SMALL GTPASES AND LUNG BETA RECEPTOR REGULATION

哮喘治疗可能变得无效，部分原因是脱敏 的β 2-肾上腺素能反应的持续治疗， 激动剂β 2-肾上腺素能受体（β 2AR）的脱敏将 作为一个模型进行研究，为7跨膜G蛋白的调节 偶联受体，并用于调节信号转导的内吞作用 一般来说，受体。脱敏发生在几个不同的 机制等激动剂结合数秒内，受体被磷酸化 从G-S中分离出来在绑定后的几分钟内， 受体通过内化进入内吞区室而发生。 被隔离的受体可能在它们回到细胞前重新敏感。 面长期暴露于激动剂（小时）会导致细胞损失， 受体（下调），并可能通过溶酶体受体 降解和受体合成减少。这项提案的目的是 使用rab家族的小GTPases，调节细胞的内吞作用， 表面受体，以发现β 2 AR的分子机制 封存和回收。工作假设是rab GTP酶 是调节β 2AR所必需的， 其细胞分布响应激动剂。小型GTP，如 rab是分子开关，其结合并水解GTP，然后解离 在两个活动状态之间进行受监管的穿梭。Rab 4和Rab 5 与内吞区室有关，可能在 囊泡运输的准确方向。这些研究将决定 rab 4和rab 5在人类β 2 AR贩运中的作用，以及 发现参与这一过程的其他蛋白质。野生型rab 4和rab 5， 以及它们的显性抑制突变，会在细胞中过度表达 表达表位标记的人β 2 AR的细胞系。受体变化 由rab 5过表达引起的隔离将使用 全细胞和膜组分中的放射性配体结合测定，和 通过免疫荧光和激光共聚焦显微镜。回收利用的变化 并在Rab 4过表达后测量再敏化。其他 内吞装置的组分将被鉴定为蛋白质， 在酵母中使用遗传测定与rab 5相互作用，然后表征 生化和亚细胞定位。这些研究将阐明 β 2 AR的细胞内运输及rab的调节作用 蛋白质在这个过程中此外，内吞机器的组件 将被确定，从而导致开发治疗方法的潜力 基于调节β 2AR的内吞作用。

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.