynorphin A; Embryo; Epitopes; Esters; Event; Exposure to; Familiarity; Family; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutathione S-Transferase; Hemagglutinin; Horseradish Peroxidase; Human; Human Cloning; Kidney; Knowledge; Ligand Binding; Lysosomes; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular; Mus; Mutagenesis; N-ethylmaleimide-sensitive protein; Opioid; Opioid Receptor; Organelles; Pathway interactions; Peptides; Pertussis Toxin; Pharmaceutical Preparations; Pharmacology; Phosphoproteins; Phosphorylation; Play; Polyacrylamide Gel Electrophoresis; Potassium; Property; Proteasome Inhibitor; Proteins; Rattus; Receptor Signaling; Recycling; Regulation; Research; Rhodopsin; Role; Screening procedure; Signal Transduction; Site; Sodium Dodecyl Sulfate-PAGE; Sorting - Cell Movement; Surface; Techniques; Therapeutic Agents; Tubulin; Ubiquitination; Water; Yeasts; base; desensitization; dysphoria; ezrin; kappa opioid receptors; kidney cell; moesin; multicatalytic endopeptidase complex; radixin protein; receptor; receptor coupling; receptor recycling; sodium-hydrogen exchanger regulatory factor; trafficking; yeast two hybrid system

DESCRIPTION (provided by applicant): Activation of (kappa) opioid receptors produces many effects, including analgesia, dysphoria and water diuresis and kappa agonists may be useful as analgesics, water diuretics and antipruritic drugs. Kappa opioid receptors are coupled through pertussis toxin-sensitive G proteins to affect a variety of effectors. Repeated or continuous administration of Kappa agonists leads to tolerance and dependence. Part of tolerance can be accounted for at the receptor level. The applicant's long term objectives are to understand at the molecular level the biochemical events that occur following chronic exposure to opioid drugs. After exposure to the selective agonist U50, 488H, the human kappa opioid receptor (hkor) undergoes internalization and the internalized receptors are recycled to plasma membranes or sorted to lysosomes and proteasomes for degradation. Whether there are signals directing the sorting of the internalized receptors is not well understood. The central hypothesis of this application is that the hkor is associated with EBP50/NHERF-1 and GABARAPL1 and undergoes ubiquitination, all of which play important roles in the regulation, trafficking and signaling of the receptor. The specific aims are as follows. (1) To investigate the interaction of the hkor with EBP50/NHERF-1, a PDZ domain-containing protein. The residues in the hkor involved in the interaction with EBP50/NHERF-1 will be determined by mutagenesis studies. Effects of receptor phosphorylation and ubiquitination on its association with EBP50/NHERF-1 will be assessed and the role of EBP50/NHERF in the U50,488H-induced stimulation of Na+,H+-exchange will be examined. (2) To examine the interaction of the hkor with GABARAPL 1. Whether there are interactions between GABARAPL1 and tubulin, N-ethylmaleimide-sensitive factor, EBP50/NHERF-1, rab5 or rab7 will be investigated. The regions of GABARAPL 1 and the hkor involved in the interaction will be determined. The functional significance of the hkor-GABARAPL1 interaction will be studied in terms of trafficking, surface expression, ligand binding and signal transduction of the hkor. (3) To delineate the role of ubiquitination in the signaling and trafficking of the hkor. Effects of different agonists and receptor phosphorylation on ubiquitination of the hkor will be examined. The sites of ubiquitination in the hkor will be determined by mutagenesis. The impact of ubiquitination on the functional properties, regulation and trafficking of the hkor will be investigated. Elucidation of the functional significance of EBP50/NHERF-1 and GABARAPL 1 and receptor ubiquitination in the trafficking and signaling of the hkor will provide better understanding of cell biology of the hkor and will have implications for development of kappa agonists and antagonists as therapeutic agents. In addition, such understanding will have important ramifications for other G protein coupled receptors since these molecules may play similar roles for other receptors.

描述（由申请人提供）：（κ）阿片受体的激活产生许多作用，包括镇痛、烦躁不安和利尿，κ激动剂可用作镇痛药、利尿剂和抗利尿药。κ阿片受体通过百日咳毒素敏感性G蛋白偶联，影响多种效应物。重复或连续给予κ激动剂导致耐受性和依赖性。部分耐受性可以在受体水平上解释。申请人的长期目标是在分子水平上了解长期暴露于阿片类药物后发生的生化事件。暴露于选择性激动剂U 50，488 H后，人κ阿片受体（hkor）发生内化，内化的受体再循环至质膜或分选至溶酶体和蛋白酶体进行降解。是否存在指导内化受体分选的信号尚不清楚。该应用的中心假设是hkor与EBP 50/NHERF-1和GABARAPL 1相关并经历泛素化，所有这些在受体的调节、运输和信号传导中起重要作用。具体目标如下。 (1)研究hkor与EBP 50/NHERF-1（一种含PDZ结构域的蛋白）的相互作用。通过诱变研究确定hkor中参与与EBP 50/NHERF-1相互作用的残基。将评估受体磷酸化和泛素化对其与EBP 50/NHERF-1结合的影响，并将检查EBP 50/NHERF在U 50，488 H诱导的Na+，H+交换刺激中的作用。(2)研究香港红十字会与GABARAPL的相互作用1。将研究GABARAPL 1与微管蛋白、N-乙基马来酰亚胺敏感因子、EBP 50/NHERF-1、rab 5或rab 7之间是否存在相互作用。GABARAPL 1和hkor参与相互作用的区域将被确定。hkor与GABARAPL 1相互作用的功能意义将从hkor的运输、表面表达、配体结合和信号转导等方面进行研究。(3)阐明泛素化在hkor信号转导和运输中的作用。将检查不同激动剂和受体磷酸化对hkor遍在化的影响。hkor中的泛素化位点将通过诱变来确定。泛素化对hkor的功能特性、调控和运输的影响将被研究。阐明EBP 50/NHERF-1和GABARAPL 1的功能意义以及受体泛素化在hkor的运输和信号传导中的作用，将提供对hkor细胞生物学的更好理解，并将对kappa激动剂和拮抗剂作为治疗剂的开发产生影响。此外，这种理解将对其他G蛋白偶联受体产生重要的影响，因为这些分子可能对其他受体起类似的作用。