From the Departments of Anesthesiology and
Genetics, Washington University School of Medicine, St. Louis,
Missouri 63110 and the ¶ Department of Biological Chemistry,
University of Michigan Medical School,
Ann Arbor, Michigan 48109-0636
Received for publication, November 16, 2000, and in revised form, March 15, 2001
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ABSTRACT |
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In comparison with the The G protein Experiments focusing on the specific role of individual G protein
subunit types have provided evidence for a certain level of selectivity
in the interaction of Among the five Materials--
PIP2 and
phosphatidylethanolamine were obtained from Avanti Polar Lipids.
[3H]PIP2 was from PerkinElmer Life Sciences.
Where a source is not stated, reagent was obtained from Sigma.
Construction of Recombinant
Baculoviruses--
Expression and Purification of Purification of G Protein PLC- Adenylyl Cyclase Assays--
Sf9 cells were infected with
baculovirus expressing adenylyl cyclase type II, and Sf9 cell
membranes were then prepared as described previously (29). Membranes
expressing adenylyl cyclase type II were used in adenylyl cyclase
assays performed according to the procedure of Smigel (30).
Preparation of Purified and Reconstituted Recombinant M2--
A
detailed description of M2 purification, reconstitution, and
measurement of G protein stimulation has been published elsewhere (31).
His6-tagged M2 was expressed in Sf9 cells by using
the recombinant baculovirus (kind gift from Dr. E. Ross). The receptor was purified using the CoCl2 affinity column according to
previously published procedures with some modifications (32). The
purified His-M2 was reconstituted into brain lipids and characterized
by binding to an antagonist,
[3H]N-methylscopolamine, as described
previously with some modifications (33). The Kd for
reconstituted M2 binding to
[3H]N-methylscopolamine was determined to be
0.25 nM (34).
GTP Hydrolysis--
Heterotrimeric G protein subunits were
formed by incubating 10 nM concentrations of Measurement of G Protein Heterotrimer Formation--
To measure
the formation of heterotrimer, a fixed concentration of Stimulation of Effectors by
AC-II is stimulated by the G protein M2 Receptor Activation of Go Containing the
The M2 stimulation of Efficiency of Heterotrimerization of
Because the G protein subunit of G proteins,
the role of the
subunit in signaling is less well understood.
During the regulation of effectors by the
complex, it is known
that the
subunit contacts effectors directly, whereas the role of
the
subunit is undefined in receptor-G protein interaction. Among the five G protein
subunits known, the
4
subunit type is the least studied. We compared the ability of
complexes containing
4 and the well characterized
1 to stimulate three different effectors:
phospholipase C-
2, phospholipase C-
3, and adenylyl cyclase
type II.
4
2 and
1
2 activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting
o
4
2 was
stimulated significantly more by the M2 muscarinic receptor compared
with
o
1
2. Because
o forms heterotrimers with
4
2 and
1
2
equally well, these results show that the
subunit type plays a
direct role in the receptor activation of a G protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
complex regulates the activity of a diverse
set of effectors, including phospholipases, adenylyl cyclases, and ion
channels (1). There is evidence that the
subunit in the complex
interacts directly with effectors (2-5). There are five
subtypes
(
1-
5) as well as an alternatively
spliced version of
5 (known as
5-long)
(6-11).
1-
4 share over 80% identity with one another, whereas
5 shares only ~50%
identity with the other
subunits (12). The divergence between
5 and the other
subunits is consistent with the
functional differences between
5 and
1
observed in effector regulation in a variety of systems (4, 13, 14).
The high sequence similarity of
1-
4
suggests that their functions are conserved. Although some experiments
indicated little difference in effector modulating capability among
these
subunit types, other experiments suggest otherwise. The G
protein-coupled receptor kinase GRK3 binds
complexes consisting
of
1,
2, and
3, but only
1 and
2 bind to the related kinase GRK2
(15). Other results indicate the selective mediation of cross-talk
between G proteins and protein kinase C modulation of N-type channels
by the
1 subunit type (16).
subunit types with receptors (17). Evidence
for similar selectivity of interaction between
subunit types and
receptors also exists (18-20). In contrast there is limited evidence
for
subunit type selectivity in receptor interaction. Whole-cell
experiments using antisense oligonucleotides directed against specific
subunit cDNAs selectively disrupted signaling from particular
receptors (21). Although the selective interaction of
subunit types
with receptors could give rise to this result, such selectivity has not
been shown so far.
subunits,
4 is the least studied. Its
role in effector regulation and receptor interaction remains unclear. To examine its effect on the
regulation of effectors and G protein interaction with a receptor, we expressed purified
recombinant
4
2 and
1
2 complexes and compared their abilities
to regulate the activity of three different G protein effectors:
PLC-
2,1 PLC-
3, and
adenylyl cyclase type II (AC-II). Next, we examined the
abilities of heterotrimers made up of
o
1
2 and
o
4
2 to couple to the M2
muscarinic receptor in a reconstituted system containing purified M2
and G protein subunits. The results indicate that in comparison to
1, the
4 subunit does not differentially modulate effector function but does differentially affect the receptor
activation of a G protein. These results indicate that the particular
subunit type present in a heterotrimer influences the effectiveness
of the receptor activation of that G protein. Because these experiments
were performed with purified receptor and G protein subunits, these
results also indicate that the
subunit plays a direct role in the
receptor activation of a G protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4-Expressing baculovirus was
constructed using the Bac-to-Bac baculovirus expression system from
Life Technologies, Inc. Because sequencing revealed a very long
cytosine-rich region at the 5'-end of the original
4
cDNA (kind gift from Dr. M. I. Simon, California Institute of
Technology), a synthetic oligonucleotide cassette made up of DX19103
(5'-GGCCGCATGAGCGAGCTGGAGCAG) and DX19104 (5'-CTGCTCCAGCTCGCTCATGC) was
constructed to replace this region. The cassette encodes the first six
amino acids of
4. It was introduced into the
4-pFastBac construct using
NotI/PvuII sites. The methods used for the
construction of baculoviruses expressing His-PLC-
2, His-PLC-
3,
adenylyl cyclase type II, the G protein
1 subunit, and
the G protein His-
2 subunit have been published
(22-26).
Complexes from Sf9
Insect Cells--
Sf9 cells were maintained in suspension in
IPL-41 medium (Life Technologies, Inc.) containing 1% Pluronic F68,
10% heat-inactivated fetal bovine serum (Atlanta Biologicals), and 50 µg/ml gentamicin. Cells were grown at 27 °C with constant shaking
at 125 rpm. For initial expression studies, Sf9 cells were
infected with the
4 virus for varying lengths of times.
Cell lysates were examined by immunoblotting with
B4-specific B4-2 antibody (27) used at 1:600
dilution. The purification of
subunits was performed essentially
as described before (26). Sf9 cells were simultaneously infected
with His-
2 baculovirus and either
4 or
1 baculovirus. Approximately 60 h after infection,
cells were lysed by nitrogen cavitation, and the membranes were
extracted with 1% cholate. The detergent extract was applied to a
column of nickel resin (nickel-nitrilotriacetic acid column from
Qiagen) and washed with Buffer A (20 mM Hepes, pH 8.0, 1 mM MgCl2, and 10 mM
-mercaptoethanol) containing 300 mM NaCl, 0.5%
C12E10, and 10 mM imidazole.
complex was eluted with Buffer A containing 50 mM NaCl, 1%
cholate, and 250 mM imidazole. Peak fractions were
concentrated to a final concentration of 1-2 mg/ml using centrifugal
filtering devices (Centricon YM10 from Millipore). For use in receptor
assays, G protein
complexes were exchanged into 20 mM Hepes, pH 8.0, 1 mM EDTA, 3 mM
MgCl2, 3 mM dithiothreitol, 0.1 M
NaCl, and 0.7% CHAPS by dialysis. The purity and concentration of
complexes were assessed by the separation of proteins on
SDS-polyacrylamide gel electrophoresis followed by staining with
Coomassie Blue and the quantitation of protein bands with laser
densitometry analysis. PLC-
activation assays were used to ensure
that the functional proportion of
4
2 and
1
2 complexes containing different
detergents was the same in all samples. The final preparations of all
proteins were over 95% pure.
Subunit--
Recombinant G
was
synthesized in Escherichia coli and purified as described
previously (28). Protein purity and quantity were estimated by
separation on SDS gels and densitometry. The proportion of the
functional
subunit was estimated by GTP
S binding assays.
Assays--
PLC-
2 and PLC-
3 were purified as
described previously (22, 23). The
stimulation of phospholipase
C-
was performed as described (22). Briefly, lipid substrate (50 µM PIP2, 200 µM PE, and
[3H]PIP2 (~8000 cpm/assay) was mixed,
dried, and ultrasonicated in a buffer containing 50 mM
Na-Hepes (pH 7.2), 3 mM EGTA, 80 mM KCl, and 1 mM dithiothreitol. 1 ng of PLC-
2 per reaction and 5 ng
of PLC-
3 per reaction were used. PLC-
and
complex were added to the substrate in ice. Reactions were started with the addition
of CaCl2 (final concentration 2.8 mM), and
reactions were incubated at 30 °C for 15 min. Reactions were
terminated by the addition of 10% trichloroacetic acid and
bovine serum albumin. After centrifugation, the supernatant containing
[3H]phosphatidylinositol 1,4,5-trisphosphate
was analyzed by scintillation counting as described before
(22).
s subunit was activated by incubation with 50 mM Na-Hepes (pH 8.0), 5 mM MgSO4, 1 mM EDTA, 1 mM dithiothreitol, and 400 mM GTP
S at 30 °C for 30 min; free GTP
S was removed
by gel filtration. All assays were performed for 10 min at 30 °C in
a final volume of 100 µl containing 10 mM
MgCl2 and 100 nM GTP
S-bound
s.
and
subunits on ice for 30 min in a buffer containing 25 mM
Hepes, 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 10 µM GDP, and 1 mM
dithiothreitol. The reconstituted M2 was incubated with heterotrimeric
Go on ice for 30 min with or without RGS4 protein (kind
gift from Dr. M. Linder, Washington University). The receptor-G protein
complex was then mixed with carbachol or water before
-[32P]GTP was added to initiate the reaction at
25 °C. The reaction mixture contained different concentrations of G
protein, 1 nM receptor, 0.2 µM GTP, 5 µM GDP, and 1 mM carbachol or 200 µM GTP in a buffer of 20 mM Hepes (pH 8.0),
100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml
bovine serum albumin. RGS4 concentration was 0.1 µM. Excess 200 µM GTP (1000× more than
-[32P]GTP) instead of carbachol serves as a
nonspecific control of GTP hydrolysis. Aliquots taken at the indicated
time points were diluted with 0.5 ml of ice-cold buffer containing 5%
activated charcoal and 50 mM K3PO4.
Samples were centrifuged, and radioactivity in supernatants was
quantified by scintillation counting.
complex
was mixed with various concentrations of
o subunit.
Initially, 360 nM
complex was incubated with
increasing concentrations of the
o subunit (11.25, 22.5, 45, 90, 180, and 360 nM) in ice for 30 min in a 10-µl
buffer containing 20 mM Hepes (pH 8.0), 100 mM
NaCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. Five
µl of this mixture were then diluted 10 times to a total of 50 µl
of buffer containing 50 mM Na-Hepes (pH 7.2), 3 mM EGTA, 1 mM EDTA, 5 mM
MgCl2, 100 mM NaCl, and 1 mM
dithiothreitol. 10 µl of this diluted sample containing
and
various concentrations of the
subunit were then added to a total of
60 µl of PLC reaction buffer containing [3H]PIP2 substrate and enzyme for determining
the PLC-
3 activity as described above (6 nM final
concentration of
complex).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
2 and
1
2 Protein Expression and
Purification--
To study the function and properties of the G
protein
4 subunit, we constructed a baculovirus
expressing
4. To confirm the viral expression
of
4, Sf9 cells were infected with this
virus for varying lengths of time; cells were then harvested, lysed, and checked for protein expression by immunoblotting with the B4-2
antibody against the
4 protein (Fig.
1A). To produce
4
2 dimers, we simultaneously co-infected
Sf9 cells with the
4 virus and a virus expressing
a His-tagged
2 subunit.
4
2
was purified by nickel-nitrilotriacetic acid chromatography.
1
2 was expressed and purified using a
similar approach.
4 consistently runs with slightly
faster mobility than does
1 (Fig. 1B). This
is consistent with a report from Asano et al. (35), who
studied the native
4 protein expressed in bovine
tissues. We confirmed that the purified
4
2 and
1
2
proteins contain the same concentration of detergents by using thin
layer chromatography with the appropriate detergent standards (data not
shown).
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Fig. 1.
Expression and purification of
complexes.
A, immunoblot analysis of
4
protein expression in Sf9 cells with the
4-specific antibody (B4-2). Cells were infected
with the
4-baculovirus for varying amounts of time and
then lysed as described under "Experimental Procedures." 10 µg of
proteins from each sample were loaded. Lane 1, uninfected
cells; lane 2, cells infected for 40 h; lane
3, 48 h; lane 4, 64 h; and lane 5,
72 h. B, Coomassie Blue staining of purified G protein
recombinant
1
2 and
4
2 subunits separated by
SDS-polyacrylamide gel electrophoresis on a 12% gel.
4
2 and
1
2--
To search for potential
differences between
4 and
1 in effector
regulation, we focused on three major effectors regulated by G protein
complexes: PLC-
2, PLC-
3, and AC-II. The effect of
complexes on these enzymes was examined. Fig.
2A shows the activation of
purified PLC-
2 by
4
2 and
1
2 complexes. Consistent with previous
reports where brain
was tested (36), both
complexes
stimulate PLC-
2 more than 4-fold above basal activity with similar
effectiveness. The PLC-
2 stimulatory properties of both
complexes are thus essentially identical. Although PLC-
2 and
PLC-
3 are isozymes that are both stimulated by the G protein
complex, there is evidence that the residues in the
subunit that
contact these two enzymes are distinct (37). This raised the
possibility that although the two
complexes showed little
difference in the activation of PLC-
2, they might interact
differentially with PLC-
3. We therefore examined the stimulation of
PLC-
3 by
4
2 and
1
2. Both
complexes activate PLC-
3 20-fold over basal activity (Fig. 2B). The higher
stimulation of PLC-
3 compared with PLC-
2 is consistent with
previous reports (36). As in the case of PLC-
2, the effectiveness
with which
1
2 and
4
2 activate PLC-
3 is similar. Because
detergents in the
preparations may themselves activate PLC-
(38), we also tested boiled preparations of
4
2 and
1
2.
Stimulation from boiled samples was minimal. Moreover, boiled samples
of
4
2 and
1
2 also showed essentially the same
profile of activity, indicating that detergent concentrations and any
other nonprotein stimulators of PLC activity are present at equivalent
levels (data not shown).
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Fig. 2.
4
2
and
1
2
stimulation of PLC-
2 and
PLC-
3 activities. 1 ng of purified
PLC-
2 (A) or 5 ng of purified PLC-
3 (B)
were assayed for stimulation with the indicated concentrations of
1
2 or
4
2 in
a lipid mixture containing [3H]PIP2 as
described under "Experimental Procedures." Basal activity is
defined as activity in the absence of the
complex. The fold
stimulation over basal activity is shown.
[3H]phosphatidylinositol 1,4,5-trisphosphate production
was measured by scintillation counting. Data in A are the
means of three independent experiments performed in duplicate (± S.E.). When examined with the unpaired t test, differences
in the activities between
4
2 and
1
2 at all concentrations were not
statistically significant. B is representative of two
independent experiments; each experiment was done in duplicate.
complex in the presence of
the activated
s subunit. To test whether
4
2 and
1
2 stimulate this enzyme differentially, we examined AC-II stimulation in
the presence of GTP
S-bound
s. Sf9 cell
membranes expressing AC-II were used as a source of the enzyme (29). In
the presence of GTP
S-bound purified
s,
4
2 and
1
2
activate AC-II significantly over the basal level. Fig.
3 shows that, as in the case of PLC-
, the extent of maximal stimulation (8-fold) by
4
2 or
1
2
and the effectiveness of both
complexes in stimulating AC-II are similar.
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Fig. 3.
4
2
and
1
2
stimulation of adenylyl cyclase type II. An adenylyl cyclase assay
was performed as described under "Experimental Procedures." Various
concentrations of
complex were used as indicated. Activities are
expressed as fold stimulation over control, which was measured in the
presence of 100 nM GTP
S-bound G
s
without the
complex. Data shown are the means of two independent
experiments performed in duplicate (± S.E.). Differences in the
activities elicited by
4
2 and
1
2 were not statistically significant.
The plot shown is representative of at least three other independent
experiments performed in duplicate.
1 and
4 Subunits--
The receptor
stimulation of a G protein can be measured as GTP
S binding to the
subunit or as GTPase activity of the
subunit in the presence of
an agonist. We have recently determined that in a reconstituted
system containing the purified M2 muscarinic receptor and G protein
subunits, the GTPase assay is much more sensitive and allows us to
measure G protein activation with a relatively low concentration of
receptor (1 nM) at ratios of receptor:G protein that are
close to 1:1 (34). These conditions are potentially closer to the
dissociation constant for M2 interaction with Go (which has
not been determined) than to the ratio of receptor to G protein used in
the less sensitive GTP
S assays. Subtle differences in the receptor
interaction of G protein heterotrimers are more likely to be revealed
under the conditions used in the GTPase assay. This notion is borne out
in the analysis of the G protein
subunit interaction with M2 where
differences in coupling were detected using these conditions (34).
o
1
2
and
o
4
2 was examined by
assaying GTPase activity in the presence of RGS4. The RGS4 protein, a
GTPase-activating protein for the Go/i
family, increases the pool of G protein heterotrimers available to the
receptor and considerably increases the sensitivity of the reaction (by
~10-fold). As expected, the addition of carbachol increases the GTP
hydrolysis rate significantly (Fig. 4).
More importantly, the M2-activated GTP hydrolysis of
o
4
2 was ~200% higher
than that of
o
1
2. This
difference was consistently observed at all G protein concentrations tested (Table I). Statistical analysis
with the unpaired t test indicated that these differences
are significant (p < 0.05). To further verify these
data, two independent preparations of each purified
complex
containing
1 or
4 were examined again.
These preparations provided similar results. As mentioned before, the concentration of detergent (CHAPS) in the purified
1
2 and
4
2 stocks was determined using thin layer chromatography (data not shown).
This analysis indicated that the detergent concentrations in the
samples were similar and were not the cause for the differential receptor activation. Furthermore, when the ability of the
1
2 and
4
2
complexes were examined in PLC-
3 activation assays, the results
indicated that the functional proportions in the stocks of both subunit
complexes were the same. Finally, the possibility that these
differences arose from the differential interaction of the
subunit
types with the RGS4 protein was tested by assaying the M2-stimulated
GTPase activity in the absence of the RGS protein. Again
o
4
2 was consistently
2-3-fold more active than
o
1
2 (Table I), indicating
that the differential stimulation of GTPase activity resulted from
receptor rather than RGS protein interaction.
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Fig. 4.
M2-stimulated GTPase activity of
o
1
2
and
o
4
2.
The G protein concentration was 2 nM. Other details of the
GTP hydrolysis assay are described under "Experimental Procedures."
The GTPase activity was expressed as Pi produced (fmol) in
a 5-µl reaction. Experiments have been replicated at least three
times, and representative data are shown.
M2-promoted GTPase activity of Go protein containing
1 and
4
1
2 and
4
2
Subunits with
o--
Because receptors interact
effectively only with the heterotrimer and not with the individual
subunits, the difference observed in the M2 receptor-stimulated
activity between
o
1
2 and
o
4
2 could be attributable
to the differential heterotrimer formation between
o and
these two
complexes. The residues in
1 and
4 that contact the
subunit are conserved, indicating
that the
1 and
4 affinity for
o is likely to be the same (39). However, it was
possible that heterotrimerization was differentially affected by
divergent residues in
1 and
4 that were
located at a distance from residues that contacted the
subunit. To
examine this possibility, we used a recently developed assay for
measuring G protein heterotrimer formation (34). This assay is based on
evidence that the
complex has overlapping sites for binding the
subunit and the PLC-
3 enzyme (40). Thus heterotrimerization
prevents
complex interaction with PLC-
3, leading to the
inhibition of the
complex-stimulated PLC-
3 activity. Because
the assay is sensitive, it can be used to examine the
o-
interaction at the same subunit concentrations (1-10 nM) used in the M2-stimulated GTPase assays above.
In contrast, the ADP-ribosylation assay that has been used extensively
in the past is less sensitive (requiring more than ~1
µM subunits), and in addition, it is complicated by the
lack of knowledge regarding the mechanistic basis of the
enhancement of the
subunit ADP-ribosylation by pertussis toxin. As
shown in Fig. 5, the
complex-stimulated PLC-
activity is inhibited in a
dose-dependent manner by the
o subunit. The
o concentration dependence of this inhibition of
1
2- and
4
2-stimulated PLC
activity is
similar, indicating that both complexes form a heterotrimer with
o with equal effectiveness.
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Fig. 5.
The
1
2
and
4
2
subunits form heterotrimer equally well with
o. The
subunit (6 nM) was incubated with the indicated concentration of
subunit on ice for 30 min to allow heterotrimer formation. PLC-
3
activity catalyzed by free
but not
-
was determined to
monitor the progress of complex
with the
subunit. The
activity stimulated by free
serves as a positive control
representing the maximum PLC activity (100%), whereas the enzyme
activity by
o alone was used as a negative control. The PLC
activity was assayed as described under "Experimental Procedures."
Values shown are the means (± S.E.) from three independent
experiments. Differences in inhibition between
1
2 and
4
2
at various concentrations of
o were not statistically
significant in an unpaired t test.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
complex is known to interact directly
with and modulate various effectors and the
subunit is known to
contact effectors, we first compared the relative abilities of the
4
2 and
1
2
complexes to stimulate three common effectors regulated by the
complex: PLC-
2, PLC-
3, and AC-II. Both
complexes activated
each of the effectors with similar potency. This observation is
consistent with the conservation between
1 and
4 of 93% of similar amino acids (Fig.
6A). It is unclear whether
this result indicates that residues not conserved between
1 and
4 play no significant role in the
regulation of effectors examined here. Comparing these results with
previous mutational studies of
1, which implicate
particular residues in PLC-
regulation, does not resolve this
question.
subunit mutants analyzed in three different studies
(40-42) did not involve residues that are divergent between
1 and
4. In another study (43), several residues were mutated simultaneously. It is therefore difficult to
interpret these results in terms of the divergence in the
1/
4 primary structures. Among the few
single residues that were mutated in this study, Asp-303 is the only
one that is not conserved between
1 and
4
(Fig. 6A). This mutant
1
complex stimulated PLC-
2 normally. Although very limited, this result is consistent with results presented here.
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Fig. 6.
Differences in the amino acid sequences of
the 1 and
4 subunits mapped on the
complex three-dimensional structure.
A, primary structures of
1 and
4 subunits with the differences highlighted.
Residues located in the putative receptor-interacting surface of the
complex are numbered. Residues in the other
subunit types at these loci are shown. (
subunit types from
top to bottom are
2,
3, and
5.) Arrows indicate
strands in the folded
subunit (panel B). Four
strands make up a sheet. Sheets are denoted as lines
labeled S1-S7. B, structure of the G protein
subunit complex (
t
1
1 from
Lambright et al. (50)). Dark gray,
subunit;
light gray,
subunit; and black,
subunit.
Open circles denote residues that are nonconservative
changes between the
1 and
4 subunit amino
acid sequences. The positions of these residues in the primary
structure of
1/
4 are: residue 1,
31; residue 2,
35; residue 3,
37; residue 4,
39; residue 5,
302; residue 6,
303; and residue 7,
305. In the phosducin-
complex the prenyl group, farnesyl (C-15), is buried in the pocket
between
sheets S6 and S7 (49). Prepared with Ras Top 1.3 by P. Valadon.
It has been known for many years that the complex is essential
for heterotrimeric G protein interaction with a receptor (44). The
reasons for this requirement have been less clear. There is increasing
evidence to support the interaction of the C-terminal domain of the
subunit with a receptor. This evidence comes from studies of
rhodopsin-Gt coupling using peptides and mutant
1 subunits (45, 46). It is also supported by the ability of a peptide specific to the
5 subunit type but not the
7 or
12 subunit type to inhibit
muscarinic receptor-mediated signaling in superior cervical
ganglion neurons (20). More recently, the
subunit type in a
heterotrimer has been shown to influence the M2 receptor-stimulated
nucleotide exchange (34). Here we used the same assay to detect a
consistent 2-3-fold difference in M2-stimulated GTP hydrolysis between
o
4
2 and
o
1
2. Because these
experiments were performed in the presence of an RGS protein, the
difference in activity reflects a difference in receptor-stimulated
nucleotide exchange. The measurement of heterotrimer formation between
o and
1
2 or
4
2 showed that both heterotrimers form
with equal effectiveness and ruled out the possibility that the
difference in receptor-stimulated nucleotide exchange arose from
differences in heterotrimer formation. Receptor-stimulated GTPase
assays performed in the absence of the RGS protein showed that the
differences arose at the site of receptor interaction and not from
differential interaction with the RGS protein.
The differences in receptor-stimulated activity between
o
4
2 and
o
1
2 therefore indicate
that the distinct primary structures of
1 and
4 influence the interaction of the heterotrimer with the
receptor. Evidence that the
subunit interacts with receptors and
previous evidence that the
subunit C and N termini interact with
receptors (47) help identify the surface of the G protein that contacts
the receptor (Fig. 6B). Inspection of the amino acid
sequences of the
1 and
4 subunits
indicates differences that are distributed over the sequence (Fig.
6A). For these differences to play a role in receptor
interaction, they most likely need to be accessible to the receptor and
therefore located on the outer surface of the molecule. The
location of the different amino acids between the
1 and
4 subunits on the three-dimensional structure of the
subunit indicates that two clusters of residues (31-39 and 302-305)
are located on parallel strands of the
subunit, as shown in Fig.
6B, although the two clusters are far apart in the primary
structure, located toward the N and C termini of the
subunit (Fig.
6A). These residues are on the outer surface of the
molecule. Most strikingly, these residues are on the surface that has
been inferred to contact the receptor; note the location of the C
termini of the
subunit and the
subunit (Fig. 6B). Finally, several of these residues show divergence between the various
subunit types (Fig. 6A).
Antisense oligonucleotides specific to 1 and
3 have previously been shown to selectively inhibit
somatostatin and muscarinic M4 receptor-mediated Ca2+
channel activity (21). The precise point in the signaling pathway that
was perturbed by the introduction of the oligonucleotides has not been
elucidated so far. Inferences about the relationship between those
results and the differential stimulation of
o in the
presence of
1/
4 cannot be drawn both for
this reason and because the physiological effect of the differential M2
stimulation of
o
4
2
versus
o
1
2 is
not known.
The recent crystal structure determination of the inactive form of
rhodopsin indicates that the intracellular portion of the receptor
spans a little over 40 Å. It is unclear whether the activated forms of
other receptors will expose intracellular surfaces that are
considerably larger in surface area than 40 Å in surface area. The distances between the C terminus of the subunit and several of
the residues in the
subunit clusters are much less than 40 Å, so
the
subunit domains and the C terminus of the
subunit can
interact with a receptor simultaneously. In contrast, the distances between these domains in the
subunit and the C terminus of the
subunit are over 50 Å, and it seems less likely that these
regions can interact with a receptor at the same time. However, even
domains that are far apart in the G protein can interact with the
receptor in a temporally sequential fashion. Direct contact between the
subunit region containing residues 302-305 and the receptor is
consistent with a previous study that showed the cross-linking of a
subunit peptide covering residues 281-340 with an
2-adrenergic receptor-derived peptide (48). Divergences
in the amino acid sequence between
1 and
4 may thus contribute directly to differences in
interaction through differential contact with corresponding residues in
the receptor. This would result in the G proteins containing these
subunit types being activated selectively, as seen here. Alternatively,
the residues in the two clusters, 31-39 and 302-305, may influence
the three-dimensional structure of the receptor-interacting surface
without contacting the receptor directly. The elucidation of the
crystal structure of a
complex that retains the prenyl
modification bound to phosducin indicates that this is a possibility.
It has been inferred from the elucidation of this structure that the
prenyl moiety is buried in a cavity between
sheets 6 and 7 of the
folded
subunit structure (49) (Fig. 6B). This region of
the
subunit is susceptible to conformational changes; the
conformation of this region has been inferred to be different in the
free
complex compared with
bound to phosducin (49). It is
thus possible that the differences between
1 and
4 in the two clusters of residues highlighted
in Fig. 6B can have an effect on the conformational state of
this prenyl binding cavity between
sheets 6 and 7. Because there is
evidence that the prenyl moiety and the last several C-terminal
residues of the
subunit contact the receptors, such a rearrangement
would indirectly affect the receptor interaction of the heterotrimer by
altering the accessibility of the
subunit tail. Recent results seem
to indicate that this is the less likely mechanism. In a study by Myung
and Garrison (42), mutations were introduced in the prenyl binding
cavity and in the region of the
subunit that undergoes
conformational changes on binding to phosducin. These mutant forms of
the
subunit did not affect the G protein interaction with the A1
adenosine receptor as measured by the ability of the G proteins
to stabilize the high affinity binding state of A1 receptors. It is
unclear whether the GTPase assays used here will detect differences
between the
subunit mutants and wild type used in that study.
Overall, the importance of these results is that the differential
receptor interaction of subunit types indicates a direct role for
the
subunit in receptor activation of a G protein.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. I. Azpiazu of our laboratory for
purified M2 and o proteins as well as valuable
discussions. We thank Dr. A. Smrcka for PLC-B.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants GM53645 (to R. T.) and GM46963 (to N. 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.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: Box 8054, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8568; E-mail: gautam@morpheus.wustl.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M010424200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PLC, phospholipase C;
Sf9 cells, Spodoptera frugiperda
cells;
AC-II, adenylyl cyclase type II;
PIP2, phosphatidylinositol 4,5-bisphosphate;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
GTPS, guanosine 5'-O-(3-thiotriphosphate);
RGS, regulator
of G protein signaling.
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