A Dominant-Negative Strategy for Studying Roles of G Proteins
in Vivo*
Annette
Gilchrist
§¶,
Moritz
Bünemann
§,
Anli
Li
,
M. Marlene
Hosey
§, and
Heidi E.
Hamm
§¶
From the
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 |
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
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
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 G
i, G
q, or
G
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 G
i carboxyl-terminal peptide results in specific
inhibition of GIRK activity in response to agonist stimulation of the
M2 muscarinic receptor. The G
i minigene
construct completely blocks agonist-mediated M2 mAChR K+ channel response whereas the control minigene constructs
(empty vector, pcDNA3.1, and the G
carboxyl peptide in random
order, pcDNA-G
iR) had no effect on agonist-mediated
M2 muscarinic receptor GIRK response. The inhibitory
effects of the G
i minigene construct were specific
because overexpression of peptides corresponding to the carboxyl
terminus of G
q or G
s had no effect on
M2 muscarinic receptor stimulation of the K+ channel.
 |
INTRODUCTION |
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
subunit. The exchange of GDP for GTP leads to
dissociation of the G
dimer from the G
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 G
subunits that are commonly divided into four families
based on their sequence similarity: Gi, Gs,
Gq, and G12. Similarly, multiple G
(5) and
G
(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
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 G
i, G
o,
G
q, and G
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 G
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 G
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 G
subunits show that the last
4-7 amino acids of G
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 G
carboxyl-terminal peptides can competitively block
G protein-coupled downstream events (23-26). A carboxyl-terminal peptide from G
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 G
t are critical
for high affinity binding of the G
t peptide to
rhodopsin. Similarly, a carboxyl-terminal peptide from
G
s (384-394), but not corresponding peptides from G
i1/2, inhibits the ability of
2-adrenergic receptors to activate G
s and
adenylyl cyclase (30). In addition, a carboxyl-terminal undecapeptide
from G
i1/2 can bind the adenosine A1 receptor, whereas
the corresponding peptide from G
t, which differs by only 1 amino acid residue does not (31). Thus, the carboxyl terminus of G
protein
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 G
(36), (ii) inhibiting
GPCRs by expressing the carboxyl terminus of
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 G
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
1B-adrenergic receptors
or M1 muscarinic receptors and the G
q
carboxyl-terminal minigene (residues 305-359) inhibits agonist
stimulated inositol phosphate (IP) production, whereas co-expression
with the G
q amino terminus (residues 1-54) has no
effect. Inhibition by G
q (305-359) was apparently
specific for Gq-coupled receptors because neither
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
G
q carboxyl-terminal minigene to the myocardium
resulted in a marked inhibition of
1B-adrenergic
receptor-mediated IP production and blockade of cardiac hypertrophy.
In this paper, we study the effects of several carboxyl termini G
peptides using a minigene approach. To test whether minigene constructs
encoding the carboxyl-terminal 11 amino acid residues from G
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 G
q in which the last 5 amino acids of G
q are substituted with the corresponding
residues from G
i or G
o (34), suggesting that this receptor contains domains that are specifically recognized by
the carboxyl terminus of G
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
G
-activated channel (52-54). Our experiments indicate that the
G
i carboxyl terminus minigene construct can completely
block M2 mAChR-mediated K+ channel activation.
The inhibition appears specific as constructs producing
G
s, G
q, or a scrambled G
i
carboxyl-terminal peptide had no effect.
 |
MATERIALS AND METHODS |
Construction of G
Carboxyl-terminal Minigenes--
The
cDNA encoding the last 11 amino acids of human G
subunits
(G
i1/2, G
s, G
q) or the
G
i1/2 carboxyl terminus in random order
(G
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 G
minigene
constructs used for transfection experiments (pcDNA3.1;
pcDNA-G
i; pcDNA-G
iR; pcDNA-G
q, and pcDNA-G
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):
H3-CD8
(human), 1 µg; pC1-GIRK1 (rat), 1 µg; pcDNA1-GIRK4 (rat), 1 µg; pcDNA3.1, pcDNA-G
i,
pcDNA-G
iR, pcDNA-G
q, or
pcDNA-G
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-G
i or pcDNA-G
iR constructs, their cDNA was used as the template for PCR with forward and
reverse primers that correspond to G
insert and vector,
respectively (forward: 5'-ATCCGCCGCCACCATGGGA; reverse:
5'-GCGAAAGGAGCGGGCGCTA). The primers for the G
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-G
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
subunit of G proteins have
been made in which mutations are made in regions that contact the
magnesium ion. For the
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 G
o or
G
i (5, 6). However, neither of these mutations has
resulted in effective dominant-negatives probably because the GDP
complexes of G
i, G
o, and
G
s have low affinity for Mg2+. Thus, we
looked to other regions on G protein
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
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
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
G
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
G
i1/2, G
q, or G
s (Table
I). As a control, we also made a minigene
that expressed the carboxyl terminus of G
i1/2 in random order (G
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 G
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).
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Table I
Carboxyl termini sequences
Alignment of the last 11 amino acid residues from human
G i1/2, G q, and G s subunits. Also shown
is the peptide sequence of G iR, the G i1/2
sequence in random order, used to construct the control
minigene.
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Fig. 1.
The cDNA minigene constructs.
Insert DNA, all G 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 G iR contains the
G 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-G i; lane 4 is
pcDNA-G iR; and lane 5 is
pcDNA-G 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).
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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 G
carboxyl-terminal peptide insert. Separation of the PCR
products on 1.5% agarose gels (Fig.
2A) indicates the presence of
the G
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).

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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-G i, or pcDNA-G 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 G carboxyl-terminal peptide insert. Separation of the PCR
products on 1.5% agarose gels indicates the presence of the G
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-G iR;
lane 3 is cells transfected with
pcDNA-G 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-G i, or pcDNA-G iR were
analyzed by ion mass spray analysis.
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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-G
i transfected cells, and peak 1 from cells
transfected with a vector expressing the carboxyl terminus in random
order (pcDNA-G
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-G
i or
pcDNA-G
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 G
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
G
, 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 G
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-G
i or pcDNA-G
iR DNA with 1 µM ACh revealed that cells transfected with
pcDNA-G
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-G
iR (Fig.
3A) but not in cells transfected with
pcDNA-G
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-G
i compared
with 24.1 ± 8.8 pA/pF (n = 11) in cells
transfected with pcDNA-G
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-G
iR (Fig. 3C). Basal levels
for all three conditions were equivalent (pcDNA 3.2 ± 1.8 pA/pF (n = 5); G
i 6.1 ± 0.9 pA/pF
(n = 14); G
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 G
i minigene
construct completely blocks the agonist-mediated M2 mAChR
GIRK1/4 response, whereas the control minigene constructs (empty
vector, pcDNA3.1, and the G
i1/2 carboxyl peptide in
random order, pcDNA-G
iR) had no effect on the
agonist-mediated M2 mAChR GIRK1/4 response.

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Fig. 3.
Minigenes encoding carboxyl-terminal
G 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-G i, or
pcDNA-G 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-G 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-G 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-G 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-G 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.
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The cardiac IKACh channel is activated upon stimulation of
M2 mAChR via G proteins of the Gi family. The
carboxyl-terminal region of G
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
G
q with corresponding residues from G
i
allowed receptors that signal exclusively through G
i
subunits to activate the chimeric
subunits and stimulate the
G
q effector, phospholipase C-
. 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 G
carboxyl termini for G
q or
G
s. ACh-stimulated IKACh currents from cells
transfected with pcDNA-G
q (Fig.
4B; 19.5 ± 5.5 pA/pF
(n = 6)) or pCDNA-G
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-G
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-G
i whose ACh-stimulated
IKACh currents were significantly decreased as compared
with cells transfected with pcDNA-G
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 G
i minigene, pcDNA-G
i.

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|
Fig. 4.
Transfection of the
G i carboxyl-terminal
minigene inhibits the M2 mAChR activated IKACh
response, whereas G s or
G q carboxyl-terminal minigenes do
not. Stably m2 mAChR-expressing HEK 293 cells were transiently
transfected with GIRK1, GIRK4 and pcDNA-G iR,
pcDNA-G i, pcDNA-G s, or
pcDNA-G q. A, the maximum current evoked
by 1 µM ACh was 3.7 ± 1.5 pA/pF (n = 14) in cells transfected with pcDNA-G i compared
with 24.1 ± 8.8 pA/pF (n = 11) in cells
transfected with pcDNA-G iR. B, the
maximum current evoked by ACh was 19.5 ± 5.6 pA/pF
(n = 6) in cells transfected with the
pcDNA-G q compared with 23.8 ± 10.5 pA/pF
(n = 6) in cells transfected with
pcDNA-G iR. C, the maximum current evoked
by ACh was 35.5 ± 9.7 pA/pF (n = 5) in cells
transfected with pcDNA-G s compared with 26.0 ± 8.0 pA/pF (n = 5) in cells transfected with
pcDNA-G iR.
|
|
Recent experiments targeting receptor-G protein interaction (45) by
constructing minigenes that encode the last 55 amino acids of
G
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 G
i minigene constructs that encode only the last 11 amino acids of the carboxyl terminus of
G
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 G
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
subunits is critical in
both mediating receptor-G protein interaction and in receptor
selectivity (31-35). Our results confirm that the carboxyl terminus of
G
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
G
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 G
carboxyl-terminal undecapeptide sequences for each of the
G
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.
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).
 |
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