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.
