Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals complex changes in non-targeted signaling proteins

Heterotrimeric G protein signaling specificity has been attributed to select combinations of Gα, β, and γ 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 Gα and Gβ subunits; 2) examining receptor-dependent activities of two effector systems (adenylyl cyclase and phospholipase Cβ); and 3) systematically silencing each of the Gα and Gβ 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 Gα and Gβ subunits. The most prevalent Gα proteins were one member of each Gα subfamily (Gα_s, Gα_i3, Gα₁₁, and Gα₁₃). We substantially abrogated expression of most of the Gα and Gβ proteins expressed in these cells, singly and some in combinations. As expected, agonistdependent activation of adenylyl cyclase or phospholipase Cβ was specifically eliminated following the silencing of Gα_s or Gα_q/11, respectively. We also confirmed that Gβ subunits are necessary for stable accumulation of Gα proteins in vivo. Gβ 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 Gα_q and Gα_i subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks.