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Originally published In Press as doi:10.1074/jbc.M511551200 on January 30, 2006

J. Biol. Chem., Vol. 281, Issue 15, 10250-10262, April 14, 2006
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Targeted Knockdown of G Protein Subunits Selectively Prevents Receptor-mediated Modulation of Effectors and Reveals Complex Changes in Non-targeted Signaling Proteins*

Andrejs M. Krumins and Alfred G. Gilman1

From the Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, October 25, 2005 , and in revised form, January 9, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterotrimeric G protein signaling specificity has been attributed to select combinations of G{alpha}, 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 G{alpha} and Gbeta subunits; 2) examining receptor-dependent activities of two effector systems (adenylyl cyclase and phospholipase Cbeta); and 3) systematically silencing each of the G{alpha} 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 G{alpha} and Gbeta subunits. The most prevalent G{alpha} proteins were one member of each G{alpha} subfamily (G{alpha}s, G{alpha}i3, G{alpha}11, and G{alpha}13). We substantially abrogated expression of most of the G{alpha} 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 G{alpha}s or G{alpha}q/11, respectively. We also confirmed that Gbeta subunits are necessary for stable accumulation of G{alpha} 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 G{alpha}q and G{alpha}i subfamily members. These findings provide a foundation for understanding the mechanisms that regulate the adaptability and remarkable resilience of G protein signaling networks.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha} subunits of the G proteins (G{alpha}). A ligand-bound receptor catalyzes the exchange of GDP for GTP on a cognate G{alpha}, and as a result, the (at least partial) dissociation of G{alpha} from a complex of Gbeta and G{gamma} 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{alpha} 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 {alpha}, beta, and {gamma} 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 G{alpha}oa,Gbeta3, and G{gamma}4, whereas similar inhibition initiated by somatostatin receptors requires G{alpha}ob,Gbeta1, and G{gamma}3 subunits (1-3). 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 siRNA2-mediated silencing of the expression of genes encoding members of the G{alpha}s, G{alpha}i, G{alpha}q, and Gbeta subfamilies of G protein subunits.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 coding regions of known human gene sequences. Single-stranded oligonucleotides were annealed as described (4). The sequences of the RNAi sense strands and the targeted sites in the open reading frames are shown in Table 1. Transient transfections of RNAi duplexes were accomplished using Oligofectamine transfection reagent (Invitrogen) according to manufacturer's instructions. Briefly, HeLa cells were seeded at 44 x 104/100-mm dish 24 h before transfection. The cells were transfected using 19 µl of Oligofectamine and a total of 190 pmol of RNAi duplex(es) in a final volume of 3.8 ml. Two days later the cells were trypsinized, counted, plated at 55 x 104/100-mm dish, and subjected to a second transfection. One day after the second transfection the cells were trypsinized, pooled, and distributed to 12-well plates. Three days after the second transfection, the cells were used for experiments or harvested for immunoblot analyses and determinations of protein concentration.


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  		TABLE 1
Reagents used in the present study to silence or identify proteins

 
cAMP Accumulation—cAMP accumulation assays were conducted using cells seeded into 12-well plates (7-10 x 104 cells/well) and incubated with 10 µCi/ml of [2,8-3H]adenine (1 mCi/ml, PerkinElmer Life Sciences) for 24 h in culture medium. Cells were incubated with cyclic nucleotide phosphodiesterase inhibitors (100 µM Ro-20-1724 and 500 µM 3-isobutyl-1-methylxanthine) for 5 min at 30 °C prior to the initiation of ligand-stimulated cAMP synthesis. Experiments were terminated by addition of ice-cold 5 mM ATP, 0.2 mM cAMP, and 5% trichloroacetic acid containing [32P]cAMP (to monitor recovery during cAMP purification). Radiolabeled cAMP was separated from other labeled compounds by successive Dowex (AG 50W-X4, Bio-Rad) and alumina chromatography as described (5).

Total Inositol Phosphate Accumulation—Total inositol phosphate (IP) accumulation was determined by incubating HeLa cells for 48 h with 10 µCi/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. Cells were incubated for 20 min at 37 °C with bicarbonate-free Dulbecco's modified Eagle's medium, 20 mM Na-HEPES (pH 7.4) containing 20 mM LiCl prior to the initiation of ligand-dependent IP synthesis. Reactions were terminated by addition of ice-cold 10 mM formic acid to precipitate protein. The soluble lysate was applied to a 1-ml Dowex AG (1-X8) column, which was washed successively with 15 ml of 10 mM formic acid containing 10 mM myo-inositol and 15 ml of 5 mM Na-tetraborate, 60 mM ammonium formate, prior to elution with 2.0 ml of 1.0 M ammonium formate, 0.1 M formic acid. Total [3H]IP was determined by scintillation counting and normalized to the amount of acid-precipitated protein. Protein was quantified as described by Bradford (Bio-Rad) (6).

Radioligand Binding—beta2-Adrenergic receptor concentrations were estimated for cells seeded in 6-well plates using 2 nM [125I]CYP (PerkinElmer Life Sciences) in the presence or absence of the beta2-adrenergic receptor-selective antagonist ICI-118,551 (10 µM) or the non-selective beta-adrenergic receptor antagonist timolol (10 µM). Each measurement was conducted in triplicate. Ligand binding reactions were quenched in ice-cold 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 100 mM NaCl and immediately filtered through Whatman GF/C filters. Radioactivity was quantified by scintillation counting. Duplicate 6-well plates were cultured to permit determinations of cell number and total protein content.

Recombinant G{alpha} and Gbeta Proteins—Recombinant bovine G{alpha}s,short, G{alpha}i1 (rat), G{alpha}i2 (rat), and G{alpha}i3 (rat) were purified after expression in Escherichia coli as described (7); the G{alpha}i proteins were coexpressed with protein N-myristoyl transferase and were thus myristoylated at their amino termini. Sf9 cells were coinfected with baculoviruses encoding human G{alpha}s,long, mouse G{alpha}11, or mouse G{alpha}13 and baculoviruses encoding Gbeta1 and His6-G{gamma}2, and the G proteins were isolated as described (8). Purified mouse G{alpha}q and G{alpha}12 were gifts from G. Tall and P. Sternweis, respectively (University of Texas Southwestern Medical Center). Recombinant bovine Gbeta1 and human Gbeta2, Gbeta3, and Gbeta4 proteins were provided by S. Gibson (University of Texas Southwestern Medical Center). Protein concentrations were determined using an Amido Black staining assay (9). Purity was >90% for all proteins as assessed by Coomassie Blue-stained SDS-PAGE gels.

Sample Preparation for Immunoblotting—Total cellular lysates were prepared by rinsing cells with phosphate-buffered saline and harvesting in SDS-PAGE lysis buffer (62.5 mM Tris-HCl, pH 6.8, 1.25% (w/v) SDS, 12.5% (v/v) glycerol, 0.2% (w/v) bromphenol blue, 25 mM dithiothreitol, and 1.25% (v/v) beta-mercaptoethanol). The lysates were centrifuged for 1 h (100,000 x g, 4 °C, Beckman TLA 45 rotor) to pellet viscous DNA.

Plasma membrane fractions from HeLa cells were prepared essentially as described (10). Briefly, cells were disrupted by nitrogen cavitation (600 p.s.i. for 30 min). Nuclei and unbroken cells were removed by centrifugation for 5 min (600 x g, 4 °C). The resulting supernatant fraction was layered onto a 23 and 43% sucrose step gradient and centrifuged (100,000 x g, 1 h, 4°C, Beckman SW 41 rotor). The enriched plasma membranes were collected at the gradient interfaces and recovered by centrifugation (200,000 x g, 30 min, 4 °C, Beckman TLA 100.3 rotor). The membranes were suspended in 20 mM Na-HEPES (pH 7.4), 150 mM NaCl, flash frozen in liquid N2, and stored at -80 °C. 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 µg) or plasma membranes (5-20 µg) were solubilized in SDS-PAGE gel buffer (11). For detecting adenylyl cyclase proteins, plasma membranes (50-100 µg) were treated with 1% SDS, 0.2 mM dithiothreitol, and 5 mM N-ethylmaleimide (5 min at 80 °C) prior to the addition of SDS-PAGE buffer.

Antibody specificities, dilutions, and sources are shown in Table 1. Following incubation with primary antibody, immunoblots were washed with phosphate-buffered saline containing 0.1% Tween 20. Secondary anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase were diluted in wash buffer and incubated with the blots for 1 h. The blots were subsequently washed and immunoreactive bands were detected by enhanced chemiluminescence (PerkinElmer Life Sciences).

Densitometry—Immunoblots were scanned into Adobe Photoshop 5.0, and the pixel intensities (arbitrary units) of the immunoreactive bands were quantified. For quantitative immunoblots, endogenous HeLa cell protein levels were interpolated from the pixel intensities of the known amounts of purified G{alpha} or Gbeta subunit standards loaded on the same gel. For relative protein determinations, the pixel intensity of immunoreactive bands in samples prepared from RNAi-transfected cells was normalized to the corresponding immunoreactive band intensity identified in an equal amount of lysate prepared from mock-transfected cells.

Quantitative Real-time PCR—Relative quantification of mRNA levels was achieved using quantitative RT-PCR. Single component amplification reactions were conducted using the relative standard curve or comparative Ct methods and TaqMan Gene Expression Assays (Applied Biosystems) according to manufacturer's instructions. Briefly, a TaqMan gene expression probe specific for amplifying a particular G{alpha} or Gbeta subunit cDNA sequence was mixed together with a One-Step TaqMan RT-PCR master mixture and 100 ng of total RNA/reaction. Total RNA was isolated from HeLa cells using the RNAqueous-4PCR kit (Ambion) and quantified spectro-photometrically (A260). Fluorescence data were collected following each amplification cycle using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Ct values were calculated from threshold values set to manual.


Figure 1
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  		FIGURE 1.
Identification of G protein subunits in HeLa cells. A, Western blots of endogenous G{alpha} subunits in total lysates of unperturbed HeLa cells. Proteins detected by immunoblotting are indicated at the left of each panel. Antisera specificity was verified by blotting total lysates of cells where expression of individual subunits was silenced by RNA interference (shown across the top of each panel). Positions of the long (52 kDa) and short (46 kDa) G{alpha}s splice variants, as well as immunodetectable bands corresponding to other G{alpha} subunits, are indicated by lines. G{alpha}q (42 kDa) and G{alpha}i1 (40 kDa) are each shown migrating below a nonspecific immunoreactive band. Equal amounts (30 µg) of total lysates were loaded in each lane of gels used for Western immunoblotting of G{alpha} subunits. G{alpha}12 antiserum did not cross-react with purified G{alpha}13 standard (not shown). B, Western blots of endogenous Gbeta subunits in total lysates of unperturbed HeLa cells. Gbeta subunits targeted for RNAi silencing are shown across the top of each panel, and proteins identified by Western blotting are indicated at the left. Gbeta1 migrated as a 36-kDa protein, whereas Gbeta2 and Gbeta4 migrated as 35 kDa. Thirty µg of each total lysate was loaded per lane for individual Gbeta isoform examination by Western blotting and 5 µg/lane of each total lysate was loaded for Western blotting examination of total Gbeta1-4 subunit expression. C, results of quantitative Western blotting for G{alpha} and Gbeta subunits in unperturbed HeLa total cell lysates. Five to 30 µg/lane of each lysate preparation was separated by SDS-PAGE along with purified G{alpha} or Gbeta subunit standards (1-10 ng range). Western blots and densitometry analysis were conducted as described under "Experimental Procedures." Each bar represents the mean ± S.D. of 10 different total cell lysates. G{alpha} protein amounts are represented by open bars and Gbeta proteins by closed bars.

 
Data Analysis—Differences between measured values (mean ± S.D.) were determined using a one-tailed t test or single analysis of variance (p < 0.05, InStat, Prism).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous G Protein Subunits in HeLa Cells—Ten proteins were identified as products of the 16 mammalian G{alpha} genes by both RT-PCR (data not shown) and immunoblotting (see control lanes in Fig. 1A): G{alpha}s (long and short splice variants), G{alpha}q,G{alpha}11,G{alpha}i1,G{alpha}i2,G{alpha}i3,G{alpha}o,G{alpha}12, and G{alpha}13. All positive RT-PCR signals were confirmed by cDNA sequencing, and antibody specificity was verified by target-specific RNAi-mediated silencing of antigens (also shown in Fig. 1A). Transcripts of two additional G{alpha} genes, G{alpha}z and G{alpha}16, were identified by RT-PCR, but the corresponding proteins were not detected despite multiple attempts to blot 100 µg of enriched plasma membrane fractions with sensitive antibodies (detection limit of <1 ng of purified protein). No mRNA or protein was detected for G{alpha}14. We did not address possible expression of G{alpha} subunits that are primarily found in specialized sensory tissues (G{alpha}olf, G{alpha}t (1 and 2), and G{alpha}g).

Of the five Gbeta genes, we detected expression of four in HeLa cells: Gbeta1, Gbeta2, Gbeta4, and Gbeta5; we found no evidence for expression of Gbeta3. Because Gbeta5 subunits appear to form selective heterodimers with RGS proteins of the R7 family (which contain G{gamma}-like domains) (12) and there is little credible evidence for the existence in vivo of heterodimers containing Gbeta5 and G{gamma} subunits, we focused our efforts on Gbeta1, Gbeta2, and Gbeta4 subunits (Fig. 1B).

We also examined the expression of G{gamma} subunits in HeLa cells. Perhaps surprisingly, RT-PCR analysis positively identified transcripts of all 12 G{gamma} genes, including G{gamma}1, whose expression is largely restricted to the retina (13). Because of the lack of specific and sensitive antisera to most G{gamma} proteins, we have limited our studies to date to G{alpha} and Gbeta.


Figure 2
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  		FIGURE 2.
Effects of silencing G{alpha}s,G{alpha}i1-3,orG{alpha}q/11 on ligand-modulated effector responses. cAMP responses in control cells (closed square) and cells lacking G{alpha}s (closed triangle) were examined following exposure to either: A,10 µM INE, or B,10 µM PGE1 for the indicated times. Each data point represents the average ± the range of duplicate measurements. The figure is representative of at least three independent experiments. C, total IP accumulation in whole cells lacking G{alpha}q (closed triangle), G{alpha}11 (inverted closed triangle), or G{alpha}q and G{alpha}11 (G{alpha}q/11, closed diamond). Total IP responses were examined in cells exposed to 100 µM histamine for the indicated times. Each point represents the average of duplicate measurements of the amount of [3H]IP (cpm) recovered from Dowex column chromatography normalized to total protein content ± the range. The data are representative of at least three independent experiments. D, inhibition of cAMP synthesis in cells lacking G{alpha}i subunits. [3H]cAMP production in cells was monitored over time following the addition of either: D, 200 µM forskolin or forskolin (200 µM) and the synthetic {alpha}2-adrenergic agonist UK 14,304 (10 µM), or E, 10 µM PGE1 in the presence and absence of 10 µM UK 14,304. Data are presented as the percent of forskolin-stimulated cAMP synthesis that was inhibited by UK 14,304 following 5 min of ligand treatment. Each bar represents the mean percent inhibition ± S.D. from three independent experiments.

 
Prior to examining the effects of silencing individual G protein subunits, we quantified the amounts of G{alpha} and Gbeta proteins in total lysates from control cells (Fig. 1C). Quantitative immunoblotting revealed that 80% of the entire G{alpha} subunit protein pool was accounted for by one member of each of the four subfamilies of G{alpha} proteins: G{alpha}i3, G{alpha}11, G{alpha}13, and the sum of long and short splice variants of G{alpha}s.Gbeta1 and Gbeta2 were expressed at similar levels and together accounted for 80% of the total Gbeta subunit pool; Gbeta4 comprised the remaining 20%. There was no significant difference between the total amount of Gbeta protein detected with a pan Gbeta antibody (Gbeta1-4) and the sum of Gbeta subunits calculated from data obtained using isoform-specific Gbeta antibodies. Transformation of the data in Fig. 1C to molar quantities reveals that the sum of the G{alpha} subunits (22 ± 2 pmol/mg lysate) closely approximates that of the Gbeta proteins (21 ± 3 pmol/mg of lysate), consistent with the notion that most or all of these subunits exist as oligomers.

Knockdown of G Protein Subunits—Immunoblots showed remarkable (more than 90%) knockdown of endogenous protein expression for each G{alpha} and Gbeta subunit (Fig. 1, A and B) after silencing by siRNAs, either individually or in combination. Greater than 90% knockdown of G{alpha} subunits was deemed necessary to test changes in receptor-modulated effector responses, because the reported stoichiometry of G protein to effector can, in some instances, exceed 10:1 (14). Thus, multiple RNAi duplexes (range: 2 to 10) were designed and generated toward each target; in general, most duplexes silenced target protein expression more than 75%.

In several instances (particularly with Gbeta subunits) treatment with RNAi oligonucleotides not only silenced the intended target but also caused cell death. Interestingly, our most lethal RNAi duplex targeted green fluorescent protein using a sequence with no significant (>12 nucleotide) overlap with the open reading frame of any known human gene, indicating that cell death may be caused by mechanisms unrelated to specific mRNA sequence recognition by RISC complexes. We rigorously avoided using siRNAs that had adverse effects on cell growth or viability.

Knockdown of G{alpha}s: Anticipated Effect on Ligand-stimulated Activation of Adenylyl Cyclases—G{alpha}s proteins mediate activation of adenylyl cyclases by so called Gs-coupled G protein-coupled receptors. As anticipated, knockdown of G{alpha}s completely abrogated ligand-dependent increases in cAMP synthesis (Fig. 2, A and B) stimulated by either INE, a beta-adrenergic receptor agonist, or PGE1, an EP prostanoid receptor agonist. A combination of RT-PCR, radioactive ligand-binding assays, and RNAi-mediated silencing of cell surface receptors (see Table 1) demonstrated that INE stimulation of cAMP synthesis occurred exclusively via the beta2-adrenergic receptor, whereas PGE1 signaling was mediated via two EP receptor isoforms, EP2 and EP4 (data not shown). We found no evidence of expression of beta1-or beta3-adrenergic receptors or EP1 or EP3 prostanoid receptors in HeLa cells. The functional effects of loss of G{alpha}s were specific. Thus, silencing of G{alpha}s did not prevent UK 14,304-dependent inhibition of cAMP synthesis or stimulation of phospholipase Cbeta activity by histamine (data not shown). These data confirm the functionality and specificity of G{alpha}s-mediated activation of adenylyl cyclases using RNAi-mediated gene suppression and illustrate the utility of this approach to study G protein signaling in a population of intact cells.

Silencing of G{alpha}q and G{alpha}11: Anticipated Effects on Activation of Phospholipase Cbeta—G{alpha}q and G{alpha}11 (along with G{alpha}14 and G{alpha}16) mediate cell surface receptor activation of PLCbeta, which catalyzes the synthesis of inositol (1,4,5)-trisphosphate from phosphatidylinositol (4,5)-bisphosphate. Inositol phosphate (IP) accumulation in HeLa cells was increased following activation of the H1-histamine receptor by agonist. The simultaneous knockdown of G{alpha}q and G{alpha}11 (denoted as G{alpha}q/11) eliminated histamine-stimulated IP accumulation (Fig. 2C). Interestingly, the silencing of either G{alpha}q or G{alpha}11 individually decreased histamine-dependent IP accumulation by ~50%, despite the fact that concentrations of G{alpha}11 in HeLa cells exceed those of G{alpha}q by 10-fold (Fig. 1C). The amount of [3H] incorporated into acid-precipitable material was unchanged in cells lacking G{alpha}q/11, suggesting that phosphatidylinositol kinase and phosphatase activities were unaffected. Silencing G{alpha}q/11 modestly (but significantly) increased ligand-dependent cAMP responses (~30%) without affecting UK 14,304-mediated inhibition of adenylyl cyclase (data not shown).

Knockdown of G{alpha}i/o: Incomplete Effects on Inhibition of Adenylyl Cyclase—G{alpha}i/o subunits transduce signals from cell surface receptors to a variety of intracellular pathways (see Ref. 15 for review), including inhibition of adenylyl cyclase activity. cAMP synthesis in HeLa cells can be inhibited by {alpha}2a- and {alpha}2b-adrenergic receptors.3 Despite silencing each of the G{alpha}i1-3 subunits, individually or in combinations of two, we observed no significant change in the ability of the synthetic {alpha}2-adrenergic receptor agonist UK-14,304 to inhibit INE-(data not shown) or forskolin-stimulated cAMP synthesis (Fig. 2D). We observed a 60% reduction in the magnitude of such inhibition only when all three G{alpha}i subunits were silenced (denoted G{alpha}i1-3) (Fig. 2D). Thus, {alpha}2-adrenergic receptors displayed no absolute specificity for G{alpha}i1-3 isoforms for the inhibition of INE or forskolin-stimulated cAMP synthesis.

Interestingly, knockdown of G{alpha}i1 did specifically interfere with UK 14,304-mediated inhibition of PGE1-stimulated cAMP synthesis (Fig. 2E), suggesting that a PGE1-stimulated adenylyl cyclase may preferentially localize with or couple to this G protein. It is most puzzling, therefore, that simultaneous silencing of G{alpha}i1-3 subunits did not abrogate inhibition of PGE1-activated cAMP synthesis by UK 14,304. Residual inhibition of adenylyl cyclase activity in all experiments was eliminated by treatment of cells with pertussis toxin, suggesting that the G{alpha}i subunit expression may have been insufficiently silenced and/or that G{alpha}o subunits, in the absence of G{alpha}i1-3 subunits, may have contributed to the inhibition of cAMP synthesis (16). Individual silencing of G{alpha}o (both a and b splice variants) had no effect on UK-14,304-mediated inhibition of cAMP synthesis. Recall that G{alpha}z was not detected in these cells.

Silencing of G{alpha} Subunits: Alterations in Accumulation of Other G{alpha} Proteins—Silencing of individual G protein {alpha} subunits routinely resulted in loss of more than 90% of that protein, detected by immunoblotting (Table 2, highlighted in yellow). Quantification of non-targeted G{alpha} proteins revealed that the concentrations of G{alpha}i3 and G{alpha}11 (Table 2) and G{alpha}13 (data not shown) were not significantly altered by such perturbations. However, we observed modest (~2-fold) increases in concentrations of G{alpha}i1 protein following knockdown of G{alpha}i2 or G{alpha}i3 (Table 2, highlighted in green) and increased G{alpha}i2 protein after silencing of G{alpha}i3. Thus, loss of the major isoform of G{alpha}i resulted in increased accumulation of the other two isoforms of G{alpha}i. Increased accumulation of G{alpha}i1 protein was associated with a dramatic increase in detectable mRNA for this protein (Fig. 3A). The level of G{alpha}o protein also increased modestly, but significantly, after combined knockdown of all three G{alpha}i subunits (Table 2). These data indicate that reductions in the concentrations of G{alpha}i isoforms are associated with compensatory changes in the levels of other proteins within the G{alpha}i subfamily.


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  		TABLE 2
Relative G{alpha} and Gbeta protein expression following select G protein subunit silencing

G protein subunits targeted for siRNA silencing are shown across the top of the table and proteins identified by Western blotting are indicated at the left. Western blots and densitometry analyses were conducted as described under "Experimental Procedures." Each value corresponds to the pixel intensity of a particular antigen identified in siRNA-transfected total cell lysates normalized against the pixel intensity obtained for the same antigen in unperturbed control cell lysates loaded on the same gel. Data are represented as the mean ± S.D. for a minimum of three separate total lysate preparations. Expression data were analyzed using single-sided analysis of variance (p {Delta} 0.05) comparison of log (base 2) transformation of relative antigen expression for a particular perturbation against log transformation of the variance of protein expression for the same antigen determined from multiple control lysate preparations (range 5-12). Effects of siRNA transfection directed at the antigen are highlighted in yellow. Significant increases in non-targeted protein expression following a knockdown of G protein subunits are highlighted in green, and significant decreases are highlighted in red.

 


Figure 3
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  		FIGURE 3.
Relative mRNA and protein expression of G protein subunits following select G{alpha} silencing. A, quantitative PCR were conducted using total RNA isolated from control cells and cells lacking G{alpha}s, G{alpha}q, G{alpha}11, G{alpha}i1, G{alpha}i2, G{alpha}i3, or G{alpha}o. Relative amounts of G{alpha} subunit mRNA expression (G{alpha}s (black), G{alpha}q (pale blue), G{alpha}11 (orange), G{alpha}13 (purple), G{alpha}i1 (pink), G{alpha}i2 (pale green), G{alpha}i3 (yellow), and G{alpha}o (blue)) are shown for cells lacking selected G{alpha} protein expression (shown across the bottom). -Fold differences were calculated using the D{Delta}Ct method. Ct values for each probe were corrected for the amount of endogenous 18 S rRNA in each sample and normalized using Ct values determined for each probe in unperturbed cell total RNA. Each bar represents the mean ± S.D. from three separate total RNA preparations. B, Western blots of G{alpha}o, G{alpha}i1, and G{alpha}q following knockdown of select G{alpha} subunits. G{alpha} proteins targeted for RNAi silencing are shown across the top of each panel and proteins identified by Western blot are indicated to the left of each panel. Migration of G{alpha}q, indicated by the line, is below a nonspecific band. The data are representative of at least three separate experiments. C, relative Gbeta mRNA levels following knockdown of G{alpha} subunits. The relative Gbeta subunit mRNA expression (Gbeta1 (pale red), Gbeta2 (green), and Gbeta4 (brown)) is shown using the same total RNA preparations described in A. Each bar represents the average ± S.D. of three separate preparations.

 
Unexpectedly, we detected substantially increased concentrations of G{alpha}i1 and G{alpha}o protein following the knockdown of either G{alpha}q, G{alpha}11, or both of these proteins (denoted as G{alpha}q/11) (Fig. 3B and Table 2, highlighted in green). These changes in protein concentrations were accompanied by increases in mRNA for both G{alpha}i1 and G{alpha}o (Fig. 3A). Taken together with the fact that the absolute amounts of G{alpha}11 and G{alpha}q are so different (and observed effects are thus less likely to be caused by alterations of ratios of G{alpha} to Gbeta{gamma}), these data suggest that G{alpha}q/11 proteins regulate (directly or indirectly) the rate of synthesis of G{alpha}i1 and G{alpha}o. Although knockdown of G{alpha}q and G{alpha}11 increased accumulation of G{alpha}i1 and G{alpha}o, the reciprocal was not generally true. However, a modest increase in G{alpha}q protein level was observed following knockdown of G{alpha}i3 (Fig. 3B and Table 2, highlighted in green). Amounts of G{alpha}12 mRNA were also increased about 4-fold in cells lacking G{alpha}q/11 protein (data not shown), but the effects on protein concentrations were not examined because of lot variability of the anti-G{alpha}12 antibody.

G{alpha}i1 stands out in examination of all of the effects of G{alpha} protein knockdowns on G protein expression. Although apparently a minor player in HeLa cells in terms of protein concentration (Fig. 1C), levels of G{alpha}i1 message and protein increased significantly when expression of G{alpha}s, G{alpha}q, G{alpha}11,G{alpha}i2,orG{alpha}i3 was compromised (Fig. 3, A and B, and Table 2).

We also examined the effects of silencing G{alpha} protein expression on Gbeta subunits (Fig. 3C and Table 2). Interesting and in some cases specific effects were observed, although they were not predictable by consideration of the relative concentrations of the proteins involved or other facts known to us. Thus silencing of G{alpha}s, G{alpha}11, or individual G{alpha}i isoforms (G{alpha}i2 and G{alpha}i3) significantly decreased accumulation of Gbeta4, the least prevalent Gbeta subunit. Knockdown of G{alpha}s also decreased levels of Gbeta2, whereas silencing G{alpha}i2 decreased levels of Gbeta1 substantially, while having only modest effects on other Gbeta proteins. The simultaneous knockdown of the three G{alpha}i proteins (denoted as G{alpha}i1-3) decreased the total immunodetectable Gbeta subunit pool by more than 50%. Decreased accumulation of Gbeta protein was associated with decreased Gbeta mRNA levels following knockdown of G{alpha}s and G{alpha}i3. However, the effect of G{alpha}i2 knockdown was not correlated with changes in levels of Gbeta mRNA.

Knockdown of Single and Paired Gbeta Isoforms: Alterations in Expression of Other G Protein Subunits—Gbeta subunits were targeted for silencing in attempts to observe the coordinated disappearance of combinations of G{alpha} and Gbeta subunits that might be prevalent in vivo, isoform-specific functions of Gbeta subunits, and regulation of Gbeta subunit expression. siRNA-mediated targeting of Gbeta1, Gbeta2, or Gbeta4 resulted in loss of more than 90% of immunodetectable protein (Fig. 1B and Table 2, highlighted in yellow). No dramatic changes were observed in non-targeted Gbeta proteins or mRNA (not shown) following the silencing of individual Gbeta proteins, although Gbeta1 and Gbeta2 levels appeared to rise modestly following silencing of Gbeta4.NoGbeta3 protein was detected after Gbeta1, Gbeta2, or Gbeta4 knockdown. Regulation of expression of individual Gbeta isoforms thus appears to occur largely independently of other individual isoforms.


Figure 4
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  		FIGURE 4.
Protein and relative mRNA expression of G{alpha} subunits following silencing of Gbeta isoforms. A, Western blots of G{alpha}s, G{alpha}11, G{alpha}13, G{alpha}i1, G{alpha}i2, and G{alpha}i3 using total lysates prepared from unperturbed cells and cells lacking Gbeta1,Gbeta2, Gbeta4,Gbeta1/2, or Gbeta1/2/4. The data are representative of four independent preparations for each condition. B, relative G{alpha} mRNA expression in cells lacking Gbeta protein expression. Relative G{alpha} mRNA amounts (G{alpha}s (black), G{alpha}11 (orange), G{alpha}13 (purple), G{alpha}i1 (pink), G{alpha}i2 (pale green), and G{alpha}i3 (yellow)) were determined as described previously using total RNA prepared from cells lacking Gbeta1, Gbeta2, Gbeta4, Gbeta1/2, and Gbeta1/2/4. Each bar represents the average ± range of relative mRNA levels calculated from two independent preparations. C, amount of G{alpha} and Gbeta subunit protein expression in total lysates prepared from cells lacking Gbeta1/2 expression. Total cell lysates were prepared from Gbeta1/2 RNAi-transfected cells and quantitative Western blots were obtained for each G{alpha} and Gbeta subunit as described previously. Each bar represents the average ± S.D. from six independent preparations.

 
Because G{alpha} proteins are stabilized by association with Gbeta{gamma} dimers (17), we hypothesized that loss of a single Gbeta subunit might reduce the level of cognate G{alpha} proteins. Because three Gbeta subunits must "service" 10 G{alpha} proteins, overlapping relationships are mandatory. Silencing of Gbeta1 resulted in reduced accumulation of G{alpha}s and G{alpha}i3; knockdown of Gbeta2 decreased G{alpha}s,G{alpha}i1, and G{alpha}i3; and silencing Gbeta4 diminished G{alpha}q and G{alpha}o while increasing G{alpha}i1 and G{alpha}i2 (Fig. 4A and Table 2). Reduced accumulation of G{alpha} proteins was not well correlated with changes in mRNA levels (Fig. 4B and not shown), consistent with the notion that Gbeta{gamma} complexes promote stability of G{alpha}. In examining these data, G{alpha}i1 again stands out. Silencing of any Gbeta provoked an increase in G{alpha}i1 mRNA; this is especially true of Gbeta4 and correlates with the increase in G{alpha}i1 protein seen with this perturbation. These Gbeta subunit-specific effects on accumulation of G{alpha} protein are consistent with the existence of isoform-specific combinations of G{alpha} and Gbeta subunits in unperturbed HeLa cells.

Silencing of individual Gbeta isoforms did not decrease concentrations of G{alpha}i2, G{alpha}11, and G{alpha}13, suggesting that G{alpha}s, G{alpha}i1, G{alpha}i3, and G{alpha}o are less stable than G{alpha}i2, G{alpha}11, and G{alpha}13 in the absence of Gbeta or that the latter group more readily redistributes among other Gbeta subunits. To shed further light on these questions, we evaluated the effects of simultaneous silencing of multiple Gbeta subunits.

Simultaneous knockdown of Gbeta1 and Gbeta2 was accomplished with either of two distinct RNAi duplexes, targeting site 865 (denoted as Gbeta1/2(a)) or 866 (denoted as Gbeta1/2(b)), both of which contain 19-nucleotide identities with the Gbeta1 and Gbeta2 open reading frames. Concomitant silencing of Gbeta1 and Gbeta2 eliminated nearly all G{alpha}i family protein, in contrast with a recent study (18) in which the G{alpha}i subfamily protein was maintained in a cultured mouse J774A.1 macrophage cell line following the knockdown of Gbeta1 and Gbeta2.

Surprisingly, G{alpha}s protein expression was maintained at 50% of control values despite the loss of Gbeta1/2 (Fig. 4C and Table 2), as were G{alpha}q, G{alpha}11,G{alpha}13, and G{alpha}o. Interestingly, each G{alpha} subfamily was represented despite the knockdown of 80% of the initial Gbeta pool. Sustained expression of some G{alpha} proteins in our Gbeta1/2 ablated cells could not be attributed to greater stability of these free G{alpha} subunits because expression of nearly all G{alpha} subunits was lost following the silencing of all Gbeta isoforms (Fig. 4A and Table 2). Decreased G{alpha} protein concentrations following knockdown of multiple Gbeta subunits occurred independently of changes in levels of G{alpha} subunit mRNA (Fig. 4B), likely indicating that Gbeta subunits are requisite for the stability of virtually all G{alpha} proteins.


Figure 5
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  		FIGURE 5.
Relative Gbeta mRNA and protein expression as a function of time following concomitant knockdown of Gbeta1 and Gbeta2 (Gbeta1/2(a)). Relative mRNA levels for: A, Gbeta1; B, Gbeta2; and C,Gbeta4 are represented by dashed lines (open square for control and open triangle for Gbeta1/2 silenced preparations) and relative protein expression is represented by solid lines (closed square for control and closed triangle for Gbeta1/2 silenced preparations). Each point is normalized against mRNA or protein expression in 1-h mock-transfected cells and represents the average ± range of two independent preparations.

 
The retention of some G{alpha} subunits in cells lacking the Gbeta1/2 protein was likely because of formation of new associations with increased concentrations of Gbeta4 (Fig. 1B, Table 2, and Fig. 5). No Gbeta3 protein was detected in cells where Gbeta1 and Gbeta2 were eliminated. Increased Gbeta4 protein was detected 48-72 h after transfection with siRNA directed against Gbeta1/2 until a new steady-state level of Gbeta4 protein was established (compare the relative Gbeta4 level 72 h after transfection (Fig. 5C) with that after 144 h (Table 2)). Interestingly, there was no detectable increase in Gbeta4 mRNA during the same time period.

Knockdown of Single and Multiple Gbeta Subunits: Functional Effects— Silencing of Gbeta1 (by either of two RNAi duplexes) substantially reduced PGE1-dependent activation of cAMP synthesis by 70% (Fig. 6A). Targeted knockdown of Gbeta2 and/or Gbeta4, on the other hand, had little effect on PGE1-dependent activity. The data indicate that at least one of the EP2 and EP4 prostanoid receptors primarily stimulates cAMP synthesis via G{alpha}s associated with Gbeta1 in HeLa cells. This interesting example of specificity is perhaps reminiscent of the observation (above) that G{alpha}i1 was specifically required for {alpha}2-adrenergic receptor-mediated inhibition of PGE1 (but not INE- or forskolin-)-stimulated adenylyl cyclase activity.

By contrast INE-stimulated cAMP synthesis was mostly unaffected following the silencing of Gbeta1, Gbeta2, or Gbeta4 (Fig. 6B). Radioligand binding studies suggest that possible loss of INE-stimulated activity in cells lacking Gbeta1 may have been offset, at least in part, by a compensatory increase in the beta2-adrenergic receptor density: control = 23,200 ± 10,200; knockdown of Gbeta1 = 53,600 ± 17,100, Gbeta2 = 29,200 ± 4,300, and Gbeta4 = 28,200 ± 10,100 (receptors/cell for n = 3 independent experiments measured in triplicate). Cells lacking both Gbeta1 and Gbeta2 showed no difference in receptor density (26,500 ± 12,100) compared with control cells.

Knockdown of individual Gbeta subunits did not affect histamine-dependent activation of PLCbeta (Fig. 6C), and little requirement for specific Gbeta isoforms was observed for {alpha}2-adrenergic receptor-dependent inhibition of ligand- or forskolin-activated cAMP synthesis (Fig. 6, D and E, and data not shown).

We then examined the effects of simultaneous elimination of Gbeta1 and Gbeta2 on ligand-dependent effector activity; these proteins account for 80% of the total cellular Gbeta in unperturbed cells. Histamine-dependent accumulation of IP was unaffected (Fig. 6C), whereas {alpha}2-adrenergic receptor-mediated inhibition of ligand-activated cAMP synthesis was reduced by at least 90% (Fig. 6, D and E). These data indicate that Gbeta4 could fully sustain both the stability and function of the G{alpha}q subfamily of G{alpha}s proteins but not the G{alpha}i subfamily, whose members largely disappear under this condition. Coincidental silencing of all three Gbeta subunits largely abrogated ligand-dependent accumulation of cAMP and IP. No Gbeta3 protein was detected in cells lacking Gbeta1, Gbeta2, and Gbeta4.

Very curiously, silencing of both Gbeta1 and Gbeta2 did not cause the loss of PGE1-dependent cAMP synthesis that occurred following the knockdown of only Gbeta1 (Fig. 6A). It also did not impair INE-stimulated adenylyl cyclase activity, which is not unanticipated because G{alpha}s concentrations are reasonably well maintained under this circumstance. Instead, PGE1- and INE-dependent activities were augmented nearly 2-fold in cells transfected with one siRNA duplex directed at both Gbeta1 and Gbeta2 (Gbeta1/2(b)), but not the other (Gbeta1/2(a)). Immunoblots revealed that G{alpha}s protein concentrations were 45% lower in cells transfected with Gbeta1/ 2(a) than in those transfected with Gbeta1/2(b). The difference may be attributed to a modestly better Gbeta subunit silencing efficiency of the Gbeta1/2(a) RNAi duplex compared with the Gbeta1/2(b) RNAi duplex (data not shown). No significant differences in relative protein expression were observed for any other G{alpha} subunit (data not shown).

We addressed a number of mechanistic possibilities that might account for the augmented cAMP response seen after silencing Gbeta1/2 with the Gbeta1/2(b) siRNA. Of interest, forskolin, a direct activator of adenylyl cyclases, caused a 3-5-fold greater cAMP response in cells lacking Gbeta1/2 (regardless of which Gbeta1/2 RNAi duplex was transfected) compared with control cells (Fig. 7). No significant difference was noted in the concentration of forskolin required to elicit a half-maximal response (control EC50 = 110 ± 60 µM and Gbeta1/2 EC50 = 60 ± 20 µM). Cells lacking Gbeta1/2 that were exposed to a maximally effective concentration of forskolin demonstrated up to a 10-fold greater rate of cAMP accumulation than control cells (Fig. 7, inset).

This forskolin-sensitized cAMP response could not be attributed to increased beta2-adrenergic receptor density or decreased phosphodiesterase activity (data not shown). Differences in basal and maximal cAMP responses (assayed with a mixture of INE, PGE1, and forskolin at saturating concentrations) in cells lacking Gbeta1/Gbeta2 expression and cells transiently transfected with a constitutively active mutant of G{alpha}s (G{alpha}s Q227L) (19) were not consistent with augmented cAMP activity because of an increased pool of free (unassociated with Gbeta{gamma})G{alpha}s subunits.

We also explored whether decreased expression of G{alpha}i isoforms in cells lacking Gbeta1 and Gbeta2 contributed to the augmented cAMP response. Concomitant knockdown of all three G{alpha}i subunits (G{alpha}i1-3) did not increase the cAMP response; instead, agonist-dependent and forskolin-stimulated adenylyl cyclase was modestly reduced by 20% (not shown). Individually silencing G{alpha}i1 or G{alpha}i3 did not significantly affect INE- or PGE1-stimulated cAMP synthesis; however, forskolin-dependent adenylyl cyclase activity was increased 90% in cells lacking G{alpha}i3 expression. Interestingly, silencing of G{alpha}i2 augmented PGE1-, INE-, and forskolin-dependent cAMP synthesis by 50, 70, and 200%, respectively (data not shown). However, silencing G{alpha}i2 concomitantly with Gbeta1/2 caused additive effects on forskolin-dependent cAMP responses (data not shown), consistent with independence of the mechanisms involved. Thus it is unlikely that the loss of G{alpha}i contributed significantly to the augmented adenylyl cyclase activity in cells lacking Gbeta1 and Gbeta2 expression.


Figure 6
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  		FIGURE 6.
Effects of silencing Gbeta subunit protein expression on ligand-modulated effector activity. A, prostaglandin E1 (10 µM PGE1), and B, isoproterenol (10 µM INE) stimulation of cAMP response. Ligand-dependent cAMP responses were examined in cells lacking an individual Gbeta subunit (Gbeta1, Gbeta2, or Gbeta4), two Gbeta subunits (Gbeta1/2), or three Gbeta subunits (Gbeta1/2/4). Each bar corresponds to the relative cAMP response in cells lacking Gbeta subunit expression compared with control cell activity following a 2-min exposure to ligand and represents the mean ± S.D. of at least eight independent experiments. C, histamine (100 µM)-dependent total IP accumulation. Each bar corresponds to the relative total IP response in cells lacking Gbeta expression compared with control cell activity following a 15-min exposure to histamine and represents the mean ± S.D. of three independent experiments. D and E, inhibition of cAMP activity in cells lacking Gbeta subunit expression. The percent UK 14,304-mediated inhibition of either: D, prostaglandin E1 (10 µM PGE1), or E, isoproterenol (10 µM INE)-stimulated activity was determined as described previously for cells lacking G{alpha} subunit expression. Each bar represents the average ± the range of two independent experiments.

 
Lastly, we examined whether the lack of Gbeta1/2 affected concentrations of adenylyl cyclase proteins. There are nine known genes that encode adenylyl cyclases; they are all activated by G{alpha}s but they are differentially regulated by G{alpha}i, Gbeta{gamma}, Ca2+, Ca2+-binding proteins, and/or phosphorylation (for review, see Ref. 20). RT-PCR analyses revealed that HeLa cells express AC types I, III, VI, VII, and IX; there was no evidence of AC types II, IV, V, or VIII using a variety of RT-PCR primers and total RNA isolated from control cells or cells lacking Gbeta1/2.

However, immunoblots of enriched plasma membranes (Fig. 8) revealed that concentrations of AC type VI (glycosylated plus non-glycosylated) and AC type III were increased 250 and 60%, respectively, in fractions prepared from cells lacking Gbeta1/2 compared with controls. Lack of reliable antibodies precluded examination of AC types I, VII, and IX. No changes in mRNA levels were observed for any of the five AC isotypes expressed in HeLa cells (data not shown). Increased concentrations of AC types VI and III, in conjunction with the fact that increased accumulation of Gbeta4 permits retention of significant amounts of G{alpha}, provide an explanation for the robust forskolin- and ligand-stimulated cAMP responses in cells lacking Gbeta1 and Gbeta2.

Knockdown of G{gamma} Subunits—HeLa cells express all 12 G{gamma} isoforms. We sought to silence the expression of all 12 endogenous G{gamma} subunits using siRNA while monitoring the effects on ligand-dependent cAMP responses. The efficacy and specificity of RNAi duplexes targeting G{gamma} subunits were determined by co-transfection of siRNAs with expression plasmids encoding G{gamma}-GFP fusion proteins (Table 1). Silencing of individual G{gamma} subunits reduced PGE1-stimulated cAMP synthesis for 7 of the 9 G{gamma} subunits tested, and the sum of the individual percentage decreases was significantly greater than 100% (data not shown). Further examination of G{gamma} contribution to signaling specificity will require specific and sensitive antibodies to all 12 G{gamma} proteins.


Figure 7
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  		FIGURE 7.
Forskolin dose response for cAMP accumulation in cells lacking Gbeta1/2 protein expression (closed triangle) and unperturbed cells (closed square). Inset, cAMP accumulation monitored as a function of time using water-soluble forskolin (1 mM). Each point represents the average ± the range of duplicate measurements. The data are representative of at least two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study brings appreciation of the fact that concentrations of G protein subunits are highly inter-related by a variety of mechanisms and that these effects can extend as well to cognate receptors and effectors. It also sounds a clear warning that those who undertake other than short-term perturbations of such networks must be willing to search broadly for the consequences of their actions, focusing on the proteins themselves and not just on more easily measured mRNA transcripts. Our best example of these two conclusions: simultaneous knockdown of Gbeta1 and Gbeta2 in HeLa cells results in a compensatory, non-transcriptionally mediated increase in Gbeta4; this is sufficient to stabilize an adequate (but reduced) amount of G{alpha}s, which can activate substantially increased levels of at least two isoforms of adenylyl cyclase. The result is an enhanced response to agonist when severe impairment was anticipated. This perturbation also resulted in loss of essentially all G{alpha}i protein, despite a large increase in mRNA for G{alpha}i1. Substantial changes in G{gamma} subunits certainly occurred as well, but were not measured. How much further were these changes felt in these normal appearing viable cells?

Concentrations of G{alpha} and Gbeta in HeLa Cells—We know of no other study where there has been reasonably comprehensive quantification of G protein {alpha} and beta subunits in a homogeneous cell population. HeLa cells contain very similar total amounts of G{alpha} and Gbeta proteins, suggesting that all subunits are present largely as heterotrimers. Stabilization of G{alpha} bybeta{gamma} is likely a major mechanism for the maintenance of the stoichiometric equivalence between G{alpha} and Gbeta{gamma}, because silencing of the Gbeta subunits present in these cells results in very substantial loss of all G{alpha} proteins.

Four G{alpha} subunits (G{alpha}s,G{alpha}11,G{alpha}i3, and G{alpha}13), each representing one of the four G{alpha} subfamilies, were the most prevalent G{alpha} proteins and together accounted for >80% of the total G{alpha} pool. Contrary to the prevalent notion that levels of G{alpha}i proteins usually exceed that of either G{alpha}s or G{alpha}q/11, HeLa cells contain comparable amounts of G{alpha}s, G{alpha}11, and total G{alpha}i1-3, with somewhat lower amounts of G{alpha}12/13. The ratios of the amounts of the family members are at least to some extent cell-type or tissue-specific; the amounts of G{alpha}i proteins in brain are very high.

Functions of G{alpha} Proteins: Implications for Signaling Specificity—Certain tenets of G protein signaling, such as the specific requirement for G{alpha}s to activate adenylyl cyclase, G{alpha}q/11 to stimulate PLCbeta, and Gbeta{gamma} to stabilize G{alpha} were verified herein. The effects of silencing G{alpha}s on agonist-dependent signaling were straight forward: one gene and one functional response.

By comparison, activation of PLCbeta through H1-histamine receptors is more complex. Two G{alpha} proteins, G{alpha}q and G{alpha}11, are coupled to the same type of receptor. Of interest, silencing of G{alpha}q or G{alpha}11 caused indistinguishable phenotypes, loss of half of histamine-stimulated PLCbeta activity, despite the fact that concentrations of G{alpha}11 exceed those of G{alpha}q by 10-fold. Because biochemical studies indicate that recombinant G{alpha}q and G{alpha}11 are equally effective at stimulating PLCbeta (21), it is possible that specific localization or organization could be invoked to explain this observation. For example, perhaps only a fraction of the highly expressed G{alpha}11 in HeLa cells is accessible to H1 receptors and/or PLCbeta.

No specificity was detected among the G{alpha}i proteins when {alpha}2-adrenergic receptor-mediated inhibition of INE- or forskolin-stimulated adenylyl cyclase activity was examined, but silencing of G{alpha}i1 did specifically interfere with {alpha}2 receptor-mediated inhibition of PGE1-stimulated adenylyl cyclase. Speculatively related, silencing of Gbeta1 interfered with stimulation of cAMP accumulation by PGE1 but not INE, and we observed no effect of this perturbation on other signaling pathways. There appears to be preferential communication between at least one EP-receptor, G{alpha}s, G{alpha}i1, and Gbeta1, although the mechanism for these apparent relationships are obscure.


Figure 8
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  		FIGURE 8.
Adenylyl cyclase protein expression in plasma membranes prepared from cells lacking Gbeta1/2 subunits. One hundred µg/lane of enriched plasma membranes were blotted for: A, type VI; or B, type III adenylyl cyclase. Glycosylated adenylyl cyclase bands migrated as large smears with apparent Mr values slightly larger than 150,000. Glycosylation of both type VI and III adenylyl cyclases was confirmed by observing increased mobility following enzymatic removal of glycosyl groups with peptide: N-glycosidase F (data not shown). Non-glycosylated type III adenylyl cyclase could not be detected, whereas non-glycosylated type VI adenylyl cyclase co-migrated with a 100-kDa standard (Precision Plus Protein Standards, Bio-Rad). Antibody specificity was verified following isotype-specific siRNA-mediated knockdown of select adenylyl cyclases. No cross-silencing effects were observed with either isotype-specific RNAi duplex. Data are representative of three independent preparations.

 
Knockdown of Gbeta Isoforms Affects G{alpha} Protein Stability—Concomitant knockdown of all three Gbeta subunits expressed in HeLa cells resulted in near complete loss of each G{alpha} without affecting G{alpha} mRNA levels (except for an increase in G{alpha}i1). Thus, Gbeta{gamma} subunits stabilize endogenous G{alpha} proteins in intact cells. A nearly G protein-free and viable cell is produced, but little G protein-coupled receptor-mediated signaling remains.

Our data demonstrating the loss of G{alpha} protein in cells lacking Gbeta subunits are in contrast to a recently published study demonstrating the apparent stability of free G{alpha} subunits in a mouse J774A.1 macrophage cell line (18). The authors hypothesized and subsequently demonstrated that G{alpha}i proteins modified with both myristate and palmitate were more stable than G{alpha} subunits carrying only one lipid modification. We speculate that the differences observed between the two cell types may be because of the existence of proteins that control the fate of free (i.e. unassociated with Gbeta{gamma})G{alpha} subunits or that the stoichiometry of palmitoylation of the G{alpha}i proteins may differ substantially in the two cells.

We found that modest amounts of G{alpha}11 and G{alpha}13 remain after silencing of Gbeta1, Gbeta2, and Gbeta4. We speculate that this may be a reflection of the intrinsic rates of nucleotide exchange that characterize these G{alpha} proteins. Their relatively slower rates of nucleotide exchange minimizes time spent in the nucleotide-free state, which is unstable. Other explanations are possible, including high intrinsic affinity for residual Gbeta{gamma} or interactions with other proteins.

Unanticipated Functional Effects of Silencing Gbeta Isoforms—No requirement for a specific Gbeta isoform was revealed for beta2-adrenergic, H1-histaminergic, or {alpha}2-adrenergic receptor-dependent modulation of effector activities. However, as noted above, some degree of functional specificity was exhibited for Gbeta1 coupling of prostanoid receptors to adenylyl cyclase. It was thus surprising that prostanoid-dependent activation of cAMP synthesis was re-established when Gbeta2 was silenced along with Gbeta1. There are a number of possibilities that could explain the retention of PGE1-mediated signaling under this circumstance. The simplest of these involves the increase in adenylyl cyclase concentrations noted after silencing of Gbeta1 and Gbeta2. Alternatively, severe perturbation of the system, such as the knockdown of Gbeta1 and Gbeta2, may have disrupted and redistributed prostenoid-dependent adenylyl cyclase signaling components from specialized plasma membrane compartments (22).

Our study also revealed some interesting features about Gbeta4, the least well studied of the conventional Gbeta subunits. The amino acid sequence of Gbeta4 is more than 90% identical to those of Gbeta1 and Gbeta2, it is ubiquitously expressed (23), and it behaves similarly to Gbeta1 and Gbeta2 in vitro (23, 24). We show that endogenous Gbeta4 has the ability to maintain G protein-coupled receptor-dependent signaling by interacting functionally with and supporting the coupling of G{alpha}s,G{alpha}q, and G{alpha}11 to effectors in cells that lack Gbeta1 and Gbeta2.

There appear to be differences in the regulation of expression of Gbeta4 compared with that of Gbeta1 and Gbeta2. Gbeta4 was the only Gbeta subunit whose protein expression was significantly reduced (50% or more) following knockdown of more than one G{alpha} subfamily. With the exception of G{alpha}i2, loss of Gbeta4 protein was correlated with decreased Gbeta4 mRNA, indicating that G{alpha}s, G{alpha}i3, and G{alpha}11 each, in some manner, regulated expression of Gbeta4 mRNA. Moreover, elimination of the functionally important (but not prevalent) Gbeta4 increased accumulation of Gbeta1 and Gbeta2, whereas only the elimination of both Gbeta1 and Gbeta2 had a significant effect on Gbeta4.

Alterations of Non-targeted Proteins—Knockdown of a specific G protein subunit often resulted in increased accumulation of a member of the same subfamily. Thus, loss of the prevalent G{alpha}i3 doubled the levels of G{alpha}i1 and G{alpha}i2, whereas loss of G{alpha}i2 doubled G{alpha}i1. Silencing of all G{alpha}i1-3 proteins impacted the accumulation of G{alpha}o. The relationships between expression of Gbeta1,Gbeta2, and Gbeta4 isoforms is noted above. Some of these effects appeared to be mediated transcriptionally; some did not. The exception is G{alpha}q and G{alpha}11, where compensatory increases were not observed following loss of either protein.

Less expected was observation of an apparently reciprocal relationship between expression of members of the G{alpha}q and G{alpha}i subfamilies. Loss of G{alpha}q or G{alpha}11 caused increased accumulation of G{alpha}i1 and G{alpha}o; loss of G{alpha}i3 resulted in increased accumulation of G{alpha}q. The significance is not clear.

It is also notable that levels of G{alpha}i1 and its mRNA increased following the knockdown of almost every G{alpha} and Gbeta subunit, which is suggestive of some as yet unknown common mechanism. Little is known about the specific signaling pathways affected by G{alpha}i1. It is widely expressed, particularly in brain, but there has been no obvious phenotype associated with G{alpha}i1 knock-out mice (25).

The impact of Gbeta knockdowns went beyond G protein family members. We documented changes in the amounts of beta-adrenergic receptors with the knockdown of Gbeta1 and increased levels of adenylyl cyclase when Gbeta1 and Gbeta2 were silenced simultaneously. Increased beta2-adrenergic receptor expression correlated with a 2-3-fold increase in mRNA for the receptor in cells lacking Gbeta1, whereas, AC type VI protein appeared to increase independently of changes in mRNA. We do not know if the changes in adenylyl cyclases are a direct result of loss of Gbeta1 and Gbeta2, increase in Gbeta4, partial loss of G{alpha}s, or other factors; however, knockdown of G{alpha}s increased adenylyl cyclase (types III and VI) expression 60-75%, suggesting that G{alpha}s likely contributes to the regulation of adenylyl cyclase expression.

We cannot comment on the generality of the specific network interactions that are revealed by these knockdown experiments in HeLa cells. Our analyses revealed many changes in non-targeted G protein subunit expression and specific network interactions are likely to be dependent on the specific cell type, the presence of different regulatory mechanisms, and relative stoichiometries of G protein subunits. Future studies using different cells and tissues will reveal the generality of such relationships.

It is clear from this study that G protein signaling pathways in HeLa cells are elastic and resilient in their ability to maintain robust regulation of effectors in the face of change. The mechanisms used to achieve this are varied and complex. It will be a daunting challenge to understand how and to what extent the concentrations and activities of the many interacting components of such networks are regulated to resist and respond to the forces that impinge upon them.


   FOOTNOTES
 
* This work was supported by National Institutes of Health NIGMS Grant GM-34497 and the Raymond and Ellen Willie Chair in Molecular Neuropharmacology (to A. G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 6001 Forest Park, Dallas, TX 75390-9041. Tel.: 214-645-6128; Fax: 214-645-6131; E-mail: Alfred.Gilman{at}UTSouthwestern.edu.

2 The abbreviations used are: siRNA, small-interfering RNA; RNAi, RNA interference; INE, isoproterenol; PGE1, prostaglandin E1; IP, total inositol-phosphate; AC, adenylyl cyclase; PLCbeta, phospholipase C beta; RT, reverse transcriptase. Back

3 S. K. Gibson, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Susanne M. Mumby and Paul C. Sternweis for generously supplying antisera and Linda Hannigan and Kevin Vale for providing excellent technical assistance. We also acknowledge Richard B. Clark, T. Kendall Harden, Susanne M. Mumby, and Gregory G. Tall for critical reading of the manuscript and Madhusudan Natarajan for help with statistical analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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