Heterotrimeric G Protein Signaling In Allergic Inflammat

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Abstract

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 subunits by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP-(active) bound forms. 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, almost nothing is known about the physiological role of these proteins in native mammalian systems. RGS13, a GAP for Gi and Gq, was found also to be expressed in murine and human mast cells and B lymphocytes. RGS13 deficient mice have been generated. A vector was constructed that replaces the RGS13 gene with LacZ. RGS13 was expressed in cultured cell lines and was found to inhibit Gi- and Gq-coupled signaling pathways. Unexpectedly, RGS13 inhibited cAMP generation induced by stimulation of a Gs-coupled receptor. Recombinant RGS13 protein was produced from bacteria, which exhibited GAP activity toward purified Gi and Gq. Polyclonal antibodies were raised against RGS13 peptides; these antisera specifically recognized RGS13 and not closely related RGS proteins. RGS13 was found to be expressed in human B lymphocytes and mast cells by immunoblotting. A Another RGS protein, RGS16, is highly expressed in activated T lymphocytes. Its role in T cell activation and chemotaxis is being studied using transgenic mice that overexpress RGS16 in CD4+ T lymphocytes. RGS16 transgenic mice express 2-3 fold higher levels of RGS16 protein in resting CD4+ and CD8+ cells than wild type controls. In a mouse model of allergic airway inflammation induced by ovalbumin (ova), RGS16 transgenic T lymphocytes exhibited abnormal activation responses. Lymphocytes in lung fluid were found to secrete increased amounts of IL-4 and IL-5 cytokines after ova challenge. This effect was replicated in vitro after recall with ova. In addition, transgenic T lymphocytes adoptively transferred to ova challenged and sensitized mice migrated poorly to the site of airway inflammation after allergen challenge and accumulated in lymph nodes as compared to wild type controls.