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Proc Natl Acad Sci U S A. 2005 Jul 5;102(27):9493-8. Epub 2005 Jun 27.

Silencing the expression of multiple Gbeta-subunits eliminates signaling mediated by all four families of G proteins.

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  • 1Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

Abstract

The Gbetagamma-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 Gbeta in G protein-coupled receptor signaling. The expression of Gbeta1 and Gbeta2, the major subtypes present in J774A.1 cells, was eliminated by sequential treatment with small hairpin RNA expressing lentivirus. These betagamma 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 Gbeta-subunits. In addition, stabilization and membrane localization of several Galpha- and Ggamma-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 Gbeta-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 Gbeta-subunits destabilized Galpha- and Ggamma-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.

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