32-adrenergic receptors ((32ARs) relax airway smooth muscle causing bronchodilation. p-agonists are commonly used drugs in asthma, but the response is erratic due to regulation from genetic variation, chronic treatment, other asthma therapies, and asthmatic inflammation. This MERIT extension will continue studies with the overall goal of understanding the basis of P2AR regulation in asthma.
In Aim 1 interactions of P2AR polymorphisms, cell-type, and modulating conditions on receptor phenotype will be ascertained, with whole- gene transfections of the (32AR representing the major human haplotypes into airway cells under various clinically relevant conditions. Studies will be carried out to define haplotype-expression relationships and their mechanisms. This will lead to a comprehensive understanding of their biology, and the fundamental basis for pharmacogenomic approaches to asthma therapy. The mechanisms of P2AR desensitization /resensitization relative to airway function in asthma remain obscure.
In Aim 2 the roles of GRK-mediated phosphorylation of P2AR on airways, particularly agonist-promoted desensitization (clinically manifested as tachyphylaxis), and adapter-protein trafficking with """"""""signal remodeling"""""""" (related to adverse airway effects of chronic treatment), will be determined. Transgenic mice will be generated with targeted expression to airway smooth muscle of the human P2AR and multiple mutated (32AR lacking phosphorylation sites for the 5 GRK isoforms. Signaling is studied in airway smooth muscle cells and correlated with airway physiology studies of contraction/relaxation under various conditions.
In Aim 3 the roles of PKA- and PKC-mediated P2AR phosphorylation on airway function will be determined, examining homologous and heterologous desensitization and receptor crosstalk, processes potentially relevant to signal dampening and dysregulated smooth muscle function. Transgenic mice will be generated with targeted expression to airway smooth muscle of mutated p2AR lacking the phosphorylation sites for PKA or PKC, and studied as in Aim 2. While we know that p-agonists, corticosteroids, and asthmatic inflammation alter airway receptors, G-proteins, or sffectors (R:G:E), the rate limiting elements are unclear, and thus mechanisms of regulation, and the nterface(s) to intervene to enhance therapy, are not known.
In Aim 4, this R:G:E stoechiometry will be ascertained by genetic manipulation of the mouse genome to increase and decrease these elements in relevant bronchodilatory and contraction circuits. Signaling at the cellular level will be correlated with function to bridge the gap between these events and relevant airway physiology. Asthma remains a serious disease affecting ~15 million in the US. Treatment with p-agonists is a mainstay of therapy, but with a better fundamental understanding of the mechanisms of receptor function and regulation, outcome can be mproved, adverse effects minimized, and new therapeutic targets defined.
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