SMALL GTPASES AND LUNG BETA RECEPTOR REGULATION

G蛋白偶联受体的一个特征是激活后脱敏，如人β2肾上腺素能受体(Beta2ARs)。这些受体在与儿茶酚胺激动剂结合后刺激腺苷环化酶，并介导诸如气道平滑肌松弛等生理作用。因此，Beta2ARs是治疗哮喘和慢性阻塞性肺疾病药物的重要靶点。β2受体的脱敏首先通过受体磷酸化而发生，导致与G蛋白解偶联，然后通过受体内化离开细胞表面进入分选内小体。大多数内化的受体是去磷酸化的，并循环到细胞表面，但受体也被分类到溶酶体以进行降解(下调)或到核周围的“循环”内小体。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室到另一个细胞室的运动是如何调节的，以及这些运动与受体活性的关系。细胞间的转运由ras相关的GTP酶RABS以及与RABS相互作用的蛋白质调控。Rab5及其相互作用的蛋白控制内吞小泡与分选内小体的融合，并参与分选和回收内小体之间的运输。Rab4和Rab11也参与内体隔间的分选。利用酵母双杂交方法，我们鉴定了一种新的膜相关Rab5相互作用(Rab5ip)，它似乎定位于内膜。本研究的具体目标是确定Rab蛋白如何通过细胞内隔室之间的运动来调节β2AR的活性和数量，并确定Rab5ip如何调节内体融合。转基因细胞将通过共聚焦显微镜和放射配基结合来确定RABS和Rab5ip如何调节分选内小体、循环内小体、质膜和溶酶体之间的交通。这些运动也将与Beta2AR去磷酸化相关。将通过Rab5ip突变体和体外内含体融合实验来研究rab5ip的功能。将确定与Rab5相互作用所需的Rab5ip结构域，并将评估与内体融合的其他调节因子的潜在相互作用。最后，将对与rab5ip相互作用的其他新蛋白进行筛选。这些研究将深入了解内吞转运事件的机制，以及它们如何调节一个重要的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.