From the Department of Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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The diversity in the heterotrimeric G protein
,
, and
subunits may allow selective protein-protein
interactions and provide specificity for signaling pathways. We
examined the ability of five
subunits (
i1,
i2,
o,
s, and
q) to associate with three
subunits
(
1,
2, and
5) dimerized to
a
2 subunit containing an amino-terminal
hexahistidine-FLAG affinity tag (
2HF). Sf9 insect
cells were used to overexpress the recombinant proteins. The
hexahistidine-FLAG sequence does not hinder the function of the
1
2HF dimer as it can be specifically
eluted from an
i1-agarose column with GDP and
AlF4
, and purified
1
2HF dimer stimulates type II adenylyl
cyclase. The
1
2HF and
2
2HF dimers immobilized on an anti-FLAG
affinity column bound all five
subunits tested, whereas the
5
2HF dimer bound only
q.
The ability of other
subunits to compete with the
q
subunit for binding to the
5
2HF dimer was
tested. Addition of increasing amounts of purified, recombinant
i1 to the
q in a Sf9 cell extract
did not decrease the amount of
q bound to the
5
2HF column. When G proteins in an
extract of brain membranes were activated with GDP and
AlF4
and deactivated in the presence of equal amounts
of the
1
2HF or
5
2HF dimers, only
q bound
to the
5
2HF dimer. The
q-
5
2HF interaction on the
column was functional as GDP, and AlF4
specifically eluted
q from the column. These
results indicate that although the
1 and
2 subunits interact with
subunits from the
i,
s, and
q families, the
structurally divergent
5 subunit only interacts with
q.
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INTRODUCTION |
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All cells possess multiple signaling pathways that transmit
signals from the hormones, autacoids, neurotransmitters, and growth factors in their environment. Complex biochemical mechanisms exist to
discriminate, integrate, and modulate a cell's response to this
constantly changing set of stimuli. One of the best characterized signal transduction systems is the pathway used by receptors
coupled to heterotrimeric G
proteins1 (1, 2). Our current
understanding of this signaling pathway shows it to be surprisingly
complex with large families of proteins comprising the receptors, G
proteins, and effectors (1, 3, 4) and important roles for both the and
subunits of the heterotrimer in activating effectors (2, 4,
5). Moreover, some ligands activate multiple G proteins (1, 2, 6), and
certain receptors activate the MAP kinase pathway (6, 7) and/or other
tyrosine kinase signaling pathways (8). Thus, an important unsolved
question in cell signaling is how a cell selects a response from the
multiple possibilities available.
Current evidence holds that specificity is determined at many levels.
In addition to the tissue-specific expression of receptors, G proteins,
or effectors (3), there are important protein-protein interactions
involving the and
subunits of the G protein heterotrimer
that determine specificity. For example, the
t subunit couples selectively to rhodopsin and the
s subunit to
the
-adrenergic receptor (1, 2, 6). Furthermore, it is clear that
the
dimer is required for efficient coupling of the
subunit
to receptors (9, 10), and there is growing evidence supporting specific
interactions of receptors with the
dimer (11-13). Both the
and
subunits of transducin appear to contact rhodopsin (14, 15),
and the presence of the
dimer significantly increases the
affinity of the Gt
subunit for rhodopsin (14). In this regard, the carboxyl terminus of the
subunit and its prenyl modification have emerged as important determinants of the interaction of G proteins with receptors (12, 13). Experiments using antisense RNA
to selectively remove G protein subunits in GH3 cells also support a role for the diversity of the G protein
subunits in
determining signaling specificity. In these cells, the
G
o1
3
4 heterotrimer couples
preferentially to the muscarinic receptor, G
o1
2
2 to the galanin
receptor, and the G
o2
1
3
combination to the somatostatin receptor (16, 17). Similar experiments using rat basophilic leukemia cells suggest that the m1 muscarinic receptor couples selectively to
q,
11,
1,
4, and
4 (18). Thus,
the existence of multiple isoforms of the
,
, and
subunits and the participation of both
and
subunits in receptor
coupling implies that the diversity of the subunits in the G
protein heterotrimer could play an important role in signal
transduction.
Although a large number of studies have focused on specific
interactions of the and/or
subunits with effectors (4, 19),
there are few investigations of the role of the
-
interaction in cell signaling. Perhaps it has been assumed that because of the
similarity of the known
subunits, all
subunits would associate with all
subunits. Recently, two more divergent members of the
subunit family,
5 and
5L, have been
described (20, 21). Whereas the amino acid sequences of
1,
2,
3, and
4 are 80-90% identical,
5 is only 52%
identical and 64% similar to
1 (20). In addition,
5 has an eight amino acid extension near the amino terminus and three short amino acid insertions within the WD repeat regions of the molecule. The
5 subunit is expressed
predominantly in the brain, with only trace amounts detected by
Northern analysis in the kidney. The
5L subunit appears
to be expressed only in retina. Both the
5 and
5L subunits can stimulate PLC-
2 activity when transiently transfected into COS-7 cells with the
2
subunit (20, 21). However, the
5
2 dimer
fails to activate the MAP kinase pathway when transfected into these
cells (22). This observation suggests that dimers containing the
5 subunit may have different functions from those
containing other
subunits. In the experiments reported here, we
have tested the ability of several
subunits to interact with
dimers containing the
1,
2, or
5 subunit to determine if the variations in amino acid sequence observed for the
subunits are manifested as differences in
affinity for
subunits. Sf9 cells were co-infected with an affinity tagged
2 subunit and various
subunits. The
resulting
2HF dimers were immobilized via the
affinity tag and allowed to interact with a variety of recombinant
subunits expressed in Sf9 cells. The results show that the
1
2HF and
2
2HF dimers interact with five different
subunits from three families, whereas the
5
2HF subunit only interacts with the
q subunit.
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EXPERIMENTAL PROCEDURES |
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Construction of Recombinant Baculoviruses for the
2HF and
5 Subunits--
The polymerase
chain reaction was used to modify the cDNA encoding the
2 subunit (23) by adding XbaI and
BamHI restriction sites to the 5
and 3
ends of the
2 coding region, respectively. The primers used were
Sense primer: 5
-AACTCTAGAATGGCCAGCAACAACACCGC-3
XbaI; and Antisense primer:
5
-CCTGGATCCTTAAAGGATAGCACAGAAAAACTTC-3
BamHI. The products of the polymerase chain reaction were
digested with XbaI and BamHI and ligated into the
pDoubleTrouble (pDT) vector (24) to add the nucleotide sequences for
the hexahistidine and FLAG affinity tags to the 5
end of the
2 coding region. To construct useful restriction sites
for subcloning into the baculovirus transfer vector pVL1393, the
2HF coding sequence was excised from pDT with
KpnI and BamHI and subcloned into the pCNTR
shuttle vector using the Prime Efficiency Blunt-End DNA Ligation Kit (5 Prime
3 Prime). The
2HF coding region was excised from pCNTR with BamHI and ligated into the BamHI
site of pVL1393 to place the ATG of the hexahistidine sequence 75 bases
downstream of the polyhedron promoter. The mouse
5
cDNA in a Bluescript SKII vector was kindly provided by Dr. Melvin
I. Simon of the California Institute of Technology. The 1803-base pair
BamHI-XbaI fragment of the
5
cDNA was subcloned into the BamHI-XbaI sites of pVL1393. To ensure fidelity, both completed transfer vectors were
sequenced in the forward and reverse directions using dye terminator
sequencing on an automated sequencer (Applied Biosystems, model 377).
Recombinant baculoviruses were isolated following co-transfection of
the transfer vector and linearized BaculoGold viral DNA into Sf9
cells using the PharMingen BaculoGold® kit. Briefly,
2 × 106 Sf9 cells were co-transfected with 1 µg of linear BaculoGold DNA and 3-5 µg of recombinant baculovirus
transfer vector DNA using calcium phosphate/DNA precipitation.
Following a 4-h incubation at 27 °C, the co-transfection medium was
removed, and the monolayer was rinsed with fresh TNM-FH medium (25)
supplemented with 10% fetal bovine serum, 50 µg/ml gentamicin
sulfate, and 2.5 µg/ml amphotericin B. The plates were incubated at
27 °C with 5 ml of fresh medium for 4-6 days. Recombinants were
detected by observing swollen, extremely large cells associated with a
low cell density and a large amount of cell debris. Recombinant
baculoviruses were purified by one round of plaque purification using
standard techniques (25). The construction of the recombinant
baculoviruses coding for the
i1,
i2,
o,
s, and
q subunits and
the
1 and
2 subunits has been described
(26-28). The baculovirus encoding the avian
11 protein (29) was the
kind gift of Dr. T. K. Harden.
Expression and Purification of Recombinant G Protein and
Subunits--
Recombinant G protein subunits were overexpressed
in suspension cultures of Sf9 insect cells as described (26, 27,
30). In most experiments, the recombinant
and
subunits were
extracted from cell pellets using the detergent Genapol C-100 at a
concentration of 0.1% (w/v). All steps were performed at 4 °C.
Frozen pellets were thawed in 15 × their wet weight in lysis
buffer containing 20 mM Hepes, pH 7.5, 150 mM
NaCl, 3 mM MgCl2, 1 mM EDTA, 17 µg/ml phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine, and 2 µg/ml each of aprotinin, leupeptin, and pepstatin, and burst by
nitrogen cavitation (600 p.s.i. for 20 min) at 0 °C. The crude
lysate was mixed with an equal volume of lysis buffer supplemented with
0.2% (w/v) Genapol C-100 and stirred for 1 h. The Genapol extract
was centrifuged at 100,000 × g for 60 min and the
supernatant decanted, and aliquots were frozen in liquid
N2. The
s subunit used in the adenylyl
cyclase assays was prepared as above except 0.1% (w/v) CHAPS was
substituted for 0.1% (w/v) Genapol C-100. Partially purified
i1 subunits were used in some experiments. Sf9
cell pellets overexpressing the
i1 subunit were
extracted without detergent; a 100,000 × g supernatant
was prepared and the protein purified on a DEAE column exactly as
described (26). This preparation of
i1 subunit is
approximately 95% pure, as determined by quantitation of a
silver-stained gel.
Preparation of the -Anti-FLAG Affinity Column--
All
column steps were carried out at 4 °C. Typically, 1 ml of the
Genapol C-100 extract of Sf9 cells overexpressing the desired
2HF dimer was applied to a 0.5-ml anti-FLAG M2
affinity gel column equilibrated with column buffer (lysis buffer
containing 0.1% Genapol C-100 and 1 mM
-mercaptoethanol) at a flow rate of 0.2 ml/min. The resulting
2HF-anti-FLAG affinity gel column (
2HF affinity column) was washed three times with 3 ml of column buffer. This procedure resulted in a highly pure
preparation of
dimers immobilized on the column (see Fig. 1).
The amount of
dimer immobilized on the column was about 6 µg/0.5 ml of resin as judged by silver staining of the
dimer
eluted from the column with 0.1 M glycine, pH 3.5. This
represents about 5% of the nominally available FLAG binding sites.
Usually the interaction of an
subunit with a particular
subunit
was measured by applying 2 ml of a Genapol C-100 extract of Sf9
cells expressing the desired
subunit to a
2HF
affinity column at a flow rate of 0.2 ml/min. The resulting
2HF affinity column was washed 4 times with 4 ml
of column buffer. In some experiments, the
2HF
heterotrimer was eluted with 0.1 M glycine, pH 3.5. In the
experiment presented in Fig. 5A, the procedure was modified
such that 2 ml of
i2 extract and 2 ml of
q extract were mixed and applied to the
2HF affinity column. In the experiment presented in
Fig. 6, a range of 17.5-525 µg of partially purified
i1 was mixed with 2 ml of
q extract and
applied to the
2HF affinity column.
Specific Elution of Subunits from
2HF-Anti-FLAG Affinity Gel--
To demonstrate a
functional interaction between
subunits and the immobilized
subunits, a
2HF affinity column (0.5 ml) was prepared
at 4 °C as described above, washed three times with 3 ml of column
buffer, and then equilibrated in
subunit binding buffer (20 mM Hepes, pH 8.0, 100 mM NaCl, 1 mM
MgCl2, 0.3% (w/v) C12E10, 10 mM
-mercaptoethanol, and 5 µM GDP). Then,
17.5 µg of purified
i1 subunit diluted in 1 ml of
subunit binding buffer was applied to the
2HF
affinity column at a flow rate of 0.2 ml/min. The
2HF
affinity column was washed 4 times with 4 ml of
subunit binding
buffer and twice with 2 ml of
subunit binding buffer containing 300 mM NaCl. The column was incubated at room temperature for
15 min. The
i1 subunit was eluted with 4 × 0.5 ml
of 20 mM Hepes, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1.0% cholate, 10 mM
-mercaptoethanol, 10 mM MgCl2, and 100 µM GTP
S, also at room temperature. The column was
washed twice with 2 ml of
subunit binding buffer containing 300 mM NaCl, before final elution with 0.1 M
glycine, pH 3.5.
Extraction of G Proteins from Bovine Brain Membranes and
Activation with GDP-AMF--
Frozen bovine brains were obtained from
PelFreeze and membranes prepared according to the method of Sternweis
and Robishaw (31), with the addition of 0.2 µg/ml aprotinin to all
buffers. The membrane preparations were stored at 80 °C. Membranes
were thawed, washed once with ice-cold 20 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, and pelleted at
40,000 × g for 30 min at 4 °C. The washed membrane
pellet (1 g of protein) was extracted for 1 h at 4 °C with 200 ml of 0.5% (w/v) C12E10 in 50 mM
Tris, pH 8.0. The extract was clarified by centrifugation at
143,000 × g for 60 min and stored at
80 °C. To
examine the interaction of the G proteins in the membrane extracts with
the various
subunits, the extracts were thawed and mixed with the
different
2HF Genapol extracts from Sf9 cells
and activated by addition of concentrated stocks to give final
concentrations of 3 mM MgCl2, 5 mM
NaF, 15 µM AlCl3, and 2.5 µM
GDP. The mixture was incubated at 30 °C for 45 min (32). The
subunits were deactivated by addition of 500 mM EDTA to a
final concentration of 20 mM EDTA and incubated for an
additional 30 min at 30 °C. Typically, 4 ml of the deactivated mixture was applied to 0.5 ml of anti-FLAG affinity gel equilibrated with 20 mM Hepes, 0.5% (w/v)
C12E10, 5 µM GDP, pH 8.0. The
loaded affinity column was washed with 5 ml of equilibration buffer
containing 400 mM NaCl and eluted with 1.0 ml of 0.1 M glycine, pH 3.5, as described above.
Silver Staining, Immunoblotting, and Quantitation--
Samples
were prepared for electrophoresis, loaded on 0.75 mm, 12% acrylamide
gels, subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and the gels stained with silver according to the
method of Bloom et al. (33). Purified bovine brain
Gi/o heterotrimer (31) was used as a standard. The protein
concentrations in the gel were compared with ovalbumin concentration
standards and quantitated following silver staining using a BioImage
scanning densitometer and the Whole Band® software
(BioImage, Ann Arbor, MI). For Western blots, gels were transferred to
nitrocellulose and immunoblotted using the following primary
antibodies: an anti-G protein subunit antibody (NEN Life Science
Products, catalog number 808), 1:1000 dilution; an anti-G protein
subunit antibody (Calbiochem, catalog number 371737), 1:1000 dilution;
and an anti-
q/
11 antibody (Santa Cruz, catalog number sc-392), 1:100 dilution. The primary antibodies were
detected using goat anti-rabbit IgG(Fc) alkaline phosphatase conjugate
(Promega) or donkey anti-rabbit IgG F(ab
)2 horseradish peroxidase conjugate (Amersham Corp.). The density of the bands on
autoradiographs obtained following ECL detection was also estimated using the Whole Band® software.
Adenylyl Cyclase Assays--
Recombinant baculovirus encoding a
FLAG epitope-tagged rat type II adenylyl cyclase was kindly provided by
Dr. Ravi Iyengar, Mount Sinai School of Medicine. Sf9 cells were
infected with the cyclase baculovirus and harvested 72 h later,
when viability was approximately 80%. The cell pellet was washed three
times with 6.8 mM CaCl2, 55 mM KCl,
7.3 mM NaH2PO4, 47 mM
NaCl, pH 6.2, and membranes prepared according to the procedure of
Taussig et al. (34). The washed membrane pellet was
resuspended in 20 mM Hepes, pH 8, 200 mM
sucrose, 1 mM EDTA, 2 mM DTT, 17 µg/ml
phenylmethylsulfonyl fluoride, 16 µg/ml
N--p-tosyl-L-lysine chloromethyl
ketone, 16 µg/ml N-tosylphenylalanyl chloromethyl ketone,
2 µg/ml leupeptin, and 3 µg/ml lima bean trypsin inhibitor at a
final total protein concentration of 1.5 mg/ml, as determined by the
method of Bradford (35). Aliquots were frozen in liquid N2
and stored at
80 °C. A 0.1% (w/v) CHAPS extract of Sf9
cells overexpressing
s (see above) was activated with
100 µM GTP
S in 5 mM MgSO4, 1 mM DTT, and 1 mM EDTA, pH 8, for 30 min at
30 °C (34). Excess GTP
S was removed by centrifugation through P6
resin (Bio-Rad) equilibrated in 50 mM Hepes, pH 8, 150 mM NaCl, 5 mM MgSO4, 1 mM DTT, 1 mM EDTA, 0.1% CHAPS, as described
previously (13). The first elution fraction, containing activated
s, was held on ice. Reaction tubes containing a total of
25 µl of type II cyclase membranes (12 µg of protein/assay tube),
activated
s,
, and/or buffer were prepared at room
temperature. The reaction was begun by addition of 75 µl of reaction
mix pre-equilibrated at 30 °C. The standard reaction mixture
contained 25 mM Hepes, pH 8, 10 mM
phosphocreatine, 10 units/ml creatine phosphokinase, 0.4 mM
3-isobutyl-1-methylxanthine, 10 mM MgSO4, 0.5 mM ATP, and 0.1 mg/ml bovine serum albumin. Reactions were
carried out for 7 min at 30 °C. Cyclic AMP production was stopped by
the addition of 1.0 ml of 0.11 N HCl and cyclic AMP quantified by radioimmunoassay (36).
Expression of Results-- Experiments presented under "Results" are representative of three or more similar experiments.
Materials--
All reagents used in the culture of Sf9
cells and for the expression and purification of G protein and
subunits have been described in detail (26, 30). The baculovirus
transfer vector, pVL1393, was purchased from Invitrogen; the
BaculoGold® kit was from PharMingen; 10% Genapol C-100
and the anti-
common subunit antibody were from Calbiochem; Prime
Efficiency blunt-end DNA Ligation Kit was from 5 Prime
3 Prime;
anti-FLAG® M2 affinity gel was from Eastman Kodak;
polyoxyethylene 10 lauryl ether (C12E10) was
from Sigma; the anti-
q/
11 antibody was
from Santa Cruz; the NEN-808 anti-
subunit antibody was from NEN
Life Science Products, and nitrocellulose was from Schleicher and
Schuell. All other reagents were of the highest purity available.
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RESULTS |
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The objective of this study was to determine if selectivity in
-
interactions could be observed in vitro with
recombinant G protein subunits isolated from baculovirus-infected
Sf9 cells. We first constructed a recombinant baculovirus
encoding sequential affinity tags on the amino terminus of the
2 subunit, a hexahistidine tag followed by a FLAG
epitope tag (24). When used in conjunction with an anti-FLAG antibody
covalently linked to agarose beads, the FLAG epitope tag provides a
convenient method for separating
subunits bound to
2HF from
subunits free in solution. Previous work
has shown that addition of a hexahistidine or FLAG affinity tag to the
amino terminus of the
subunit does not prohibit association with
the
subunit (37-39) or the subsequent association of the
dimer with
subunits (37). The heterotrimeric G protein crystal
structure also suggests that an extension of the amino terminus of the
subunit would be unlikely to interfere with
-
interactions
(40, 41).
The silver-stained SDS-polyacrylamide gel in Fig.
1 illustrates the steps involved in the
preparation of and
-
affinity columns. Sf9 cells
were co-infected with recombinant baculovirus encoding for the
1 and
2HF subunits, and the recombinant
1
2HF protein was extracted from membranes
of cells harvested 48 h post-infection. Crude detergent extracts
were applied to anti-FLAG M2 affinity gel columns and washed with 5-10
column volumes. The resulting product of this one-step purification is
shown in lane 3 of Fig. 1. The FLAG epitope tag on the
2HF subunit is available for binding to the anti-FLAG
antibody and produces a dramatic increase in purity in a single step.
The presence of the hexahistidine tag and FLAG epitope results in
reduced electrophoretic mobility of the
2HF subunit
relative to
2 (lane 1 versus 3).
Approximately 12 µg of
1
2HF were
captured per ml of anti-FLAG M2 affinity gel suspension, as determined
by quantitation of the eluted
subunit on a silver-stained gel. In
subsequent experiments designed to monitor
-
interaction, the
1
2HF was captured as before and the
resulting
2HF affinity column used to specifically
bind partially purified
i1 subunits. To determine first
the amount of
i1 necessary for stoichiometric binding to
immobilized
1
2HF, replicate
1
2HF affinity columns were prepared and
then varying amounts of
i1 subunit applied.
Stoichiometric binding was achieved at a 3-7-fold excess (w/w) of
i1 over immobilized
1
2HF
(data not shown). Lane 4 shows the
i1
preparation used for these experiments. Lane 5 shows the
resulting
i1
1
2HF eluted
with 0.1 M glycine after an excess of
i1 was
applied to the
1
2HF affinity column.
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To demonstrate that the immobilized 1
2HF
was properly folded and functional, a 0.5-ml
i1
1
2HF affinity column,
prepared identically to that shown in lane 5, was treated
with 100 µM GTP
S. The
i1 eluted
specifically, in a volume of 0.5 ml, as shown in lane 6.
Lane 7 shows the elution of the remaining
1
2HF dimer with glycine. Thus, the
i1 subunit can be dissociated from the
1
2HF subunit with GTP
S treatment,
analogous to activation of the native heterotrimer in solution (42).
Heterotrimeric G proteins can also be activated with GDP-AMF resulting
in dissociation of the
dimer (43). When an
i1
1
2HF affinity column
similar to that described above was activated with GDP-AMF, the
i1 subunit was specifically eluted (data not shown).
To test the functionality of the
i1
1
2HF interaction in
another way, we subjected a detergent extract of
1
2HF to our normal
purification
strategy, DEAE ion exchange chromatography followed by
i1-agarose affinity chromatography (30). The
-agarose
affinity chromatography exploits the ability of GDP-AMF to dissociate
the
subunit from the
subunit. Fig.
2A shows a Western blot,
developed with an anti-
antibody, of the DEAE pool (PL)
applied to the
i1 column and the
i1
column pass-through (PT). Comparison of lanes 1 and 2 shows a typical result for
1
2. A very high proportion of the
1
2 present in the DEAE pool binds to the
i1-agarose. Lanes 3 and 4 show a
very similar result obtained when a DEAE pool containing
1
2HF was applied to the
i1-agarose. This observation is consistent with the
result obtained with immobilized
1
2HF and
i1 free in solution, as described above. To obtain
further evidence of functional
i1
1
2HF interaction, we
treated the
1
2HF-loaded
i1-agarose column with GDP-AMF. Fig. 2B shows
a silver-stained SDS-polyacrylamide gel of the
i1-agarose column pass-through (lane 2), wash
fractions (lanes 3-5), and subsequent elution of
1
2HF by treatment with GDP-AMF
(lanes 6-8). Thus,
1
2HF binds tightly to immobilized
i1 and elutes upon activation of
i1 with GDP-AMF.
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We next tested the ability of 1
2HF to
stimulate one effector, type II adenylyl cyclase. It is known that
1
2 is a potent activator of type II
cyclase in the presence of an
s subunit activated with
GTP
S (44). The data in Table I compare
the stimulation of type II cyclase by
1
2
and
1
2HF in the concentration range
0-100 nM. At 100 nM, the
1
2HF dimer is capable of a 15-fold stimulation of cyclase over the effect of GTP
S-
s
alone. However, at each concentration tested, the
1
2 dimer activates adenylyl cyclase to a
significantly greater extent than does the
1
2HF dimer. This reduced stimulation
could be due to a decreased effective concentration of
1
2HF relative to
1
2 at the adenylyl cyclase-containing membrane surface, or to a specific interference between the affinity tags on the
subunit's amino terminus and type II cyclase. This matter is under further investigation. We conclude that the presence of
the hexahistidine and FLAG epitopes on the amino terminus of the
2 subunit does not abrogate interaction of
1
2HF with at least one effector, type II
adenylyl cyclase.
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Previous work with the adenosine A1 receptor showed little difference
between the ability of i1,
i2, and
i3 to support high affinity binding of agonist in the
presence of
1
2 (13, 45). Since this
observation implies similar affinity of the three
i
subunits for
1
2, we tested the ability of
another
i isoform, the
i2 subunit, to
bind to a
1
2HF affinity column. Two ml of
a crude detergent extract of Sf9 cells infected with recombinant
baculovirus encoding the
i2 subunit was applied to a
0.5-ml
1
2HF affinity column as described
above, washed extensively, and the bound
i2 and
1
2HF eluted with glycine. A
silver-stained polyacrylamide gel of the product is shown in Fig.
3A, lane 2. Thus, the
immobilized
1
2HF was also able to bind
i2, and a 2-ml volume of crude extract containing
i2 subunit was sufficient excess to obtain
stoichiometric binding of
i2 to immobilized
1
2HF.
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Having demonstrated functional activity for
1
2HF and its ability to bind both
purified
i1 and a crude detergent extract of the
i2 subunit, we tested the ability of other
2HF dimers to bind
i2. The
1-,
2-, and
5
2HF columns were first constructed by
application of appropriate crude cell extracts to anti-FLAG M2 affinity
gel. Pilot experiments were performed to ensure that equivalent amounts
of
1-,
2-, and
5
2HF were bound to anti-FLAG columns by
applying a sufficient excess of each
2HF detergent extract to saturate the available FLAG binding sites. Equal volumes of
a crude detergent extract of Sf9 cells infected with recombinant baculovirus encoding the
i2 subunit were then applied to
each
2HF affinity column. The columns were washed
extensively to remove any nonspecifically bound
i2.
Finally, the
i2
2HF was eluted with
glycine. The elution products were analyzed by gel electrophoresis
followed by silver staining and Western blotting with an anti-
common antibody (Fig. 3, A and B). Under these conditions, the
1
2HF and
2
2HF columns captured equivalent, and
roughly stoichiometric, amounts of
i2 (Fig. 3A,
lanes 2 and 3). Since the
5 subunit
co-migrates with the
i2 subunit under the
electrophoresis conditions employed, it is not possible to determine
from the silver-stained gel whether
5
2HF
captured
i2 (Fig. 3A, lanes 4 and
5). However, the Western blot in Fig. 3B
demonstrates clearly that
5
2HF bound
little, if any,
i2 under conditions where
1
2HF and
2
2HF bound amounts of
i2 easily detectable with the same primary antibody (compare lanes 2 and 3 with 4).
To determine if 5
2HF was unable to bind
i2 due to steric constraints of immobilization, we
tested the ability of
5
2HF free in
solution to bind to an
i1-agarose column, an experiment analogous to the
1
2HF-
i1-agarose system
illustrated in Fig. 2. The concentration of
5
2HF in the DEAE pool applied to the
i1-agarose column was compared with the concentration of
5
2HF in the column pass-through. Both the
DEAE pool and the
i1 column pass-through gave similar
intensity when developed with an anti-
subunit antibody (data not
shown), indicating little or no binding. Furthermore, no
5
2HF product was detected on
silver-stained polyacrylamide gels after treatment of the
i1-agarose with GDP-AMF. Therefore, the low affinity of
5
2HF for
i1 is not due to
immobilization of the
2HF.
We then selected representatives of three subfamilies,
i/o,
s, and
q (46), to
investigate the ability of
5 to interact with other
subunits. Crude detergent extracts of Sf9 cells infected with
recombinant baculovirus encoding the appropriate
subunit isoform
were applied to three
2HF affinity columns
constructed as before. After extensive washing, the specifically bound
subunits were eluted, along with their
2HF
counterparts, by treatment with 0.1 M glycine. The
resulting products were analyzed by Western blotting with either an
anti-
common antibody in the case of
i2,
o, and
s or with an anti-
q
specific antibody for
q. The results are shown in Fig.
4. As observed earlier using
i2 (Fig. 3),
1
2HF and
2
2HF bind easily detectable amounts of
i2 under conditions where
5
2HF does not (Fig. 4, lanes
1-3). This pattern is repeated with respect to binding
o and
s (lanes 4-9). However, when detergent extracts containing recombinant
q were
applied to each
2HF column,
1
2HF,
2
2HF,
and
5
2HF all bound
q equally (lanes 10-12). Thus,
5
2HF appears to bind the
q
subunit selectively. To determine if the
5
2HF dimer was able to interact with
other members of the Gq family, we have performed pilot
experiments with a recombinant, avian
11 subunit (29).
This protein is 96% identical in amino acid composition to the mouse
11 subunit (47) and 100% identical in the 20 amino
acids shown to contact the
subunit in the x-ray structure of the
heterotrimer (41). Preliminary results indicate that crude detergent
extracts containing
11 bind equally well to all three
2HF dimers (data not shown). Thus, at least one other
member of the Gq family binds to the
5
2HF dimer. The interaction of the other
members of the family (the G14-16
subunits) with the
5 subunit is currently under investigation.
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The observation of selective
q-
5
2HF interaction raises
the issue of relative affinities. One approach to this question is to
determine the ability of other
subunits to compete with
q for binding to
5
2HF.
Since the
q preparation was from a crude cell extract,
we first selected a similar preparation of the
i2
subunit for competition experiments. The
1
2HF and
5
2HF columns were constructed as before,
and then a mixture of equal volumes of
i2 and
q detergent extracts were applied to each. After
extensive washing, the
2HF complexes were eluted
with 0.1 M glycine. The
i2/
q
mixture applied to the columns (L) was compared with the
glycine elution fractions (E) by Western blot using anti-
common or anti-
q antibodies. Lanes 1-4 of
Fig. 5A show the result
obtained with
1
2HF. As expected from the
individual
subunit experiments, the
1
2HF affinity column captured both
i2 and
q subunits (lane 2 versus
4). The
5
2HF affinity column also
bound
q (lane 8) at a level roughly
comparable to that bound by
1
2HF
(lane 4 versus 8). However,
5
2HF bound no detectable
i2 subunit (lane 2 versus 6).
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Since all the above experiments were performed with recombinant
proteins, we tested the subunit selectivity of the
affinity columns with native
subunits. As bovine brain membranes are known
to contain a complex mixture of
subunits, including
i,
o, and
q (3, 48), we
used an extract of bovine brain membranes as a starting material for
these experiments. Crude detergent extracts of Sf9 cells
infected with recombinant baculovirus encoding for the
1
2HF or
5
2HF dimers were mixed with brain
membrane extract and the
subunits activated by treatment with
GDP-AMF as described under "Experimental Procedures." The mixtures
were deactivated by addition of excess EDTA, resulting in the
association of a fraction of the brain
subunits with the
recombinant
2HF dimers. The deactivated mixture was
applied to an anti-FLAG affinity column and the
2HF complexes eluted. The proteins in the mixtures applied to the affinity columns and the glycine elution fractions were
resolved on acrylamide gels, transferred to nitrocellulose, and probed
with anti-
subunit antibodies. The
1
2HF dimer bound
subunits which gave
positive signals with anti-
common antibodies and
anti-
q/11 antibodies (Fig. 5B, lanes 2 and
4). However, the
5
2HF dimer
only associated with
subunits detected by the
anti-
q/11 antibody (lane 6 versus 8), in
agreement with the selectivity observed with recombinant
subunits
(Fig. 5A). Interestingly, the
5
2HF dimer binds a clearly resolved
doublet from the brain extract (Fig. 5B, lane 8). The
anti-
q/11 antibody used in these experiments does not
cross-react with
i/o subunits, and therefore this
doublet is most likely
q and/or
11. Thus,
when the
5
2HF dimer was presented with a
complex mixture of native heterotrimeric G proteins, it selectively
bound the
q/11 subunits. Moreover, it did not appear to
interact with the
i or
o subunits which are present at high concentrations in brain membranes.
Because the q,
i2, and bovine brain
preparations are all crude detergent extracts, it is not possible to
estimate the molar ratio of competing
subunit to
q
subunit applied to the immobilized
2HF. To address
this issue in part, we employed the purified
i1
preparation described previously. The
i1 subunit in this preparation represents approximately 95% of the intensity on a silver-stained gel. Increasing amounts of this
i1 stock
were diluted into a fixed, larger volume of
q crude
extract. Based on the amount of
5 subunit immobilized on
the column as estimated from silver-stained gels, a 3-100-fold excess
of
i1 was added to the
q extract. These
mixtures were applied to immobilized
5
2HF, washed, and eluted with 0.1 M glycine. The loading mixture, the last wash, and the
elution fractions were examined by Western blot using an
anti-
q/11 antibody (Fig.
6A) and an anti-
common antibody (Fig. 6B). Even at the largest excess of
i1 over the immobilized
5
2HF, there was no detectable competition
by
i1 for
q binding to
5
2HF. The ECL signal representing bound
q in Fig. 6A was quantitated on a scanning
densitometer. Fig. 6C shows a plot of this integrated
intensity versus excess
i1 present. Note that
there is no apparent diminution of
q binding at ratios of
i1 to
5
2HF far in
excess of the ratio required for stoichiometric binding of
i1 by
1
2HF (about
3:1).
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To demonstrate that the
q
5
2HF interaction was
functional, we constructed an
q
5
2HF affinity column as
described in Fig. 4 and treated the immobilized heterotrimer with
GDP-AMF to activate and thereby dissociate
q. Fig.
7A presents a silver-stained
gel of the GDP-AMF elution product (E1-E3-AMF, lanes 4-6).
Because
q and
5 co-migrate under these
electrophoresis conditions, we verified the identity of the GDP-AMF and
glycine elution products by Western blot with an
anti-
q/11 antibody (Fig. 7B). Comparison of
lanes 4 and 7 in Fig. 7B shows that
the majority of the
q bound to
5
2HF eluted specifically with GDP-AMF.
Thus, the
q subunit is associating with the immobilized
5
2HF in a manner that permits the
q subunit to be activated and to dissociate from the
5
2HF.
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DISCUSSION |
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The data presented in this report provide clear evidence that the
diversity of the subunits in the G protein heterotrimer can have
important functional consequences for the interaction of certain and
subunits. Although all the
subunits examined interact with
the
1 or
2 subunit, the structurally
different
5 subunit interacts selectively with the
q subunit and the nearly identical
11
subunit. We inspected two heterotrimeric crystal structures for sites
of intersubunit contact which might be responsible for the observed
selectivity (40, 41). These structures show that nine locations
involving 16 amino acids on the
1 subunit are primarily
responsible for interacting with the Switch I, Switch II, and the
amino-terminal regions of the
subunit. Of these 16 amino acids,
only 3 are different in the
5 subunit (Leu55
Gly, Tyr59
Leu, and Ser98
Thr,
based on the
1 sequence). Although the essential
residues necessary for a WD repeat (20, 49) are conserved in the
5 subunit, the overall amino acid sequence of the
protein is only 52% identical and 62% similar to that of the
1 subunit. Thus, there are amino acid differences in the
sequences surrounding the direct
subunit contact sites and other
regions of dissimilarity distributed throughout the entire
5 sequence. Similarly, examination of the sequences of
the
subunits shows multiple differences in the amino acids
contacting the
subunits in the
i,
o,
s, and
q subunits, but there is only one
site where the
q/11 subunit is unique (41). The
i,
o, and
s subunits have
a Phe at position 195 in the beginning of the Switch II region, and the
q/11 subunit share a Val at this position (41). Since
there are multiple differences in sequence in both the
and
isoforms under consideration relative to the isoforms that have been
crystallized, it is not possible to suggest a molecular basis for the
selective interaction of the
5
2HF dimer
with the
q subunit. However, the net effect of the
various differences in
-
contacts must be substantial, as we have
found that a large excess of the
i1 subunit does not measurably compete with the
q subunit for binding to the
5 subunit (see Fig. 6).
In evaluating the selectivity of the 5
2HF
dimer for
subunits in the Gq family, it is important to
consider the fidelity with which Sf9 cells modify recombinant
proteins. The
subunits of most G proteins are modified with
myristoyl and/or palmitoyl groups at their amino terminus, and the
subunits are modified with a prenyl group at their carboxyl terminus
(50). These modifications markedly affect the affinity of the
subunits for the
dimers (51). The available evidence suggests
that the proteins used in this work are properly modified. Recombinant
Gi and Go
subunits have been shown to be
myristoylated (26), and the Gq and G11
subunits are able to activate phospholipase C-
equally with native
proteins (52). The Gs
subunit produced in Sf9
cells fully activates adenylyl cyclase and is 50-fold more potent than the protein expressed in Escherichia coli but is not as
potent as
s purified from liver (53). The carboxyl
terminus of the
2 subunit expressed in Sf9 cells
appears to be properly and fully processed (54). Thus the available
experimental evidence supports the hypothesis that recombinant proteins
isolated from Sf9 cells are properly modified, and therefore the
interactions reported here with recombinant proteins mimic those in
intact cells. Most importantly, the major result of the study is
considerably strengthened by the data shown in Fig. 5B
demonstrating that the
5
2HF dimer also
selectively associates with the
q/11 subunits in a
mixture of native G proteins extracted from brain membranes.
Little is known about the biological role of the six different subunits in determining the specificity of cellular signaling. The
1-
4 subunits are widely expressed, each
contain 340 amino acids and are 80-90% identical in sequence (3). In
contrast, Northern analysis of various murine tissues shows the
5 subunit to be expressed predominantly in the brain
(20), but more recently
5 subunit expression has been
detected in rat portal vein (55). Expression of the similar
5L subunit which has a 42-amino acid amino-terminal
extension appears restricted to certain areas of the retina (21). These
two
subunits do appear to be localized to the membrane (21) and are
thus presumed to be involved in G protein-mediated signaling in sensory
and nervous tissue. The data in this report suggest that the
5 subunit (and possibly the
5L subunit)
participates in signaling via
q-linked receptors. Interestingly, treatment of rod outer segment membranes with GTP
S failed to release the
5L subunit (21). Because members
of the
q family are slow to exchange guanine nucleotides
and are more readily activated by AMF (52), this observation is
consistent with our finding of a specific interaction between the
5 and
q subunits.
The biological implications of the restricted tissue distribution and
the divergent sequences of the two 5 subunits are not fully understood. The
dimer has multiple roles in G
protein-mediated signaling. In addition to being required for the
subunit to couple to receptors (9, 10, 14), the dimer can regulate the
activity of multiple effectors including certain isoforms of PLC-
,
K+, and Ca2+ channels, phosphatidylinositol
3-kinase, adenylyl cyclase, the MAP kinase pathway and can help
translocate receptor kinases to the plasma membrane (4). The functional
roles of the two
5 subunits have not been fully
explored, but they have been demonstrated to form functional dimers
with the
2,
3,
4,
5, and
7 subunits (20, 21). Analysis of
the interaction of the
and
subunits using the yeast two-hybrid
technique also shows an interaction between the
5
subunit and multiple
subunits (56). Moreover, the
5
2 and
5L
2
dimers markedly increase inositol phosphate breakdown in COS-7 cells
transfected with the cDNAs for either
5 subunit, the
2 subunit, and PLC-
2 (20-22). Although
the
5
2 dimer can activate
PLC-
2 in transfected COS cells, it does not activate the
MAP kinase or JNK kinase pathways in these cells (22). In contrast,
transfection of the
1
2 dimer is able to activate both PLC-
and the kinases (22, 57, 58). Our preliminary experiments show that the
5
2HF dimer is
not able to activate type II adenylyl cyclase. Thus, the
5 subunit (and possibly the
5L subunit)
may not interact with certain important effectors.
The data described above combined with the data in this report suggest
a number of possibilities for the biological role of the
5 subunits in signaling. First, heterotrimers containing the
5 subunit are most likely to couple to the
q subunit, and thus only
q-linked
receptors may generate a
5
dimer to regulate effectors. The ability of other members of the Gq family to
couple to the
5 subunit needs to be explored. Second,
dimers containing the
5 subunit may only be
capable of interacting with a subset of the effectors regulated by
other
dimers. In the retina, the
q-linked
pathways have been assumed to play a minor role in visual signal
transduction (59), but recent studies of mouse retina using
immunological techniques have demonstrated the presence of the
11 subunit and PLC-
4 (60). Thus, a
function for this signaling pathway may emerge. A wide variety of
q-linked receptors exist in neural tissue (61). One
interesting pathway regulated by m1 or bradykinin receptors via the
q subunit involves inhibition of M-type potassium
currents (62, 63). The known ability of the
dimer to regulate
K+ and Ca2+ channels via multiple mechanisms
(4, 61, 64) suggests interesting potential roles for dimers containing
the
5 subunit in the regulation of ion channel activity.
As multiple G protein-mediated signals are often integrated by a single
neuron (61, 64), selective inputs by different
dimers may allow
distinct cellular responses. The observation that the
5
2 dimer does not appear to activate the
MAP kinase pathway (22) reinforces this possibility and indicates that
dimers containing the
5 subunit may regulate a limited
range of effectors. Thus, there may be an advantage to a more
restricted
signal in retina and neurons. Since recombinant
dimers of defined composition have not been tested against all
the known effectors regulated in this manner, it will be important to
determine which effectors are regulated by dimers containing the
5 subunit. This information may help explain the
restricted tissue distribution of these proteins.
In summary, the data in this report provide partial understanding for
the large diversity of the proteins comprising the G protein
heterotrimer. The finding that the 5 subunit interacts selectively with the
q subunit suggests that it will be
important to examine this issue in a number of signaling systems using
recombinant proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. Joel M. Linden and Anna Robeva
for the pDoubleTrouble (pDT) expression vector, Dr. Melvin I. Simon for
the cDNA for the 5 subunit, Dr. Ravi Iyengar for the
baculovirus encoding type II adenylyl cyclase, and Dr. T. K. Harden for the baculovirus encoding the
11 subunit. We
also acknowledge Kate Kownacki for technical assistance, the University
of Virginia Biomolecular Research Facility for DNA sequencing, and the
Diabetes Core Facility for cAMP assays.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants PO1-CA-40042 and RO1-DK-19952.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Box 448 Health
Sciences Center, University of Virginia, Charlottesville, VA 22908. Tel.: 804-924-5618; Fax: 804-982-3878; E-mail:
jcg8w{at}virginia.edu.
1
The abbreviations used are: G proteins, guanine
nucleotide-binding regulatory proteins; Sf9 cells,
Spondoptera frugiperda cells (ATCC number CRL 1711); DTT,
dithiothreitol; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; C12E10, polyoxyethylene 10 lauryl ether;
Genapol C-100, polyoxyethylene, 10 dodecyl ether; ECL®,
enhanced chemiluminescence; TLCK,
N--p-tosyl-L-lysine chloromethyl ketone; FLAG antibody, anti-FLAG® M2 antibody; GTP
S,
guanosine 5
-3-O-(thio)triphosphate; GDP-AMF, a mixture of
GDP, MgCl2, NaF, and AlCl3, at the indicated
concentrations; MAP kinase, mitogen-activated protein kinase; PLC,
phospholipase C.
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REFERENCES |
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