SMALL GTPASES AND LUNG BETA RECEPTOR REGULATION

哮喘治疗可能部分由于脱敏而变得无效 持续使用β-2-肾上腺素能反应 激动剂。β2-肾上腺素能受体(β2AR)的脱敏将 作为7-膜跨G蛋白调节模型的研究 偶联受体，以及信号的调节内吞作用 一般情况下，受体。脱敏是由几个不同的 机械装置。在激动剂结合的几秒钟内，受体就被磷酸化了 从G-S中分离出来。在绑定后的几分钟内，封存 受体通过内化进入胞内隔室而发生。 隔离的受体可能在它们返回到 浮出水面。长期接触激动剂(小时)会导致细胞丢失 受体(下调)，并可能通过溶酶体受体发生 降解和受体合成减少。这项提议的目标是 使用Rab家族的小GTP酶，调节细胞的内吞作用 表面受体，以揭示β2受体的分子机制 封存和回收。工作假说是Rab GTP酶 是调节Beta2AR所必需的，因为它引起了 它的细胞分布对激动剂的反应。小的GTP酶，如 RAB是一种分子开关，可以结合和水解GTP，然后解离 来自两个活动州之间受监管的穿梭班车中的GDP。Rab4和Rab5 与内细胞室相关，并可能在 准确的水泡流量方向。这些研究将确定 Rab4和Rab5在人β2AR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Rab4和Rab5， 以及它们的显性抑制突变，将在一个细胞中过度表达 表达表位标记的人Beta2AR的行。受体的变化 将使用以下方法研究Rab5过表达导致的隔离 全细胞和膜部分的放射性配基结合分析，以及 用免疫荧光和激光共聚焦显微镜观察。回收利用的变化 过表达Rab4后进行复敏实验。其他 内吞细胞器的成分将被鉴定为 使用酵母中的遗传测试与Rab5相互作用，然后表征 通过生物化学和亚细胞定位。这些研究将澄清 β2AR的细胞内转运及Rab的调节作用 在这一过程中的蛋白质。此外，内吞细胞机的部件 将被确定，从而带来开发治疗学的潜力 在调控β2AR内吞作用的基础上。

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.