Asthma is one of the most common respiratory diseases world-wide; 300 million people are currently receiving treatment and global economic cost exceeds those of tuberculosis and HIV/AIDS combined. Thus, asthma remains an area of considerable unmet medical need. Allergic asthma is caused by an overzealous Th2 immune response to allergens in which immunoglobulin E (IgE) and mast cells play critical roles. Thus, aggregation of high affinity IgE receptor (Fc?RI) by allergen on mast cells results in histamine release and the generation of lipids and cytokines, which are responsible for the manifestations of asthma. The focus of our laboratory has been to study the regulation of G protein coupled receptor (GPCR) signaling in mast cells. Functions of GPCRs are regulated via their phosphorylation by a family of protein kinases known as GPCR kinases (GRKs) and the recruitment of the scaffolding molecule -arrestin (-arr). The receptor/-arr complex serves to attenuate G protein activation leading to receptor desensitization. -arr also promotes downstream MAPK signaling pathways independent of G protein activation. To the best of our knowledge, the role of -arr on Fc?RI signaling in mast cells has never been reported. Based on our unexpected preliminary data, we hypothesize that -arr2 serves as a scaffolding protein to promote Fc?RI- mediated cofilin dephosphorylation in mast cells. We further hypothesize that this -arr2-mediated cofilin dephosphorylation contributes to mast cell recruitment in the lung to promote the development chronic asthma.
In aim 1, retrovirus will be used to transduce different domains of -arr2 into -arr2-/- mast cells and their ability to promote Fc?RI-mediated cofilin dephosphorylation and mast cell chemotaxis will be determined. We will then overexpress different sub-domains of -arr2 into mast cells endogenously expressing -arr2 and their ability to interfere with Fc?RI-mediated cofilin dephosphorylation and mast cell chemotaxis will be determined.
In aim 2, we will delete -arr2 in mast cells by breeding -arr2-floxed mice and carboxypeptidase-3 (Cpa-3) cre mice. We will also overexpress -arr2 in mast cells in vivo by engrafting -arr2-transduced BMMCs into mast cell-deficient Wsh/Wsh mice. These complementary approaches will be used to test the hypothesis that -arr2 expressed in mast cells contributes to their recruitment in the lung and promotes the development of chronic asthma. Finally, we will express cofilin and peptide domains of -arr2 in mast cells in vivo. These strategies will be used to determine if mast cell-specific expression of cofilin and -arr2 domains modulate chronic asthma. This study will likely generate new information regarding the roles of -arr2 and cofilin in mast cell chemotaxis in vitro and in vivo and may provide a better rationale for the development of novel therapeutics for chronic asthma.
Chronic asthma is a major cause of morbidity in the United States and is caused by the release of inflammatory mediators from mast cells. This study is based on the identification of a novel protein that is important for mast cell recruitment to the lung. We believe that proposed studies will generate new information on the mechanism of mast cell recruitment and may offer new approaches for the treatment of asthma.
