From the Departments of Pharmacology and
¶ Internal Medicine/Hypertension, University of Michigan,
Ann Arbor, Michigan 48109-0632
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ABSTRACT |
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G protein heterocomplex undergoes dissociation
and association during its functional cycle. Quantitative measurements
of and
subunit binding have been difficult due to a very
high affinity. We used fluorescence flow cytometry to quantitate
binding of fluorescein-labeled Gi1
(F-
) to
picomolar concentrations of biotinylated G
. Association in Lubrol
solution was rapid (kon = 0.7 × 106 M
1 s
1), and
equilibrium binding revealed a Kd of 2.9 ± 0.8 nM. The binding showed a complex dependence on
magnesium concentration, but activation of F-
with either
GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of
the heterocomplex (Kd > 100 nM). The
binding was also influenced by the detergent or lipid environment.
Unlabeled
reconstituted in biotinylated phospholipid vesicles
(pure phosphatidylcholine or mixed brain lipids) bound F-
~2-3-fold less tightly (Kd = 6-9
nM) than in Lubrol. In contrast,
in ionic detergents such as cholate and
3-[(cholamidopropyl)diethylammonio]-1-propanesulfonate exhibited
substantially lower affinities for F-
. Dissociation of F-
from
reconstituted in lipid vesicles was observed upon addition of
aluminum fluoride or excess unlabeled
subunit, indicating that
myristoylated
subunit has only a weak interaction with lipids
without the
subunit. The kinetics of aluminum
fluoride-stimulated dissociation were slower than those of the
subunit conformational change detected by intrinsic fluorescence. These
results quantitatively demonstrate G protein subunit dissociation upon
activation and provide a simple but powerful new approach for studying
high affinity protein/protein interactions in solution or in a lipid
environment.
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INTRODUCTION |
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Receptor-mediated activation of heterotrimeric GTP-binding
proteins is a common mechanism for transducing biological signals. Cell-surface receptors regulate G proteins by inducing a conformational change that catalyzes release of GDP from the subunit, allowing GTP
to bind and activate the G protein. It is generally accepted that G
protein activation in vitro involves
subunit
dissociation from
subunits and that dissociated
and
subunits interact with effector proteins to modulate cellular responses
(1-5). G protein deactivation is mediated by an intrinsic
subunit
GTPase activity that can be enhanced by GTPase-accelerating proteins (6, 7), followed by
and
subunit reassociation. While G
protein subunit association and dissociation have been extensively studied in detergent solutions, it has been difficult to obtain precise
affinities for the subunits. The subunit interactions in a lipid
environment are even less well understood.
Several areas of uncertainty remain in the mechanism of G protein
subunit interactions with themselves and with membranes. The role of
lipids and G protein subunit modifications in /
interactions
has not been defined quantitatively. The sequence of events in subunit
activation and dissociation has recently been called into question (8).
Also, it has been suggested that unphysiologically high magnesium
concentrations are required for Gs subunit dissociation
(9). Thus, we wished to test in a quantitative manner the binding of
the
and
subunits in both detergent solution and in a lipid
environment to study factors regulating their affinity. One of the
major limitations to the study of very high affinity protein/protein
interactions is that the concentrations of proteins needed to evaluate
affinities are often greater than the Kd for the
binding interaction. This leads to artifacts and inaccurate
Kd determinations. Sklar and co-workers (10) have
used flow cytometry to study high affinity binding of fluorescent
ligands to the surface of cells. More recently, Nolan et al.
(11) used bead-based systems to study protein/DNA interactions in
vitro. In this report, we describe the use of biotinylated G
(b-
)1 bound to
avidin-coated polystyrene beads and fluorescein-labeled Gi1
(F-
) to measure equilibrium constants and rates
for
and
binding in the picomolar range by flow cytometry. In
addition, biotinylated lipids permit measurements of the binding of
unlabeled
with F-
in a lipid environment. Mechanisms of
/
interactions and factors that modify those interactions are
described. This simple and quantitative approach to the study of
protein/protein interactions provides new information about G protein
function and has great potential for the study of the assembly of
membrane protein complexes in general.
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EXPERIMENTAL PROCEDURES |
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Materials--
Fluorescein 5-isothiocyanate was obtained from
Molecular Probes, Inc. (Eugene, OR). Avidin-coated 6-µm polystyrene
particles (catalog No. SVP-60-5) were purchased from Spherotech, Inc.
(Liberville, IL). Maleimidobenzoyl-N-hydroxysulfosuccinimide
ester and
N-(6-((biotinoyl)amino)-hexanoyl)dipalmitoyl-L--phosphatidylethanolamine triethylammonium salt (EZ-LinkTMBiotin-LC-DPPE) were
obtained from Pierce. Phosphatidylcholine (catalog No. A-30) was
obtained from Doorsan Serdary Research Laboratories (Englewood Cliffs,
NJ). Biotinamidocaproate N-hydroxysuccimide ester,
-aminobutyl-agarose, and bovine brain membrane extract (catalog No.
B-3635) were purchased from Sigma. The expression vectors pQE6
containing rat cDNA sequence for Gi1
and pBB131
containing yeast N-myristoyltransferase (12) were generously
provided by Drs. Maurine Linder and Jeffrey Gordon (Washington
University, St. Louis, MO), respectively.
Purification of r-myr-i1 and Brain
Subunits--
The pQE6 Gi1
and pBB131
N-myristoyltransferase vectors were cotransformed into the
JM109 or BL21(DE3) strain of Escherichia coli. The resulting
recombinant myristoylated G protein
i1 subunit (r-myr-
i1) was expressed and purified according to Mumby
and Linder (13) with minor modifications. The specific activity was 15 pmol/µg based on [35S]GTP
S binding (14). Bovine
brain G
subunits were isolated from purified
Go/Gi as described by Katada et
al. (15).
Labeling of and
Subunits--
Purified
r-myr-
i1 was exchanged into DTT-free buffer containing
20 mM HEPES, pH 8.5, 1 mM EDTA, 100 mM NaCl, 0.5% cholate, 10 µM GDP, and AMF
(20 µM AlCl3, 10 mM
MgCl2, and 10 mM NaF) through consecutive
Centricon-30 concentration and dilution. In a 5-ml reaction mixture, 50 nmol of r-myr-
i1 was incubated with a 10-fold molar
excess of fluorescein 5-isothiocyanate for 2 h at room temperature in the dark with rocking. The reaction was terminated by addition of
0.5 ml of 1 M glycine, pH 8.5, and the majority of free dye was removed by dialysis using a Slide-A-Lyzer-10 (Pierce) overnight in
20 mM HEPES, pH 8.0, 1 mM EDTA, and 3 mM DTT in the dark at 4 °C. Active fluorescein-labeled
r-myr-
i1 (F-
) was affinity-isolated on a
-agarose column (16). This yielded ~64% of the originally loaded F-
with a specific activity of 11 pmol/µg of
[35S]GTP
S binding and 0.9 mol of dye/mol of protein
incorporated.
Reconstitution of into Biotinylated Lipid
Vesicles--
Biotinylated phospholipid vesicles containing
subunits were prepared by a gel filtration procedure as described
previously (19). EZ-LinkTMBiotin-LC-DPPE (biotin-labeled lipid),
featuring a 2.7-nm spacer arm to avoid steric hindrance, was mixed with phospholipids in a 1:100 molar ratio to permit attachment of the vesicles to the avidin-coated beads. Phosphatidylcholine or bovine brain membrane extract (0.8 mg) was mixed with 8 µg of biotin-labeled lipid in chloroform/methanol (2:1), dried under N2, and
resuspended in 0.2 ml of a 1.2% solution of sodium cholate. Mixtures
were vortexed for 5 min at room temperature and sonicated for 30 min at
4 °C to clarity. Vesicles were prepared by gel filtration through a
Sephadex G-25-50 column equilibrated with 20 mM HEPES, pH
8.0, 1 mM EDTA, 1 mM DTT buffer of the
lipid/cholate solution with or without 80 µg of purified bovine brain
subunits. Fractions containing
were pooled, snap-frozen
in aliquots in liquid nitrogen, and stored at
80 °C. The amount of
vesicle-bound
accessible on the beads was estimated by
immunostaining with
-specific polyclonal antibody (MS/1,
amino-terminal
-specific rabbit antisera, NEN Life Science
Products), followed by staining with a secondary fluorescein
5-isothiocyanate-conjugated antibody and detection by flow
cytometry.
Flow Cytometric Analysis--
Flow cytometric analyses of G
protein subunit interactions were performed on a Becton Dickinson
FACScan equipped with an air-cooled 15-watt argon ion laser. b-
was prebound to the avidin-coated beads within 3 h of each
experiment. Beads (2 × 106/ml) were added to 2 nM b-
, incubated for 30 min at room temperature, washed to remove unbound b-
, and diluted in HEDNML (with 0.2 mM free Mg2+) to make the final
concentration 0.02-0.5 nM. For studies with lipids,
vesicles were allowed to bind to avidin-coated beads in HEDNM (HEDNML
without Lubrol) with 0.1% BSA and no detergent for 30 min at room
temperature. In equilibrium binding experiments, 0.1-40 nM
F-
was incubated in a 0.2-ml reaction volume with 105
beads/ml for 30 min at room temperature (21-24 °C) in the dark. Samples were run through the flow cytometer, and data were collected on
the forward scatter (FSC), side scatter (SSC), and fluorescein (FL-1)
channels. Data were gated on the singlet population of beads, which
represents ~80% of all scattering events, and histograms of FL-1
fluorescence were obtained (see Fig. 1). Mean channel numbers for FL-1
were calculated from 3000 to 5000 events.
G Protein Activation Kinetics--
The intrinsic fluorescence of
i(GDP)·
was measured using a stopped-flow
spectrofluorometer (SX-17MV, Applied Photophysics Ltd., Leatherhead,
United Kingdom). Purified r-myr-
i1 and bovine brain
subunits were mixed in equimolar concentrations in HEDNML and
incubated for 20 min at 21 °C. Equal volumes of heterotrimer (200 nM) and a 2× stock of AMF (40 µM
AlCl3, 20 mM MgCl2, and 20 mM NaF) in the same buffer were mixed, and the continuous
time course of the fluorescence increase was measured with a
photomultiplier tube behind a WG 320 filter (exciting at 290 nm with
9-nm slits).
Calculation of Free Magnesium-- All binding experiments were done with 0.2 mM free Mg2+ unless otherwise indicated. The final buffer composition was 50 mM HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, and 1.2 mM MgSO4. The concentration of free magnesium was calculated from total added MgCl2 using WinMAXC software (Stanford University, Pacific Grove, CA) using the BERS parameter set. The concentration of chelators, ionic strength, pH, and temperature of the buffer are taken into account by the analysis.
Data Analysis and Statistics-- Prism (GraphPad Software, San Diego, CA) was used for unweighted nonlinear least-squares fitting of all of the data.
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RESULTS |
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The use of flow cytometry for measuring high affinity F-
binding to b-
on avidin-coated beads is illustrated in Fig.
1A. Binding of 40 nM fluorescein-labeled Gi1
to 0.5 nM b-
(medium solid line) can be readily
distinguished from nonspecific binding (thick solid line) in
the histograms shown in Fig. 1B. Because the measured result
is the mean of ~3000-5000 events, the mean channel numbers are very
accurate, and replicates generally agreed within 2%. Nonspecific
binding was only 15-20% of total binding even at 40 nM
F-
. Avidin-coated beads without
gave essentially the same
binding as
-bound beads in the presence of excess
subunit
(data not shown). Accurate measurements of binding were routinely made
with
concentrations as low as 100 pM (see Figs. 2-5), and even 20 pM
gave clear binding signals.
The binding could be substantially decreased by preincubation of F-
with either GTP
S or AMF (Fig. 1B).
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The time course of association and dissociation of 1 nM
F- with 100 pM b-
is illustrated in Fig.
2. The data were generally fit well by a
single exponential association and dissociation, but in some
experiments, two kinetic phases of dissociation were evident. The
koff was 0.047 ± 0.005 min
1
(0.00078 s
1) with a half-time of 14.8 min. The
dissociation upon addition of AMF was much faster, being nearly
complete by the first time point at 1 min. The apparent on-rate
(kon(app)) was measured at several
concentrations of F-
, and individual rate constants were determined.
A secondary plot of kon(app) versus
[F-
] (Fig. 2A, inset) gave a
kon of 0.7 × 106
M
1 s
1 and a
koff of 0.0013 s
1. This yields a
kinetic Kd of 1.9 nM.
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Since magnesium profoundly affects the interaction of and
subunits, we examined the effect of free magnesium on
/
binding in Lubrol. As expected, there was greater binding at lower magnesium concentrations (Fig.
3A). There appeared to be a
biphasic curve with a plateau at the normal physiological magnesium
concentration of 0.1-1 mM. Saturation curves with 0.2 or
9.8 mM free magnesium showed equivalent maximum binding
capacities with an ~2-fold difference in Kd,
2.9 ± 0.8 (n = 7) and 4.4 ± 1.1 (n = 5), respectively (Fig. 3B). Other
experiments with 20 ppm Lubrol or the monodisperse form
C12E10 showed no significant difference in
affinity from 0.1% Lubrol (data not shown).
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There have been questions raised recently about whether GTP analogs can
fully dissociate subunits in low magnesium solutions (9). In our
system, the binding of F- was completely eliminated after full
occupancy with GTP
S at 0.2 mM free Mg2+
(Fig. 4). Initial experiments showed
evidence of some residual affinity, but this was due to incomplete
occupancy of the
subunit with
nucleotide.2 Similarly,
aluminum fluoride in the presence of 10 mM magnesium resulted in a Kd for
/
association of >100
nM.
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Interestingly, the affinity of subunits changed significantly with
different detergents. Preliminary saturation experiments showed
Kd values for F- of 3, 30, and 50 nM
for Lubrol, CHAPS, and cholate, respectively. Due to the lower affinity
and higher nonspecific binding, the saturation data with cholate and CHAPS were less reliable. Thus, IC50 values for
r-myr-
i1 were measured in competition with F-
(Fig.
5 and Table
I). As in the direct binding studies,
r-myr-
i1 competed ~10 and 30 times less well in CHAPS
and cholate, respectively. Although the ratio of affinities in the
three detergents were conserved, IC50 values for
competition by r-myr-
i1 were consistently lower than
Kd values determined in saturation experiments for
F-
binding to
. This may reflect slightly weaker
binding by the labeled
subunit or a heterogeneity of the
pool purified from bovine brain tissue. In the latter case, there could
be a higher affinity in competition experiments due to the selective
occupancy of high affinity
sites by the low labeled ligand
concentrations.
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To examine G protein subunit binding affinity in a lipid environment,
we prepared bovine brain lipid vesicles containing 1 mol % biotin-labeled lipid with and without unmodified subunit. Incorporation of
into the lipid vesicles and vesicle binding to
avidin beads were monitored by immunostaining with a
-specific polyclonal antibody. There was very little competable binding of F-
to the beads with biotin-labeled lipids alone, but lipids containing
showed substantial specific binding (Fig.
6A). Similarly, when the
biotin-labeled lipids were left out of the reconstitution mixture,
there was no specific binding of F-
to the beads (provided BSA was
included in the incubation and wash buffers). This demonstrates the
requirement for biotin-labeled lipids and rules out nonspecific absorption of lipid vesicles to the avidin-coated beads. The affinity for specific F-
binding to
in brain lipid vesicles
(Kd = 8.6 ± 1.8 nM; Fig.
6B) was slightly lower than the affinity in Lubrol
(Kd = 2.9 ± 0.8 nM). The affinity
of F-
for
in vesicles prepared from pure
dimyristoylphosphatidylcholine was similar to that in bovine brain
lipids (Kd = 6.8 ± 1.4 nM; data
not shown). In competition studies, the IC50 of r-myr-
i1 for
in brain lipids was also a factor of
2 greater than that in Lubrol (Table I).
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With the ability to measure G protein subunit interactions in lipid
membranes, we wanted to determine whether G protein subunit dissociation would lead to rapid G dissociation from the membrane. Heterotrimer was assembled by preincubation of F-
with the
-containing biotinylated bovine brain lipid vesicles coupled to
the beads. Dissociation of F-
was initiated by adding a 50-fold
excess of unlabeled r-myr-
i1 or by activation with
aluminum fluoride. Both treatments resulted in F-
dissociation from
the vesicles (Fig. 7). In agreement with
the dissociation rates for the heterotrimer in Lubrol solution,
AMF-induced release of F-
was rapid (koff = 0.047 min
1). As expected from the lower affinity between
subunits in the lipid environment than in Lubrol, the dissociation rate
from the surface of the vesicles was ~2-5-fold faster than that in
Lubrol (koff = 0.28 min
1). These
data show that r-myr-
i1 can rapidly dissociate from a
lipid vesicle surface following G protein activation. The incomplete dissociation of F-
upon AMF activation may be due to some small residual affinity of AMF-bound
subunits for
or lipids (Figs. 4 and 6A).
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By combining two real-time measurements, namely spectroscopic analysis
of intrinsic tryptophan fluorescence of the heterotrimer and flow
cytometric detection of physical separation of the subunits, the
sequence of the events involved in G protein activation could be
resolved. The increase in G tryptophan fluorescence has been used as
a measure of conformational changes accompanying G protein activation
(20). Rapid kinetics of the
i1 intrinsic fluorescence increase were recorded using a stopped-flow spectrofluorometer. Addition of AMF resulted in a 10-15% increase in fluorescence of the
i1·
heterotrimer. Controls using HEDNML instead
of AMF or mixing of boiled r-myr-
i1·
with AMF
showed no change in the intensity of the fluorescence. The AMF
activation-induced dissociation of F-
from the F-
·b-
heterotrimer assembled on the avidin-coated beads was analyzed by
continuous acquisition mode on the flow cytometer. The rate of G
activation monitored by intrinsic fluorescence was ~3-fold faster
than the rate of subunit dissociation by flow cytometry (Fig.
8). This indicates that an activating
conformational change in the
subunit precedes subunit dissociation
(see "Discussion"). This was not a difference between labeled and
unlabeled protein as similar rates of AMF-induced fluorescence change
were seen with F-
·
. If there was any difference, F-
gave
slightly faster kinetics (data not shown).
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DISCUSSION |
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The results described here extend our knowledge of G protein
subunit interactions and how they relate to the lipid environment and G
protein activation mechanisms. This novel flow cytometric approach
permitted us to address quantitatively the role of detergent, lipid
environment, and regulatory factors affecting G protein and
subunit binding. Also, we show that G
conformational changes precede
subunit dissociation and that upon G protein activation in lipid
vesicles, a myristoylated
subunit rapidly dissociates from the
membrane.
Previous attempts to determine affinities between G protein subunits
using different experimental approaches have resulted in a range of the
dissociation constants. Kohnken and Hildebrandt (21) used Western blots
to detect binding of the subunit to biotinyl-
immobilized on
streptavidin-agarose. The apparent Kd values in
0.05% Lubrol-PX were ~20 nM for
41 and ~350 nM for
39. The last value was at
least three times larger that the EC50 measured indirectly
from the
-supported ADP-ribosylation of
39 by
pertussis toxin (22, 23). Helmreich and co-workers (24) used
steady-state fluorescence energy transfer to examine the interaction
between fluorescein-labeled Go
and rhodamine-labeled brain
subunits. In 0.25% Lubrol, the apparent
Kd for specific GTP
S-sensitive binding was 10 nM. The signal-to-noise ratio in these studies was limited
as only 20% quenching of fluorescence was seen. In comprehensive
studies by Ueda et al. (25), the inhibitory effect of six
different
forms on the steady-state GTPase activity of
subunits was determined in 0.001-0.05%
C12E10. Both Gs
and
Go
showed EC50 values of 2 nM
for
1
1 and 0.2-0.5 nM for
all other
complexes. Gi2
GTPase was inhibited
with an EC50 of ~0.4 nM by all
subunits tested. In our analysis of resonance energy transfer between
fluorescein-labeled Go
and eosin-labeled
by
stopped-flow kinetics, we observed only 5% quenching of F-
fluorescence and could only say that the equilibrium Kd was <60 nM (26). Thus, the present
data showing a dissociation constant of 1-3 nM in
0.002-0.1% Lubrol are generally consistent with previous literature
reports. The advantages of this approach are as follows: 1) direct
measurement of the binding interaction rather than an indirect
functional measure; 2) the simplicity of the method and the
reproducibility of results; and 3) its easy application to a wide
variety of experimental conditions, which has allowed us to test the
effects of many factors that regulate subunit interaction.
Flow cytometry is equally suitable for continuous analysis of real-time
binding or dissociation kinetics (10, 11, 27). We defined the on- and
off-rate constants for -GDP and
, and the binding is quite
fast; kon is 0.7 × 106
M
1 s
1. The dissociation of the
i·
complex upon AMF activation has been shown
previously by its ability to reverse the aggregation of
i in the presence of
(28). Cerione and co-workers
(8) proposed that the dissociation of transducin subunits by AMF
precedes the tryptophan fluorescence change usually attributed to
activation. A potential problem with that study is the indirect
measurements of dissociation by subunit exchange and fluorescence
energy transfer. Our direct determination of the dissociation rate of
the
i·
heterotrimer found it to be 3-fold slower
than the rate of the G protein conformational change accompanying
activation. This suggests a simpler model in which the conformational
change occurs first, followed by dissociation.
G protein subunit interactions are strongly influenced by magnesium
(21, 24, 29-31). We observed a 2-fold reduction in /
binding
affinity by increasing the free MgCl2 concentration from
0.2 to 10 mM, and binding was reduced even further in 40 mM MgCl2. This nucleotide-independent
dissociation at very high magnesium is important to keep in mind in
evaluating G protein subunit interactions. Because of these
observations, all other studies in this paper used a physiologically
relevant free Mg2+ concentration of 200 µM.
The effect of GTP
S to completely prevent formation of the
F-
·b-
heterocomplex even in the presence of physiological
concentrations (0.2 mM free MgCl2) is at odds
with a recent report for
s that indicated that very high
magnesium concentrations were required for subunit dissociation (9). It
will be interesting to examine
s/
interactions
with the flow cytometric method to further evaluate this possible
discrepancy.
One aim of this study was to quantitate interactions between G protein
subunits in detergent micelles and in lipid vesicles under similar
conditions. Several frequently used detergents had significant effects
on the affinity of heterocomplex formation. Sodium cholate (0.4%) and
CHAPS (0.7%) reduced the affinity of and
by >10-30-fold.
Reconstitution of
into biotinylated phospholipid vesicles
appeared to preserve the high affinity interaction with fluorescently
labeled r-myr-
i1 with an excellent signal-to-noise ratio. The affinity for
/
binding on the surface of lipid
vesicles was 2-3-fold lower than that observed in 0.1% Lubrol, which
is consistent with the difference previously reported for
o using resonance energy transfer (Kd = 10 nM in 0.2% Lubrol versus 30 nM
in lipid vesicles) (24).
Dissociation of myristoylated F- from the membrane surface following
G protein activation by AMF is consistent with the earlier observations
where activation of the heterotrimer led to a slow, partial release of
o and
i from the cell membranes (32, 33) or phospholipid vesicles (28). The incomplete dissociation of F-
shown in Fig. 7 could indicate that a fraction of the F-
pool is not
activated by AMF, or it could reflect low affinity binding of F-
to
lipid-incorporated
in the presence of AMF. The failure of
GTP
S treatment to induce
release in other systems (34, 35)
suggested that
subunits are tightly attached to cell membranes
through a mechanism that may be independent of interaction with the
complex. Recent data indicate that dual fatty acid modification
of
contributes to a membrane association of the subunit, with
palmitoylation providing stronger association with membrane lipids
(36-38) and myristoylation enhancing the affinity of
for
subunits (12, 39). The discrepancy in the binding of
to native
membranes versus lipid vesicles could be explained by
differences in lipid modification of
subunits or by binding to some
other proteins, distinct from the
subunit, which could anchor
more tightly to the membrane.
The simplicity and sensitivity of the flow cytometric approach for measuring G protein subunit interactions in both detergent and lipid environment permit testing of several important questions. The ability to analyze the dynamics of heterotrimer complex formation and disassembly upon activation with guanine nucleotides and receptors in reconstituted systems is an exciting future direction.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs. Larry A. Sklar
(University of New Mexico) and John P. Nolan (Los Alamos National
Laboratory) for the suggestion of flow cytometry and consultation
support from the National Flow Cytometry Resource supported
by National Institutes of Health Grant RR01315. We are grateful to Drs.
Maurine E. Linder and Jeffrey I. Gordon for generously providing
plasmids for the Gi1 protein and
N-myristoyltransferase. We thank Dr. Latham J. Claflin
(Department of Microbiology, University of Michigan) for help in flow
cytometry.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM 39561 (to R. R. N.).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 Research Fellowship Award 25F967 from the American Heart Association, Michigan Affiliate.
To whom correspondence should be addressed: Dept. of
Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Tel.: 734-763-3650; Fax: 734-763-4450; E-mail:
RNeubig{at}umich.edu.
1
The abbreviations used are: b-,
biotinylated G
; F-
, fluorescein-labeled Gi1
;
r-myr-
i1, recombinant myristoylated G protein
i1 subunit; GTP
S, guanosine
5'-O-(3-thiotriphosphate); DTT, dithiothreitol; BSA,
bovine serum albumin; CHAPS,
3-[(cholamidopropyl)diethylammonio]-1propanesulfonate.
2
Even with 90% saturation of the subunit
with GTP
S, there was sufficient unoccupied
subunit to give an
apparent Kd of 20 nM (versus
2 nM for GDP-bound
subunit).
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REFERENCES |
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