A Dominant-Negative Strategy for Studying Roles of G Proteins in Vivo*

Annette GilchristDagger §, Moritz BünemannDagger §, Anli LiDagger , M. Marlene HoseyDagger §, and Heidi E. HammDagger §parallel

From the Dagger  Institute for Neuroscience, § Department of Molecular Pharmacology and Biochemistry, Northwestern University, Chicago, Illinois 60611 and  Department of Pharmacology, University of Illinois, Chicago, Illinois 60612

    ABSTRACT
Top
Abstract
Introduction
References

G proteins play a critical role in transducing a large variety of signals into intracellular responses. Increasingly, there is evidence that G proteins may play other roles as well. Dominant-negative constructs of the alpha  subunit of G proteins would be useful in studying the roles of G proteins in a variety of processes, but the currently available dominant-negative constructs, which target Mg2+-binding sites, are rather leaky. A variety of studies have implicated the carboxyl terminus of G protein alpha  subunits in both mediating receptor-G protein interaction and in receptor selectivity. Thus we have made minigene plasmid constructs that encode oligonucleotide sequences corresponding to the carboxyl-terminal undecapeptide of Galpha i, Galpha q, or Galpha s. To determine whether overexpression of the carboxyl-terminal peptide would block cellular responses, we used as a test system the activation of the M2 muscarinic receptor activated K+ channels in HEK 293 cells. The minigenes were transiently transfected along with G protein-regulated inwardly rectifying K+ channels (GIRK) into HEK 293 cells that stably express the M2 muscarinic receptor. The presence of the Galpha i carboxyl-terminal peptide results in specific inhibition of GIRK activity in response to agonist stimulation of the M2 muscarinic receptor. The Galpha i minigene construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha carboxyl peptide in random order, pcDNA-Galpha iR) had no effect on agonist-mediated M2 muscarinic receptor GIRK response. The inhibitory effects of the Galpha i minigene construct were specific because overexpression of peptides corresponding to the carboxyl terminus of Galpha q or Galpha s had no effect on M2 muscarinic receptor stimulation of the K+ channel.

    INTRODUCTION
Top
Abstract
Introduction
References

Many biologically active molecules transduce their signals through heptahelical receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins).1 G proteins play important roles in determining the specificity and temporal characteristics of a variety of cellular responses. Upon activation, G protein-coupled receptors (GPCRs) interact with their cognate heterotrimeric G protein, inducing GDP release with subsequent GTP binding to the alpha  subunit. The exchange of GDP for GTP leads to dissociation of the Gbeta gamma dimer from the Galpha subunit, and both initiate unique intracellular signaling responses (for review, see Refs. 1 and 2). Molecular cloning has resulted in the identification of 18 distinct Galpha subunits that are commonly divided into four families based on their sequence similarity: Gi, Gs, Gq, and G12. Similarly, multiple Gbeta (5) and Ggamma (11) subunits have been identified.

In all G proteins studied GTP is bound as a complex with Mg2+, and the GTP- and Mg2+-binding sites are tightly coupled. Dominant-negative constructs of the alpha  subunit of G proteins have been made in which mutations are made in residues that contact the magnesium ion. Although this approach was quite successful with p21ras and other small G proteins (3, 4), dominant-negative Galpha i, Galpha o, Galpha q, and Galpha 11 have been less effective (5-10). This is probably because of the degree to which Mg2+ is necessary to support GDP binding. p21ras forms a tight and nearly irreversible GDP·Mg2+ complex, whereas Galpha subunits bind Mg2+ in the GDP·Mg2+ complex with lower affinity than in the GTP·Mg2+ complex (11-14).

Specific determinants of receptor-G protein interaction have been under investigation for many years. It is thought that there are multiple sites of contact between the activated receptor and the G protein. Studies using ADP-ribosylation by pertussis toxin, site-directed mutagenesis, peptide-specific antibodies, and chimeric proteins indicate that the carboxyl terminus of the Galpha subunit is not only an essential region for receptor contact, but is also important for determining G protein receptor specificity (reviewed in Refs. 1 and 15). The crystal structures of various Galpha subunits show that the last 4-7 amino acids of Galpha were not observed (16-22) indicating that the region is conformationally flexible in the absence of other interactions.

In vitro assays, as well as microinjection studies of intact cells, indicate Galpha carboxyl-terminal peptides can competitively block G protein-coupled downstream events (23-26). A carboxyl-terminal peptide from Galpha t not only binds, but will also directly stabilize photoactivated rhodopsin (27, 28). Using a combinatorial peptide library Martin et al. (29) have shown that specific residues within the carboxyl terminus of Galpha t are critical for high affinity binding of the Galpha t peptide to rhodopsin. Similarly, a carboxyl-terminal peptide from Galpha s (384-394), but not corresponding peptides from Galpha i1/2, inhibits the ability of beta 2-adrenergic receptors to activate Galpha s and adenylyl cyclase (30). In addition, a carboxyl-terminal undecapeptide from Galpha i1/2 can bind the adenosine A1 receptor, whereas the corresponding peptide from Galpha t, which differs by only 1 amino acid residue does not (31). Thus, the carboxyl terminus of G protein alpha  subunits is critical in both mediating receptor-G protein interactions and in receptor selectivity (31-35).

"Minigene" plasmid vectors are constructs designed to express relatively short polypeptide sequences following their transfection into mammalian cells. Minigenes have been used by investigators to look at a variety of responses related to G proteins including (i) binding of pleckstrin homology (PH) domains to Gbeta gamma (36), (ii) inhibiting GPCRs by expressing the carboxyl terminus of beta 2 adrenergic receptor kinase (37-39), and (iii) identifying intracellular domains of GPCRs critical for G protein coupling (40-44). Experiments using minigenes that express the last 55 amino acids of Galpha q to target the receptor-Gq interface to achieve class-specific inhibition were recently published by Akhter et al. (45). Transient transfection of COS-7 cells with alpha 1B-adrenergic receptors or M1 muscarinic receptors and the Galpha q carboxyl-terminal minigene (residues 305-359) inhibits agonist stimulated inositol phosphate (IP) production, whereas co-expression with the Galpha q amino terminus (residues 1-54) has no effect. Inhibition by Galpha q (305-359) was apparently specific for Gq-coupled receptors because neither alpha 2A-adrenergic receptor-mediated IP production (Gi-coupled), nor dopamine D1A receptor-mediated cAMP production (Gs-coupled) were inhibited. In addition, transgenic mice made by targeting the Galpha q carboxyl-terminal minigene to the myocardium resulted in a marked inhibition of alpha 1B-adrenergic receptor-mediated IP production and blockade of cardiac hypertrophy.

In this paper, we study the effects of several carboxyl termini Galpha peptides using a minigene approach. To test whether minigene constructs encoding the carboxyl-terminal 11 amino acid residues from Galpha subunits could effectively inhibit G protein-coupled receptor-mediated cellular responses, we chose a system in which 1) the importance of the carboxyl terminus and 2) the downstream effector system had been well established. Numerous studies (46-49) have shown that the M2 muscarinic receptor (mAChR) couples exclusively to the Gi/Go family. The M2 mAChR can efficiently couple to mutant Galpha q in which the last 5 amino acids of Galpha q are substituted with the corresponding residues from Galpha i or Galpha o (34), suggesting that this receptor contains domains that are specifically recognized by the carboxyl terminus of Galpha i/o subunits. The effector system that we selected was the M2 mAChR-activated inwardly rectifying K+ channel (IKACh). In cardiac cells, the IKACh channel is formed as a heterotetramer of G protein-regulated inwardly rectifying K+ channels (GIRK), with two GIRK1 and two GIRK4 subunits (50, 51). This channel is activated upon stimulation of M2 mAChR in a manner that is completely pertussis toxin-sensitive and is the prototype for a direct Gbeta gamma -activated channel (52-54). Our experiments indicate that the Galpha i carboxyl terminus minigene construct can completely block M2 mAChR-mediated K+ channel activation. The inhibition appears specific as constructs producing Galpha s, Galpha q, or a scrambled Galpha i carboxyl-terminal peptide had no effect.

    MATERIALS AND METHODS

Construction of Galpha Carboxyl-terminal Minigenes-- The cDNA encoding the last 11 amino acids of human Galpha subunits (Galpha i1/2, Galpha s, Galpha q) or the Galpha i1/2 carboxyl terminus in random order (Galpha iR) were synthesized (Great American Gene Co.) with newly engineered 5'- and 3'-ends (Fig. 1). The 5'-end contained a BamHI site followed by the ribosome binding consensus sequence (5'-GCCGCCACC-3'), a methionine (ATG) for translation initiation, and a glycine (GGA) to protect the ribosome binding site during translation and the nascent peptide against proteolytic degradation. A HindIII site was synthesized at the 3'-end immediately following the translational stop codon (TGA).

The DNA was brought up in sterile ddH2O (stock concentration 100 µM). Complimentary DNA was annealed in 1× NEBuffer 3 (50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol; New England Biolabs) at 85 °C for 10 min then allowed to cool slowly to room temperature. The annealed cDNA were ligated for 1 h at room temperature into pcDNA 3.1(-) plasmid vector (Invitrogen) previously cut with BamHI and HindIII. After digestion with each restriction enzyme, the pcDNA 3.1 plasmid vector was run on an 0.8% agarose gel, the appropriate band cut out, and the DNA purified (GeneClean II Kit, Bio101). For the ligation reaction the ratio of insert to vector was approximately 25 µM to 50 ng, respectively. Following ligation, the samples were heated to 65 °C for 5 min to deactivate the T4 DNA ligase.

Ligation reaction (1 µl) was electroporated into 50-µl competent ARI814 cells (Bio-Rad) (Escherichia coli Pulsar; 29) and there were cells immediately placed into 1 ml of SOC (Life Technologies, Inc.). After 1 h at 37 °C, 100 µl was spread on LB/Amp plates and incubated at 37 °C for 12-16 h. To verify that insert was present, several colonies were grown overnight in LB/Amp and their plasmid DNA purifed (Qiagen SpinKit). The plasmid DNA was digested with NcoI (New England Biolabs, Inc.) for 1 h at 37 °C and run on a 1.5% agarose gel. Vector alone produced 3 bands (3.4, 1.3, and 0.7 kilobases), whereas vector with insert resulted in 4 bands (3.4, 1.0, 0.7, and 0.3 kilobases). DNA with the correct pattern was sequenced (Northwestern University Biotechnology Center) to confirm the appropriate sequence. The Galpha minigene constructs used for transfection experiments (pcDNA3.1; pcDNA-Galpha i; pcDNA-Galpha iR; pcDNA-Galpha q, and pcDNA-Galpha s) were purified from 500-ml cultures using endotoxin-free maxi-prep kits (Qiagen).

Cell Culture and Transfection-- Human embryonic kidney (HEK) 293 cells, stably expressing the M2 mAChR (~400 fmol receptor/mg protein) (55) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.), streptomycin/penicillin (100 units each; Life Technologies, Inc.) and G418 (500 mg/liter; Life Technologies, Inc.). Cells were grown under 10% CO2 at 37 °C. In all transfections for electrophysiological studies the CD8 reporter gene system was used to visualize transfected cells (56). Dynabeads coated with anti-CD8-antibodies were purchased from Dynal. A standard calcium phosphate procedure was used for transient transfection of HEK cells. The following amounts of cDNA were used for transient transfections (unless otherwise indicated): pi H3-CD8 (human), 1 µg; pC1-GIRK1 (rat), 1 µg; pcDNA1-GIRK4 (rat), 1 µg; pcDNA3.1, pcDNA-Galpha i, pcDNA-Galpha iR, pcDNA-Galpha q, or pcDNA-Galpha s, 4 µg. Typically the total amount of cDNA used for transfecting one 10-cm dish was 7 µg. All assays were performed 48-72 h posttransfection. The cDNAs for the GIRK1 and GIRK4 were gifts from F. Lesage and M. Lazdunski (Nice, France) and cDNA for CD8 was from G. Yellen (Harvard University, Boston, MA).

To determine minigene RNA expression, transiently transfected cells were washed twice with phosphate-buffered saline, lysed with 350 µl of RLT lysis buffer (Qiagen, Rneasy Mini Kit), homogenized using a QIAshredder column (Qiagen), and total RNA was processed according to the manufacturer's protocol. Total RNA was eluted in diethyl pyrocarbonate-treated water, quantified, and stored at -20 °C. cDNA was made from total RNA using a reverse transcribed polymerase chain reaction (RT-PCR) (CLONTECH Advantage RT-for-PCR kit) according to the manufacturer's protocol. To verify the presence of insert in cells transfected with pcDNA-Galpha i or pcDNA-Galpha iR constructs, their cDNA was used as the template for PCR with forward and reverse primers that correspond to Galpha insert and vector, respectively (forward: 5'-ATCCGCCGCCACCATGGGA; reverse: 5'-GCGAAAGGAGCGGGCGCTA). The primers for the Galpha minigenes amplify a 434-bp fragment only if the insert carboxyl termini oligonucleotides are present; no band is observed in cells transfected with empty vector (pcDNA3.1). As controls, PCR was also performed using T7 forward with the vector reverse primer, which amplified a 486-bp fragment in all cDNA tested or G3DPH primers (CLONTECH), which amplified a 983-bp fragment in all cDNA tested.

Additionally, transiently transfected cells were trypsinized, pelleted, washed twice with phosphate-buffered saline, and stored at -80 °C. Cellular extracts were prepared by homogenizing the cell pellets for 15 s (ESGE Bio-homogenizer M133/1281-0) in fractionation buffer (10 mM HEPES, pH 7.3, 11.5% sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 3,000 × g for 20 min, and the supernatant centrifuged at 100,000 × g for 30 min. The cytosolic fraction from the resulting supernatant was collected; and the fractions stored at -80 °C until needed. For high pressure liquid chromatography analysis, 100 µl of cytosolic extract was loaded onto a C4 column (Vydac) equilibrated with 0.1% trifluoroacetic acid in ddH2O. Elution of the peptide was performed using 0.1% trifluoroacetic acid in acetonitrile. The amount of acetonitrile was increased from 0 to 60% over 45 min. Peaks were collected, lyophilized, and analyzed using ion mass spray analysis (University of Illinois-Urbana Champagne).

Measurement of IKACh Currents-- For the measurement of inwardly rectifying K+ current, whole cell currents were recorded as described previously (57, 58). The extracellular solution contained 120 mM NaCl, 20 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM Hepes-NaOH, pH 7.4. The solution for filling the patch pipettes was composed of 100 mM potassium glutamate, 40 mM KCl, 5 mM MgATP, 10 mM Hepes-KOH, pH 7.4, 5 mM NaCl, 2 mM EGTA, 1 mM MgCl2, and 0.01 mM GTP. All standard salts as well as acetylcholine were from Sigma.

To minimize variations caused by different transfections or culture conditions, control experiments (transfection with pcDNA-Galpha iR) were done in parallel. Membrane currents were recorded under voltage clamp, using conventional whole cell patch techniques (59). Patch pipettes were fabricated from borosilicate glass capillaries, (GF-150-10, Warner Instrument Corp.) using a horizontal puller (P-95 Fleming & Poulsen) and were filled with the solutions listed above. The DC resistance of the filled pipettes ranged from 3 to 6 mega-ohms. Membrane currents were recorded using a patch-clamp amplifier (Axopatch 200, Axon Instruments). Signals were analog filtered using a low pass Bessel filter (1-3 kHz corner frequency). Data were digitally stored using an IBM compatible PC equipped with a hardware/software package (ISO2 by MFK, Frankfurt/Main, Germany) for voltage control, data acquisition, and data evaluation.

To measure K+ currents in the inward direction, the potassium equilibrium potential was set to about -50 mV and the holding potential was -90 mV as described (57, 58). Agonist-induced currents were evoked by application of acetylcholine (ACh; 1 µM) using a solenoid-operated superfusion device, which allowed for solution exchange within 300 ms. Linear voltage ramps (from -120 mV to + 60 mV within 500 ms) were applied every 10 s. By subtracting nonagonist-dependent currents we were able to resolve the current voltage properties of the agonist-induced currents. For analysis of the data the maximal current density (peak amplitude) of ACh-induced inwardly rectifying K+ currents were measured at -90 mV and compared.

Data Analysis-- Data are presented as mean ± S.E. The statistical differences were determined using the Student's t test (GraphPad Prism; version 2.0).

    RESULTS AND DISCUSSION

Dominant-negative constructs of the alpha  subunit of G proteins have been made in which mutations are made in regions that contact the magnesium ion. For the alpha  subunit of G proteins, this includes mutations of the Gly residue within the invariant sequence (G203T,G204A), as well as mutations of a Ser residue in the effector loop, switch I region (S47C) in either Galpha o or Galpha i (5, 6). However, neither of these mutations has resulted in effective dominant-negatives probably because the GDP complexes of Galpha i, Galpha o, and Galpha s have low affinity for Mg2+. Thus, we looked to other regions on G protein alpha  subunits that could serve to block receptor-G protein interactions, and consequently serve as dominant-negatives. A variety of studies have implicated the carboxyl terminus of G protein alpha  subunits in mediating receptor-G protein interaction and selectivity (for review, see Refs. 14 and 15). We have shown that carboxyl termini from G protein alpha  subunits are important sites of receptor binding, and peptides corresponding to the carboxyl terminus can be used as competitive inhibitors of receptor-G protein interactions (27, 29, 30). This interaction is quite specific as we found that a difference in 1 amino acid can annul the ability of the Galpha i1/2 peptide to bind the A1 adenosine receptor-G protein interface (31).

To determine whether we could selectively antagonize G protein signal transduction events in vivo by expressing peptides that block the receptor-G protein interface, we generated minigene plasmid constructs that encode carboxyl-terminal peptide sequences from Galpha i1/2, Galpha q, or Galpha s (Table I). As a control, we also made a minigene that expressed the carboxyl terminus of Galpha i1/2 in random order (Galpha iR, Table I). The minigene insert DNA were made by synthesizing short complimentary oligonucleotides corresponding to the peptide sequences from the carboxyl terminus of each Galpha with BamHI and HindIII restriction sites at the 5' and 3' ends, respectively. Complementary oligonucleotides were annealed and ligated into the mammalian expression vector pcDNA 3.1(-). The DNA was cut with NcoI, and separated on a 1.5% agarose gel to determine whether the insert was present. As shown in Fig. 1, when insert is present there is a new NcoI site resulting in a shift in the band pattern, such that the digest pattern goes from three bands (3345, 1352, and 735 bp) to four bands (3345, 1011, 735, and 380 bp).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Carboxyl termini sequences
Alignment of the last 11 amino acid residues from human Galpha i1/2, Galpha q, and Galpha s subunits. Also shown is the peptide sequence of Galpha iR, the Galpha i1/2 sequence in random order, used to construct the control minigene.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1.   The cDNA minigene constructs. Insert DNA, all Galpha carboxyl-terminal peptide minigenes contain a BamHI restriction enzyme site at the 5'-end followed by a ribosomal binding site sequence, a methionine for translation initiation, a glycine for stabilization of the peptide, the peptide sequence, a stop codon, and a HindIII restriction enzyme site at the 3'-end. The Galpha iR contains the Galpha i1/2 carboxyl peptide sequence in random order. Vector, following annealing, complimentary oligonucleotides were ligated into BamHI/HindIII cut pcDNA 3.1 plasmid vector, and the ligated insert/vector DNA was electroporated into competent cells. NcoI digest, plasmid DNA was purified, digested with NcoI, and separated on a 1.5% agarose gel to determine whether insert was present. Lane 1 is a 1-kilobase pair DNA ladder; lane 2 is pcDNA3.1; lane 3 is pcDNA-Galpha i; lane 4 is pcDNA-Galpha iR; and lane 5 is pcDNA-Galpha q. When insert is present there is a new NcoI site resulting in a shift in the band pattern, such that the digest pattern goes from three bands (3345, 1352, and 735 bp) to four bands (3345, 1011, 735, and 380 bp).

As our minigene approach depends on competitive inhibition, a key element for success is the expression of adequate amounts of peptides to block intracellular signaling pathways. To confirm the presence of the minigene constructs in transfected cells, total RNA was isolated 48 h posttransfection, cDNA made with RT-PCR, and PCR analysis was performed using the cDNA as template with primers specific for the Galpha carboxyl-terminal peptide insert. Separation of the PCR products on 1.5% agarose gels (Fig. 2A) indicates the presence of the Galpha carboxyl terminus peptide minigene RNA by a single 434-bp band. Control experiments were done using a T7 forward primer with the vector reverse primer to verify the presence of the pcDNA3.1 vector, and G3DPH primers (CLONTECH) to approximate the amount of total RNA (data not shown).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Transient transfection of mingene vectors. HEK 293 cells that stably express M2 mAChR were transiently transfected with DNA from GIRK1/4 and pcDNA3.1, pcDNA-Galpha i, or pcDNA-Galpha iR. A, to confirm the presence of the minigenes in the transiently transfected HEK 293 cells, total RNA was isolated 48 h posttransfection (Qiagen Rneasy Kit with QIAshredder). The cDNA was made through RT-PCR (CLONTECH). The PCR analysis was completed using the cDNA as template with primers specific for the Galpha carboxyl-terminal peptide insert. Separation of the PCR products on 1.5% agarose gels indicates the presence of the Galpha carboxyl terminus peptide minigene RNA by a single 434-bp band. Lane 1 is a 1-kilobase pair DNA ladder; lane 2 is PCR products from cells transfected with pcDNA-Galpha iR; lane 3 is cells transfected with pcDNA-Galpha i; lane 4 is cells transfected with pcDNA3.1. B, to verify that the peptide was being produced in the transiently transfected cells, the cells were lysed 48 h posttransfection, homogenized, and cytosolic extracts analyzed by HPLC. Peaks from cells transfected with pcDNA3.1, pcDNA-Galpha i, or pcDNA-Galpha iR were analyzed by ion mass spray analysis.

To verify that the peptide was being produced in the transfected cells, 48 h posttransfection, cells were lysed and homogenized. Cytosolic extracts were analyzed by high pressure liquid chromatography, and peaks (Fig. 2B) were analyzed by ion mass spray analysis. The mass spectrometer analysis for peak 1 from the pcDNA-Galpha i transfected cells, and peak 1 from cells transfected with a vector expressing the carboxyl terminus in random order (pcDNA-Galpha iR) indicate that a 1450 molecular weight peptide was found in both cytosolic extracts. This is the expected molecular weight for both 13 amino acid peptide sequences. The fact that they were the major peptides found in the cytosol from cells transiently transfected with the pcDNA-Galpha i or pcDNA-Galpha iR vectors strongly suggests that the vectors are producing the appropriate peptide sequences. Therefore, analysis of the transiently transfected HEK 293 cells indicates (1) minigene vectors are present, and (2) the corresponding peptides are being expressed.

We examined whether the presence of the Galpha i carboxyl-terminal peptide minigene would result in a significant inhibition of a downstream functional response following agonist stimulation of the transiently transfected cells. G protein-regulated inwardly rectifying K+ channels modulate electrical activity in many excitable cells (for review, see Refs. 60-62). Because the channel opens as a consequence of a direct interaction with Gbeta gamma , whole cell patch clamp recording of inwardly rectifying K+ currents can be used as a readout of G protein activity in single intact cells. Thus, we tested whether the Galpha carboxyl-terminal peptide minigenes could inhibit M2 mAChR activation of inwardly rectifying K+ currents. Superfusion of HEK 293 cells transiently transfected with GIRK1/GIRK4 and either pcDNA-Galpha i or pcDNA-Galpha iR DNA with 1 µM ACh revealed that cells transfected with pcDNA-Galpha i DNA have a dramatically impaired response to the M2 mAchR agonist (Fig. 3). Fig. 3, A and B shows representative recordings of whole cell membrane currents at -90 mV. Superfusion of the cells with ACh activates inward currents in cells transfected with pcDNA-Galpha iR (Fig. 3A) but not in cells transfected with pcDNA-Galpha i (Fig. 3B). The inwardly rectifying IV-curve for the ACh-induced current from the experiment shown in Fig. 3A is illustrated in Fig. 3D. The strong inwardly rectifying properties of this current is characteristic of IKACh channels. Summarized data for the maximum amplitude of ACh-evoked currents are shown for three different transfection conditions as indicated by the black bars. The maximum current evoked by ACh was 3.7 ± 1.5 pA/pF (n = 14) in cells transfected with the pcDNA-Galpha i compared with 24.1 ± 8.8 pA/pF (n = 11) in cells transfected with pcDNA-Galpha iR. As a control we transfected cells with empty vector (pcDNA3.1). The ACh responses in these cells (16.5 ± 7.7 pA/pF (n = 5) was not significantly different from responses measured in cells transfected with pcDNA-Galpha iR (Fig. 3C). Basal levels for all three conditions were equivalent (pcDNA 3.2 ± 1.8 pA/pF (n = 5); Galpha i 6.1 ± 0.9 pA/pF (n = 14); Galpha iR 5.6 ± 2.0 pA/pF (n = 10)). To exclude experiments in which we recorded currents from cells that may not have expressed the functional channel, only those cells that exhibited a basal nonagonist-dependent Ba2+ (200 µM) sensitive inwardly rectifying current were used for analysis. Thus, it appears that the Galpha i minigene construct completely blocks the agonist-mediated M2 mAChR GIRK1/4 response, whereas the control minigene constructs (empty vector, pcDNA3.1, and the Galpha i1/2 carboxyl peptide in random order, pcDNA-Galpha iR) had no effect on the agonist-mediated M2 mAChR GIRK1/4 response.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Minigenes encoding carboxyl-terminal Galpha i peptides inhibit M2 mAChR activated IKACh. HEK 293 cells stably expressing the M2 mAChR were transiently transfected with DNA from GIRK1/4 and pcDNA3.1, pcDNA-Galpha i, or pcDNA-Galpha iR. A, a representative example of the activation of inwardly rectifying K+ currents upon superfusion of 1 µM ACh in a HEK 293 cell transiently transfected with GIRK1, GIRK4, and pcDNA-Galpha iR DNA. B, a representative example of the activation of inwardly rectifying K+ currents upon superfusion of 1 µM ACh in a HEK 293 cell transiently transfected with GIRK1, GIRK4, and pcDNA-Galpha i DNA. C, the maximum current evoked by Ach (black bars) was 3.7 ± 1.5 pA/pF (n = 14) in cells transfected with the pcDNA-Galpha i compared with 16.5 ± 7.7 pA/pF (n = 5) in cells transfected with empty vector (pcDNA3.1) or 24.1 ± 8.8 pA/pF (n = 11) in cells transfected with pcDNA-Galpha iR. The white bars represent the basal Ba2+-sensitive currents. D, the current voltage relation of the ACh-induced current shows the characteristic inward rectification and a reversal potential near the potassium equilibrium potential typical for IKACh.

The cardiac IKACh channel is activated upon stimulation of M2 mAChR via G proteins of the Gi family. The carboxyl-terminal region of Galpha has also been shown to be critical in determining the specificity of GPCR-G protein interactions (34, 63). Substitution of 3-5 carboxyl-terminal amino acids from Galpha q with corresponding residues from Galpha i allowed receptors that signal exclusively through Galpha i subunits to activate the chimeric alpha  subunits and stimulate the Galpha q effector, phospholipase C-beta . To determine whether carboxyl-terminal peptides from other classes of G proteins could inhibit the agonist-mediated M2 mAChR GIRK1/4 response, we transiently transfected HEK 293 cells stably expressing the M2 mAChR with GIRK1/GIRK4 and with minigene constructs encoding Galpha carboxyl termini for Galpha q or Galpha s. ACh-stimulated IKACh currents from cells transfected with pcDNA-Galpha q (Fig. 4B; 19.5 ± 5.5 pA/pF (n = 6)) or pCDNA-Galpha s (Fig. 4C; 35.5 ± 9.7 pA/pF (n = 5)) were not significantly different from those of cells transfected with the control minigene vector, pCDNA-Galpha iR (23.7 ± 10.5 pA/pF (n = 6) and 26.0 ± 7.9 pA/pF (n = 5), respectively). This is very different from cells transfected with pcDNA-Galpha i whose ACh-stimulated IKACh currents were significantly decreased as compared with cells transfected with pcDNA-Galpha iR (Fig. 4A; 3.7 ± 1.5 pA/pF (n = 14) versus 24.1 ± 8.8 pA/pF (n = 11)) These findings confirm the specificity of the inhibition of M2 mAChR-activated G protein-coupled IKACh responses by expression of the Galpha i minigene, pcDNA-Galpha i.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Transfection of the Galpha i carboxyl-terminal minigene inhibits the M2 mAChR activated IKACh response, whereas Galpha s or Galpha q carboxyl-terminal minigenes do not. Stably m2 mAChR-expressing HEK 293 cells were transiently transfected with GIRK1, GIRK4 and pcDNA-Galpha iR, pcDNA-Galpha i, pcDNA-Galpha s, or pcDNA-Galpha q. A, the maximum current evoked by 1 µM ACh was 3.7 ± 1.5 pA/pF (n = 14) in cells transfected with pcDNA-Galpha i compared with 24.1 ± 8.8 pA/pF (n = 11) in cells transfected with pcDNA-Galpha iR. B, the maximum current evoked by ACh was 19.5 ± 5.6 pA/pF (n = 6) in cells transfected with the pcDNA-Galpha q compared with 23.8 ± 10.5 pA/pF (n = 6) in cells transfected with pcDNA-Galpha iR. C, the maximum current evoked by ACh was 35.5 ± 9.7 pA/pF (n = 5) in cells transfected with pcDNA-Galpha s compared with 26.0 ± 8.0 pA/pF (n = 5) in cells transfected with pcDNA-Galpha iR.

Recent experiments targeting receptor-G protein interaction (45) by constructing minigenes that encode the last 55 amino acids of Galpha q also indicate that class-specific inhibition can be achieved. However, transient transfection experiments with this construct resulted in only a 27-48% inhibition of inositol phosphate accumulation. Our Galpha i minigene constructs that encode only the last 11 amino acids of the carboxyl terminus of Galpha i1/2 resulted in an 85% inhibition of the IKACh response. This difference may be caused by variations in the length of the expressed minigene (55 versus 11 residues). The longer peptide may fold in such a way that the critical carboxyl-terminal region is partly buried. We have shown that shorter peptides can effectively bind to receptors (27, 29-31). Because the extreme carboxyl terminus of Galpha subunits in their GDP-bound conformation is disordered in crystal structures (16, 20), the smaller peptide may be able to fit into its binding site more effectively. Alternatively, the difference may be in the amount of peptide being expressed because of differences in methods of transfection and cell type being studied.

Molecular determinants other than the carboxyl terminus are also involved in the recognition between heterotrimeric G proteins and their cognate receptors (27, 64). However, a variety of studies have shown that the carboxyl terminus of G protein alpha  subunits is critical in both mediating receptor-G protein interaction and in receptor selectivity (31-35). Our results confirm that the carboxyl terminus of Galpha i is able to block agonist-mediated responses completely and thus is important in receptor selectivity and specificity. Most importantly, this method appears to be a promising approach for completely turning off G protein-mediated responses in transfected cells and in vivo. Transfection of different Galpha carboxyl-terminal peptide should allow us to selectively block signal transduction through any G protein and thus provides a novel dominant-negative strategy. We have now made minigene constructs encoding Galpha carboxyl-terminal undecapeptide sequences for each of the Galpha subunits. These minigenes should provide an effective dominant-negative approach that will allow us to define new roles of G proteins in vivo. The approach may also allow us to explore the coupling mechanisms of receptors that interact with multiple G proteins and tease out the downstream responses mediated by each G protein.

    ACKNOWLEDGEMENTS

We thank Dr. Robert Ten Eick for providing equipment for patch clamp studies and Drs. Nikolai Skiba, Teresa Vera, Maria Mazzoni, and Hyunsu Bae for advice and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by Grants EY06062 and EY10291 (to H. E. H.), HL50121 (to M. M. H.), a Distinguished Investigator Award from the National Alliance for Research on Schizophrenia and Depression (to H. E. H.), Research Fellowship award Bu 1133/1-1 from the Deutsche Forschungsgemeinschaft (to M. B.), and by a postdoctoral training Grant HL07829 (to A. G.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Northwestern University, Institute for Neuroscience, 320 E. Superior 5-555 Searle, Chicago, IL 60611.

    ABBREVIATIONS

The abbreviations used are: G proteins, guanine nucleotide-binding proteins; GPCR, G protein-coupled receptor; IP, inositol phosphate; HEK, human embryonic kidney; mAChR, muscarinic receptor; ACh, acetylcholine; IKACh, inwardly rectifying K+ channel; GIRK, G protein-regulated inwardly rectifying K+ channel; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s).

    REFERENCES
Top
Abstract
Introduction
References
  1. Hamm, H. E., and Gilchrist, A. (1996) Curr. Opin. Cell Biol. 8, 189-196[CrossRef][Medline] [Order article via Infotrieve]
  2. Hamm, H. E. (1998) J. Biol. Chem. 273, 669-672[Free Full Text]
  3. John, J., Rensland, H., Schlichting, I., Vetter, I., Borasio, G. D., Goody, R. S., and Wittinghofer, A. (1993) J. Biol. Chem. 268, 923-929[Abstract/Free Full Text]
  4. Quilliam, L. A., Kato, K., Rabun, K. M., Hisaka, M. M., Huff, S. Y., Campbell-Burk, S., and Der, C. I. (1994) Mol. Cell. Biol. 14, 1113-1121[Abstract]
  5. Slepak, V. Z., Quick, M. W., Aragay, A. M., Davidson, N., Lester, H. A., and Simon, M. I. (1993) J. Biol. Chem. 268, 21889-21894[Abstract/Free Full Text]
  6. Slepak, V. Z., Katz, A., and Simon, M. I. (1995) J. Biol. Chem. 270, 4037-4041[Abstract/Free Full Text]
  7. Osawa, S., and Johnson, G. L. (1991) J. Biol. Chem. 266, 4673-4676[Abstract/Free Full Text]
  8. Hermouet, S., Merendino, J. J., Gutkind, J. S., and Spiegel, A. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10455-10459[Abstract]
  9. Carrel, F., Dharmawardhane, S., Clark, A. M., Powell-Coffman, J. A., and Firtel, R. A. (1994) Mol. Biol. Cell 5, 7-16[Abstract]
  10. Winitz, S., Gupta, S. K., Qian, N.-X., Heasley, L. E., Nemenoff, R. A., and Johnson, G. L. (1994) J. Biol. Chem. 269, 1889-1895[Abstract/Free Full Text]
  11. Higashijima, T., Ferguson, K. M., Sternweis, P. C., Smigel, M. D., and Gilman, A. G. (1987) J. Biol. Chem. 262, 762-766[Abstract/Free Full Text]
  12. Higashijima, T., Ferguson, K. M., Smigel, M. D., and Gilman, A. G. (1987) J. Biol. Chem. 262, 757-761[Abstract/Free Full Text]
  13. Lee, E., Taussig, R., and Gilman, A. G. (1992) J. Biol. Chem. 267, 1212-1218[Abstract/Free Full Text]
  14. Sprang, S. R. (1997) Annu. Rev. Biochem. 66, 639-678[CrossRef][Medline] [Order article via Infotrieve]
  15. Bourne, H. R. (1997) Curr. Opin. Cell Biol. 9, 134-142[CrossRef][Medline] [Order article via Infotrieve]
  16. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 372, 276-279[CrossRef][Medline] [Order article via Infotrieve]
  17. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628[CrossRef][Medline] [Order article via Infotrieve]
  18. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319[CrossRef][Medline] [Order article via Infotrieve]
  19. Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374[CrossRef][Medline] [Order article via Infotrieve]
  20. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058[Medline] [Order article via Infotrieve]
  21. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 269, 1405-1412
  22. Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Science 270, 954-960[Abstract]
  23. Hamm, H. E., Deretic, D., Hofman, K., Schleicher, A., and Kohl, B. (1987) J. Biol. Chem. 262, 10831-10838[Abstract/Free Full Text]
  24. Takano, K., Yasufuku-Takano, J., Kozasa, T., Nakajima, S., and Nakajima, Y. (1997) J. Physiol. 502, 559-567[Abstract]
  25. Aragay, A. M., Collins, L. R., Post, G. R., Watson, A. J., Feranisco, J. P., Brown, J. H., and Simon, M. I. (1995) J. Biol. Chem. 270, 20073-20077[Abstract/Free Full Text]
  26. Jones, S., Brown, D. A., Milligan, G., Willer, E., Buckley, N. J., and Caulfield, M. P. (1995) Neuron 14, 399-405[Medline] [Order article via Infotrieve]
  27. Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B., and Hoffmann, K. P. (1988) Science 241, 832-835[Medline] [Order article via Infotrieve]
  28. Osawa, S., and Weiss, E. R. (1995) J. Biol. Chem. 270, 31052-31058[Abstract/Free Full Text]
  29. Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366[Abstract/Free Full Text]
  30. Rasenick, M. M., Watanabe, M., Lazarevic, M. B., Hatta, S., and Hamm, H. E. (1994) J. Biol. Chem. 269, 21519-21525[Abstract/Free Full Text]
  31. Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Proctor, W. R., Lupica, C. R., Dunwiddie, T., and Hamm, H. E. (1998) J. Biol. Chem 273, 14912-14919[Abstract/Free Full Text]
  32. Blahos, J. N., Mary, S., Perroy, J., de Colle, C., Brabet, I., Bockaert, J., and Pin, J. P. (1998) J. Biol. Chem. 273, 25765-25769[Abstract/Free Full Text]
  33. Kostenis, E., Conklin, B. R., and Wess, J. (1997) Biochemistry 36, 1487-1495[CrossRef][Medline] [Order article via Infotrieve]
  34. Liu, J., Conklin, B. R., Blin, N., Yun, J., and Wess, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11642-11646[Abstract]
  35. Onrust, R., Herzmark, P., Chi, P., Garcia, P. D., Lichtarge, O., Kingsley, C., and Bourne, H. R. (1997) Science 275, 381-384[Abstract/Free Full Text]
  36. Luttrell, L. M., Hawes, B. E., Touhara, K., van Biesen, T., Koch, W. J., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 12984-12989[Abstract/Free Full Text]
  37. Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 6193-6197[Abstract/Free Full Text]
  38. Dickenson, J. M., and Hill, S. J. (1998) Eur. J. Pharmacol. 355, 85-93[CrossRef][Medline] [Order article via Infotrieve]
  39. Fedorov, Y. V., Jones, N. C., and Olwin, B. B. (1998) Mol. Cell. Biol. 18, 5780-5787[Abstract/Free Full Text]
  40. Hawes, B. E., Luttrell, L. M., Exum, S. T., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 15776-15785[Abstract/Free Full Text]
  41. Luttrell, L. M., Ostrowski, J., Cotecchia, S., Kendall, H., and Lefkowitz, R. J. (1993) Science 259, 1453-1457[Medline] [Order article via Infotrieve]
  42. Carlson, S. A., Chatterjee, T. K., and Fisher, R. A. (1996) J. Biol. Chem. 271, 23146-23153[Abstract/Free Full Text]
  43. Thompson, J. B., Wade, S. M., Harrison, J. K., Salafranca, M. N., and Neubig, R. R. (1998) J. Pharmacol. Exp. Ther. 285, 216-222[Abstract/Free Full Text]
  44. Ulloa-Aguirre, A., Stanislaus, D., Arora, V., Vaananen, J., Brothers, S., Janovick, J. A., and Conn, P. M. (1998) Endocrinology 139, 2472-2478[Abstract/Free Full Text]
  45. Akhter, S. A., Luttrell, L. M., Rockman, H. A., Iaccarino, G., Lefkowitz, R. J., and Koch, W. J. (1998) Science 280, 574-577[Abstract/Free Full Text]
  46. Lai, J., Waite, S. L., Bloom, J. W., Yamamura, H. I., and Roeske, W. R. (1991) J. Pharmacol. Exp. Ther. 258, 938-944[Abstract]
  47. Thomas, E. A., and Ehlert, F. J. (1994) J. Pharmacol. Exp. Ther. 271, 1042-1050[Abstract]
  48. Offermanns, S., Wieland, T., Homann, D., Sandmann, J., Bombien, E., Spicher, K., Schultz, G., and Jakobs, K. H. (1994) Mol. Pharmacol. 45, 890-898[Abstract]
  49. Dell'Acqua, M. L., Carroll, R. C., and Peralta, E. G. (1993) J. Biol. Chem. 268, 5676-5685[Abstract/Free Full Text]
  50. Corey, S., Krapivinsky, G., Krapivinsky, L., and Clapham, D. E. (1998) J. Biol. Chem. 273, 5271-5278[Abstract/Free Full Text]
  51. Krapivinsky, G., Gordon, E. A., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141[CrossRef][Medline] [Order article via Infotrieve]
  52. Krapivinsky, G., Krapivinsky, L., Wickman, K., and Clapham, D. E. (1995) J. Biol. Chem. 270, 29059-29062[Abstract/Free Full Text]
  53. Krapivinsky, G., Kennedy, M. E., Nemec, J., Medina, I., Krapivinsky, L., and Clapham, D. E. (1998) J. Biol. Chem. 273, 16946-16952[Abstract/Free Full Text]
  54. Sowell, M. O., Ye, C., Ricupero, D. A., Hansen, S., Quinn, S., Vassilev, P., and Mortensen, R. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7921-7926[Abstract/Free Full Text]
  55. Pals-Rylaarsdam, R., Xu, Y., Witt-Enderby, P., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270, 29004-29011[Abstract/Free Full Text]
  56. Jurman, M. E., Boland, L. M., Liu, Y., and Yellen, G. (1994) BioTechniques 17, 876-881[Medline] [Order article via Infotrieve]
  57. Bünemann, M., Brandts, B., zu Heringdorf, D. M., van Koppen, C. J., Jakobs, K. H., and Pott, L. (1995) J. Physiol. 489, 701-777[Abstract]
  58. Bünemann, M., and Pott, L. (1995) J. Physiol. 482, 81-92[Abstract]
  59. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflugers Arch. 391, 85-100[Medline] [Order article via Infotrieve]
  60. Jan, L. Y., and Jan, Y. N. (1997) Curr. Opin. Cell Biol. 9, 155-160[CrossRef][Medline] [Order article via Infotrieve]
  61. Breitwieser, G. (1996) J. Membr. Biol. 152, 1-11[CrossRef][Medline] [Order article via Infotrieve]
  62. Wickman, K. B., and Clapham, D. E. (1995) Curr. Opin. Neurobiol. 5, 278-285[CrossRef][Medline] [Order article via Infotrieve]
  63. Conklin, B. R.., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. R. (1993) Nature 363, 274-276[CrossRef][Medline] [Order article via Infotrieve]
  64. Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 2071-2077


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.