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

Jong-Ik Hwang, Sangdun Choi, I. D C Fraser, Mi Book Chang, Melvin I. Simon

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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 DTP 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.

Original languageEnglish
Pages (from-to)9493-9498
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number27
DOIs
Publication statusPublished - 2005 Jul 5
Externally publishedYes

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Keywords

  • Cell signaling
  • RNA interference

ASJC Scopus subject areas

  • General
  • Genetics

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