SMALL GTPASES AND LUNG BETA RECEPTOR REGULATION

G蛋白偶联受体的一个特点是激活后脱敏，如人类β 2-肾上腺素能受体（β 2- ars）。这些受体结合儿茶酚胺激动剂后刺激腺苷酸环化酶，介导气道平滑肌松弛等生理作用。因此，Beta2ARs是用于治疗哮喘和慢性阻塞性肺疾病的药物的重要靶点。β 2ars的脱敏首先通过受体磷酸化发生，导致与g蛋白的解偶联，然后通过受体内化远离细胞表面并进入分选内体。大多数内化受体被去磷酸化并再循环到细胞表面，但受体也被分类到溶酶体进行降解（下调）或核周围的“循环”内体。因此，内吞作用和细胞内分选事件是调节配体激活的信号转导受体的关键机制。我们的长期目标是确定受体从一个细胞区室到另一个区室的运动是如何被调节的，以及这些运动与受体活性的关系。细胞间的运输是由与ras相关的gtp酶（称为rabs）以及与rabs相互作用的蛋白质来调节的。Rab5及其相互作用蛋白控制内吞囊泡与分选核内体的融合，并参与分选核内体与再循环核内体之间的交通。Rab4和rab11也参与内体室间的分选。利用酵母双杂交方法，我们发现了一种新的膜相关rab5相互作用（rab5ip），它似乎定位于内体膜。本研究的具体目标是确定rab5ip蛋白如何通过细胞内区室之间的运动调节β 2ar活性和数量，以及rab5ip如何调节核内体融合。转染后的细胞将通过共聚焦显微镜和放射配体结合检查，以确定rabs和rab5ip如何调节分选核内体、再循环核内体、质膜和溶酶体之间的交通。这些运动也将与β 2ar去磷酸化相关。rab5ip的功能将通过rab5ip突变体和体外核内体融合实验进行研究。将确定与rab5相互作用所需的Rab5ip结构域，并评估与内核体融合的其他调节因子的潜在相互作用。最后，将筛选与rab5ip相互作用的其他新蛋白。这些研究将深入了解内吞运输事件的机制，以及它们如何调节重要信号转导受体的活性和数量。

A characteristic of G protein-coupled receptors is their desensitization after activation, as exemplified by human beta2- adrenergic receptors (beta2ARs). These receptors stimulate adenylyl cyclase after binding catecholamine agonists, and mediate such physiologic actions such as the relaxation of airway smooth muscle. Beta2ARs thus are important targets for drugs used to treat asthma and chronic obstructive pulmonary disease. Desensitization of beta2ARs occurs first by receptor phosphorylation, causing uncoupling from G-protein, then by internalization of receptors away from the cell surface and into sorting endosomes. Most internalized receptors are dephosphorylated and recycle to the cell surface, but receptors are also sorted to lysosomes for degradation (downregulation) or to perinuclear 'recycling' endosomes. Endocytosis and intracellular sorting events are thus critical mechanisms for the regulation of ligand-activated signal transducing receptors. Our long-term goals are to determine how receptor movements from one cell compartment to another are regulated, and the relationship of these movements to receptor activity. Trafficking between cellular compartments is regulated by ras-related GTPases called rabs, and by proteins interact with rabs. Rab5 and its interacting proteins control the fusion of endocytic vesicles with the sorting endosome, and re implicated in the traffic between sorting and recycling endosomes. Rab4 and rab11 also participate in sorting among endosomal compartments. Using the yeast two-hybrid method we identified a novel, membrane associated rab5 interacting (rab5ip) that appears to be localized to endosomal membranes. The specific goals of the present study are to determine how rab proteins regulate beta2AR activity and number by movements between intracellular compartments, and to determine how rab5ip regulates endosome fusion. Transfected cells will be examined by confocal microscopy and radioligand binding to determine how rabs and rab5ip regulate traffic among the sorting endosome, the recycling endosome, the plasma membrane and lysosomes. These movements also will be correlated with beta2AR dephosphorylation. The function of rab5ip will be studied with rab5ip mutants and an endosome fusion assay in vitro. Rab5ip domains required for interaction with rab5 will be determined, and potential interactions with other regulators of endosome fusion will be assessed. Finally, a screen will be performed for other novel proteins that interact with rab5ip. These studies will provide insight into the mechanisms of endocytic trafficking events, and how they regulate the activity and number of an important signal-transducing receptor.