 |
INTRODUCTION |
Signal transduction involving heterotrimeric G
proteins1 is a universal
mechanism for the integration of extracellular stimuli such as
hormones, neurotransmitters, odorants, and light (1, 2). The components
involved in this signaling cascade are diverse, including a large
number of receptors, G protein
and 
subunits and effectors.
Even though the diversity of the proteins in this system could
potentially account for the known specificity of signaling in
differentiated cells, the mechanisms for determining specificity are
not completely defined. The
-adrenergic receptor is one of the most
well characterized seven transmembrane spanning receptors, and provides
an excellent example of selective coupling to a particular
subunit,
Gs. When activated, Gs can stimulate all nine
adenylyl cyclase isoforms (3, 4). The G protein 
dimer, when
released after receptor activation, is also able to regulate adenylyl
cyclase (5). However, the regulation of the various isoforms of
adenylyl cyclase by the 
dimer is much more selective;
apparently, only AC2, AC4 (6, 7), and AC7 (8) are stimulated by 
,
whereas the neuronal-specific AC1 (4) and possibly AC5 and AC6 are
inhibited by the dimer (9). Moreover, there is evidence that AC2 does
not respond well to dimers composed of certain
and
subunits
(10) or to dimers containing the phosphorylated
12
subunit (11). Thus, to understand fully the regulation of adenylyl
cyclase by a Gs-coupled receptor, one needs to know which

dimers are most likely to support receptor G protein coupling
and the effects of 
dimers on the various isoforms of adenylyl cyclase.
The number of functionally distinct 
dimers is potentially very
large, with seven G protein
isoforms (including two splice variants) and 12
isoforms characterized to date (12-14). Most in vitro studies involving coupling of receptors to
Gs
or regulation of adenylyl cyclases by distinct

dimers have used dimers containing the
1,
2, or
5 subunits (15, 16). The ubiquitous
cellular and tissue distribution of Gs
provides the
potential for interaction with all five
isoforms and underscores
the importance of understanding the role of the different
isoforms
on signaling pathways involving Gs
. For example, the
antisense studies of Kleuss et al. (17-19) suggest that
specific isoforms of the heterotrimer couple to different receptors,
and a number of in vitro studies imply that defined 
dimers may be released upon receptor activation (16, 20, 21). In
addition, isolation of G protein heterotrimers from a variety of
tissues using chromatography or immunoprecipitation has shown that
certain
and
subunits preferentially associate with one another
as well as with distinct
O isoforms (22, 23). These data
suggest that specific combinations of G protein subunits do exist
in vivo and may have specialized roles in various signaling cascades.
To examine the roles of the various
subunits in
receptor-Gs coupling, and in regulating adenylyl cyclase,
recombinant Gs
and 
dimers containing
1-5 complexed with
2 were expressed in
baculovirus-infected Sf9 insect cells and purified. Proteins were then reconstituted into partially purified Sf9 cell
membranes overexpressing either the
1-adrenergic
receptor, the A2a adenosine receptor, AC1 or AC2. The effects of the
1-5
2 combinations were measured in four
assays as follows: 1) the ability to couple the Gs
subunit to the
1-adrenergic receptor; 2) the ability to
couple the Gs
subunit to the A2a adenosine receptor; 3)
the ability to stimulate AC2; and 4) the ability to inhibit AC1. Clear differences were observed among the five 
dimers in both receptor coupling and effector regulation, suggesting that the diversity of the
subunit contributes extensively to signaling specificity.
 |
EXPERIMENTAL PROCEDURES |
Construction of Recombinant Baculoviruses--
Construction of
baculoviruses encoding the
1,
2,
5,
2,
2FH, the
Gs
and, Gi1
subunits has been described
(11, 24-26). The viruses encoding AC1 and AC2 were the kind gift of R. Iyengar (27, 28). Baculoviruses encoding the rat
1-adrenergic receptor and the human A2a adenosine
receptor were gifts from E. Ross (University of Texas, Southwestern
Medical Center) and J. Linden (University of Virginia), respectively
(29). The human
3 cDNA (30), a gift from S. R. Ikeda (Guthrie Institute), was excised from pCI with EcoRI
and NotI; the mouse
4 cDNA (31), a gift
from W. F. Simonds (National Institutes of Health) was excised
from pcDNA3 with BamHI and XbaI. The human
3s cDNA, a gift from D. Rosskopf (Institute for
Pharmacology, Essen, Germany), is a truncated variant of the
full-length
3 cDNA, in which the
3s
protein product has a deletion of amino acids 168-208 (32); excision
of the
3s cDNA from pGEMT was accomplished with
BamHI and PstI. The existing restriction sites
were used to ligate digestion products into the multiple cloning site
immediately downstream of the polyhedron promoter in the baculovirus
transfer vector, pVL1393. All clones were sequenced to confirm the
fidelity of the cDNA in pVL1393. Recombinant baculoviruses for
3,
3s, and
4 were prepared
by co-transfection of linear wild type BaculoGold® viral
DNA (PharMingen) with pVL1393 transfer vectors containing the specific
sequences into Sf9 cells as described (26) and purified with
one round of plaque purification (33).
Expression and Purification of Recombinant G Protein
and

Dimers--
Sf9 cells were infected with recombinant
baculoviruses encoding the desired
and/or 
dimer combinations
at a multiplicity of infection of three and harvested 48-60 h after
infection. 
dimer combinations containing
1-4 and
2 were purified by Gi1
affinity
chromatography as described (34). The dimer containing
5
was expressed with a
2 subunit engineered to have a
hexahistidine and FLAG tag (26) at the N terminus (
2HF)
and purified from isolated Sf9 cell membranes by FLAG affinity
and Ni2+ affinity chromatography, followed by anion
exchange chromatography (16).
Mass spectrometry was used to examine post-translational processing of
the
2 subunit. Purified 
dimers were analyzed by matrix-assisted laser desorption ionization-mass spectrometry to obtain
masses of the
subunits as described in Lindorfer et al.
(35). For 
dimers with a protein concentration of less than 150 ng/µl, acetone precipitation was used to concentrate the protein
before mass analysis (36). Post-translational processing of the
2 isoform includes cleavage of the N-terminal
methionine, acetylation of the resulting N-terminal alanine,
geranylgeranylation at the cysteine four residues from the C terminus,
cleavage of three C-terminal residues, and carboxymethylation of the
resulting C-terminal geranylgeranylated cysteine. These
post-translational modifications have been observed in
subunits
isolated from bovine brain (37) and in Sf9 cells (35). The
predicted mass of the properly processed
2 isoform is
7750 Da; insertion of a His-Flag (HF) tag at the N terminus increases
the predicted mass to 10,013 Da. Mass spectra of the purified 
isoforms containing either
2 or
2HF
demonstrated that the major mass in each spectrum was compatible with
these predicted masses within the accuracy of the instrument (35). For
example, in one set of purified 
dimers, the experimental masses
of the
2 subunits were as follows:
1
2, 7758 Da;
2
2, 7764 Da;
3
2, 7760 Da;
4
2, 7759 Da; and
5
2FH, 10,020 Da.
Attempts were made to purify
3s combined with various
subunits. A protein with the appropriate molecular weight was
expressed in Sf9 cells as judged by immunoblotting with a
-common antibody (PerkinElmer Life Sciences 808); however, the major
barrier to purification was that it was not possible to solubilize the
protein from the Sf9 cell pellet. For example, soluble extracts
of whole cell pellets prepared using 1% (v/v) Genapol, 1% (w/v)
CHAPS, or 1% (w/v) Cholate contained little
3s protein
in supernatant fractions that could be detected with the
-common
antibody. Expression of various
and or the
2FH,
5, and
7 subunits with
3s
in Sf9 cells did not affect the solubility of the protein (data
not shown), and thus characterization of this protein was not pursued.
Gs
was overexpressed with a
1 subunit
engineered to have a hexahistidine and FLAG tag (11) at the N terminus
(
1HF), along with
2HF, and purified using
a modification of the method described by Kosaza and Gilman (38).
Briefly, harvested cells were resuspended in half the infection volume
with cell lysis buffer (20 mM Tris, pH 8.0, 10 µM GDP, 17 µg/ml PMSF, and 2 µg/ml pepstatin,
leupeptin, and aprotinin). After resuspension, cells were lysed by
nitrogen cavitation (25), and membranes were collected by
centrifugation at 28,000 × g for 20 min at 4 °C. A
Potter-Elvehjem homogenizer was used to resuspend the pellets in a
quarter of the original resuspension volume (~63 ml) of cell lysis
buffer containing 10 µg/ml DNase. After a 15-min incubation on ice,
membranes were collected again by centrifugation at 28,000 × g for 20 min at 4 °C and resuspended with a
Potter-Elvehjem homogenizer in a volume of extraction buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM
MgCl2, 0.5% (v/v) Genapol, 1 mM
-mercaptoethanol, 50 µM GDP, 17 µg/ml PMSF, and 2 µg/ml of leupeptin, aprotinin, and pepstatin) equivalent to 10 times
the weight of the original cell pellet. Membranes containing expressed
G protein were resuspended and stirred with extraction buffer for
1 h at 4 °C, followed by centrifugation at 142,000 × g for 1 h at 4 °C; the solubilized Gs
/
1FH
2FH supernatant extracts
(typically about 100 ml) were flash-frozen in liquid nitrogen and
stored at
80 °C.
To begin the Gs
purification, the extract was diluted
with an equal volume of Ni2+ column buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM
MgCl2, 0.2% (v/v) Genapol, 1 mM
-mercaptoethanol, 10 µM GDP, 5 mM
imidazole, 17 µg/ml PMSF, and 2 µg/ml pepstatin, leupeptin, and
aprotinin) and loaded onto a Ni2+-NTA Superflow column at 2 ml/min. Unless otherwise noted, all steps were performed at 4 °C.
The volume of the column bed was ~5% of the volume of the Genapol
extract. The column was washed with 6 column volumes of
Ni2+ column buffer, 6 column volumes of Ni2+
column buffer containing 300 mM NaCl, and 3 more column
volumes of Ni2+ column buffer. At this point, the column
and buffers were warmed to room temperature for 10-20 min, and
Gs
was activated and eluted with 4 column volumes of
activation buffer (Ni2+ column buffer containing 50 mM MgCl2, 10 mM NaF, and 30 µM AlCl3) also warmed to room temperature.
Although the increased temperature facilitates activation of the
subunit, this step should be completed as quickly as possible, as
functional activity of
decreases with prolonged elevation of
temperature. Pilot experiments using SDS-PAGE to identify the
Gs
subunit indicated that the first 8 ml of eluate
after the void volume contained the protein. Thereafter, these
fractions were collected on ice and pooled. The fractions containing
Gs
were diluted 5-fold with 15Q buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 1 mM
MgCl2, 0.1% (w/v) CHAPS, 1 mM DTT, 10 µM GDP) and loaded onto a 200-µl 15Q anion exchange
column. This dilution facilitates adsorption of the protein to the
column by reducing of the Cl
concentration to ~50
mM. In addition to concentrating the protein, the 15Q step
is necessary to remove AlF
, imidazole, and Genapol, which would itself be concentrated along with
the protein in the next concentration step. After the protein was
loaded, the column was washed with 15Q buffer containing 10 mM NaCl (15Q buffer A) for 20 min at 1 ml/min. Protein was
then eluted with 15Q buffer containing 600 mM NaCl (15Q
buffer B) in a linear gradient of 0-50% 15Q buffer B over 15 min.
One-ml fractions were collected, and 12% SDS-PAGE followed by either
immunoblotting or silver staining with purified Gs
as a standard
was used to determine which fractions contained Gs
.
Fractions from the 15Q column containing Gs
were pooled
and concentrated with a Centricon 30 that had been passivated with a
1% BSA solution as described (16). The concentrated protein was
diluted 10-fold with 15Q buffer containing 100 mM NaCl to reduce the high NaCl concentration that resulted from the elution from
the 15Q column, and then concentrated once more to a volume of 100-200
µl, aliquoted, and stored at
80 °C. The yield of purified Gs
from 10 g of Sf9 cell pellet (wet
weight) was typically 10-20 µg. All protein estimates were
determined using scanning densitometry of silver-stained gels as
described previously (26), with standard curves generated from
ovalbumin standards.
Gi1
was purified by a similar method with the following
exceptions. The Ni2+ column buffer contained 20 mM Tris, pH 8.0, 150 mM NaCl, 0.2% (w/v)
CHAPS, 1 mM
-mercaptoethanol, 10 µM GDP, 5 mM imidazole, 17 µg/ml PMSF, and 2 µg/ml pepstatin,
leupeptin, and aprotinin. Protease inhibitors and imidazole were
removed from the elution step, and the Gi1
was taken
directly to a Centricon 30 where it was concentrated and diluted
10-fold with Ni2+ column buffer supplemented with 2 mM MgCl2. This step was repeated, and 100-200
µl of Gi1
at 100-200 ng/µl were stored in aliquots at
80 °C. As one criterion for the viability of the Gs
and Gi1
subunits, the ability of the proteins to bind
GTP
S in solution was measured. The stoichiometry of nucleotide
binding of two preparations of Gs
averaged 0.9 mol/mol.
The Gi1
subunit bound GTP
S at a stoichiometry of
~0.3 mol/mol and also coupled effectively to the A1 adenosine
receptor in assays similar to the one shown in Fig. 2A (data
not shown).
Preparation of Membranes Containing Recombinant
1-Adrenergic Receptors, A2a Adenosine Receptors, or
Adenylyl Cyclases--
Sf9 cells were infected with recombinant
baculoviruses encoding either the rat
1-adrenergic
receptor, the human A2a adenosine receptor, and either type I or type
II adenylyl cyclase (27, 28). In the case of the
1-adrenergic receptor, harvested cells were resuspended
in membrane homogenization buffer (20 mM HEPES, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 17 µg/ml
PMSF, and 2 µg/ml leupeptin and aprotinin), and cells were lysed by
nitrogen cavitation. The cell lysate was centrifuged at 750 × g to pellet unbroken cells and nuclei. Membranes were
prepared from the supernatant of the low speed spin by centrifugation
at 28,000 × g for 20 min at 4 °C. Endogenous G
proteins were then stripped from the membranes or inactivated by
incubation with urea. Membranes containing the
1-adrenergic receptor were homogenized with resuspension
buffer (50 mM HEPES, pH 7.5, 3 mM
MgSO4, 1 mM EDTA, 17 µg/ml PMSF, and 2 µg/ml leupeptin and aprotinin) containing 7 M urea and
allowed to incubate for 30 min at 4 °C. Resuspension buffer was used
to dilute the membranes to 4 M urea prior to centrifugation
at 142,000 × g for 30 min at 4 °C. Membranes were
washed twice with resuspension buffer, and homogenization buffer was
used to resuspend the membranes, which were stored in aliquots at
80 °C. Membranes containing the A2a adenosine receptor were
prepared using essentially the same method, except that homogenization
buffer consisting of 25 mM HEPES, pH 7.5, 100 mM NaCl, 1% (w/v) glycerol, 17 µg/ml PMSF, and 2 µg/ml
leupeptin, and aprotinin was used throughout the preparation. Radioligand binding experiments with
[125I]iodocyanopindolol and 125I-ZM-241385
were used to determine receptor number and affinity for the
1-adrenergic and A2a adenosine receptors, respectively. Stripping membranes with urea did not greatly affect the
pharmacological properties of these two receptors (data not shown). The
GTP
S binding experiments presented under "Results" were obtained
using a single preparation of membranes expressing either the
1-adrenergic or the A2a adenosine receptor. Membranes
expressing AC1 or AC2 were prepared as described previously (28). Total
membrane protein concentration was determined by BCA assay using bovine
serum albumin as a standard, and aliquots of membranes were stored at
80 °C.
Measurement of Agonist-stimulated GTP
S Binding to
Gs
after Reconstitution with 
into Membranes
Expressing either the
1-Adrenergic Receptor or the A2a
Adenosine Receptor--
Kinetic parameters of agonist-stimulated
binding of [35S]GTP
S to Gs
in the
presence of different concentrations of
1
2 were established with time course
experiments. Aliquots of Sf9 cell membranes containing the
1-adrenergic receptor were pelleted by centrifugation
and resuspended in 100-400 µl of GTP
S binding buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM
EDTA, 5 mM MgCl2, 1 mM DTT, 0.1%
BSA, 0.5 µM GDP, and 1 µM AMP-PNP) with a
28-gauge needle. The membrane suspension was reconstituted with 5 nM Gs
subunit such that the Gs
:receptor ratio was 26:1; different concentrations of
1
2 were then added and allowed to
incubate for 30 min at 4 °C. The incubation temperature was
increased to 25 °C for 10 min to equilibrate the reconstituted
system to the reaction temperature; additions of
[35S]GTP
S (final concentration 10 nM) and
isoproterenol (final concentration 1 mM) initiated the time
course. The binding of [35S]GTP
S to receptor-activated
Gs
was measured at 1-min intervals by vacuum
filtration. Increasing concentrations of 
increased the rate of
receptor-catalyzed exchange of GDP for GTP
S on Gs
.
The observed rates were relatively linear (see Fig. 2A),
thus the effect of 
was quantified by the amount of
[35S]GTP
S binding measured at a reaction time of 7 min. Seven minutes were chosen as a compromise that allowed a
measurable signal before the reaction rate slowed.
By using this protocol, 10 different 
concentrations, ranging
from 0.08 to 20 nM
x
2, were
examined to determine the efficiency of coupling Gs
to
receptor. Membranes were reconstituted with 5 nM
Gs
and the indicated 
concentrations in a total volume of 30 µl per tube. The concentrations of 
were prepared by serial dilution with GTP
S binding buffer containing 0.1% CHAPS; 2 µl of each 
concentration was diluted to the final incubation volume of 40 µl, giving a CHAPS concentration of 0.005% for all but
the highest concentrations of 
. After the incubation protocols described above, 8 µl of buffer containing [35S]GTP
S
(~1,000,000 dpm) and isoproterenol were added to each tube to start
the 7-min reaction. The reaction was terminated as described above, and
efficiency of coupling was determined by plotting
[35S]GTP
S binding as a function of 
concentration (Fig. 2B). Receptor specificity for
Gs
was demonstrated using the same protocol by
reconstitution of Gi1
with the
1-adrenergic receptor (Fig. 2B).
A slight modification of this protocol was used to obtain dose response
experiments with 
and the A2a adenosine receptor. The
Gs
:receptor ratio was 1.3:1, and in order to break down endogenous adenosine that is continuously generated in membrane preparations, adenosine deaminase was added to the membrane suspension before the 30-min incubation at a concentration of 14 units of activity/ml. The A2a adenosine receptor was activated with 100 nM 5'-N-ethylcarboximide adenosine.
Measurement of Adenylyl Cyclase Activity--
In addition to
activated Gs
, the diterpene forskolin can stimulate all
isoforms of adenylyl cyclase, whereas Ca2+/calmodulin can
stimulate AC1, AC3, and AC8 (3). Gs
and forskolin were
natural choices for this study, as they both activated AC1 and AC2,
whereas Ca2+/calmodulin will not activate AC2. Forskolin
was used to activate successfully AC1, but inhibition of the activity
by 
was not as robust as in the case of the Gs
-activated AC1 (data not shown); Gs
was therefore
chosen as the activator for both AC1 and AC2. GTP
S-activated
Gs
was prepared by incubation of protein with gel
filtration buffer (50 mM HEPES, pH 8.0, 150 mM
NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, and 0.1% CHAPS) with the addition of 5 mM MgCl2 (10 mM total) and 10 µM GTP
S for 30 min at 30 °C. Unbound GTP
S was
removed by centrifugation with a 2-ml P6 desalting column equilibrated
with gel filtration buffer using the method described in Yasuda
et al. (39). Reconstitutions of Gs
and 
were performed as described by Lindorfer et al. (16).
Maximal activity of AC1 and AC2 was confirmed by stimulation with
increasing concentrations of GTP
S-activated Gs
.
Cyclic AMP production was measured using a radioimmunoassay (40).
Enzymatic activity reached a maximal rate of ~5 nmol of cAMP/min/mg
protein for both AC1 and AC2, which is consistent with previous
preparations from this laboratory (16), and with published data
(41).
For experiments involving the effect of different 
isoforms on
adenylyl cyclase, activated Gs
is required at
concentrations below what is necessary for maximal stimulation of ACII;
therefore, 10 nM activated Gs
was chosen as
the concentration suitable for co-activation of AC2. Complete
activation of AC1 is required to observe inhibition by 
; for this
reason, 50 nM Gs
was used for activation of
AC1. Adenylyl cyclase experiments utilized a single preparation of each
membrane type.
Calculation and Expression of Results--
In experiments using
the
1-adrenergic and A2a adenosine receptors, data from
at least three experiments and two different sets of 
dimers were
normalized as a percentage of maximal GTP
S binding as determined by
the one-site binding curves generated by GraphPad Prism. After
normalization, the data were averaged for each 
isoform, and
GraphPad Prism was used to obtain estimates of the EC50
values and statistical analysis of the binding curves. These data are
presented in Table I.
GraphPad Prism was used to estimate EC50 and
Vmax values for the potentiation of
Gs
-stimulated AC2 activity by 
. At least three
experiments using data from two different sets of 
were analyzed,
and average values were reported in Table I. For AC1, GraphPad Prism
was used to generate inhibition curves for each of the experiments with
the different 
dimers; the data were then normalized as percent
inhibition of the estimated rate of cAMP production with 50 nM GTP
S-activated Gs
in the absence of

. Normalized data from at least three experiments and two different sets of 
dimers were averaged and analyzed by GraphPad Prism to obtain IC50 estimates (Table I). Statistical
significance for differences among binding curves for both receptors
and AC1 and AC2 was determined using the F-statistic; this technique is able to discern small but significant differences between two binding
curves (42).
Materials--
Reagents for Sf9 cell culture and
purification of 
dimers has been described previously (16, 25,
26, 34). 125I-ZM-241385 was a kind gift from J. Linden,
University of Virginia; baculovirus transfer vector was from
Invitrogen; the BaculoGold kit was from PharMingen; DNase, GDP,
imidazole, isoproterenol, and HEPES were from Sigma; adenosine
deaminase, CHAPS, and GTP
S were from Roche Molecular Biochemicals;
P-6 desalting gel was from Bio-Rad; 10% Genapol C-100 was from
Calbiochem; Ni2+-NTA Superflow resin was from Qiagen;
[35S]GTP
S and [125I]iodocyanopindolol
from PerkinElmer Life Sciences; Source 15Q anion exchange resin was
from Amersham Pharmacia Biotech; type HA 0.45-µm nitrocellulose
filters and Centricon 30 concentrators were from Millipore. All other
materials were of the highest available purity.
 |
RESULTS |
Preparation of G Protein
and 
Subunits--
Fig.
1A presents a silver-stained
gel showing the purity of the five 
dimers used in this study.
Fig. 1B presents a similar gel showing the purity of the
Gs
used. Significantly, both the 
dimers and the
Gs
subunit were purified using biological affinity
columns. The dimers containing
1-4 were purified with a
Gi1
-agarose column ensuring that the proteins bound to
subunits with high affinity and that the C terminus of the
subunit was properly modified (see "Experimental Procedures"). Even
so, a
doublet was occasionally observed in the SDS-PAGE analysis of
3
2. The reasons for this behavior are not
understood. The
5
2HF dimer was purified
from the membrane fraction of Sf9 cells using
Ni2+/FLAG chromatography. Mass spectrometry was used to
demonstrate that the
subunit in this dimer was modified to the same
extent as dimers purified on the Gi1
-agarose column,
and the biological activity of
5
2HF was
vetted by determining its ability to activate PLC-
(10, 16) to the
same extent as
1
2 (data not shown). Gs
was purified by
AlF
elution from a Gs
:
1HF
2HF heterotrimer bound to a
Ni2+-NTA-agarose column. The activity of the Gs
preparation was verified by its ability to activate AC1 and AC2.
Fig. 1, C and D, shows that the
subunit
activated these cyclases 4-8-fold, with EC50 values of 1.9 nM for AC2 and 8.5 nM for AC1. These results are similar to other values reported in the literature for
Gs
expressed in Sf9 cells (43), validating the
quality of both the purified Gs
and the membrane
preparations of AC1 and AC2.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Purity of G protein and subunits. A, the
five isoforms of the subunit were overexpressed in Sf9
insect cells with the 2 or 2FH subunit
and purified by Gi1 affinity chromatography
( 1-4 2) or Ni2+-NTA affinity
chromatography ( 5 2FH). B,
Gs was overexpressed in Sf9 insect cells with a
1 2 dimer containing a hexahistidine tag.
The heterotrimer was adsorbed to a Ni2+-NTA column, and
Gs was eluted specifically with
AlF . Purity of  dimers (250 ng
of each isoform) and Gs (150 ng) was visualized by silver staining
after separation by 12% SDS-PAGE; positions of molecular weight
markers are indicated at the right. C, Sf9
cell membranes expressing AC2 were incubated with increasing
concentrations of Gs activated with GTP S, and cAMP
levels were determined using a radioimmunoassay; the calculated
EC50 for the experiment shown is 1.9 nM.
D, Sf9 cell membranes expressing AC1 were
characterized as in C; the calculated EC50 for
the experiment shown is 8.5 nM.
|
|
The Ability of Different 
Subunits to Support Coupling of
Receptors to Gs
--
A major goal of this study was to
examine the possibility that different
subunits interact
selectively with certain G protein-coupled receptors. Since exchange of
GDP for GTP is the first step of G protein signaling subsequent to
receptor activation, an agonist-dependent GTP
S-binding
assay was used. Fig. 2A
presents an experiment performed with membranes expressing the
1-adrenergic receptor reconstituted with Gs
and two concentrations of the 
dimer. Note that the rate of
isoproterenol-stimulated GTP
S binding is nearly linear and highly
dependent on the concentration of 
dimer. The
triangles in Fig. 2A represent a basal rate of
GTP
S binding to membranes reconstituted with Gs
;
this rate was observed in the absence of 
(as illustrated in the
figure) or with a fully reconstituted system in the absence of
isoproterenol. Coupling of receptor to G protein is a composite of many
biochemical interactions, the most important being the interactions of
-
and receptor-
-
. Receptor-
interactions were
probed with a variation of the protocol designed to be poised on the
concentration of the 
dimer. To define precisely the ability of
1
2 to support coupling of Gs
to the
1-adrenergic receptor, a reaction time of 7 min was chosen to generate concentration-response curves with the
different 
isoforms. A representative concentration-response
curve performed with
1
2 is presented in
Fig. 2B; the apparent EC50 estimated from this
curve is 0.7 nM. The fidelity of receptor-
interactions was confirmed by the reconstitution of the
1-adrenergic
receptor with Gi1
, which demonstrates that even high
concentrations of
1
2 (20 nM)
do not support isoproterenol-dependent GTP
S binding (Fig. 2B) to the Gi1
subunit. In contrast,
when the same Gi1
was reconstituted with
1
2 into membranes containing the A1 adenosine receptor, robust agonist-dependent GTP
S
binding was observed (data not shown, but see Ref. 39).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration dependence of
1 2
on coupling Gs to the
1-adrenergic receptor.
A, time course of [35S]GTP S binding to
Sf9 cell membranes containing the 1-adrenergic
receptor and reconstituted with 5 nM Gs and
0, 0.5, or 10 nM 1 2. Ten
nM [35S]GTP S and 1 mM
isoproterenol (Iso) were added at time 0, and bound
nucleotide was determined at 1-min intervals by vacuum filtration.
B, increasing concentrations of
1 2 were reconstituted with Gs
or Gi1 into membranes containing the
1-adrenergic receptor, and the reaction was initiated as
described in C; the amount of [35S]GTP S
bound was determined at the 7-min time point. The EC50 for
 supported coupling of Gs to receptor was 0.7 nM as determined by fitting the data to a one-site
model.
|
|
The experiment performed with Gs
in Fig. 2B
was repeated using each of the five 
isoforms, and the normalized
data are shown in Fig. 3A.
Highest in coupling efficiency was
4
2,
with an EC50 of 0.5 nM;
2
2 was considerably less efficient with
an EC50 of 2.7 nM. There were slight but
statistically significant differences in most of the 
isoforms,
with only
1
2 and
3
2 showing no differences and an
EC50 of 1.0 nM. Poorest of all at coupling
Gs
to the
1-adrenergic receptor was
5
2 with an EC50 of 17.1 nM (see Table I).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of ability of different
 isoforms to couple
Gs to the
1-adrenergic and A2a adenosine
receptors. A, five x 2
isoforms were reconstituted with 5 nM Gs and membranes containing the 1-adrenergic receptor and
the efficiency of coupling measured as described under "Experimental
Procedures." Data from three experiments were normalized as a percent
of maximal binding of [35S]GTP S, and the averaged data
plotted; error bars, most of which were within ±10%, were
omitted for clarity. B, five x 2
isoforms were tested as in A, but with the A2a adenosine
receptor. Data from at least three experiments were normalized and
plotted as in A. EC50 estimates of the data sets
for each  dimer can be found in Table I. C-G, data
from A and B were replotted to highlight
differences in each particular x 2 isoform
between the 1-adrenergic receptor ( ) and the A2a
adenosine receptor (A2a). Dotted lines indicate 
concentrations of 1 and 10 nM on the x axis.
C, 1 2; D,
2 2; E,
3 2; F,
4 2; G,
5 2FH.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Comparison of the ability of five different  isoforms to couple
Gs to the 1-adrenergic receptor, the A2a
adenosine receptor, or to potentiate the activation of ACII by
Gs , or inhibit stimulation of ACI by Gs
EC50 and IC50 values were determined by fitting the
averaged data (n 3) to single site binding or
competition curves as described under "Experimental Procedures";
bold numbers indicate EC50 or IC50 values from the
statistical fit, and numbers in parentheses represent the range of
values within the 95% confidence interval. Statistical significance
(indicated by the superscript) was determined using the F
statistic.
|
|
To determine if the rank order of affinities determined with the
1-adrenergic receptor was the same with another
Gs-linked receptor, we examined the ability of the panel of

dimers to support coupling of Gs
to the A2a
adenosine receptor. Normalized data for each of the 
isoforms and
the A2a adenosine receptor are shown in Fig. 3B; the
coupling efficiencies and statistical analysis are presented in Table
I. Highest in affinity was
4
2 with an
EC50 of 1.3 nM;
2
2 and
3
2
were lower in affinity, both with EC50 values around 6 nM. Strikingly,
1
2 was much
less efficient at coupling to the A2a adenosine receptor, with an
EC50 of 15.7 nM. Lowest in coupling efficiency
was
5
2, with an EC50 >100
nM.
Fig. 3, C-G, illustrates that there are striking
differences in the ability of the two Gs-linked receptors
to couple to the five different 
isoforms. Perhaps the most
dramatic differences occurred with the
1
2
isoform, which coupled 15-fold more efficiently to the
1-adrenergic receptor than to the A2a adenosine receptor (Fig. 3C). Similarly,
3
2
demonstrated a 7-fold difference between the two receptors (Fig.
3E). Note that
4
2 was the most
effective at coupling Gs
to either receptor (Fig.
3F), and that
5
2 coupled poorly (Fig. 3G). In contrast, there are minimal differences
in the ability of
2
2 to couple to either
receptor (Fig. 3D). It is important to stress that the only
differences in these five sets of experiments are the types of
recombinant receptor expressed in the Sf9 membranes. The G
protein
and 
subunits reconstituted into the membranes were
identical in each case. Thus, the data clearly demonstrate that
specific G protein
subunits exhibit distinct preferences for
different receptors. Importantly, these preferences are a result of
interactions of receptor with the type of
subunit in the dimer,
since the Gs
-
interactions are presumably
identical for both receptors.
Activation of AC2 by 
Isoforms--
The dramatic differences
in the ability of the panel of 
dimers to couple to
Gs-linked receptors imply that different 
dimers
might be released by receptor activation to act on downstream effectors. Since 
is a known potentiator of Gs
-stimulated AC2 activity, and differences have been observed in the
ability of dimers containing the
1 or
5
subunits to stimulate AC2 (9, 16), the ability of all five
subunits
to activate AC2 was compared. The role of 
in the activation of
AC2 is particularly interesting in that 
can increase the rate of
cAMP production approximately 5-fold over the maximal effect of
Gs
(Fig. 4), suggesting

can regulate cAMP levels in vivo. Ten nM
GTP
S-activated Gs
and increasing concentrations of
the five purified 
isoforms were reconstituted with Sf9
membranes expressing AC2, and cAMP production was measured. A
representative experiment is presented in Fig. 4. Dimers containing
1-4 were similar in their ability to potentiate AC2
activity in the presence of activated Gs
, with all
dimers increasing cAMP production 4-6-fold
(Vmax values ranging from 30 to 40 nmol of
cAMP/min/mg of protein). Consistent with previous reports (16),
5
2 was significantly less active. Data
from at least three similar experiments were normalized and averaged to
determine the EC50 values for each 
dimer, and the
results are summarized in Table I. The EC50 values for
1-4 range from 3.5 to 13 nM, and the value
for
5
2 is significantly higher at 76 nM. Careful analysis of the data indicates that the
2
2 dimer is 3-fold less potent than the
1
2 and
4
2
dimers. Also of interest is the observation that dimers containing
3 and
4, which had not been previously tested on adenylyl cyclase, were as effective at stimulation of AC2 as
dimers containing
1 and
2. These results
suggest that all four
isoforms can effectively participate in
Gs
signaling pathways affecting the regulation of cAMP
via AC2.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4.
Comparison of the potency of five
x 2
isoforms in the activation of AC2. Increasing concentrations of
the indicated x 2 dimers were incubated with
10 nM GTP S activated Gs and membranes
containing AC2. Data shown are representative experiments where each
point is the average of duplicates; the calculated EC50
values for the data are as follows: 1 2,
8.6 nM; 2 2, 14.0 nM; 3 2, 7.3 nM;
4 2, 2.2 nM; and
5 2FH, 76.8 nM.
EC50 estimates from the averaged data sets for each 
dimer can be found in Table I.
|
|
Inhibition of AC1 by 
Isoforms--
AC1 is in very high
concentration in neuronal tissue and is notable among the adenylyl
cyclase isoforms in that it can be markedly inhibited by the 
dimer. The activity of AC1 expressed in membranes was demonstrated with
purified Gs
, which stimulated cAMP production with an
EC50 of 8.5 nM (Fig. 1D). This
activation of AC1 by Gs
provided the opportunity to
compare the inhibitory properties of all five 
dimers. Increasing
concentrations of purified 
isoforms were reconstituted with 50 nM GTP
S-activated Gs
into Sf9
membranes expressing AC1. A representative experiment illustrating the
robust inhibition of AC1 elicited by
1
2
is presented in Fig. 5A, where
the dimer reduced cAMP production by over 50%. Similar experiments
were performed with the full panel of 
dimers. The data were
normalized and are presented in Fig. 5B. Dimers containing
the
1,
2, and
4 subunits
were not different in their ability to inhibit AC1, with
IC50 values ranging from 10 to 17 nM. In
contrast, the
3
2 dimer had an almost 10-fold higher IC50 of 110 nM, and
surprisingly, no inhibition was observed with
5
2 at the concentrations tested (Fig.
5B and Table I). These data suggest that 
dimers
composed of different
isoforms, especially those containing
3 and
5, may differentially regulate the
type I and type II adenylyl cyclase isoforms.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Comparison of the potency of five
x 2
isoforms at inhibiting Gs
-stimulated AC1 activity. A,
representative experiment where increasing concentrations of
1 2 were reconstituted with 50 nM GTP S-activated Gs and membranes
containing AC1; each point is the average of duplicates. The calculated
IC50 for the data shown is 8.1 nM.
B, increasing concentrations of five
x 2 isoforms were reconstituted with 50 nM GTP S-activated Gs and membranes as in
A. The y axis is expressed as percent of maximal
cAMP production in the absence of  . Each point is the average of
at least three separate experiments, ± S.E. Average values for all of
the experiments can be found in Table I.
|
|
 |
DISCUSSION |
Although considerable research has focused on the role of the
subunit in receptor signaling via G proteins, it is also clear that the

dimer is required for coupling the
subunit to receptors (16,
39, 44, 45), and both the
and
subunits appear to contribute to
the interaction. Current evidence indicates that the C-terminal 10 amino acids of the
subunit and the nature of the prenyl group
(farnesyl versus geranylgeranyl) are very important
determinants of the coupling of G proteins to receptors (20, 21, 39).
Compared with
and
, less is known about the important domains in
the
subunit, but cross-linking experiments indicate that the C
terminus of the
subunit is able to interact directly with the
receptor (46). However, the regions close to the C terminus of the
subunit are likely to be quite discrete because mutations in amino
acids His311, Arg314, and Trp332
have no effect on the ability of the dimers to support receptor coupling but cause a major disruption in the ability of the 
dimer to activate PLC-
or AC2 (47).
Whereas the diversity of the
and
subunits offers attractive
possibilities for determining the specificity of cellular signaling,
the functional significance of this heterogeneity has not been
completely elucidated. Some insight has come from an elegant set of
experiments in which antisense mRNAs to various G protein
and

subunits were injected into the nuclei of GH3 cells, leading to
the observation that specific receptors couple to distinct isoforms of
the G protein heterotrimer. These experiments indicated that the
M4 muscarinic receptor preferred a heterotrimer composed of
O1:
3
4, whereas the
somatostatin receptor preferred a heterotrimer composed of
O2:
1
3 (17-19). These
results have been extended to other systems using the antisense
approach (48), where it has been demonstrated that selectivity of
receptor coupling to
subunits of the Gi (49),
Gq (50), and G12/13 (51) families depends upon
the composition of the 
dimer. However, few experiments have
examined the composition of 
dimers that couple receptors to
Gs
. Moreover, reproduction of this signaling
specificity using in vitro systems has proven difficult.
A major contribution of the experiments presented in this report is
that a panel of highly active recombinant 
dimers was used in a
sensitive assay to compare the ability of five
subunits to couple
to two different Gs-linked receptors. The ability of a
defined heterotrimer to support coupling of an
subunit to a
particular receptor depends primarily on the affinity of the receptor
for the heterotrimer (48), and presumably on the affinity of the
interaction between the
and 
subunits (16, 26). Since the
order of coupling efficiencies in the present experiments was quite
different between the
1-adrenergic and the A2a adenosine receptors, the possibility that the differences observed here were due
to differences in the
:
affinity seems unlikely. Thus, the
order of EC50 values for each receptor is most likely a
reflection of its preference for the particular
subunit examined.
The
5
2 dimer, which couples
M1 muscarinic receptors efficiently to the Gq
subunit (16), was particularly ineffective at coupling the
Gs
subunit to either receptor. In contrast, the
4
2 dimer was consistently highest in
coupling efficiency for either receptor. Even more surprising was the
finding that the
1
2 dimer, which in most
assays of 
function is potent and efficacious, was very poor at
coupling Gs
to the A2a adenosine receptor. These
results lead to the following conclusions: 1) receptors can
discriminate among the G protein heterotrimers based on the
isoform
alone; 2) all
1-4 isoforms can function in signaling
cascades involving Gs
; and 3) dimers containing
5 are not likely to be released from
Gs-coupled receptors. Finally, since different 
dimers may be released upon receptor activation, the downstream effects
of the distinct
isoforms may have different signaling properties.
One of the immediate downstream targets for 
is the family
of receptor kinases that phosphorylate G protein-coupled receptors upon
recruitment to the membrane by the dimer (52). Experiments have
examined the ability of defined 
dimers to interact with the
receptor kinases. For example, dimers containing
1 and
2 interact with GRK2, the kinase responsible for
down-regulation of the
-adrenergic and A2a adenosine receptors (53)
far better than dimers containing
3 (54). Another study
examined the ability of a variety of defined 
dimers to promote
the phosphorylation of both the
2-adrenergic receptor
and rhodopsin by GRK2. These results indicate a significant difference
in the ability of the various 
dimers to promote phosphorylation
of the
2-adrenergic receptor or rhodopsin and suggest
that the type of
subunit could determine selectivity between the
two receptors (55). Thus, even though
1-4 are over 80%
identical, the accumulating evidence suggests the type of
subunit
in the dimer may have a larger role in determining signaling
specificity than previously appreciated.
Another immediate downstream target for the 
subunit is the
effector adenylyl cyclase (3). Results presented here demonstrate that
dimers containing
1,
2, or
4 were able to regulate either type of adenylyl cyclase
effectively. In contrast,
5
2 was not particularly effective at inhibiting AC1 or in stimulating AC2 (16).
Intriguingly,
3
2 was almost 10-fold
weaker at inhibiting AC1 as compared with the
1-4
isoforms (Table I), whereas it was equally effective on AC2. These
results suggest that upon stimulation of certain Gs-linked
receptors, co-activation of AC2 by 
is relatively nonspecific,
whereas inhibition of AC1 by 
is more selective and may be
receptor-dependent. This specificity of interaction between
AC1 and the different
isoforms suggests that dimers containing the
3 subunit have signaling roles distinct from those
containing
1,
2, or
4.
The regions of the 
dimer that are thought to interact with AC1
and AC2 have been examined using competition experiments with synthetic
peptides and alanine mutagenesis. Peptides identical to residues
86-105 and 115-135 of the
1 subunit were able to inhibit stimulation of AC2 by the 
dimer, implicating these residues of the
subunit (56), as well as others (57), as sites on
the molecule that interact with AC2. Moreover, the QEHA peptide, which
represents a sequence from AC2 thought to interact with the 
dimer, was able to bind directly to the
subunit. Molecular modeling
of the QHEA peptide-
interaction also identified the region of
defined by residues 75-165 as a potentially important effector-binding domain (58). Mutagenesis experiments have suggested that three residues in the
subunit involved in
:
interface, Asp228, Asp246, and Trp332, are
important for the activation of AC2 but have no effect on the ability
of the dimer to inhibit AC1 (59). Studies of the outer surface of the
torus show that Asn132 in blade two of the protein is
important for inhibition of AC1, but seems to have little effect on
activation of AC2 (60). The observation that ADP-ribosylation of
Arg129 of
1, a residue also present in
2-4, prevents the inhibition of AC1 by the 
dimer
supports the argument that this is an important domain in the
interaction of the dimer with AC1 (61).
Examination of the regions identified by the experiments discussed
above in the
1-4 subunits shows minimal differences in
the amino acid sequence. This is consistent with the observation that
these four 
dimers activated AC2 equally. A similar conclusion applies to the ability of three of the dimers to inhibit AC1; the
intriguing exception was
3
2, which was
far less effective. Unfortunately, there are no obvious differences in
the amino acid sequence of the
3 subunit in these
regions to explain the differences in activity, suggesting that some
other region in the molecule is also involved in the interaction with
AC1. There are, however, sequence variations that could explain the
lack of activity of
5
2 on either isoform
of cyclase. In contrast to the near identity of the
1-4
subunits in the regions outlined above that are thought to interact
with AC2 and AC1, the
5 subunit has 13 amino acid
differences in these regions when compared with the
1
subunit. Moreover, there is a two-amino acid insert in the region
between residues 130-132, a site identified by ADP-ribosylation experiments as being important for the inhibition of AC1 (61). Although
not definitive, these differences provide a reasonable starting point
for future experiments.
Despite the homology in amino acid sequence among these
isoforms,
differences in localization have been observed. For example, in
contrast to the other
isoforms that were localized to the membrane,
3 was observed predominantly in cytoplasmic fractions in
both heart (62) and retina (63). This property of
3 does not seem likely to be responsible for the observed differences in
inhibition of AC1, since
3
2 was just as
effective as the other 
dimers at activating the membrane-bound
protein AC2 and supporting coupling to the
1-adrenergic
and A2a adenosine receptors. A more reasonable explanation is that
certain residues unique to
3 impart specificity either
through altering the contacts with an effector molecule such as AC1, or
those unique residues slightly influence the conformation of the
subunit, thereby altering interactions with other proteins. Whatever
the reason, one important conclusion is that differences in AC1
inhibition may result from the release of different 
dimers by
receptor activation.
This concept appears to apply especially well to dimers containing the
5 subunit. Even though the current data suggest that the
5
2 dimer is unlikely to be released from
Gs-linked receptors, it clearly can be released by
activation of Gq-linked receptors (16). However,
accumulating evidence shows that the
5
2
dimer does not regulate a variety of effectors, including AC1, AC2, phosphatidylinositol 3-kinase, PLC-
3 and the mitogen-activated protein kinase pathway (this report and Refs. 64 and 65). Even though
the
5
2 dimer did not regulate adenylyl
cyclase in our experiments, transfection of the dimer into COS-7 cells
caused an inhibition of both AC1 and AC2 (66). These conflicting data may be explained by other potential partners for
5, such
as RGS 6, 7, 9, and 11 (67, 68), which may impinge upon the adenylyl cyclase cascade in vivo. This evidence of multiple partners
for the
5 subunit suggests the
5 protein
may have functions not normally associated with
subunits and makes
the physiological role of
5 on effectors unclear.
Especially interesting is the possibility that the
5
subunit may exist as a monomer and exchange between
subunits and
RGS proteins (69). Although the role of
5 in signaling
is clearly complex, one conclusion from this information is that
receptors that couple to and release dimers containing the
5 subunit are less likely to generate cross-talk between
signaling systems because of the limited effect of
5 containing dimers on downstream effectors.
Brain is one tissue where all of the signaling components studied in
this report are expressed at high levels (70-73). Thus, the
differential effects of the
isoforms demonstrated in this report
could have major effects on signaling cascades in the brain. Some
experimental support for this concept comes from experiments showing
that small amounts of 
derived from Gs activation can inhibit the neuronal-specific AC1 in vivo (74). Further
information needed to corroborate these proposed differences in 
signaling includes cellular and subcellular localization of these
molecular components. Once the subcellular architecture in these
tissues is better understood with respect to G proteins, signaling
models based on specific G protein
subunits can be refined with precision.