The 132-adrenergic receptor (132AR) plays a major role in the """"""""fight-or-flight"""""""" response including mediation of bronchodilation. Eliciting bronchodilation with agonists of the 132AR is, along with steroids to treat inflammation, a major treatment of chronic asthma. The effectiveness of the bronchodilators is known to decrease over time (desensitize), and as such the characterization of agonist desensitization of the 132AR has been the focus of a multitude of studies, and the evidence derived from these has made it a paradigm for the study of G protein coupled receptors (GPCRs). However, many questions remain concerning the molecular mechanisms of desensitization, and importantly. how the 132AR signaling complex resensitizes following removal of stimulation. At the systems biology level, there is a need for a dynamic modeling of the complex inhibitory feedback loops involving 132AR phosphorylation by PKA and multiple G protein coupled receptor kinase (GRK) subtypes, and their downstream sequelae such as arrestin binding, internalization, and activation of phosphodiesterase. Since most studies of 132AR desensitization have been performed with cell lines overexpressing the 132AR, there is a need for studies of desensitization in primary human cells expressing endogenous levels of the 132AR. Another aspect that has received little attention has been the development of inhibitors of desensitization. Our group has made significant inroads in ongoing studies of three areas of the 132AR desensitization process;characterization of GPCR activation of GRKs, development of a panel of inhibitors of GRK activity, and systems modeling of desensitization and resensitization, leading to the following specific aims: (1) characterization of 132AR desensitization in both HASM and model cell systems with a focus on quantitative systems modeling of the processes that control loss of both 132AR efficacy and downstream actions of the second messenger cAMP through phosphodiesterase hydrolysis;(2) determination of the mechanism of activation of GRKs by the 132AR receptor and the related GPCR rhodopsin through detailed structure/function studies of evolutionarily important GRK residues;and (3) development of peptide inhibitors that disrupt the GPCRlGRK interaction based on knowledge gained of important GRK and 132AR domains involved in the interaction.
The goal of this proposal is to characterize the mechanisms that lead to the desensitization of hormone and drug stimulation of the ?2-adrenergic receptor in human airway cells, to determine the structural basis for the receptor interaction with the major desensitization machinery, and to develop inhibitors of desensitization based on mimicry of the interaction. Our findings will provide improved rationale for treatment of airway diseases such as asthma.
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