J Biol Chem, Vol. 273, Issue 26, 16265-16272, June 26, 1998
Sites for G
Binding on the G Protein 
Subunit Overlap with
Sites for Regulation of Phospholipase C and Adenylyl Cyclase*
Ying
Li
,
Pamela M.
Sternweis§,
Sara
Charnecki,
Temple F.
Smith¶,
Alfred G.
Gilman§,
Eva J.
Neer
, and
Tohru
Kozasa§
From the Department of Medicine, Harvard Medical
School and Brigham and Women's Hospital, Boston, Massachusetts 02115, the ¶ Biomolecular Engineering Research Center, Boston University,
Boston, Massachusetts 02111, and the § Department of
Pharmacology, University of Texas, Southwestern Medical Center, Dallas,
Texas 75235
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ABSTRACT |
Heterotrimeric G proteins, composed
of 
and 
subunits, forward signals from transmembrane
receptors to intracellular effector enzymes and ion channels. Free
activates downstream targets, but its action is terminated by
association with GDP-liganded subunits. Because can inhibit
activation of many effectors by , it is likely that the subunit binding surfaces on overlap the surfaces necessary for
effector activation. To test this hypothesis, we mutated residues on
shown to contact in the recently published crystal structures
of the heterotrimer (Wall, M. A., Coleman, D. E.,
Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm,
H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The subunit binds to the flat, top surface of the
toroidal subunit and also extends a helix along the side of the subunit at blade 1. We mutated four residues on the top surface of (H1[L117A], H1[D228R], H1[D246S], and H1[W332A]) and two
residues on the side of that contacts (H1[N88A/K89A]). Each of the mutant proteins was able
to form dimers, but they differed in their ability to bind and to activate phospholipase C 2 (PLC2),
PLC3, and adenylyl cyclase II. Mutation of residues
along the side of the torus at blade 1 diminish affinity for but do
not prevent activation of any of the effectors. Mutations on the binding surface differentially affected PLC2,
PLC3, and adenylyl cyclase II. Residues that affect
PLC and adenylyl cyclase II activity are found on opposite sides of
the central tunnel, suggesting that PLC and adenylyl cyclase, like the
subunit, make many contacts on the top surface. None of the
mutations affected the ability of to inhibit adenylyl cyclase I. We conclude that , PLC2, PLC3, and
adenylyl cyclase II share an interaction on the top surface of . The
importance of individual residues is different for binding and for
effector activation and differs even between closely related isoforms
of the same effector.
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INTRODUCTION |
Heterotrimeric G proteins composed of and subunits
forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Activation of the G protein through the
receptor causes dissociation of from . Each of the subunits is then able to regulate downstream targets. All known effectors are
regulated only by the dissociated or subunits and not by the
heterotrimer. When GTP bound to is cleaved to GDP, the
subunits reassociate. Reassociation with is not obligatory for
deactivation of because the conformational change in that accompanies hydrolysis of GTP to GDP contributes to the termination of
signaling (for a recent review, see Ref. 1). There are at least two
potential explanations for the universal finding that formation of the
heterotrimer turns off any signal transmitted through .
First, there may be a major conformational change in induced by
binding to . Second, all effectors may share a part of the binding site on , so that the inhibition of effector activation
by would be primarily steric. The recent publication of the crystal
structure of the heterotrimer (2, 3) and of the isolated
subunit (4) suggests that the latter explanation is correct. The
structure seems to be quite rigid, and no major conformational
differences were seen between the subunit in a
heterotrimer versus the free subunit. These observations
suggest that a place to look for effector contact sites is on a surface
of that interacts with .
The subunit consists of a symmetrical seven-bladed propeller
structure with four kinds of surfaces: the flat surface at the narrow
end that we call the "top," the flat surface at the wide end (the
"bottom"), the outer surface of the torus, and the surface that
lines the tunnel through the middle of the molecule (Fig. 1). The subunit contacts in two of these regions. The first /
interface is between the amino-terminal helix of and the first
blade of the propeller. The amino terminus of has long been
known to be important for the formation of heterotrimers (5-7), and
the crystal structure beautifully reveals why this is so. The major
points of contact along this interface include residue
Lys89 that contacts residues Leu15 and
Leu19 on (2, 3). The second interface between and
is made up of residues on the top surface of the torus.
These residues that contact are located in turns between the short
strands that make up the blades of the propeller.
In order to test the hypothesis that residues in the subunit that
are important for interaction with may overlap with residues
important for activation of effectors, we have analyzed the
consequences of mutating residues on the surface of that interacts
with the switch II region of and residues on the sides of blade 1 of that contact the amino terminus of the subunit. The
positions of the mutated residues are shown in Fig. 1. We compared the
ability of the mutants to activate two isoforms of phospholipase C
and to regulate two isoforms of adenylyl cyclase. The function of
subunits containing these mutations were analyzed in three
different expression systems: in vitro translation,
transient expression in COS-7 cells, and in vitro
reconstitution with proteins purified from baculovirus-infected
Sf9 cells. None of the mutations interfered with the ability of
subunits to form dimers. As expected, some, but not all,
mutations affected the ability of mutant dimers to interact with
. Most importantly, the results show that the contact surface on
the flat, narrow end of the propeller is important for effector
activation. Moreover, the mutations did not have equal consequences for
the effectors tested, nor even between closely related subtypes of the
same effector.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Transfection was done with LipofectAMINE according to the
manufacturer's protocol using 6-7 µg of LipofectAMINE/ml of culture
medium (Opti-MEM, Life Technologies, Inc.) for 5 h or overnight.
The medium containing LipofectAMINE was removed, and cells were washed
twice with Dulbecco's modified Eagle's medium/5% fetal bovine
serum.
Mutagenesis and Plasmid Construction--
Site-directed
mutagenesis was done in the pAlter vector (Promega) according to the
manufacturer's instructions. The cDNA of the 1
subunit was cloned into pAlter vector as described (8, 9). The
single-stranded 1 DNA was produced and used as a
template for mutagenesis. To facilitate the transfer of mutants among
different vectors, a silent mutation corresponding to amino acids 144 and 145 was introduced into 1 to create a unique
KpnI site. To construct a hexahistidine-tagged
1 subunit
(H1),1 the
initial methionine was mutated into glutamine, and at the same time, a
HindIII site and a PstI site were introduced. An annealed double-stranded DNA encoding the first methionine and six
histidines was synthesized and ligated between this new
HindIII site and the EcoRI site from the pAlter
vector. The amino acid sequence added to the amino-terminally
His-tagged 1 subunit is MSHHHHHHGSLLQ. For expression in
COS-7 cells, the EcoRV-KpnI fragment of
H1 was transferred to the pCDNA3 (Invitrogen)
1 vector using the blunted HindIII and the
KpnI sites. The H61 in pAlter was used as a
template for the creation of the mutated subunit N88A/K89A (AACAAG/GCCGCG); L117A (CTG/GCG); D228R (GAG/AGA); D246S (GAC/AGC); and
W322A (TGG/GCG). The mutation of H61 was confirmed by
double-stranded sequencing. The mutated part of the 1
subunit was transferred into pCDNA3 containing H61,
using the HindIII and KpnI sites, or
KpnI and BamHI as appropriate.
PLC3 (obtained from S. G. Rhee) was transferred to
the pCDNA3 vector between the EcoRV and XhoI sites. The EcoRV site was abolished. PLC2 was used in the
pMT2 vector as obtained from M. Simon. The construction of
HA2 (2 tagged at the amino terminus with
hemagglutinin epitope) was described in Ref. 10.
Transient Expression in COS-7 Cells and PLC Assay in COS-7
Cells--
COS-7 cells (1.5-3 × 105/well) in
six-well plates were transfected with 1.8 µg of DNA and 10 µg of
LipofectAMINE in Opti-MEM (Life Technologies, Inc.) per well. Forty to
48 h after transfection, cells were starved for 1 h in
methionine- and cysteine-deficient RPMI 1640 medium containing 5%
dialyzed fetal bovine serum for 30 min and then labeled with 100 µCi/ml Trans[35S]-Label (ICN) for 3 h. Labeled
cells were lysed at 4 °C in 0.7 ml of HMSDET buffer. All further
steps were at 4 °C. The lysates were incubated with 10-15 µl of
packed protein A-Sepharose for 20-30 min and centrifuged at 14,000 rpm
in a microcentrifuge for 10 min. The supernatants were incubated 1 h or overnight with 0.5-2 µl of 12CA5 monoclonal antibody (Babco)
against the hemagglutinin epitope on the 2 subunit and
then incubated with 40 µl of protein A-Sepharose (50% v/v) for 30 min and centrifuged for 30 s. The pellets were washed twice with
HMSDET and once with phosphate-buffered saline and then buffer for
ADP-ribosylation (100 mM Tris-HCl, pH 7.6, 2 mM
MgCl2, 1 mM EDTA, and 10 mM DTT).
ADP-ribosylation was carried out in a 40-µl volume containing 1 mM ATP, 1 mM NADP, 10 µM NAD, 100 µM GTP, 7.5 mM thymidine, 60 mM
Tris, pH 7.6, 1.2 mM MgCl2, 0.6 mM
EDTA, 6 mM DTT, 0.5 µCi [32P]NAD, and 0.25 µg of activated pertussis toxin. The reaction proceeded at 37 °C
for 30 min. The final products were analyzed by 11% SDS-PAGE. For
exposure of the 32P signal without a contribution from
35S, a piece of black film was placed between the gel and
the film to be exposed.
[3H]Inositol phosphate formation was measured by
modifications of the methods described in Refs. 11 and 12. COS-7 cells (0.5-1.5 × 105/well) in 12-well plates were
transfected with PLC2 (pMT2 vector) or
PLC3 (pcDNA3) and wild-type
1, histidine-tagged 1
(H1), or histidine-tagged 1 mutants. A
mixture of 0.8-0.9 µg of DNA and 3.5 µg of LipofectAMINE in 0.5 ml
of Opti-MEM (Life Technologies, Inc.) was added to the cells. The day
after transfection, cells were incubated with 2 µCi/ml
myo-[2-3H]inositol in inositol-deficient
Dulbecco's modified Eagle's medium with 4% fetal bovine serum.
Fifteen min later, LiCl2 (final concentration, 10 µM) was added to each well, and cells were incubated
overnight at 37 °C. The cells were extracted twice with 0.5 ml of 20 mM formic acid. The extracts were combined, neutralized to
pH 7.5 with 30 mM ammonium hydroxide, and loaded on 0.5 ml
AG1-X8 anion exchange columns. Prior to use, the columns were washed
with 2 ml of 1 M NaOH and 2 ml of 1 M formic
acid and equilibrated with water to neutrality. The columns were washed
with 10 bed volumes of water and 10 bed volumes of 5 mM
borax and 60 mM sodium formate. The inositol phosphates
were eluted with 10 bed volumes of 1 M ammonium formate and
0.1 M formic acid. A 2-ml aliquot of the eluates was
counted in a scintillation counter.
In Vitro Translation, Immunoprecipitation, and
Cross-linking--
All subunits were transcribed and translated using
the TNT-coupled reticulocyte lysate system (Promega). Typically, 1 µg
of plasmid DNA and 20 µCi of [35S]methionine were used
in a 50-µl reaction. In all cases, transcription was directed by the
T7 promoter. Synthesis of the desired product was routinely verified by
running 5 µl of the translation mixture in a small 11 or 13%
polyacrylamide gel (13), followed by autoradiography with overnight
exposure. Independently translated and subunits were mixed
together and incubated at 37 °C for 90 min to allow dimer formation.
Because translation was usually more efficient, 10-15 µl of translation mixture was typically added to 50 µl of translation
mixture. Fifty µl of the mixture was passed over an 8 ml AcA
34 column (Sepracor) equilibrated with HMSE plus 0.05% Lubrol PX at
4 °C in order to remove DTT and to separate the dimers from
undimerized . The fractions containing were concentrated
5-10-fold using a Centricon-30 concentrator (Amicon).
For cross-linking, 30 µl of this sample was mixed with 10 µl of
o (2-5 µg) purified from bovine brain (14) in HMSE or
10 µl of HMSE buffer alone, and the reaction was initiated by the addition of 1.6 µl of freshly prepared 50 mM bismaleimide
hexane (BMH) (Pierce) in Me2SO (8, 9). In control
un-cross-linked samples, 20 mM DTT was added prior to BMH.
After 40 min at 4 °C, DTT (20 mM) and/or Laemmli sample
buffer containing 15% -mercaptoethanol was added, and the samples
were boiled and resolved by SDS-PAGE on 9% polyacrylamide gels (13).
Dried gels were soaked in Enhance and then used for autoradiography.
The radioactive bands could be visualized after 2-7 days of exposure
at 
70 °C.
Sf9 Cell Culture and Construction of Recombinant
Baculovirus--
Sf9 cells were cultured in suspension in
IPL-41 medium containing 1% Pluronic F68, 10% heat-inactivated fetal
bovine serum, and 50 µg/ml gentamicin at 27 °C with constant
shaking (125 rpm).
To generate recombinant 1 mutant viruses, the mutated
1 cDNAs (K89A, L117A, D228R, D246S, and W332A) were
subcloned into pVL 1392 transfer vector, and the resulting plasmids
were cotransfected into Sf9 cells with BacPac6 viral DNA
linearized with Bsu361 (CLONTECH) using Lipofectin
(Life Technologies, Inc.). Two viruses, one with and one without
His6 tag, were generated for [L117A], [D228R], or
[W332A], and the one that gave higher protein expression was used for
subsequent studies. The 1[D228R] and
1[D246S] mutants were used with a hexahistidine
(His6) tag at the amino terminus; the other mutants were
used without a tag. Recombinant viruses were plaque-purified and
amplified as described (15). Recombinant baculoviruses encoding
1, 2, and
His-6-2 have been described previously (16,
17).
Purification of Mutant and Other Proteins from Sf9
Cells--
Sf9 cells (1 liter; 1.5 × 106
cells/ml) were coinfected with amplified recombinant baculoviruses
encoding 1 (K89A, L117A, and W332A) and
His6-2 or His6-1
(D246S and D228R) and 2. Cells were harvested after
48-66 h, and membranes were prepared as described (17). Sodium cholate
was added to a final concentration of 1%, and the mixture was stirred
on ice for 1 h, followed by centrifugation at 100,000 × g for 40 min. The supernatant was diluted 3-fold with Buffer
A (20 mM NaHEPES, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 10 mM
-mercaptoethanol, 0.5% polyoxethylene 10-lauryl ether) and loaded
onto a Ni-NTA (Qiagen) column (0.5 ml) that had been equilibrated with
Buffer A. The column was washed with 5 ml of Buffer A containing 400 mM NaCl and 10 mM imidazole and 5 ml of Buffer
B (20 mM NaHEPES, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 10 mM -mercaptoethanol, 0.3% octyl -D-glucopyranoside, 10 mM imidazole). Recombinant was eluted with 2 ml of
Buffer B containing 1% octyl -D-glucopyranoside and 150 mM imidazole. The eluate was concentrated and exchanged
into 20 mM NaHEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, and 1% octyl -D-glucopyranoside
with a Centricon-30 (Amicon). Because of low expression, the eluate
from the Ni-NTA column containing 1 (W332A) was further
purified over Mono Q 5/5 by fast protein liquid chromatography
(Amersham Pharmacia Biotech) in the presence of 1% octyl
-D-glucopyranoside with a gradient (0-400
mM) of NaCl. The peak fractions were combined and processed
as above. The capacity to support ADP-ribosylation of i1
by pertussis toxin was performed as described (18).
Recombinant q and wild-type
12 were purified from Sf9 cells as
described (17). Myristoylated i1 was purified from
Escherichia coli as described (19). PLC2 was
purified from Sf9 cells and kindly provided by Dr. Paul C. Sternweis (University of Texas Southwestern Medical Center). Protein
was measured as described in Ref. 20.
In Vitro Assays for Phospholipase C and Adenylyl Cyclase
Activity--
Phospholipase C activity was measured using sonicated
micelles containing 50 µM phosphatidylinositol
4,5-bisphosphate, 500 µM phosphatidylethanolamine, and
inositol-[2-3H] phosphatidylinositol 4,5-bisphosphate
(NEN Life Science Products) (2,500 cpm/assay) in a solution containing
50 mM NaHEPES, pH 7.5, 0.42 mM EDTA, 3 mM EGTA, 2 mM MgCl2, 1.7 mM CaCl2, 42 mM NaCl, 47 mM KCl, 4 µM GDP, 0.125 mg/ml bovine serum
albumin, 1 mM DTT, and 0.375% octyl
-D-glucopyranoside with 0.1 nM
PLC-2 and the indicated amount of . The mixture
(60 µl) was incubated at 30 °C for 8 min, and the amount of
IP3 generated was quantitated as described (21).
To measure adenylyl cyclase activity, purified mutants were
reconstituted with 10 µg of membranes from Sf9 cells
expressing type I or type II adenylyl cyclase for 3 min at 30 °C in
a final volume of 20 µl. Assays were then performed as described (22) for 7 min at 30 °C in a total volume of 50 µl containing 4 mM MgCl2 and 0.2% octyl
-D-glucopyranoside. The presence of the hexahistidine
tag at the amino terminus of either 1 or
2 did not affect any of the enzymatic assays (data not
shown).
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RESULTS |
Formation of Dimers by Mutant H1 and Their
Association with --
To evaluate the relationship between
surfaces of that bind and that activate PLC, we mutated four
residues on the flat, top surface of and two residues on the side
of blade 1 (see Fig. 1). The mutations
were introduced into the background of rat 1 tagged at
the amino terminus with six additional histidine residues. Addition of
the hexahistidine tag was extremely important for assays in COS-7 cells
because the size difference between H1 and wild-type
1 allowed us to discriminate transfected, mutated subunits from endogenous subunits. We could detect no differences in these assays between the hexahistidine 1
(H1) and wild-type 1 (see below). Before
we could assess the ability of mutated subunits to activate PLC, it
was essential to establish that they could form dimers. We expected
that some, but not all, mutations would also affect the ability of
to form heterotrimers with . To evaluate these two issues,
transfected COS-7 cells were labeled with
Trans[35S]-Label and H1 or the mutant
proteins were immunoprecipitated through cotransfected
HA2 (10). Because we immunoprecipitated through one
subunit (HA2) but measure the other, this assay measured only the amount of dimers that accumulate, and not the total synthesis of . When HA2 is transfected, it dimerizes
with both wild-type endogenous and transfected H1
subunits. Therefore, both types of subunits were immunoprecipitated
with the anti-HA2 antibody, but the two can be readily
distinguished by their mobility in SDS-PAGE (Fig.
2A).
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Fig. 1.
Structure of . Space-filling model
of seen from the surface of that interacts the . The subunit is shown in yellow, and the subunit is shown in
red. The residues mutated in these studies are indicated in
blue, and the residues cross-linked to by BMH are shown
in white. The figure was drawn from coordinates kindly
provided by Dr. Stephen Sprang, University of Texas Southwestern
Medical Center (Dallas, TX).
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Fig. 2.
Coimmunoprecipitation and ADP-ribosylation of
i2 with dimers of HA2 and
H1 mutants. A, COS-7 cells in 6-well plates
were transfected with a combination of 0.6 µg of i2,
0.6 µg of HA2, and either 0.6 µg of
H1, H1 mutants or 0.6 µg of
HA2, 0.6 µg of i2, and 0.6 µg of
vector DNA (for the left two lanes). 35S-Labeled
cell lysates were immunoprecipitated with the monoclonal antibody
directed against the HA epitope on 2. One sample of each
duplicate pair was [32P]ADP-ribosylated by pertussis
toxin after immunoprecipitation as described under "Experimental
Procedures." The top panel shows duplicate samples, the
first labeled with 35S, the next with 35S and
32P (in only). ADP-ribosylation slightly slows the
mobility of , so the 32P band is slightly above the
35S band. The bottom panel shows the
radioautogram of 32P only. The film was shielded from
35S during exposure. B, the amount of
i2 in the experiments of the type shown in Fig. 3 was
quantitated by densitometry. H1 was defined as 1.0, and
each H1 mutant was expressed as a fraction of H1 ± S.E. (n = 3). In each case, the amount of accounted
for by coprecipitation with endogenous has been subtracted (see
text). Open bars show data from experiments with
35S only, and hatched bars show data from
experiments in which 35S was shielded and only
32P exposed the film.
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We also used immunoprecipitation to measure the ability of mutant
H12 dimers to associate with transfected
i2. Cotransfection of i2 had no
reproducible effect on the amount of dimer formed. Because
antibody to HA2 precipitates both endogenous and
transfected , it was essential to be able to subtract the amount
of i2 coprecipitating with a dimer containing endogenous
and HA2 from the amount coprecipitating with a dimer
of transfected H1 mutants and HA2. The
amount of coprecipitated with endogenous was determined from
the lysates of cells transfected with HA2, i2, and vector but no additional . From the relative
density of the and the bands (taking into account the number of
methionine and cysteine residues), we calculated that 0.6-0.8 mol of
were precipitated per mol of endogenous
.2
To determine whether the i2 immunoprecipitating with
H1HA2 dimers was associating correctly,
we measured the ability of coimmunoprecipitated to be
[32P]ADP-ribosylated by pertussis toxin. Although only
the subunit is ADP-ribosylated, the substrate for the toxin is
the heterotrimer (14). Alternate lanes in the top
panel of Fig. 2 show 35S label only or 35S + 32P. The bottom panel shows 32P
only. These experiments are quantitated and summarized in Fig. 2B and Table I. The results
show that mutation H1[W332A] has little effect on the
ability of to interact with , but each of the other mutations
diminishes coimmunoprecipitation of through . For each
mutant, the results were the same whether we measured the amount of by 35S or by ADP-ribosylation. This correlation suggests
that there are no dramatic differences in the ability of mutant
to support ADP-ribosylation of once a complex has formed.
Another way to assess the interaction of mutant dimers with is chemically to cross-link mutant dimers to using the cysteine-specific reagent, BMH. We have previously shown that this
reagent specifically cross-links cysteine 215 of o
either to cysteine 204 or cysteine 271 of , giving two cross-linked products (8, 9). In the wild-type , the two cross-linked products
are formed approximately equally (Fig.
3). From the crystal structure, we know
that the distance between the residues on o and on is very close to that of the fully extended cross-linking reagent. The
ability of the reagent to reach to one or the other of the cysteines on
depends on a correct orientation of with respect to . The
formed from one of the mutants on the top surface of (H1[D246S]) gave the same two cross-linked products as
wild-type in the same ratio. In containing each of three other
mutants, the ratio of cross-linked products was different. The upper
band produced by cross-linking Cys215 to Cys204 was decreased in dimers containing
H1[W332A] and H1[L117A]. In
H1[D228R], the lower band (produced by cross-linking
Cys215 to Cys271) was missing. Mutating
the two residues that contact the amino-terminal helix of (H1[N88A/K89A]) greatly diminished the affinity of
for . Indeed, containing this mutation produced barely detectable cross-linked products, although both bands were faintly visible. The cross-linking reaction is an irreversible reaction and is
therefore able to reveal even low affinity interactions between the
subunits. We explain the altered cross-linking pattern of
H1[W332A], H1[L117A] and
H1[D228R] by suggesting that the subunit is still
able to interact with the mutated , but that it is tilted on its
binding site. It is unlikely the changes in the cross-linking pattern
are due to local effects of the mutations themselves, because no
mutated residue is adjacent to the cysteine whose cross-linking it
affects.
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Fig. 3.
Cross-linking of in vitro
synthesized 1 mutants to purified brain
o. In vitro translated wild-type 1
or H1 mutants were dimerized with 2-HA
and cross-linked in the presence of o as described under
"Experimental Procedures." Both treated (+BMH) and
untreated (BMH (20 mM DTT added before BMH))
samples were analyzed by 9% SDS-PAGE followed by autoradiography.
Cross-linked products were visualized after a 2-day exposure. The
positions of the molecular mass markers are indicated at the
left. Shown is a representative of three experiments for
each mutant.
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Activation of Phospholipase C Isoforms by 1
Mutants--
PLC2 and PLC3 are two
isoforms of PLC that are activated by the dimer (11, 23, 24). We
used two methods to compare the ability of mutant proteins to
interact with and to activate the two isoforms of PLC. First, we
cotransfected COS-7 cells with wild-type and mutant H1
subunits, HA2 and PLC2, or
PLC3 and measured the increase in inositol phosphate
production. Second, we synthesized the proteins in Sf9 cells,
purified them, and measured activation of PLC2 in
vitro. Transfection of into COS-7 cells did not
significantly affect basal PLC activity (probably because the
level is elevated only in the fraction of the cells that took up the
cDNA, whereas all cells contribute to the basal activity) (data not
shown). Transfection of PLC, together with and , caused a
3-fold increase in inositol phosphate production compared with
transfection of PLC alone (Fig. 4A). Neither nor alone increased the activity of transfected PLC (data not shown).
Addition of the hexahistidine tag had no effect on the activity of ,
and HA2 was as effective as 2. As was
previously shown by Katz et al. (11), we found that
dimers that contain a mutant that cannot be prenylated at the
carboxyl terminus do not activate PLC in the COS-7 cells (data not
shown). Finally, activation of PLC by was blocked by
cotransfection of i2 (see below). Taken together, these
controls, together with published in vivo and in
vitro studies (11, 25-29), support the interpretation that the
elevation of inositol phosphates that we measured reflects activation
of PLC by . This interpretation is further strengthened by
agreement of the data obtained in transfected cells with those obtained
with purified proteins.
As shown in Table I, mutation in residues on the side of the torus
(H1[N88A/K89A] had little effect on the ability of the
subunit to activate PLC2 or PLC3,
despite a profound effect on the affinity for i2 as
measured in solution. In contrast, three of the four mutations on the
top surface of markedly diminished the ability of the mutant
to activate phospholipase C2. The ability of
H1[L117A] to activate PLC2 was equal to
that of the wild-type. Another mutation in a known contact point
(H1[W332A]) had little effect on binding of to
but diminished stimulation of PLC2. Mutations
that affect the ability of the subunit to activate
PLC2 do not always have similar effects on the ability to activate PLC3. For example,
H1[D246S] activated PLC3 almost as well
as wild-type, but was blunted in its ability to activate PLC2. In contrast, H1[L117A] was fully
active with respect to PLC2 but inactive with respect to
PLC3. These results are consistent with a model in which
the interaction interfaces of with or with different
effectors overlap, but the importance of specific residues for each
function is different.
Cotransfection of i2 blocks PLC activation by
(Fig. 4A), even when the
has a diminished affinity for in solution (for example,
H1[L117A] or H1[D246S]). Of the
mutations we made, changes in residues on the side of the torus
(H1[N88A/K89A]) had the most profound effect on the
affinity for i2 in solution, as measured by
immunoprecipitation. Nevertheless, in cells, expression of
i2 blocked activation of PLC2 by
H1[N88A/K89A] with a dose-response curve similar to
its inhibition of wild-type (Fig. 4B). Analysis of
i2 expression by Western blot at each cDNA
concentration showed that the i2 levels rose
approximately equally in cotransfections with PLC2 and
wild-type or mutant (data not shown).
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Fig. 4.
Cotransfection of i2 inhibits
PLC activation by wild-type and mutant . A,
inhibition of PLC3 by coexpression of i2.
COS-7 cells in 12-well plates were transfected with the indicated
cDNAs in the following amounts: 0.2 µg of H1 or
mutant H1, 0.2 µg of 2, and 0.2 µg of
i2. In all cases, vector DNA was added to give a final
DNA concentration of 0.8 µg/well. PLC3 activation was measured as
described under "Experimental Procedures." The data shown are
representative of two experiments. The error bars indicate
the range of duplicate assays. Where there are no error bars, the range
was too small to display. Filled columns, without
i2; open columns, with i2.
B, cotransfection of i2 inhibits
PLC2 activation by H1 [N88A/K89A].
COS-7 cells were transfected and assayed as described under
"Experimental Procedures." The x axis represents the
logarithm of ng of i2 cDNA transfected, and the
y axis shows activation of PLC by H1 or
H1 mutants. Shown is a representative of three
experiments. Activation of PLC2 by
H12 or
H1[N88A/K89A]2 was taken as the 100%
value for each.  , H1[N88A/K89A]2;
 , H12.
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To confirm the results in COS-7 cells, recombinant mutant
proteins were synthesized in Sf9 cells and purified; SDS-PAGE analysis of these samples is shown in Fig.
5. All five 1 mutants were
purified as complexes with the 2 subunit. In these
studies, we used a single 1[K89A] rather than a double
H1[N88A/K89A] mutant at the side of the
1 torus. All of the complexes supported ADP-ribosylation
of i1 by pertussis toxin, although the potency of
H1[K89A]2 was about half that of the
wild-type protein, presumably reflecting the lower affinity of this
mutant for i, consistent with the properties of the
double mutant (data not shown).
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Fig. 5.
SDS-polyacrylamide gel electrophoresis of
purified mutant subunits. Purified subunits made in
insect cells were subjected to SDS-PAGE on a 15% gel, which was then
stained with Coomassie Blue. The amounts loaded ranged from 0.7 to 1.6 µg. Lane 1, 1[K89A]H2;
lane 2, 1[L117A]H2;
lane 3, H1[D228R]2;
lane 4, H1[D246S]2;
lane 5, 1[W332A]H2;
lane 6, wild type 1.
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Activation of PLC2 by purified wild-type and mutant
complexes is shown in Fig. 6.
Consistent with results in COS-7 cells, H1[K89A]2 and
H1[L117A]2 were approximately equal to
wild-type in activating PLC2, but the other three
mutations on the top surface of 1 were severely blunted
in their ability to activate PLC2. Although there are
quantitative differences in the degree of impairment of D228R, D246S,
and W332A in the two experimental systems, the conclusion that mutating
each of the three residues diminishes PLC2 activation is
consistent in both. In analyzing a large number of mutations at various
sites in , we have sometimes observed differences in the ability of
mutant proteins to fold correctly, depending on the expression system,
with the most native state achieved when the protein is made in
mammalian cells (28). It is possible that the final conformation of the
mutant subunit is slightly different when they are made in
mammalian COS-7 cells as opposed to insect cells.
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Fig. 6.
Activation of PLC2 by purified
mutant subunits. The indicated amount of each mutant
was reconstituted with 0.1 nM PLC2, and the
synthesis of IP3 was measured over 8 min at 30 °C as
described under "Experimental Procedures."  , K89A; , L117A;
, D228R;  , D246S;  , W332A;  , wild-type. Data shown are the
average of duplicate determinations from a single experiment that is a
representative of three such experiments.
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Activation and Inhibition of Adenylyl Cyclase by
1 Mutants--
The effects of mutant
12 complexes on adenylyl cyclase
activities are shown in Fig. 7.
activates type II adenylyl cyclase in the presence of s,
but it inhibits type I adenylyl cyclase (29). The apparent
affinities of H1[D246S]2 and
1[W332A]2 for type II adenylyl cyclase
are clearly diminished; we were unable to assess unequivocally their
maximal capacities to activate the enzyme because of our inability to
achieve higher concentrations of these proteins in the assay (Fig.
7B). Within a similar range of concentrations,
H1[D228R]2 did not activate type
II adenylyl cyclase. The 1[K89A]2 and
1[L117A]2 mutants were
indistinguishable from wild-type complex. In contrast, all five mutant
complexes inhibited type I adenylyl cyclase (Fig.
7A). These inhibitory activities were lost after
inactivation of the proteins at 95 °C for 5 min (data not shown).
The observation that H1[D228R]2 is able
to inhibit type I adenylyl cyclase (albeit with the lowest apparent
potency of the group tested), whereas it is inactive in
PLC2, PLC3, and type II adenylyl cyclase
assays, confirms the conclusion, based on coimmunoprecipitation and
cross-linking studies, that the protein is not grossly misfolded (Figs.
2 and 3).
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Fig. 7.
Inhibition of type I adenylyl cyclase or
stimulation of type II adenylyl cyclase by purified mutant
subunits. The indicated amount of each mutant was
reconstituted with 10 µg of Sf9 cell membranes from cells
expressing type I adenylyl cyclase (A) or type II adenylyl
cyclase (B) in the presence of 50 nM
GTPS-s. Adenylyl cyclase activity was measured as
described under "Experimental Procedures." , K89A; , L117A;
, D228R; , D246S; , W332A; , wild-type. Data shown are the
average of duplicate determinations from a single experiment that is
representative of three such experiments.
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The inability of any of the mutations on the top surface of to
interfere with the inhibition of type I adenylyl cyclase raises the
possibility that inhibition of type I adenylyl cyclase would not
require the binding surface and would be an exception to the rule
that association with blocks interaction of with all
effectors. However, incubation of wild-type with
GDP-q interfered with both activation of type II
adenylyl cyclase and inhibition of type I adenylyl cyclase (Fig.
8), suggesting overlap of with these
interacting surfaces. The interface between and the two adenylyl
cyclases must require different parts of the top surface.
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Fig. 8.
GDP-q interferes with the
ability of to inhibit type I adenylyl cyclase or to stimulate
type II adenylyl cyclase. The indicated concentration of
was incubated with () or without () 100 nM
GDP-q on ice for 10 min. These samples were then
reconstituted with 10 µg of Sf9 cell membranes from cells
expressing type I adenylyl cyclase (A) or type II adenylyl
cyclase (B) in the presence of 50 nM
GTPS-s. Adenylyl cyclase activity was measured as
described under "Experimental Procedures." Data shown are the
average of duplicate determinations from a single experiment that is
representative of two such experiments.
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DISCUSSION |
The interpretation of the functional consequences of mutation
introduced into a protein structure depends on demonstrating, as far as
possible, that the mutation produces only a local change and not a
global one. We have mutated some of the residues in known to
contact (2, 3) in order to test the hypothesis that and
effectors share a common surface. We have analyzed the properties of
the mutant subunits in three kinds of expression systems, which
allows us to evaluate different aspects of their function. None of the
mutant proteins reported in this paper appeared to have global effects
on structure or its ability to assemble with
Top
Abstract
Introduction
