MECHANISMS OF BETA-2 ADRENERGIC RECEPTOR INTERNALIZATION

这个项目的目标是确定激动剂诱导的细胞 β2-肾上腺素能受体内化的机制。 β2-肾上腺素能受体明显内化至少在 不同的独立程序、下调监管和自动减支，其中 可能是所有G蛋白偶联受体所共有的。自动减支一直是 参与了受体的短期脱敏和 再敏化。任何一种疾病都会导致正常受体功能的丧失 隔离途径的组成部分可能与疾病有关 在慢性心力衰竭中，正常的心脏收缩能力可能依赖于 关于正常受体再增敏或异常受体的预防 脱敏。各种突变的β2-肾上腺素能受体已经 产生了介于野生和野生之间的隔离行为 通过在基序中进行点突变来实现类型和非功能受体 第七跨膜、胞质区的连接 受体。这个基序是大多数已知的G蛋白偶联的共同基序 受体，只有少数例外。管理Beta-的机制- 肾上腺素能受体内化可能是共同的或起作用的 解释激动剂诱导的其他成员行为的范式 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 细胞的组成部分，包括BetaARK1，一种可能发挥作用的蛋白激酶 在慢性心脏病中起着重要作用。此外，这一点的一般性 范例将通过与其他G-蛋白偶联的比较来评估 感受器。修饰的受体已经在它们的 氨基末端将以暂时或永久的方式在 用不同的细胞株构建表达载体。受体行为 分布将通过与放射性配体结合来研究 适当的拮抗剂或单抗使用任一流 用于免疫荧光细胞定量或数字分析的细胞术 图像。激动剂依赖和独立的受体分布将是 使用已知受体相关蛋白的抗体进行关联，例如 β-arrestin、betaARK和其他膜蛋白，如 用免疫荧光法在整个细胞中检测到网状蛋白和小窝蛋白 纯化的细胞裂解物。使用免疫共沉淀技术 野生型和隔离缺陷受体，将尝试 确定隔离可能需要的其他细胞成分 正常发生正常发生。识别这些组件可能有助于 旨在逆转异常受体行为的治疗方法的开发。

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.