Silencing the expression of multiple Gβ-subunits eliminates signaling mediated by all four families of G proteins
The Gβγ-subunit complex derived from heterotrimeric G proteins can act to regulate the function of a variety of protein targets. We established lentiviral-based RNA interference in J774A.1 mouse macrophages to characterize the role of Gβ in G protein-coupled receptor signaling. The expression of Gβ1 and Gβ2, the major subtypes present in J774A.1 cells, was eliminated by sequential treatment with small hairpin RNA expressing lentivirus. These βγ complex-deficient cells lost the ability to respond to G protein-mediated signals. Chemotaxis and the phosphorylation of Akt in response to C5a were both blocked. Similarly, C5a-mediated actin polymerization, C5a- and UTP-stimulated intracellular calcium mobilization, and the stimulation of cAMP formation by isoproterenol were all eliminated in the absence of the Gβ-subunits. In addition, stabilization and membrane localization of several Gα- and Gγ-subunit proteins was strongly effected. Furthermore, in DNA microarray analysis, regulation of gene expression stimulated by prostaglandin E2 and UTP was not observed in cells lacking Gβ-subunits. In contrast, phagocytotic activity, serum-dependent cell growth and the patterns of gene expression induced by stimulating the Toll receptors with LPS were similar in wild-type cells and small hairpin RNA-containing cells. Thus, ablation of the Gβ-subunits destabilized Gα- and Gγ-subunits and effectively eliminated G protein-mediated signaling responses. Unrelated ligand regulated pathways remained intact. These cells provide a system that can be used to study signaling in the absence of most G protein-mediated functions.
Additional Information© 2005 by The National Academy of Sciences of the USA. Contributed by Melvin I. Simon, April 27, 2005. Published ahead of print June 27, 2005. This work was supported by the National Institutes of Health Grant GM34236 (to M.I.S.). I.D.C.F. and S.C. are supported in part by a National Institutes of Health Grant GM 62114 (to the Alliance for Cellular Signaling Cooperative Agreement).
Published - PNAS-2005-Hwang-9493-8.pdf