MECHANISMS OF BETA-2 ADRENERGIC RECEPTOR INTERNALIZATION

BETA-2 肾上腺素受体内化机制

• 批准号: 2702087
• 负责人: LAWRENCE S. BARAK
• 金额: $ 8.24万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2702087
• 关键词: CHO cells; G protein; adenylate cyclase; arrestins; beta adrenergic agent; beta adrenergic receptor; beta adrenergic receptor kinase; biological signal transduction; caveolins; clathrin; embryo /fetus tissue /cell culture; flow cytometry; immunofluorescence technique; immunoprecipitation; isozymes; membrane proteins; monoclonal antibody; plasmids; protein kinase; receptor coupling; receptor expression; receptor sensitivity; transfection /expression vector

The goal of this project is to determine the agonist induced cellular mechanisms responsible for beta2-adrenergic receptor internalization. Beta2-adrenergic receptors apparently internalize during at least two different independent processes, down regulation and sequestration, which may be common to all G-protein coupled receptors. Sequestration has been implicated in the processes of short term receptor desensitization and resensitization. Loss of normal receptor function from failure of any component part of the sequestration pathway may contribute to disease as in chronic heart failure where normal cardiac contractility may depend upon normal receptor resensitization, or prevention of abnormal receptor desensitization. Various mutant beta2-adrenergic receptors have been produced which exhibit sequestration behavior intermediate between wild type and nonfunctional receptors by making point mutations in a motif at the junction of the seventh transmembrane, cytoplasmic region of the receptor. This motif is common to most of the known G-protein coupled receptors with only a few exceptions. The mechanisms governing beta- adrenergic receptor internalization then may be common to or serve as a paradigm to explain the agonist induced behavior of other members of the G-coupled protein receptor family. This study will test the ability of wild type and sequestration abnormal receptors to interact with other components of the cell, including betaARK1, a protein kinase that may play a role in chronic heart disease. Additionally, the generality of this paradigm will be assessed by comparison to other G-protein coupled receptors. Modified receptors that have been epitope tagged at their amino termini will be expressed in a transient or permanent manner in different cell lines using plasmid expression vectors. Receptor behavior and distribution will be studied either by radioligand binding with appropriate antagonists or monoclonal antibodies using either flow cytometry for quantitation or digital analysis of immunofluorescence cell images. Agonist dependent and independent receptor distribution will be correlated using antibodies to known receptor associated proteins such as beta-arrestin, betaARK and against other membrane proteins such as clathrin and caveolin in either whole cells using immunofluorescence or in purified cell lysates. Using the technique of co-immunoprecipitation with wild type and sequestration defective receptor, attempts will be made to determine other cell constituents that might be required for sequestration to occur normally. Identification of these components may facilitate the development of therapies aimed at reversing abnormal receptor behavior.

该项目的目的是确定激动剂诱导的细胞 负责β2-肾上腺素受体内在化的机制。 beta2-肾上腺素能受体显然至少在两个期间内化 不同的独立过程，下调和隔离，这些过程 所有G蛋白偶联受体可能是常见的。 隔离已经 与短期受体脱敏和 复敏。 任何失败因失败而失去正常受体功能 隔离途径的组成部分可能会导致疾病 在慢性心力衰竭中，正常心脏收缩率可能取决于 在正常的受体脱位或预防异常受体的情况下 脱敏。 各种突变β2-肾上腺素受体已经 产生的表现出隔离行为中间的野生 通过在基序中进行点突变的类型和非功能受体 第七跨膜的连接，胞质区域的连接 受体。 对于大多数已知的G蛋白耦合而言，此主题是常见的 受体只有少数例外。 管理β-的机制 然后，肾上腺素能受体内在化可能是常见或用作 范式解释激动剂引起的其他成员的行为 G偶联蛋白受体家族。 这项研究将测试 野生型和隔离异常受体与其他受体相互作用 细胞的成分，包括betaark1，一种可能发挥的蛋白激酶 在慢性心脏病中的作用。 此外，这是一个普遍性 将通过与其他G蛋白耦合进行比较来评估范式 受体。 在其上标记为表位的修饰受体 氨基终端将以瞬态或永久方式表示 使用质粒表达向量的不同细胞系。 受体行为 和分布将通过放射性配置的结合来研究 使用任何一种流量的适当拮抗剂或单克隆抗体 用于定量或数字分析免疫荧光细胞的细胞仪 图像。 激动剂依赖和独立受体分布将是 使用抗体与已知受体相关蛋白的抗体相关 beta-arrestin，betaark和其他膜蛋白（例如 使用免疫荧光或在整个细胞中的网格蛋白和小窝蛋白 纯化的细胞裂解物。 使用与共免疫沉淀技术 野生型和隔离有缺陷的受体，将尝试 确定其他细胞成分可能需要的隔离 正常发生。 这些组件的识别可能有助于 旨在逆转异常受体行为的疗法的发展。

项目成果

Agonist-specific regulation of delta-opioid receptor trafficking by G protein-coupled receptor kinase and beta-arrestin.

G 蛋白偶联受体激酶和 β-抑制蛋白对 δ-阿片受体运输的激动剂特异性调节。

• DOI: 10.3109/10799899909036653
• 发表时间: 1999
• 期刊: Journal of receptor and signal transduction research
• 作者: Zhang,J;Ferguson,SS;Law,PY;Barak,LS;Caron,MG
• 通讯作者: Caron,MG

Nucleotide release by airway epithelia.

气道上皮释放核苷酸。

• DOI: 10.1007/978-94-007-1217-1_1
• 发表时间: 2011
• 期刊: Sub-cellular biochemistry
• 作者: Lazarowski,EduardoR;Sesma,JulianaI;Seminario,Lucia;EstherJr,CharlesR;Kreda,SilviaM
• 通讯作者: Kreda,SilviaM

Signaling, desensitization, and trafficking of G protein-coupled receptors revealed by green fluorescent protein conjugates.

绿色荧光蛋白缀合物揭示 G 蛋白偶联受体的信号传导、脱敏和运输。

• DOI: 10.1016/s0076-6879(99)02016-9
• 发表时间: 1999
• 期刊: Methods in enzymology
• 作者: Barak,LS;Zhang,J;Ferguson,SS;Laporte,SA;Caron,MG
• 通讯作者: Caron,MG