G protein-coupled receptors (GPCRs) play a critical role in regulating multiple airway functions including airway smooth muscle (ASM) contraction and relaxation. Dynamic regulation of GPCRs often results in their movement or sorting within the cell. We hypothesize that receptor sorting is controlled by multiple mechanisms including post- translational modifications and protein-protein interactions. We also hypothesize that GPCR localization and sorting plays an important role in regulating signaling. To directly test these hypotheses, we propose a comprehensive series of studies aimed at providing insight into the mechanisms regulating GPCR localization, sorting and signaling in ASM cells. These studies will focus primarily on the beta2-adrenergic receptor (beta2AR) but will also address additional receptor subtypes important in airway function such as H1 histamine and cysteinyl leukotriene LT, and LT2. Important features of this proposal include the use of non-transformed cell lines and characterization of both endogenous and transfected GPCRs. Specifically, we will: 1) Define the mechanisms involved in GPCR localization within micro-domains of the plasma membrane. Studies will involve the use of immunofluorescence, cell fractionation and biochemical analysis to assess GPCR localization. The role of ligands, post-translational modifications, additional proteins, targeting motifs, and oligomerization in localization will be determined. 2) Elucidate the mechanisms involved in endocytic sorting of GPCRs. Emphasis will be placed on establishing the role of post-translational modifications, targeting motifs and interacting proteins in mediating sorting of endocytosed GPCRs. 3) Characterize the roles of GPCR localization and endocytosis in signaling and cellular functions. We will utilize a variety of strategies to more clearly define the role of GPCR localization and endocytosis in regulating various signaling pathways and their associated cellular functions. These studies will rely on the use of selective inhibitors of various sorting pathways and GPCR mutants that are selectively altered in sorting. 4) Characterize the physiological role of beta2AR localization in airway function in mice. Transgenes encoding wild type or mutant beta2ARs altered in localization or sorting properties will be expressed in mice and the resultant lines will undergo ovalbumin sensitization and acute challenge. Studies will examine the effect of beta2AR mutations as well as beta2AR gene knockout on beta-agonist-mediated effects on pulmonary function, airway inflammation, and in vitro contractile properties of ASM strips. The proposed studies should provide new insight into the regulation of receptor localization and sorting, and clarify the role of localization in cellular signaling and in cellular/systems function.
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