Mast cells (MCs) and T lymphocytes are two cell types integral to development of an allergic response and asthma. The signature response of each of these cells, degranulation and cytokine production, respectively, is induced primarily by cross-linking of the receptor for antigen. In addition, both mast cells and T cells express numerous inflammation-generating receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of intracellular G-protein-coupled signal transduction in these cells and subsequent pathways to inflammation. In particular, the project focuses on the control of G protein activity in inflammatory processes by a novel family of regulators of G protein signaling (RGS proteins), which inhibit function of G alpha-i and G alpha-q, but not G alpha-s, subunits by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, little is known about the physiological role of these proteins in native mammalian systems. In the previous year's work, we identified an RGS protein, RGS13, which inhibits IgE-mediated mast cell degranulation and anaphylaxis in mice by counteracting activation of the critical downstream enzyme phosphoinositide-3 kinase (PI3 kinase). These results uncovered a new physiological function of RGS proteins with broad implications for cell growth, metabolism, and immunity: the direct inhibition of PI3 kinase. We hypothesized that abnormalities in RGS13 expression or function may exist in patients with idiopathic anaphylaxis or other disorders characterized by increased mast cell reactivity. Because we discovered during the course of this work that several RGS proteins regulated PI3 kinase, we investigated whether RGS family members homologous to RGS13 such as RGS16 behaved in the same way in different cell types. During 2009, we extended our findings by describing regulation of PI3 kinase by RGS16 in breast cancer cells. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. We found that RGS16 reduced growth of breast cancer cells by suppressing PI3 kinase-induced proliferation. We also mapped the domains on RGS16 and PI3 kinase (p85 subunit) required for direct interaction. These studies helped clarify the mechanism by which RGS proteins mitigate PI3 kinase activity. The next step will be to co-crystallize the RGS-PI3K complex to enable design of therapeutic agents that mimic the action of RGS proteins. Such compounds might be eventually tested for their ability to alleviate mast-cell mediated allergic disorders. Unexpectedly, RGS13 overexpression in an epithelial cell line inhibited cAMP generation induced by stimulation of a Gs-coupled receptor and by forskolin, a direct activator of adenylyl cyclase. The biochemical basis for this effect was investigated using downstream activators of this signaling pathway. We found that RGS13 acts in the nucleus where it binds the activated (phosphorylated) form of the transcription factor CREB, which is the target of the cAMP pathway. RGS13 overexpression inhibited CREB promoter occupancy in vivo and suppressed CREB-dependent gene expression, while siRNA-mediated knockdown of RGS13 expression had the opposite effect. RGS13-deficient B lymphocytes displayed increased CREB DNA binding and transcription of a CREB target gene, OCA-B. We are currently studying whether RGS13 deficiency affects cyclic AMP-induced IgE production by B cells, a CREB-dependent mechanism. Another major area of investigation in this project is the regulation of chemokine GPCR-mediated recruitment of inflammatory cells to sites of allergic inflammation. We found that RGS16 is expressed in activated Th1, Th2, and Th17 CD4+ lymphocytes. RGS16-deficient T cells migrate more to the Th2-specific chemokine CCL17 in vitro, and we found more Th2 cells in the lungs of allergen-challenged mice Rgs16-/- mice than in wild type counterparts. From these preliminary results, we conclude that RGS16 attenuates Th2 responses to Schistosoma antigens. We plan to confirm these results in a full model of S. mansoni infection in collaboration with Dr. Thomas Wynn (Laboratory of Parasitic Diseases, NIAID). Finally, a newer focus of this project is to identify the chemokine receptor/G protein/RGS protein axis utilized by mouse basophils in allergic inflammation. These cells have received considerable recent attention as crucial mediators of Th2 responses and anaphylaxis. This project is just underway.
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