Targeted knockdown of G protein subunits selectively prevents receptor-mediated modulation of effectors and reveals...

Heterotrimeric G protein signaling specificity has been attributed to select combinations of Galpha, beta, and gamma 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 Galpha and Gbeta subunits; 2) examining receptor-dependent activities of two effector systems (adenylyl cyclase and phospholipase Cbeta); and 3) systematically silencing each of the Galpha and Gbeta 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 Galpha and Gbeta subunits. The most prevalent Galpha proteins were one member of each Galpha subfamily (Galpha(s), Galpha(i3), Galpha(11), and Galpha(13)). We substantially abrogated expression of most of the Galpha and Gbeta proteins expressed in these cells, singly and some in combinations. As expected, agonist-dependent activation of adenylyl cyclase or phospholipase Cbeta was specifically eliminated following the silencing of Galpha(s) or Galpha(q/11), respectively. We also confirmed that Gbeta subunits are necessary for stable accumulation of Galpha proteins in vivo. Gbeta 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 Galpha(q) and Galpha(i) subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks.