Abstract
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.
Highlights
Vated subunits are capable of modulating the functional properties of effector proteins
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 ␣, , and ␥ subunits that are expressed in single cells
Endogenous G Protein Subunits in HeLa Cells—Ten proteins were identified as products of the 16 mammalian G␣ genes by both RT-PCR and immunoblotting: G proteins (G␣), G␣q, G␣11, G␣i1, G␣i2, G␣i3, G␣o, G␣12, and G␣13
Summary
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. Three days after the second transfection, the cells were used for experiments or harvested for immunoblot analyses and determinations of protein concentration. Total Inositol Phosphate Accumulation—Total inositol phosphate (IP) accumulation was determined by incubating HeLa cells for 48 h with 10 Ci/ml of myo-[2-3H]inositol (1 mCi/ml, PerkinElmer Life Sciences) in inositol-free Dulbecco’s modified Eagle’s medium (Specialty Media, Phillipsburg, NJ) supplemented with 5% fetal bovine serum. Protein concentrations for total lysates and membranes were determined with the Amido Black protein assay (9) using bovine serum albumin as standard. Immunoblotting and Antibodies—In preparation for immunoblotting, total cell lysates (5–30 g) or plasma membranes (5–20 g) were solubilized in SDS-PAGE gel buffer (11). Endogenous HeLa cell protein levels were interpolated from the pixel intensities of the known amounts of purified G␣ or G subunit standards loaded on the same gel. Data Analysis—Differences between measured values (mean Ϯ S.D.) were determined using a one-tailed t test or single analysis of variance (p Ͻ 0.05, InStat, Prism)
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