Heterotrimeric G Protein Signaling In Allergic Inflammation

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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. RGS13, a GAP for Gi and Gq, but not Gs, was found to be expressed in murine and human mast cells (MCs) and B lymphocytes. Rgs13-deficient mice were generated to determine the function of RGS13 in these cell types. Allergic (MC-dependent) physiological responses were evaluated in these mice. Surprisingly, both cutaneous and systemic anaphylaxis induced by cross-linking of MC antigen receptors with IgE antibody were markedly increased in RGS13-deficient mice. This abnormality was caused by enhanced IgE-evoked degranulation of cultured bone marrow-derived mast cells (BMMCs). Reconstitution of these BMMCs with RGS13 inhibited degranulation. In addition, the GAP activity of RGS13 was not required for this effect. Rather, subsequent detailed molecular analysis revealed that RGS13 regulates IgE-mediated allergic responses by interacting with the p85 regulatory subunit of the enzyme phosphoinositide-3-kinase (PI3K), which is a critical component of the allergen-induced signaling route leading to MC degranulation. These results have uncovered a new physiological function of RGS13 in mast cells. We hypothesize that abnormalities in RGS13 expression or function may exist in patients with idiopathic anaphylaxis or other disorders accompanied by increased mast cell reactivity. 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. An RGS highly homologous to RGS13, RGS16, is highly expressed in mouse and human activated T lymphocytes. We are studying the function of RGS16 in T cell migration and activation using RGS16-deficient mice. Lymphoid organs of these mice contain comparable numbers of lymphocytes as their wild-type counterparts. RGS16 expression was upregulated by T-cell-receptor stimulation of both murine CD4 and CD8 cells. Splenic B and T cells from RGS16 knockout mice migrated more in vitro after exposure to the chemokines CCL21, CXCL12, and CXCL13. Current studies involve examination of the immune responses after immunization with T-dependent antigens.