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 (Gαs, Gαi3, 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, agonist-dependent activation of adenylyl cyclase or phospholipase Cβ was specifically eliminated following the silencing of Gαs or Gαq/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 Gαq and Gαi subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks. 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 (Gαs, Gαi3, 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, agonist-dependent activation of adenylyl cyclase or phospholipase Cβ was specifically eliminated following the silencing of Gαs or Gαq/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 Gαq and Gαi subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks. Signal-transducing heterotrimeric G proteins are associated with the inner face of the plasma membrane, positioned as middlemen for activation by membrane-spanning, heptahelical receptors, and regulation of a variety of intracellular effectors. Interactions among these proteins are controlled by agonist-induced changes of receptor conformation and nucleotide-driven conformational changes of the α subunits of the G proteins (Gα). A ligand-bound receptor catalyzes the exchange of GDP for GTP on a cognate Gα, and as a result, the (at least partial) dissociation of Gα from a complex of Gβ and Gγ subunits. These activated subunits are then capable of modulating the functional properties of effector proteins (e.g. adenylyl cyclases and phospholipases). The intrinsic GTPase activity of Gα serves as a molecular timer, returning the protein to the GDP-bound state and allowing reformation of the inactive heterotrimer. Much remains to be learned about the specificity of G protein signaling in vivo, the relative importance of isoforms of G protein subunits with apparently redundant functions, and the qualitative and quantitative significance of the fact that many hundreds of G protein heterotrimers can be assembled from the collection of G protein α, β, and γ subunits that are expressed in single cells. There is evidence for exquisite specificity of signaling through certain pathways. For example, intranuclear injection of anti-sense oligonucleotides against specific G protein subunits revealed that the M4-muscarinic receptor-mediated inhibition of L-type Ca2+ channels requires Gαoa,Gβ3, and Gγ4, whereas similar inhibition initiated by somatostatin receptors requires Gαob,Gβ1, and Gγ3 subunits (1Kleuss C. Hescheler J. Ewel C. Rosenthal W. Schultz G. Wittig B. Nature. 1991; 353: 43-48Crossref PubMed Scopus (467) Google Scholar, 2Kleuss C. Scherubl H. Hescheler J. Schultz G. Wittig B. Nature. 1992; 358: 424-426Crossref PubMed Scopus (332) Google Scholar, 3Kleuss C. Scherubl H. Hescheler J. Schultz G. Wittig B. Science. 1993; 259: 832-834Crossref PubMed Scopus (332) Google Scholar). The generality of these and related studies has not been examined, and there has been little effort to monitor and understand the compensatory mechanisms that such perturbations may set in motion. Furthermore, studies performed in vitro do not reveal such demanding specificity, and mechanisms of such phenomena are not known. Herein we describe a more comprehensive attempt to examine these issues. We have sought information about the expression of most G protein subunits in a clonal human cell line (HeLa), and we have examined the functional and compensatory effects of siRNA 2The abbreviations used are: siRNA, small-interfering RNA; RNAi, RNA interference; INE, isoproterenol; PGE1, prostaglandin E1; IP, total inositol-phosphate; AC, adenylyl cyclase; PLCβ, phospholipase C β; RT, reverse transcriptase. 2The abbreviations used are: siRNA, small-interfering RNA; RNAi, RNA interference; INE, isoproterenol; PGE1, prostaglandin E1; IP, total inositol-phosphate; AC, adenylyl cyclase; PLCβ, phospholipase C β; RT, reverse transcriptase.-mediated silencing of the expression of genes encoding members of the Gαs, Gαi, Gαq, and Gβ subfamilies of G protein subunits. Reagents—All reagents were purchased from Sigma unless noted otherwise. Mammalian Cell Culture—HeLa cells (from ATCC) were cultured at 37 °C in Dulbecco's modified Eagle's medium with high glucose (Invitrogen) supplemented with 10% fetal bovine serum under an atmosphere of 95% air, 5% CO2. Cells were cultured in the same lot of serum and passaged twice weekly by trypsinization. Fresh cultures were established from the same frozen stock after 10 passages. Transient RNA Interference (RNAi) Transfection—Single-stranded 21-mer oligonucleotides (containing 19 ribonucleotides and 2 3′-deoxythymidine residues) targeting the open reading frames of selected proteins were designed using the Dharmacon siRNA Design Center. Candidate sequences were subjected to further BLAST analysis against the human genome data base (NCBI) and selected for further study if no more than 14 contiguous bases were identical to of human oligonucleotides were as J. W. Nature. PubMed Scopus Google Scholar). 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The of Gβ G protein members. We changes in the amounts of receptors with the of and of adenylyl cyclase and were receptor expression with a in for the receptor in cells protein to of changes in We do not if the changes in adenylyl cyclases are a of of and in of Gαs, or other of Gαs adenylyl cyclase and expression that Gαs to the regulation of adenylyl cyclase We on the generality of the specific interactions that are revealed by these in HeLa cells. revealed many changes in G protein subunit expression and specific interactions are to be on the specific cell the of and relative of G protein subunits. studies using cells and reveal the generality of such is from study that G protein signaling in HeLa cells are and in their to regulation of in the face of The mechanisms used to are and be a to understand and to the concentrations and activities of the many of such are to and to the that We and C. for and and for We also B. and G. for reading of the and for with