Heterotrimeric G Protein Signaling Allergic Inflammation

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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. RGS4, a GAP expressed highly in mouse brain, heart, and hematopoietic cells, was found also to be expressed in murine mast cells. RGS4 deficient mice are being generated. A vector was constructed that replaces the RGS4 gene with LacZ. Embryonic stem cells were transfected and are currently being screened for homologous recombination. An uncharacterized RGS protein, RGS13, was cloned from lung cDNA. RGS13 was expressed in cultured cell lines and was found to inhibit Gi- and Gq-coupled signaling pathways. 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 lung, kidney, and spleen tissue by immunoblotting and immunohistochemistry. A targeting vector in which the RGS13 gene is disrupted by LacZ was constructed in order to generate RGS13 null mice. Another RGS protein, RGS16, is highly expressed in activated T lymphocytes. Its role in T cell activation and chemotaxis is being studied using several methods. TAT-RGS16 fusion proteins were purified from bacteria and transduced into cultured T cell lines and primary splenocytes. These cells are stimulated with chemokines to study the effect of RGS16 overexpression on chemotaxis. RGS16 transgenic mice were generated that express 2-3 fold higher levels of RGS16 protein in resting CD4+ and CD8+ cells than wild type controls. These mice are currently being crossed with mice transgenic for an antigen (ova)-specific TCR to assess the effect of RGS16 on T cell activation and trafficking in a mouse model of ova-induced inflammation.