From the Department of Pharmacology, Health Sciences Center, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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Although the G protein dimer is an
important mediator in cell signaling, the mechanisms regulating its
activity have not been widely investigated. The
12
subunit is a known substrate for protein kinase C, suggesting
phosphorylation as a potential regulatory mechanism. Therefore,
recombinant
1
12 dimers were overexpressed
using the baculovirus/Sf9 insect cell system, purified, and
phosphorylated stoichiometrically with protein kinase C
. Their
ability to support coupling of the Gi1
subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or
phospholipase C-
was examined. Phosphorylation of the
1
12 dimer increased its potency in the
receptor coupling assay from 6.4 to 1 nM, changed the
Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal
efficacy by 50%. In contrast, phosphorylation of the dimer had no
effect on its ability to activate phospholipase C-
. The native
1
10 dimer, which has 4 similar amino
acids in the phosphorylation site at the N terminus, was not
phosphorylated by protein kinase C
. Creation of a phosphorylation
site in the N terminus of the protein (Gly4
Lys)
resulted in a
1
10G4K dimer which could be
phosphorylated. The activities of this
dimer were similar to
those of the phosphorylated
1
12 dimer.
Thus, phosphorylation of the
1
12 dimer on
the
subunit with protein kinase C
regulates its activity in an
effector-specific fashion. Because the
12 subunit is
widely expressed, phosphorylation may be an important mechanism for
integration of the multiple signals generated by receptor
activation.
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INTRODUCTION |
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Most cells possess multiple signaling pathways to receive signals
from the hormones, autacoids, neurotransmitters, and growth factors in
their environment. One of the best characterized signal transduction
systems is used by receptors coupled to the heterotrimeric G
proteins1 (1-5). Receptors
activate this system by stimulating the release of bound GDP from the G
protein subunit leading to exchange of GDP for GTP in the
protein's nucleotide binding site. Binding of GTP induces a
conformational change in the
subunit, simultaneously activating the
protein and markedly decreasing its affinity for the
dimer (1).
Both the GTP-bound form of the
subunit and the released
subunit are capable of activating multiple effectors to generate
intracellular messages (3, 4, 6, 7). The mechanisms that regulate the
lifetime of the active, GTP-bound form of the
subunit have been
studied extensively. All
subunits have an intrinsic GTPase
activity, which hydrolyzes bound GTP to GDP (1, 3, 4), returning the
molecule to its basal state and increasing its affinity for GDP and the
subunit (1, 3, 4, 8). Both changes induce formation of the
stable, heterotrimeric form of the G protein. Interestingly, the GTPase
activity of many
subunits can be increased by a class of proteins
termed RGS molecules (9, 10) and by certain effectors such as PLC-
(11).
Although the activity of the subunit is regulated by multiple
mechanisms, regulation of the activity of the
dimer is not well
characterized. Recently, the
12 subunit has been shown to be a substrate for protein kinase C (12, 13), suggesting that dimers
containing this
subunit may be subject to regulation by
phosphorylation. The
12 subunit is widely expressed
(12-15) and, given the extensive role of the
subunit in cell
signaling (6, 16), its phosphorylation may have important consequences. To examine the effects of
12 subunit phosphorylation on
its activity, we purified recombinant
1
12
dimers from baculovirus-infected Sf9 insect cells,
phosphorylated them with PKC
and
1, and tested their
activity in three assays of
function. We examined the ability of
phosphorylated dimers to support coupling of the Gi1
subunit to the A1 adenosine receptor and to activate two effectors, type II adenylyl cyclase or phospholipase C-
. Phosphorylation of the
1
12 subunit had no effect on its ability
to activate PLC-
, but increased its potency in the receptor coupling
assay and markedly inhibited its ability to activate adenylyl cyclase. These results suggest that phosphorylation reduces the ability of the
signal to increase cyclic AMP levels and favors activation of
other effectors.
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EXPERIMENTAL PROCEDURES |
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Construction of Recombinant Baculoviruses for the and
Subunits--
Full-length clones encoding the human
10
and
12 proteins were identified in the EST data base and
obtained from Research Genetics, Inc (
12,
GenBankTM N42722;
10, GenBankTM
U31383). To minimize the length of the construct 5' from the ATG start
codon, the end of the
12 cDNA was modified using the polymerase chain reaction (PCR). For the
12 cDNA,
the primers used were: (sense primer: 5'-CCCGGGATGTCCAGCAAAACAGCA-3';
antisense primer: 5'-ATAGAGACTGCAGAGTCCAT-3'). The PCR products were
subcloned into the pCNTR shuttle vector, the
12 coding
sequence excised from pCNTR with SmaI and XbaI,
and ligated into these sites in the baculovirus transfer vector,
pVL1393. The native
10 cDNA was excised from the
pT7T3D plasmid with EcoRI, further digested with
BanII and Asp700 and subcloned into the pCNTR
shuttle vector. The
10 coding sequence was excised from
pCNTR with BamHI and XbaI and ligated into these
sites in the pVL1393 transfer vector. The N terminus of the
10 protein was modified to have a protein kinase C
phosphorylation site by mutagenesis of the
10 cDNA
using PCR. The primers used were: (sense primer:
5'-GGATCCATGTCCTCCAAGGCTAGC-3'; antisense primer:
5'-CACTTTGTGCTTGAAGGAATTCC-3'). This modification introduced a
Gly4
Lys mutation (G4K) into the protein. The products
of the PCR reaction were subcloned into pCNTR, digested with
BamHI and EcoRI, and ligated into these sites in
the pVL1393 transfer vector. To add the hexahistidine-FLAG affinity
tags to the 5' end of the
1 subunit, the polymerase
chain reaction was used to add XbaI and BamHI
restriction sites to the 5' and 3' ends of the
1 coding region, respectively. The primers used were: (sense primer:
5'-TCTAGAATGAGTGAGCTTGACCAGTT-3'; antisense primer:
5'-GGATCCTTAGTTCCAGATCTTGAGGA-3'). The products of the reaction
were digested with XbaI and BamHI and ligated into the pDouble Trouble (pDT) vector, which adds the nucleotide sequences for the hexahistidine and FLAG affinity tags to the 5' end of
the
1 coding region (17). The
1HF coding
region was excised from pDT with HindIII and
BamHI and subcloned into the pCNTR shuttle vector. The
1HF coding sequence was excised from pCNTR with
BamHI and ligated into the BamHI site of pVL1393. The pVL1393 transfer vectors containing these four constructs were
sequenced to verify the fidelity of the
and
sequences. Recombinant baculoviruses were constructed by co-transfecting each
transfer vector with linearized BaculoGold® viral DNA into Sf9
cells using the PharMingen BaculoGold® kit as described (18). The
recombinant baculoviruses were purified by one round of plaque purification using standard techniques (19). The construction of the
recombinant baculoviruses coding for the Gi1 and
Gs
subunits and the A1 adenosine receptor have been
described (20, 21).
Expression and Purification of Recombinant G Protein and
Subunits--
G protein
and
subunits were
overexpressed by infecting suspension cultures of Sf9 insect
cells with recombinant baculoviruses (22, 23). The Gi1
subunit was purified to homogeneity as described (22). The recombinant
dimers were extracted from Sf9 cells and purified using
DEAE chromatography and an
subunit affinity column (23).
Phosphorylation of Purified Subunits by PKC--
The
purified
1HF
12 subunit was incubated for
30 min at 30 °C in 50 mM Tris, pH 7.5, 1 mM
-mercaptoethanol, 10 mM MgCl2, 0.4 mM CaCl2, 40-100 µM ATP,
recombinant PKC
or
, 40 µg/ml phosphatidylserine, and 0.8 µg/ml diolein. Usually, 25 µg of
dimer was incubated with
0.74 unit of PKC
to achieve stoichiometric phosphorylation of the
subunit. Control reactions contained deionized water in the place
of PKC. The stoichiometry of the phosphorylation reaction was measured
by including 40 µM [32P]ATP (500-2000
cpm/pmol) in the reaction mix and subjecting the phosphorylated
subunits to Tricine/SDS-PAGE (24). The resolved
subunit was cut
from the dried gel and the amount of radioactive phosphate incorporated
estimated by scintillation counting. After the phosphorylation reaction
and before use in the assays, the
subunit was repurified from
the reaction mixture by loading it onto a 0.25 ml
Ni2+-NTA-agarose column (Qiagen) and washing with 15 ml of
20 mM Hepes, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 1 mM
-mercaptoethanol,
0.6% (w/v) CHAPS, and 5 mM imidazole to remove PKC. The
dimer was eluted with 1 ml of the wash buffer containing 200 mM imidazole. To ensure that no PKC activity was carried
into the assays with the
dimer, its activity was monitored in
the elution fractions using the kinase reaction buffer described above
with 25 µg/ml histone 3S as substrate (25). As expected, the kinase
activity eluted in the void volume of the column and not with the
dimer. Controls were also performed to determine whether the
30 °C incubation with PKC and subsequent re-purification reduced the
activity of the
dimer in the functional assays. In these
experiments, a mock-phosphorylation reaction was performed in the
absence of PKC, the
dimer re-purified and its activity compared
with that of dimers subjected only to the
-agarose column (see
"Results").
Measurement of the High Affinity Ligand Binding Conformation of
the A1 Adenosine Receptor--
Sf9 insect cell membranes
overexpressing recombinant A1 adenosine receptors were prepared as
described (21) and reconstituted with G protein and
subunits
on ice for 30 min (26). The high affinity, agonist binding conformation
of the receptor was measured using the agonist ligand
125I-N6-(aminobenzyl)adenosine as
described (26). Each reaction tube contained 20 fmol of receptor, 6 nM Gi1
subunit, 0-10 nM
dimer, 50 nM GDP, and 0.3 nM
125I-N6-(aminobenzyl)adenosine.
Because this assay was incubated for 3 h before filtration, 100 nM microcystin was included to inhibit protein phosphatases
in the Sf9 cell membrane preparation (27).
Measurement of Phospholipase C- Activity--
Large
unilamellar phospholipid vesicles were prepared by extrusion into a
buffer containing 50 mM Hepes, pH 8.0, 3 mM
EGTA, 80 mM KCl, and 1 mM dithiothreitol with a
Avanti Polar Lipids mini-extruder (28). The phospholipid vesicles
contained a 4:1 molar ratio of phosphatidylethanolamine and
phosphatidylinositol 4,5-bisphosphate at final concentrations of 100 and 25 µM, respectively, and about 7000 cpm/assay of
[inositol-2-3H]phosphatidylinositol
4,5-bisphosphate. Phospholipid vesicles and
subunits were mixed
on ice in an assay buffer containing 50 mM Hepes, pH 8.0, 0.17 mM EDTA, 3 mM EGTA, 17 mM
NaCl, 67 mM KCl, 0.83 mM MgCl2, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin. The
final concentration of CHAPS contributed by the
preparations in
each assay tube was kept below 0.01% (w/v) to eliminate effects of
detergent on PLC-
activity (29). The reaction was begun by addition
of 10 ng of recombinant, turkey erythrocyte PLC-
and 3 µM free Ca2+ to each assay tube. The mixture
was incubated for 15 min at 30 °C and stopped by the addition of
ice-cold 10% trichloroacetic acid followed by the addition of 10 mg/ml
bovine serum albumin. Assay tubes were centrifuged at 4,000 × g and the [3H]inositol 1,4,5-trisphosphate
released measured by liquid scintillation counting (30).
Measurement of Adenylyl Cyclase Activity--
Sf9 insect
cell membranes overexpressing recombinant, rat type II adenylyl cyclase
(31) were prepared as described (18). The Gs subunit
was extracted from an Sf9 cell preparation with 0.1% (w/v)
CHAPS as described (18). Cyclase containing membranes (5 µg of
protein/assay tube) were reconstituted with GTP
S-activated Gs
subunit (32) and varying concentrations of
dimer on ice for 30 min. The reaction buffer (25 mM Hepes,
pH 8.0, 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) was preincubated at 30 °C for 20 min.
Production of cyclic AMP was initiated by addition of the reconstituted
membranes to the reaction buffer and the incubation continued for 10 min at 30 °C. Reactions were stopped by the addition of 0.1 N HCl and cyclic AMP measured using an automated
radioimmunoassay (33).
Electrophoresis-- Tricine/SDS-polyacrylamide gels were run according to the procedure of Schagger and von Jagow (24). The separating gel contained 16.5% total acrylamide, 0.4% bisacrylamide, and 10% (v/v) glycerol. The stacking gel contained 4% total acrylamide and 0.1% bisacrylamide. Gels were run at constant voltage (~100 volts) at 10 °C for 4-5 h. Resolved proteins were stained with silver by the method of Morrissey (34), with the modification that the dithiothreitol incubation was reduced to 15 min.
Calculations and Expression of Results-- Experiments presented under "Results" are representative of three or more similar experiments. Data expressed as dose-response curves were fit to rectangular hyperbolas using the fitting routines in the GraphPad Prizm® software. Statistical differences between the curves were determined using all the individual data points from multiple experiments to calculate the F statistic as described (35).
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 (20, 23). The baculovirus
transfer vector, pVL1393, was purchased from Invitrogen; the
BaculoGold® kit from PharMingen; 10% GENAPOL® C-100, CHAPS,
microcystin, and the
and
1 isoforms of PKC from
Calbiochem; Ni2+-NTA-agarose from Qiagen;
[3H]phosphatidylinositol bisphosphate from NEN Life
Science Products; PMA from Sigma; the pCNTR shuttle vector from 5 Prime
3 Prime, Inc. (Boulder, CO). All other reagents were of the highest
purity available.
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RESULTS |
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Stoichiometry of Phosphorylation of the 12 and the
10G4K Subunits--
Recent experiments have
demonstrated that the bovine
12 subunit is a substrate
for protein kinase C (12), but the functional significance of this
phosphorylation event has not been extensively studied. As this newly
discovered
12 subunit is widely expressed (12-15), its
phosphorylation may have important consequences. To examine the effects
of
12 subunit phosphorylation on its activity, we
purified recombinant
1
12 dimers from
Sf9 insect cells, phosphorylated them with PKC
and
, and
tested their activity in assays of
function. The human
12 subunit was rapidly phosphorylated by PKC
to a
stoichiometry of about 1 mol/mol as shown in Fig. 1, A and B. Protein
kinase C
1 was less effective than PKC
; the
stoichiometry only reached 0.5 mol/mol after 60 min of incubation. Addition of a hexahistidine-FLAG affinity tag to the
1
subunit (
1HF) facilitated removal of PKC from the
reaction mixture prior to assessing
function. Thus, the
phosphorylated
1HF
12 dimer in the
reaction mix can be applied to a Ni2+-NTA-agarose column
and pure
dimer eluted with imidazole. The left side
of Fig. 1C shows that stained PKC protein was removed by
this procedure and assay of kinase activity in the column elution fractions determined that the
dimer was free of residual kinase activity (see "Experimental Procedures"). The right side
of the figure shows that only the
12 subunit in the
dimer is phosphorylated. Addition of the hexahistidine-FLAG affinity
tag to the N terminus of the
1 subunit did not affect
the association of the
and
subunits or purification of the
dimer on an
-subunit affinity column (data not shown).
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Effect of Phosphorylation of the Subunit on Receptor
Coupling--
Having established the ability of protein kinase C to
phosphorylate the two recombinant
subunits, we examined the effects of phosphorylation on the function of the dimer. The
subunits play several important roles in the signaling mechanisms used by
receptors to activate effectors. In combination with the
subunit,
they participate in forming the high affinity agonist binding
conformation of the receptor (21, 38, 39); they stabilize the basal
state of the system by increasing the affinity of the
subunit for
GDP (7, 8); and when released from the
subunit, they activate
effectors such as type II adenylyl cyclase and phospholipase C-
(6,
7). We first examined the effect of
subunit phosphorylation on the
ability of the
dimer to support establishment of the high
affinity, agonist binding conformation of a G protein-coupled receptor.
In membranes from Sf9 cells overexpressing recombinant A1
adenosine receptors, about 90% of the receptors are in a low affinity
conformation. Reconstitution of pure Gi
and
subunits into these membranes establishes high affinity agonist binding
and provides a sensitive assay for receptor-
interactions
(21). Fig. 3 shows that the dimers used
in this study,
1
12,
1
10,
1
10G4K, and
1HF
12, were able to re-establish the high
affinity, agonist binding conformation of the receptor with a potency
and efficacy equal to the well studied and highly effective
1
2 dimer (26). All dimers tested support
coupling with a Kact of 0.5-1.0 nM
(see Fig. 3 legend). Thus, the newly discovered
10 and
12 subunits are able to couple very effectively to the
Gi1
subunit and the A1 adenosine receptor when
dimerized with the
1 subunit. Importantly, neither the
hexahistidine-FLAG tag added to the N terminus of the
1
subunit nor the G4K mutation made in the N terminus of the
10 subunit affect the ability of these recombinant
dimers to induce the high affinity conformation of the A1
adenosine receptor.
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Effect of Subunit Phosphorylation on Effector
Activity--
The data in Fig.
5A show the ability of the
native and modified
dimers used in this study to activate
PLC-
in an in vitro assay using
[3H]PIP2 incorporated into phospholipid
vesicles as substrate. Note that
1
12,
1
10,
1HF
10G4K, and
1HF
12 are equally as effective as the
1
2 dimer. All four forms of the protein
stimulated the release of [3H]inositol
1,4,5-trisphosphate with a Kact of 6-8
nM and were equally effective (~8-fold increase in
activity). The data in Fig. 5B demonstrate that there is no
difference in the ability of either the phosphorylated or
unphosphorylated
1HF
12 dimers to activate
PLC-
. The phosphorylated or unphosphorylated forms of the
1HF
10G4K dimers were also tested
and no differences were observed (data not shown). As can be seen by
comparison of the maximal activities shown in Fig. 5, A and
B, the phosphorylation protocol caused about a 40%
decrement in
activity in the PLC assay.
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DISCUSSION |
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Identification of the diversity in the family of G protein subunits has prompted studies to determine whether the differences in
these proteins translate to specificity in transmembrane signaling (42). In this regard, one major finding of this study is that, when
combined with the
1 subunit, the newly discovered
12 and
10 subunits are equal in potency
and efficacy to the well studied
1
2
dimer. Both the
1
12 and
1
10 dimers were fully effective in the
receptor coupling assay using the Gi1
subunit and the A1 adenosine receptor and able to maximally activate type II adenylyl cyclase and PLC-
. As these
subunits are widely expressed in brain and peripheral tissues (12, 14, 15, 37), they are likely to play
important roles in signaling by a large number of G protein coupled
receptors. A second important finding is that phosphorylation of the
1
12 dimer with protein kinase C has
distinct effects on the activity of the molecule, increasing its
potency in the receptor coupling assay and inhibiting its ability to
stimulate type II adenylyl cyclase. Previous studies have determined
that the phosphorylation site in
12 is Ser1
at the N terminus of the molecule (12, 13). This finding is consistent
with our observations that the phosphorylation site created in the
10G4K subunit makes it a substrate for protein kinase C
and that phosphorylation regulates its activity. Although not fully
explored, the finding that the dephosphorylation of
12
is blocked by microcystin suggests that the protein phosphatases that
dephosphorylate the protein in the intact cell are most likely protein
phosphatase 1 and/or 2A. Overall, the protein kinases and phosphatases
regulating the phosphorylation state of the
12 subunit
are those known to participate in responses to receptors generating
diacylglycerol and Ca2+ (27, 43-45), suggesting an
important role for this event in cell signaling.
The finding that phosphorylation of the subunit can reduce the
ability of the
dimer to stimulate one effector without changing
its activity on other effectors adds complexity to the regulation of
this signal. Previously, the known mechanisms for regulating
activity only involved sequestration by either G protein
subunits
or phosducin (3, 6, 7). Because the GDP bound form of the
subunit
has a higher affinity for the
subunit, an important mechanism
for regulating the activity of the
subunit is the return of the
active, GTP-bound
subunit to its basal state (1, 3, 4, 7). Indeed,
overexpression of
subunits is an effective means of decreasing the
activity of
subunits in cultured cells (46, 47). Phosducin also has a nanomolar affinity for the
subunit (48) and, whereas its
role in cell function is still emerging, it appears to inhibit the
dimer by sequestration of the protein following dissociation of
the
:
heterotrimer (49). Because the
dimer is needed for coupling the
subunit to receptors (21, 38, 39), the continued
activation of
subunits is decreased. Accordingly, overexpression of
phosducin in cultured cells can inhibit the ability of released
to activate PLC-
or type II adenylyl cyclase (50). Interestingly,
phosphorylation of Ser73 in phosducin via the cyclic
AMP-dependent protein kinase decreases its affinity for the
dimer (49, 51), and treatment of cells overexpressing phosducin
with dibutyryl cyclic AMP can relieve its inhibitory effects (50). In
this context, the finding that phosphorylation of the
12
subunit with protein kinase C can inhibit the activity of the
dimer toward certain effectors offers new paradigms for understanding
the regulation of this important signal.
The observation that phosphorylation of the 12 subunit
on Ser1 inhibits the ability of the dimer to stimulate type
II adenylyl cyclase suggests that the N-terminal region of the
subunit is important for interaction with this effector. In support of
this concept, pilot experiments demonstrated that a peptide mimicking the N-terminal 21 amino acids of the
2 protein can
inhibit the ability of
1
2 to stimulate
cyclase.2 Thus, the
negatively charged phosphate group at the N terminus of the protein may
inhibit the interaction of the dimer with the cyclase molecule. Two
other functional domains of the
subunit have been intensively
studied using both biochemical assays and site directed mutagenesis.
The C-terminal 15 amino acids and the prenyl group are important for
interaction with the plasma membrane, the
subunit, and the receptor
(4, 5, 26, 52, 53), and the central region of the molecule is important
for specific interaction with the
subunits (54, 55). The importance
of these interaction sites is clearly supported by the x-ray structures of the
heterotrimer, which indicate a stretch of 15 amino acids beginning at Arg30 of
1 that interact
with the
subunit and likely contacts between the C-terminal domain
of the
subunit and the membrane-
subunit interface (56, 57).
Overall, these findings are interesting because they indicate that
three different domains of this small protein are used for interactions
with other proteins. Comparison of the N-terminal sequences of the 11
subunits through the first helical region (Val26 in
1 (57)) shows that amino acid identity varies from 15 to 85%. Thus, the diversity of this domain may be important for
specificity in effector signaling.
The domains in the PLC- molecule that interact with the
dimer
have been examined using a number methods. Multiple lines of evidence
indicate that the
dimer binds near the Y domain in the catalytic
core of PLC-
(58). Refinement of this location using overexpression
of glutathione S-transferase fusion proteins containing
small regions of the molecule suggests that the amino acids between
Leu580 and Val641 are involved in binding the
dimer (59). Experiments using small peptides indicate a
potential
binding domain in the 10 amino acids between
Glu574 and Lys583 (60). The domains in the
subunit that interact with effectors appear to be similar to those
responsible for binding the
subunit (7). However, the domains in
the
subunits responsible for interaction with PLC-
have not been
clearly identified. The finding that the ability of the
subunit
to stimulate PLC-
is not affected by phosphorylation suggests that
the central or C-terminal regions in the protein are more likely to
interact with PLC-
than the N-terminal domain. Alternatively, the
negative charge introduced by phosphorylation of the protein may not
inhibit binding of the dimer to PLC-
.
It is especially interesting that phosphorylation of the
12 subunit increases the affinity of the
receptor-
interaction, since the structure of the heterotrimer
shows no interaction between the N terminus of the
subunit and the
subunit (56, 57). In addition, the N-terminal region of the
subunit is predicted to be some distance from the receptor and the
membrane (57). A similar situation occurs with phosducin, where
phosphorylation changes its affinity for the
dimer (49, 51), yet
the phosphorylation site is not directly in contact with either the
or
subunit (61). One possible explanation of this result is that
phosphorylation may cause an indirect effect on receptor-heterotrimer
interactions by an induced conformational change in the
dimer.
However, a definitive answer should emerge from direct structural
analysis of the receptor-
interaction.
The finding that phosphorylation of 1
12
markedly decreases its ability to stimulate type II adenylyl cyclase
and focuses the
signal toward other effectors is likely to be a
broadly important regulatory mechanism. The
12 subunit
is widely expressed and has been demonstrated to be phosphorylated by
PKC in intact cells following receptor activation (12, 13). The type II adenylyl cyclase is expressed at high levels in the brain and the type
IV adenylyl cyclase, with nearly identical regulatory properties, is
widely expressed in peripheral tissues such as lung, heart, kidney, and
liver (62). Thus, the phosphorylation of the
12 subunit
has the potential to affect the interplay of the Ca2+ and
cyclic AMP signaling networks in most cells. As one example, in
vascular smooth muscle cells where phosphorylation of the
12 subunit occurs following application of the
contractile agonists vasopressin or angiotensin II (12), this mechanism
may augment the ability of Ca2+ to cause contraction by
blunting a rise in cyclic AMP, which relaxes smooth muscle (63). A
similar mechanism could be used in neural networks to amplify the
effects of a Ca2+ signal and blunt those of cyclic AMP.
The fact that only the 12 subunit has a protein kinase C
phosphorylation site in its N terminus is intriguing. Whether other
subunits can be phosphorylated is an important issue. However, the
12 subunit is highly expressed in all regions of the
brain (14) and in most peripheral tissues (12). Thus,
12
may be the
subunit in the
dimers used in many important
signaling systems. Our findings that the
1
12 dimer is equal in activity to the
better studied
1
2 dimer support this
conclusion. However, the
12 subunit may also be used by
cells for undiscovered or specialized signaling roles where
phosphorylation is critical to its activity. In this regard, in Swiss
3T3 and C6 cells, dimers containing
12 appear to be
localized to actin stress fibers, whereas those containing the
5 subunit are found in focal adhesions. Moreover, the
12 dimers appear to bind much more tightly to purified actin filaments than do dimers containing the
5
subunit (15). These findings suggest that the cytoskeleton may be an important site for
12 function and may lead to discovery
of new roles for the
dimer in cell signaling.
The observation that phosphorylation can change the effect of the
dimer on certain effectors may have consequences for many
signaling systems not studied in this report. The
dimer is
emerging as an important regulatory signal for a large number of
effectors including: K+ and Ca2+ channels (64),
the
adrenergic receptor kinase (65), phosphatidylinositol 3-kinase
(66-68), mitogen-activated protein kinase (16), and novel kinases such
as p21-activated protein kinase (69). It will be important to determine
whether phosphorylation alters the activity of the
1
12 dimer toward any of these signaling molecules.
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ACKNOWLEDGEMENTS |
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We thank Dr. Joel M. Linden for the
125I-N6-(aminobenzyl)adenosine, the
pDT vector, and help with statistical analysis; Dr. Ravi Iyengar for
the baculovirus encoding type II adenylyl cyclase; and Dr. T. K. Harden for the turkey phospholipase C-. We also acknowledge Rimma
Khazan for technical assistance, the University of Virginia
Biomolecular Research Facility for DNA sequencing, and the Diabetes
Core Facility for [32PO4]ATP and the 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.
Supported by a fellowship from the Virginia Affiliate of the
American Heart Association. Present address: Dept. of Medicine, University of Tokyo, Tokyo 112-8688, Japan.
§ 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.
The abbreviations used are:
G proteins, guanine
nucleotide-binding regulatory proteins; Sf9 cells, Spondoptera frugiperda cells (ATCC number CRL 1711)CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonateGENAPOL®
C-100, polyoxylethylene(10)dodecyl etherPKC, protein kinase CPMA, phorbol 12-myristate 13-acetatePCR, polymerase chain reactionPLC, phospholipase CPAGE, polyacrylamide gel electrophoresisTricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineNTA, nitrilotriacetic acidGTPS, guanosine
5'-O-(thiotriphosphate).
2 H. Y. and J. C. G., unpublished experiments.
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