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J. Biol. Chem., Vol. 281, Issue 15, 10250-10262, April 14, 2006

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Targeted Knockdown of G Protein Subunits Selectively Prevents Receptor-mediated Modulation of Effectors and Reveals Complex Changes in Non-targeted Signaling Proteins^*

From the Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390

Heterotrimeric G protein signaling specificity has been attributed to select combinations of G, , and subunits, their interactions with other signaling proteins, and their localization in the cell. With few exceptions, the G protein subunit combinations that exist in vivo and the significance of these specific combinations are largely unknown. We have begun to approach these problems in HeLa cells by: 1) determining the concentrations of G and G subunits; 2) examining receptor-dependent activities of two effector systems (adenylyl cyclase and phospholipase C); and 3) systematically silencing each of the G and G subunits by using small interfering RNA while quantifying resultant changes in effector function and the concentrations of other relevant proteins in the network. HeLa cells express equimolar amounts of total G and G subunits. The most prevalent G proteins were one member of each G subfamily (G_s, G_i3, G₁₁, and G₁₃). We substantially abrogated expression of most of the G and G proteins expressed in these cells, singly and some in combinations. As expected, agonist-dependent activation of adenylyl cyclase or phospholipase C was specifically eliminated following the silencing of G_s or G_q/11, respectively. We also confirmed that G subunits are necessary for stable accumulation of G proteins in vivo. G subunits demonstrated little isoform specificity for receptor-dependent modulation of effector activity. We observed compensatory changes in G protein accumulation following silencing of individual genes, as well as an apparent reciprocal relationship between the expression of certain G_q and G_i subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks.

Received for publication, October 25, 2005 , and in revised form, January 9, 2006.

^* This work was supported by National Institutes of Health NIGMS Grant GM-34497 and the Raymond and Ellen Willie Chair in Molecular Neuropharmacology (to A. G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 6001 Forest Park, Dallas, TX 75390-9041. Tel.: 214-645-6128; Fax: 214-645-6131; E-mail: Alfred.Gilman{at}UTSouthwestern.edu.

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