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

Abstract

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 , G␣ 11 , and G␣ 13 ). We substantially abrogated expression of most of the G␣ and G␤ proteins expressed in these cells, singly and some in combinations. As expected, agonistdependent 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.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 ␣ subunits of the G proteins (G␣). A ligand-bound receptor catalyzes the exchange of GDP for GTP on a cognate G␣, and as a result, the (at least partial) dissociation of G␣ from a complex of G␤ and G␥ 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 G␣ 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 th...

Minimal Determinants for Binding Activated Gα from the Structure of a Gαi1−Peptide Dimer,

“…If modified to allow cell penetration (e.g., ref. 50), KB-1753 could effectively block Gα i ·GTP/effector signaling while preserving receptor/heterotrimer coupling and Gβγ-mediated signaling, unlike pertussis toxin which permanently impairs Gα i βγ coupling and signaling via ADP-ribosylation (51) or of siRNA-mediated Gnai transcript silencing which can result in compensatory upregulation of other Gα i/o family members (52). The selective interaction of KB-1753 with Gα i subunits over Gα o (Fig.…”

Negative feedback regulation of Wnt signaling by Gβγ-mediated reduction of Dishevelled

“…The sequences of siRNA for Gβ1 and Gβ2 were adapted from Krumins and Gilman (2006). The sequences of siRNAs; siGβ1/Gβ2, 5'-UACGACGACUUCAACUGCATT-3'/5'-UGC-AGUUGAAGUCGUCGUATT-3'; siGFP, 5'-GUUCAGCGU-G U C C G G C G A G T T-3 ' / 5 ' -C U C G C C G G A C A C G C-UGAACTT-3'.…”

“…For these experiments we confirmed that the Wnt1 signaling pathway is intact and functional as indicated by an increased level of β-catenin upon co-transfection of Wnt1 ( Figure 6A). To further confirm that Gβγ signaling is necessary for the Wnt-mediated loss of Dishevelled, siRNA that can knockdown both Gβ 1 and Gβ 2 (Krumins and Gilman, 2006) was added to HEK293T cells. The cells with that had been given siRNA had lowered Gβ 1 and Gβ 2 , but not other control, mRNAs ( Figure 6B) and had the expected diminished block in the loss of Dishevelled induced by either Wnt1 or Wnt5a ( Figure 6C).…”

Assessing the Role of Gαq/11 in Cellular Responses: An Analysis of Investigative Tools

“…These include pools of five~20 nucleotide target-specific siRNAs, pools of three to five lentiviral vector plasmids or particles each encoding target-specific short hairpin RNAs, or non-viral shRNAs specifically targeting Gαq/11 genes. One of the first reported gene knockdown studies of Gα proteins was the knockdown of Gαq and Gα11 gene expression using siRNA in HeLa cells [23]. This work demonstrated an absolute requirement of Gαq/11 to stimulate histamine-mediated PLC activity.…”