Mast cells and T lymphocytes, two cell types integral to development of an allergic response and asthma, 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 G alpha subunit function 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, was found to be expressed in murine and human mast cells (MCs) and B lymphocytes. RGS13 deficient mice have been generated. A vector was constructed that replaces the RGS13 gene with LacZ, and positive beta-galactosidase staining was observed in cultured, bone marrow-derived and tissue mast cells from knockout mice. Allergic (MC-dependent) physiological responses are currently being evaluated in these mice. In addition, human mast cell lines overexpressing RGS13 or deficient in RGS13 have been made. Activation and migration responses of these cells are currently being studied. 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 adenylyl cyclase. The biochemical basis for this effect is being investigated using downstream activators of this signaling pathway. A highly homologous RGS protein, RGS16, is highly expressed in mouse and human activated T lymphocytes. Its function in T cell activation and chemotaxis is being studied by production of conditional knockout mice lacking RGS16 in T lymphocytes. Intracellular flow cytometry confirmed RGS16 expression in unfractionated human T cells, and RGS16 levels in T cells subsets are being evaluated using surface marker analysis. Preliminary data suggest high RGS16 expression in germinal center T lymphocytes isolated from lymphoid tissue. Activation and proliferation induced by T cell receptor stimulation as well as chemokine-mediated migration of RGS16-deficient human T cells are being analyzed using siRNA.
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