Silencing the expression of multiple Gbeta-subunits eliminates signaling mediated by all four families of G proteins.
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.
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Jong-Ik
JI
Hwang
Sangdun
S
Choi
Iain D C
ID
Fraser
Mi Sook
MS
Chang
Melvin I
MI
Simon
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002633:Chemotaxis / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D016086:Lentivirus; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010587:Phagocytosis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D034622:RNA Interference; D015398:Signal Transduction / Q000502:physiology*
D000199:Actins; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019204:GTP-Binding Proteins; D002118:Calcium