SMALL GTPASES AND LUNG BETA RECEPTOR REGULATION

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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.