SMALL GTPASES AND LUNG BETA RECEPTOR REGULATION

小 GT 酶和肺 β 受体调节

• 批准号: 2226140
• 负责人: BRIAN J KNOLL
• 金额: $ 16.3万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-10 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2226140
• 关键词: asthma; beta adrenergic receptor; binding proteins; confocal scanning microscopy; endocytosis; guanosinetriphosphatases; immunofluorescence technique; intracellular transport; protein purification; protein transport; radiotracer; receptor binding; receptor coupling; receptor sensitivity; suppressor mutations; tissue /cell culture

Asthma therapy may become ineffective in part because of desensitization of the beta2-adrenergic response by continued treatment with beta2- agonist. Desensitization of the beta2-adrenergic receptor (beta2AR) will be studied as a model for the regulation of 7-membrane spanning G-protein coupled receptors, and for the regulated endocytosis of signalling receptors generally. Desensitization occurs by several distinct mechanisms. Within seconds of agonist binding, receptor is phosphorylated and uncouples from G-S. Within minutes of binding, sequestration of receptors occurs by internalization into an endocytic compartment. Sequestered receptors probably are resensitized before they return to the surface. Chronic exposure to agonist (hours) causes the loss of cellular receptor (down-regulation), and may occur by both lysosomal receptor degradation and decreased receptor synthesis. The goal of this proposal is to use small GTPases of the rab family, that regulate the endocytosis of surface receptors, to discover the molecular mechanisms of beta2AR sequestration and recycling. The working hypothesis is that rab GTPases are required for the regulation of the beta2AR by causing an alteration in its cellular distribution in response to agonist. Small GTPases such as rabs are molecular switches that bind and hydrolyze GTP, then dissociate from GDP in a regulated shuttle between two activity states. rab4 and rab5 are associated with the endocytic compartment and may play a role in the accurate direction of vesicle traffic. These studies will determine the roles of rab4 and rab5 in the trafficking of the human beta2AR, and discover other proteins involved in this process. Wild-type rab4 and rab5, and their dominant suppressive mutations, will be overexpressed in a cell line that expresses an epitope-tagged human beta2AR. Changes in receptor sequestration caused by rab5 overexpression will be studied using radioligand binding assays in whole cells and in membrane fractions, and by immunofluorescence with laser confocal microscopy. Changes in recycling and resensitization will be measured after overexpression of rab4. Other components of the endocytic apparatus will be identified as proteins that interact with rab5 using a genetic assay in yeast, and then characterized biochemically and by subcellular localization. These studies will clarify the intracellular trafficking of beta2AR and the regulatory roles of rab proteins in that process. In addition, components of the endocytic machine will be identified, leading to the potential for developing therapeutics based on regulating the endocytosis of beta2AR.

哮喘治疗可能部分是由于脱敏的 通过继续治疗beta2- beta2-肾上腺素反应 激动剂。 β2-肾上腺素能受体（BetA2AR）的脱敏 作为调节G蛋白的7个膜的调节模型 耦合受体，以及对信号的调节性内吞作用 受体一般。脱敏是通过几个不同的 机制。在激动剂结合的几秒钟内，受体被磷酸化 和g-s的未成年人。在结合的几分钟内，隔离 受体通过内部化到内吞区室。 隔离的受体可能在回到 表面。长期暴露于激动剂（小时）会导致细胞的损失 受体（下调），并且可能由两种溶酶体受体发生 降解和受体合成降低。该提议的目的是 使用Rab家族的小gtpases，以调节 表面受体，发现β2AR的分子机制 隔离和回收。工作假设是Rab GTPases 通过造成更改的beta2ar调节所需 其响应激动剂的细胞分布。小gtpases，例如 Rabs是结合并水解GTP的分子开关，然后分离 在两个活动状态之间的受管班车中的GDP。 rab4和rab5 与内吞室相关，可能在 囊泡流量的准确方向。这些研究将确定 rab4和rab5在人类beta2ar的贩运中的角色，以及 发现参与此过程的其他蛋白质。野生型rab4和rab5， 它们的主要抑制突变将在细胞中过表达 表达具有表位标记的人beta2ar的线。受体的变化 将使用RAB5过表达引起的封存 整个细胞和膜级分中的放射性结合测定，以及 通过激光共聚焦显微镜免疫荧光。回收的变化 并在Rab4过表达后测量敏化。其他 内吞仪的成分将被鉴定为蛋白质 使用酵母中的遗传测定与Rab5相互作用，然后表征 生化和亚细胞定位。这些研究将澄清 Beta2AR的细胞内贩运和RAB的调节作用 在该过程中的蛋白质。另外，内吞机器的组件 将被确定，从而有可能发展治疗剂 基于调节β2AR的内吞作用。