MECHANISMS OF BETA-2 ADRENERGIC RECEPTOR INTERNALIZATION

本项目的目标是确定激动剂诱导的细胞 负责β 2-肾上腺素能受体内化的机制。 β 2-肾上腺素能受体在至少两个周期内明显内化， 不同的独立过程，下调和封存， 可能是所有G蛋白偶联受体所共有的。 扣押一直是 参与短期受体脱敏过程， 再敏感化 由于任何一种功能的丧失而丧失正常的受体功能。 螯合途径的组成部分可能有助于疾病， 在正常心脏收缩力可能依赖于 在正常受体再敏化后，或在异常受体再敏化后， 脱敏 各种突变的β 2-肾上腺素能受体已经被发现， 所产生的化合物表现出介于野生 型和非功能性受体，使点突变的基序， 第七个跨膜的连接处，细胞质区域， 受体的 该基序是大多数已知的G蛋白偶联蛋白所共有的。 只有少数例外。 控制贝塔的机制- 肾上腺素能受体内化则可能是共同的或作为一个 范式来解释激动剂诱导的其他成员的行为， G偶联蛋白受体家族。 这项研究将测试 野生型和螯合异常受体与其他 细胞的组成部分，包括β ARK 1，一种蛋白激酶， 在慢性心脏病中的作用。 此外，这种普遍性 将通过与其他G蛋白偶联的 受体。 修饰的受体已经在它们的 氨基末端将以瞬时或永久的方式表达， 使用质粒表达载体的不同细胞系。 受体行为 和分布将通过放射性配体与 合适的拮抗剂或单克隆抗体 用于免疫荧光细胞定量或数字分析的细胞术 图像. 激动剂依赖性和非依赖性受体的分布将是 使用已知受体相关蛋白的抗体进行关联， β-抑制蛋白、β ARK和其他膜蛋白， 网格蛋白和小窝蛋白在使用免疫荧光的全细胞中，或在使用免疫荧光的全细胞中， 纯化的细胞裂解物。 采用免疫共沉淀技术， 野生型和螯合缺陷型受体，将尝试 确定隔离可能需要的其他细胞成分 正常发生。 这些组件的识别可以促进 开发旨在逆转异常受体行为的疗法。

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.