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INTRODUCTION |
The most common type of signaling pathway requires the sequential
interactions of a seven-transmembrane receptor, a G protein composed of
,
, and
subunits, and an effector. How the specificity of
this signaling pathway is encoded in the protein-protein interactions of ever increasing numbers of these signaling partners is not known. A
simple way to encode the specificity would be for each receptor to
interact with a specific G protein 

heterotrimer. In this
regard, the recent identification of 23
subunits (1), 6
subunits (2, 3), and 12
subunits (4-6) predicts the potential
existence of several hundred G protein 

heterotrimers that
could serve as intermediaries between a similarly high number of
receptors and a somewhat smaller number of effectors. Supporting this
scenario, there is increasing evidence that the subunit composition of
G protein 

heterotrimers may provide the level of selectivity that is needed to interact with the multitude of receptors and effectors that are now known to exist. Antisense (7, 8) and ribozyme
(9) strategies have proven to be most useful in identifying which of
the potential G protein 

heterotrimers are physiologically
relevant. These approaches have the advantage that they allow the
selective suppression of individual G protein subunits and the
subsequent identification of which signaling pathway(s) are impaired.
In a recent study, we described the first use of the ribozyme strategy
to suppress specifically the expression of G protein
7
subunit, thereby identifying a specific role of this subtype in
coupling the
-adrenergic receptor to stimulation of adenylyl cyclase
activity in HEK 293 cells (9). In the present study, we have explored
the use of the same ribozyme approach to identify functional
associations of the G protein
7 subunit with particular G protein
or
subunits in this signaling pathway. To this end, a
ribozyme directed against the
7 subunit was transfected
into HEK 293 cells to suppress specifically the expression of the
7 protein, and then the effects of this manipulation on
the levels of the
and
s subunits of the G proteins
were determined. Of the
subunits, only the
1 subunit
was coordinately reduced following treatment with the ribozyme directed
against the
7 subunit, thereby demonstrating a
functional association between the
1 and
7 subunits. By contrast, neither the 52- nor 45-kDa
s subunits were suppressed following treatment with the
ribozyme directed against the
7 subunit. Taken together,
the results indicated that the
7 and
1
subunits play a specific role in the
-adrenergic receptor signaling
pathway and that their role cannot be compensated for by other members
of these large, multi-gene families.
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EXPERIMENTAL PROCEDURES |
Ribozyme Design and Delivery to HEK 293 Cells--
A chimeric
DNA-RNA hammerhead ribozyme targeted against the G protein
7 subunit mRNA was chemically synthesized and
modified by adding two phosphorothioate linkages at the 3'-end, as
described previously (9). Also, HEK 293 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum and plated into 100-mm dishes, as described
previously (9). The cells were then transfected at approximately
60-80% confluence with fresh serum-free medium containing premixed
7 ribozyme (2 µM) and the cationic lipid,
LipofectAMINE (20 µg/ml, Life Technologies, Inc.). At 5 h
post-transfection, fetal bovine serum was added to a final
concentration of 6%, and sequential addition of 0.5 µM
ribozyme was supplemented to the total concentration of 4 µM at 48 h post-transfection. The control cells were
treated identically but without added ribozyme.
Immunoblot Analysis--
To determine the effect of ribozyme
treatment on G protein subunits, membranes from control and
7 ribozyme-transfected cells were extracted with 1%
sodium cholate overnight as described previously (10). The protein
concentrations were determined by Amido Black assay, and equal amounts
of proteins were resolved on 15% SDS-polyacrylamide gel and
transferred to Nitro-plus nitrocellulose (0.45-µm pore size, Micron
Separations Inc.) using a high temperature procedure described
previously (11). Following transfer to nitrocellulose, the blots were
probed with anti-G protein subtype-specific antibodies (9, 11-14).
Briefly, after blocking, the nitrocellulose blots were incubated with
the primary antibodies for 1 h in high detergent blotto at a
dilution of 1:500 for B-69 (
1); a dilution of 1:150 for
D-17-6 (
2); a dilution of 1:100 for B-24
(
3); a concentration of 1 µg/ml for C-16
(
4) (Santa Cruz Biotechnology Inc.); a dilution of
1:2000 for
5 (CytoSignal Research Products); and a
dilution of 1:500 for 584 (G
s). After three successive
washes, the blots were incubated for 1 h with
125I-labeled goat anti-rabbit F(ab')2 fragment
(1 × 105 dpm/ml, NEN Life Science Products) in high
detergent blotto. After washing, the blots were subjected to
autoradiography by exposure to BioMax MS film (Eastman Kodak Co.), and
the intensity of immunodetectable bands was quantified using the
Molecular Dynamics PhosphorImager SI.
Subcellular Fractionation--
After transfection, cells were
washed with ice-cold phosphate-buffered saline and then lysed with
ice-cold lysis buffer which contains 2 mM
MgCl2, 1 mM EDTA, 20 mM Hepes, 10 mM dithiothreitol, 1 mM
aminoethylbenzenesulfonyl fluoride, 1 µg/ml pepstatin A, and 1 mM benzamidine. The separation of the cytosolic and
particulate fractions was accomplished by centrifugation at
250,000 × g for 30 min. The particulate was extracted
with 1% sodium cholate overnight at 4 °C. Equal percentages of the
cytosolic and solubilized particulate fractions from control and
ribozyme-transfected cells were resolved by SDS-polyacrylamide gel
electrophoresis and subjected to immunoblot analysis.
Construction of FLAG-tagged Human
1 cDNA and
Hemagglutinin-tagged Human
7 cDNAs--
In order to
monitor the half-life of
1 subunit in the intact cell
setting, epitope tagging was used in this study. The FLAG mammalian
transient expression system was used for constructing FLAG-tagged human
1 subunit, and the influenza virus hemagglutinin (HA)1 tag was used for
constructing HA-tagged human
7 subunit cDNA. The
FLAG system is designed for intracellular expression of amino-terminal Met-FLAG fusion protein which relies on the fusion of the FLAG peptide
of eight amino acids (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the coding
sequence of a target gene in a pCMV5 expression vector. This FLAG
epitope tag is recognized by an anti-FLAG M2 monoclonal
antibody (Sigma). The human
1 subunit cDNA coding region was generated by polymerase chain reaction and then cloned into
the pFLAG-CMV-2 vector. The human G protein
7 subunit
cDNA coding region was generated by polymerase chain reaction with 5' primer sequence coding for nine amino acids of HA tag epitope (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) (15) and then cloned into the
pCI-neo Mammalian Expression Vector (Promega Corp., Madison, WI). A
mutant form of the HA-tagged
7m cDNA was generated
in the same way by mutating Cys to Ser in the carboxyl-terminal CAAX box, thereby preventing its modification by isoprenylation (16). This
HA epitope tag is recognized by anti-HA monoclonal antibody (Roche
Molecular Biochemicals).
Metabolic Labeling and Immunoprecipitation--
Following
transfection, the FLAG-tagged
1 protein and HA-tagged
7 protein were detected by immunoprecipitation with
anti-FLAG M2 monoclonal antibody and anti-HA high affinity
monoclonal antibody. Briefly, HEK 293 cells grown in 60-mm dishes were
transfected with a plasmid encoding either FLAG-tagged hybrid protein
1 subunit or HA-tagged
7 protein by
LipofectAMINE transfection method. At 24 h post-transfection,
cells were incubated for 45 min in methionine- and cysteine-free
Dulbecco's modified Eagle's medium and then pulse-labeled with
70-100 µCi of [35S]methionine (NEN Life Science
Products) either for 2.5 min for monomer detection or 1 h for
dimer detection, and then chased for the time points indicated in
the figure legends. The incorporation of label was stopped by addition
of complete medium supplemented with non-radioactive methionine at a
final concentration of 1 mM. Cells were lysed in the lysis
buffer (50 mM Tris-HCl, pH 7.6, 5 mM
MgCl2, 0.5% Nonidet P-40, 0.1% Lubrol, 1 mM
EDTA, 10 µg/ml leupeptin, 10 mM benzamidine, 1 mM aminoethylbenzenesulfonyl fluoride, and 10 µg/ml
pepstatin A) and then passed through 25-gauge 5/8 needle and
freeze-thawed once in
80 °C freezer. The lysates were depleted of
nuclei and cell debris by centrifugation for 10 min at 14,000 rpm in an
Eppendorf centrifuge and then pre-cleared twice with 20 µl of protein
A/G plus-agarose (Santa Cruz Biotechnology). Equal amounts of control
and transfected cells were subjected to immunoprecipitation. For
recovery of the
1 subunit in the monomer form,
immunoprecipitation was performed by overnight incubation at 4 °C on
a rocker with 15 µg/ml anti-FLAG M2 monoclonal antibody. For recovery
of the
1 subunit in the dimer form, immunoprecipitation was carried out with 5 µg/ml anti-HA high affinity monoclonal antibody. The immune complexes were precipitated by adsorption to
protein A/G plus-agarose for an additional 3 h at 4 °C followed by four washes in NET buffer (50 mM Tris-HCl, pH 7.6, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40).
Finally, the immune complexes were dissociated by heating for 10 min in
SDS sample buffer. Protein A/G plus-agarose beads were pelleted by
centrifugation, and the supernatants were resolved by
SDS-polyacrylamide gel electrophoresis. The gel was fixed and soaked
for 30 min in Amplify reagent (Amersham Pharmacia Biotech) and then
dried and exposed to Kodak BioMax MS film with Kodak Biomax
Transcreen-LE Intensifying Screen at
80 °C. The intensity of the
immunodetectable bands on the autoradiogram was quantified by
densitometry analysis.
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RESULTS |
Effect of Ribozyme-mediated Suppression of the
7
Subunit on the Levels of
Subunits in HEK 293 Cells--
Under
physiological conditions, the
and
subunits exist as a dimer
that functions as a single entity (17-20). Synthesis and assembly of
the 
dimer begins in the cytosol, with subsequent translocation
to the plasma membrane being dependent on post-translational lipid
modifications of the
subunit (21, 22). Therefore, we reasoned that
ribozyme-mediated loss of the
7 protein might limit the
formation and translocation of a specific 
dimer to the plasma
membrane. In this event, we expected that the content of one or more
subunits might be reduced in the plasma membrane. Accordingly, the
expression of the known
subunits was examined following the
treatment of HEK 293 cells with ribozyme specific for the
7 subunit.
To date, six
subunits have been identified by molecular cloning (2,
3). To determine which of these are expressed in HEK 293 cells, it was
first necessary to generate (for
1,
2,
and
3) or obtain commercially available (for
4 and
5) antibodies specific for each
subtype. Then, the specificities of these antibodies were determined by
immunoblot analysis of the recombinantly expressed
proteins. As
shown in Fig. 1, the sizes of the
recombinantly expressed
1,
2,
3/6,
4, and
5 proteins
ranged from 35 to 39 kDa and the antibodies reacted only with their
corresponding
proteins. To determine whether the ribozyme-mediated
loss of the
7 protein would affect the expression of one
or more of these
subunits, membrane proteins from HEK 293 cells
treated in the absence (C) or presence (RZ) of
the ribozyme were immunoblotted with these
subtype-specific
antibodies. As shown in Fig.
2A and quantified for three
separate experiments in Fig. 2B, the
1,
2,
4, and
5 subunits were
readily detected in membranes from HEK 293 cells treated in the absence
(C) or presence (RZ) of the ribozyme. Under the
same condition, the
3 subunit was not expressed at a
detectable level, but whether this is due to the lack of expression or
the relatively poor affinity of the
3 subtype-specific
antibody is not known. When the relative amounts of the
1,
2,
4, and
5 subunits were quantified in the ribozyme-treated membranes and then expressed as a percentage of their levels in control
membranes, the levels of the
2,
4, and
5 subunits showed no reduction. However, the level of
the
1 subunit showed a striking reduction in
ribozyme-treated membranes to 30.2 ± 6.9% of its level in
control membranes. By way of comparison, the level of the
7 subunit showed a similarly striking loss in
ribozyme-treated membranes to 21 ± 8.4% of its level in control
membranes (Fig. 2, A and B). These data showing
coordinate suppression of the
1 subunit along with the
7 subunit provide strong evidence for their functional
association in the intact cell setting.

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Fig. 1.
Specificity of G protein subtype-specific antibodies. The recombinantly expressed G
protein 1, 2, 3, and
5 subunits were resolved on a 15% SDS-polyacrylamide
gels, transferred to nitrocellulose, and then immunoblotted with subtype-specific antibodies, as described previously (11, 12). Since
recombinantly expressed G protein 4 subunit is not
available yet, a bovine brain membrane preparation containing this
protein was used as positive control. The sizes of the various subtypes ranged from 35 to 39 kDa. Br, brain.
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Fig. 2.
Ribozyme suppression of G protein
subtypes in HEK 293 cells at the protein
level. Membrane proteins from control (C) and
7 ribozyme-transfected (RZ) cells were
extracted, resolved on 15% SDS-polyacrylamide gels, transferred to
nitrocellulose, and immunoblotted as described under "Experimental
Procedures." Following transfer, the nitrocellulose was cut along the
30-kDa marker; the higher molecular blot was probed with one of the subtype-specific antibodies, and the lower molecular blot was probed
with the 7 subtype-specific antibody (A-67).
A, shows representative immunoblots demonstrating the
selective loss of the 1 and 7 subunits.
B, quantifies the loss of the 1 and
7 subunits in the 7 ribozyme-treated
cells. The intensities of the bands from three separate experimental
sets of immunoblots were determined by PhosphorImager analysis. The
relative amounts of proteins in the 7 ribozyme-treated
cells were expressed as a percentage of their levels in the control
cells. The data shown are mean ± S.E.
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Mechanism of Loss of the
1 Protein--
The
resulting decrease in the expression of the
1 protein
following ribozyme treatment indicated that the
7
protein is required for the appearance of the
1 protein
in the membrane. This requirement could reflect the need for the
7 protein to allow the translocation of the
1 protein from its place of synthesis in the cytosol to the site of its function in the plasma membrane. To examine this possibility, subcellular fractionation experiments were performed. HEK
293 cells treated in the absence or presence of ribozyme directed against the
7 subunit were fractionated and the
resulting soluble and particulate fractions were subjected to
immunoblot analysis with the
1 subtype-specific antibody
(B-69). As shown in Fig. 3A
and quantified for three separate experiments in Fig. 3B,
the amount of
1 protein in the particulate fraction was
reduced to 38 ± 2.98% (n = 3) in the
ribozyme-treated cells (RZ) compared with the control cells
(C). However, this loss in the particulate fraction did not
result in detectable accumulation of the
1 protein in
the cytosolic fraction in the ribozyme-treated cells (RZ, S) compared with the control cells (C, S). These data support
the notion that the
1 protein, without benefit of
association with
7 protein, may be rapidly degraded.

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Fig. 3.
Subcellular fractionation of
1 subunit. After transfection,
control (C) and 7 ribozyme-treated cells
(RZ) cells were lysed, separated into soluble (S)
and particulate (P) fractions by centrifugation at
250,000 × g, and then equal percentages of these
fractions were resolved on 15% SDS-polyacrylamide gels, transferred to
nitrocellulose, and immunoblotted with the 1-specific
antibody (B-69). A shows representative immunoblots
demonstrating the loss of the 1 subunit from the
particulate fraction of the 7 ribozyme-treated cells
(RZ, P) compared with the control (C, P) but no
detectable accumulation of 1 subunit in the soluble
fraction (RZ, S) compared with control (C, S).
B quantifies the change in the 1 subunit. The
intensities of the bands from three separate experimental sets of
immunoblots were determined by densitometric analysis. The relative
amounts of proteins in the particulate fraction from the
7 ribozyme-treated cells (RZ) were expressed
as a percentage of their levels in the control (C) cells.
The data shown are mean ± S.E.
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Change in Half-life of
1 Subunit in Monomeric Versus
Heterodimeric State--
To test if the stability of the
1 subunit is different in the monomeric state
versus heterodimeric state in complex with the
7 subunit, pulse-chase labeling studies were performed
for each condition. For this purpose, FLAG- and HA-tagged versions of
the human
1 and
7 subunits were
constructed, respectively. Previous studies have shown that the use of
these epitope tags does not interfere with assembly of the 
dimer
(23, 24). Moreover, these tagged proteins can be readily monitored with well characterized monoclonal antibodies whose immunoprecipitation capabilities and spectrums of cross-reactivity with non-tagged proteins
are already known.
To determine the half-life of the
1 monomer, HEK 293 cells expressing the FLAG-tagged
1 subunit alone were
labeled with [35S]methionine for 2.5 min and then chased
for various time points. Subsequently, cells were lysed and
immunoprecipitated with the anti-FLAG M2 antibody in the presence of
0.5% SDS for the recovery of
1 monomer. The immune
complexes were precipitated, denatured in SDS sample buffer, and
resolved on 15% SDS-polyacrylamide gels. As shown in Fig.
4A, the
35S-labeled
1 monomer was readily recovered
at 0 min of chase. However, essentially no labeled
1
monomer was detected by 3 h of chase. To determine the half-life
of the
1 monomer more accurately, shorter chase periods
were employed. As shown in Fig. 4B and quantified for three
separate experiments in Fig. 4C, there was progressive loss
of the 35S-labeled
1 monomer from 0 to
3 h of chase. Based on densitometric and curve fitting analysis,
the loss of the labeled
1 monomer was best fit to a
single phase exponential decay, with an estimated half-life of only
20.8 min.

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Fig. 4.
Half-life of the
1 monomer. At 24 h
post-transfection with the FLAG-tagged human 1 subunit,
HEK 293 cells were pulse-labeled with 70 µCi of
[35S]methionine for 2.5 min and chased for the indicated
time points. The 35S-labeled 1 subunit was
immunoprecipitated with anti-FLAG M2 monoclonal antibody for recovery
of 1 monomer and then resolved on 15%
SDS-polyacrylamide gels. Gels were fixed, dried, and exposed to Kodak
BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying Screen at
80 °C. The images were quantified by densitometric analysis.
A and B show representative autoradiograms of the
35S-labeled 1 monomer over longer and
shorter time points, respectively. C quantifies the loss of
the 35S-labeled 1 monomer over the shorter
time points (B). Curve fitting analysis of the results from
three separate experimental sets revealed the loss of the
35S-labeled 1 monomer was best fit to a
single phase exponential decay, with an estimated half-life of only
20.8 min.
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To determine the half-life of
1 protein in a
heterodimeric complex with the
7 protein, HEK 293 cells
expressing the
1 subunit alone or in combination with
the HA-tagged
7 subunit were labeled with
[35S]methionine for 1 h. Then cells were lysed and
immunoprecipitated with the anti-HA antibody in the absence or presence
of 0.5% SDS. Subsequently, immune complexes were resolved on 15%
SDS-polyacrylamide gels. As shown in Fig.
5, recovery of the
35S-labeled
7 subunit was the same when
immunoprecipitated with the anti-HA antibody in the presence
(lane 1) or absence (lane 2) of SDS. By contrast,
recovery of the labeled
1 subunit was very different
between these two conditions. No labeled
1 subunit was
detected when the
7 subunit was immunoprecipitated in
the presence of SDS (lane 1), but it was readily observed
when the
7 subunit was immunoprecipitated in the absence
of SDS (lane 2). These results indicated that the
1 subunit is present as a complex with the
7 subunit in the absence of SDS and, as a result, can be
brought down with the anti-HA antibody directed against the
7 subunit. Without transfection of
7
subunit (lane 3) or without addition of anti-HA antibody
(lane 4), neither the
1 subunit nor the
7 subunit were immunoprecipitated, showing that the
appearance of the
1 subunit is dependent on the presence of the
7 subunit or the anti-HA antibody,
respectively.

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Fig. 5.
Detection of the
1 7
dimer by immunoprecipitation. HEK 293 cells expressing the
1 subunit alone or in combination with the HA-tagged
7 subunit were labeled with
[35S]methionine for 1 h. Cells were then lysed and
immunoprecipitated with the anti-HA antibody in the absence or presence
of 0.5% SDS. Subsequently, immune complexes were resolved on 15%
SDS-polyacrylamide gels, and the gels were fixed, dried, and exposed to
Kodak BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying
Screen at 80 °C.
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By having validated this method for detection of labeled
1 subunit in the heterodimeric state, we sought to
determine the half-life of the
1 subunit when complexed
with the
7 subunit. For this purpose, HEK 293 cells
expressing the
1 subunit and the HA-tagged
7 subunit together were labeled with
[35S]methionine for 1 h, chased for the various time
points, and then immunoprecipitated with the anti-HA antibody in the
absence of SDS. As shown in Fig.
6A and quantified for three
separate experiments in Fig. 6B, there was progressive loss
of the 35S-labeled
1 protein from 0 to
54 h of chase. Based on densitometric and curve fitting analysis,
the loss of the majority of the labeled
1 subunit when
complexed with the wild type HA-tagged
7 subunit was
best fit to a single phase exponential decay, with an estimated half-life of 14.2 h. However, a small portion of the labeled
1 protein in the heterodimeric state was stable for at
least 54 h. Taken together, these data indicated that the
1
7 dimer is turned over >40-fold more
slowly than the
1 monomer in the intact cell
setting.

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Fig. 6.
Half-life of the
1 subunit complexed with the wild type
HA-tagged 7 subunit. HEK 293 cells co-expressing 1 subunit and HA-tagged
7 subunit were labeled with
[35S]methionine for 1 h, chased for the various time
points, and then immunoprecipitated with the anti-HA antibody in the
absence of SDS for recovery of the HA- 7 subunit
complexed with the 1 subunit. The immune complexes were
resolved on 15% SDS-polyacrylamide gels, and the gels were fixed,
dried, and exposed to Kodak BioMax MS film with a Kodak Biomax
Transcreen-LE Intensifying Screen at 80 °C. The images were
quantified by densitometric analysis. A shows a
representative autoradiogram of the 35S-labeled
1 complexed with the HA- 7 subunit.
B quantifies the loss of the 35S-labeled
1 complexed with the HA- 7 subunit. Curve
fitting analysis of the results from three separate experimental sets
revealed the loss of the 35S-labeled 1 in
the heterodimeric state was best fit to a single phase exponential
decay, with an estimated half-life of 14.2 h. For this analysis,
the "0" time point was extrapolated.
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To determine whether association with the
7 subunit is
sufficient to stabilize the half-life of the
1 subunit
or whether translocation of the
1
7
complex to the membrane is required for this stabilization, we
generated a mutant form of the HA-tagged
7 subunit by
replacing the Cys residue with a Ser residue in the carboxyl-terminal
"CAAX" motif. As a result of this substitution, the mutant
HA-tagged
7 subunit is not able to undergo prenylation or translocation to the membrane (16). HEK 293 cells expressing the
1 subunit and the mutant HA-tagged
7
subunit together were labeled with [35S]methionine for
1 h, chased for the various time points, and then
immunoprecipitated with the anti-HA antibody in the absence of SDS. As
shown in Fig. 7A and
quantified for two separate experiments in Fig. 7B, there
was a progressive and similar loss of the 35S-labeled
1 protein whether complexed with mutant or wild type HA-tagged
7 subunit (compare with Fig. 6A).
Based on densitometric and curve fitting analysis, the loss of the
majority of the labeled
1 subunit when complexed with
the mutant HA-tagged
7 subunit was best fit to a single
phase exponential decay, with an estimated half-life of 16.3 h.
These data demonstrated that association with the
7
subunit is sufficient for stabilization of the
1 subunit.

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Fig. 7.
Half-life of the
1 subunit complexed with the mutant
HA-tagged 7 subunit. A mutant
form of the HA-tagged 7 subunit was generated by
replacing the Cys residue with a Ser residue in the carboxyl-terminal
CAAX motif. HEK 293 cells co-expressing the 1 subunit
and the mutant HA-tagged 7 subunit were labeled with
[35S]methionine for 1 h, chased for the various time
points, and then immunoprecipitated with the anti-HA antibody in the
absence of SDS. The immune complexes were resolved on 15%
SDS-polyacrylamide gels, and the gels were fixed, dried, and exposed to
Kodak BioMax MS film with a Kodak Biomax Transcreen-LE Intensifying
Screen at 80 °C. The images were quantified by densitometric
analysis. A shows a representative autoradiogram of the
35S-labeled 1 complexed with the mutant
HA- 7 subunit. B quantifies the loss of the
35S-labeled 1 complexed with the mutant
HA- 7 subunit. Curve fitting analysis of the results from
two separate experimental sets revealed the loss of the
35S-labeled 1 complexed with the mutant
HA- 7 subunit was best fit to a single phase exponential
decay, with an estimated half-life of 16.3 h. For this analysis,
the 0 time point was extrapolated.
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Effect of Ribozyme-mediated Suppression of the
7
Subunit on the Levels of 45 and 52 kDa
s Subunits in HEK
293 Cells--
To determine whether a ribozyme directed against the
7 subunit would also affect the expression of one or
more of the
s subunits, membrane proteins from HEK 293 cells treated in the absence or presence of the ribozyme against
7 subunit were immunoblotted with the G protein
s subtype-specific antibody (antibody 584). As shown in
Fig. 8A, both the 45- and
52-kDa
s subunits, which are derived from alternative
splicing (25), were detected in control and ribozyme-treated cells. The
relative amounts of the two forms of
s subunits were
quantified in control and
7 ribozyme-treated cells by
densitometry and then expressed as percentages of their levels in
control cells for three separate experiments in Fig. 8B.
There were no differences in the intensities of the 45- and 52-kDa
s subunits between the control and ribozyme-treated
cells even though the intensities of the
1 and
7 subunits were markedly and coordinately suppressed in
the ribozyme-treated cells.

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Fig. 8.
Lack of ribozyme suppression of G protein as
subtypes in HEK 293 cells at the protein level. Membrane proteins
from control (C) and 7 ribozyme-transfected
(RZ) cells were extracted, resolved on 15%
SDS-polyacrylamide gels, transferred to nitrocellulose, and
immunoblotted as described under "Experimental Procedures."
A shows representative immunoblots demonstrating no loss of
the s subtypes even though selective loss of the
1 and 7 subunits occurred. B
quantifies the results in control (C) versus
7 ribozyme-treated (RZ) cells. The
intensities of the bands from three separate experimental sets of
immunoblots were determined by PhosphorImager analysis. The relative
amounts of proteins in the 7 ribozyme-treated cells were
expressed as a percentage of their levels in the control cells. The
data shown are mean ± S.E.
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 |
DISCUSSION |
The G protein 
subunits exist as a tightly associated
complex that plays a prominent role in transducing information from a
receptor to an appropriate effector in a specific fashion. Thus, the

subunit complex binds directly to receptors (26-28), where it
may act as a conformational switch to direct receptor-G protein coupling (29). Also, the 
subunit complex interacts directly with
a variety of effectors to regulate their activities (for reviews see
Refs. 4 and 30). Finally, the 
subunit complex regulates kinases
involved in desensitization of receptors (31). Recently, in
vitro (32-36) and in vivo (7-9) studies raise the strong possibility that the specific composition of the 
subunit complex contributes to the recognition of these signaling components.
With the identification of 6
subtypes (2, 3) and 12
subtypes
(4-6), it has become increasingly important to decipher which 
subunit complexes actually exist in intact cells and to identify their
roles in particular signaling pathways. In vitro strategies
have revealed that certain combinations are not possible, but most
combinations are physically able to form 
dimers (18, 37, 38).
While providing valuable information on structure-function relationships, these reconstitution approaches fall critically short of
establishing the roles of specific 
subunits in particular signaling pathways in the intact cell setting. Increasingly, reverse genetics strategies are being employed to fill this gap (7-9). In a
previous study, we used the ribozyme approach to identify a specific
role of the
7 subunit in the
-adrenergic receptor signaling pathway (9). HEK 293 cells transfected with a ribozyme directed against the
7 subunit showed a specific
reduction of the
7 protein that was associated with a
significant attenuation of isoproterenol-stimulated adenylyl cyclase
activity. In the present study, we asked whether loss of the
7 protein would have any effect on the expression and/or
membrane localization of the associated
and
s
proteins that comprise the G protein heterotrimer in the
-adrenergic
receptor signaling pathway. Our results show the first successful use
of a ribozyme approach directed against a specific
subunit to
identify a functional association with a particular
subunit.
Functional Association of
1 and
7
Subunits--
The
subunit is synthesized in the cytosol and then
translocated to the membrane upon association with the appropriately modified
subunit (21, 22). This suggested the possibility that
ribozyme-mediated loss of the
7 protein might be a
useful approach to obtain information on its functional association
with a particular
subtype. Following treatment of HEK 293 cells
with ribozyme specific for the
7 subunit, only the
1 protein showed a coordinate reduction with the
7 protein in the membranes of ribozyme-treated cells
compared with control cells (Fig. 2). Next, the mechanism underlying
the coordinate suppression of the
1 protein was
explored. Subcellular fractionation studies revealed that loss of the
1 protein in the membrane did not lead to any detectable
increase in the
1 protein in the cytosol of
ribozyme-treated cells. However, pulse-chase labeling studies showed a
dramatic difference in the half-life of the
1 monomer
(20.8 min, Fig. 4) compared with the
1
7
dimer (14.2 h, Fig. 6). These data indicate that the
1
protein is rapidly and specifically degraded when sufficient
7 protein is not available for dimerization. The
7 protein undergoes post-translational processing,
including prenylation and carboxyl methylation (16, 20, 21). To explore
the influence of processing, a mutant
7 protein was
produced by replacing the Cys residue four residues from the carboxyl
terminus with a Ser residue. The effect of this substitution is to
prevent prenylation and carboxyl methylation of the mutant
7 protein. Pulse-chase labeling studies revealed no
significant difference in the half-life of the
1 protein
when complexed with the wild type versus the mutant
7 protein (compare Figs. 6 and 7). This result indicates that dimerization rather than post-translational processing and translocation to the membrane is sufficient to increase the half-life of the
1 protein. This contrasts with a previous study
showing prenylation and carboxyl methylation of RhoA leads to an
increase in the half-life of this protein (39). While coordinate
suppression of the
1 and
7 subunits
provides strong evidence for their functional association in the intact
cell setting, these results also raise a number of questions. One
question is whether the pairing of the
1
7
subunits is specific to HEK 293 cells or is common to various types of
cells. A second question revolves around what factors govern the
preferential assembly of the
1
7 dimer in cells expressing multiple
and
subtypes. One possibility is a
structural feature that favors certain 
subunit combinations. However, in vitro studies show that the
1 and
7 subunits have the potential to interact with a wide
variety of subtypes (37, 38). Another possibility is a spatial factor
that directs selective assembly of the
1
7
dimer within a particular subcellular compartment. In this regard,
there is evidence that certain mRNAs are localized within cells,
resulting in proteins being synthesized within discrete subcellular
compartments (40, 41). That the
subtypes are localized within
different subcellular domains is increasingly clear (42, 43).
Lack of Functional Association of
s and
7 Subunits--
Ribozyme-mediated suppression of the
7 protein is associated with significant attenuation of
-adrenergic receptor-stimulated adenylyl cyclase activity (9). Since
loss of the
7 protein results in corresponding reduction
of the
1 protein, the results of the present study
provide strong evidence that the
7 and
1 subunits, together with the
s subunit, comprise the
Gs heterotrimer that couples the
-adrenergic receptor to
adenylyl cyclase (44). The
s subunit comes in multiple
forms that are generated by alternative splicing of a single gene (45,
46). Interestingly, ribozyme-mediated suppression of the
7 subunit has no effect on either the 52- or 45-kDa
s proteins (Fig. 8). This result indicates that the
s subunits associate with the membrane and are stable in
the absence of the
1
7 subunit complex.
This is consistent with studies showing that the
s
subunits contain their own membrane targeting signals (47, 48).
Assembly of G Protein Heterotrimers--
These and other studies
(49) have begun to answer very basic questions regarding the synthesis
and assembly of G protein heterotrimer. The G protein
,
, and
subunits are synthesized on ribosomes in the cytosolic compartment, and
they are directed to the plasma membrane by the way of
post-translational modification of both
(48, 50) and
(21, 22)
subunits. Increasing evidence suggests that there is a specific order
of addition proceeding from the individual monomers through the 
dimer to the 

trimer (20, 22, 47, 51). Importantly, this order
of assembly has not been examined for a specific combination of G
protein 

subunits that is known to exist in the intact cell
setting. The results of the present study showing a functional
association of the G protein
s
1
7 subunits should allow
examination of this question.