From the Cancer Center, Department of Pathology,
§ Department of Cell Biology and Physiology, University of
New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, the
¶ Ralph and Muriel Roberts Laboratory for Vision Science, Sun
Health Research Institute, Sun City, Arizona 85351, the
Department of Pharmacology, University of Michigan School of
Medicine, Ann Arbor, Michigan 48109, and the ** National Flow Cytometry
Resource, Los Alamos, New Mexico 87545
Received for publication, October 23, 2000, and in revised form, March 7, 2001
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ABSTRACT |
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Receptor based signaling mechanisms are the
primary source of cellular regulation. The superfamily of G
protein-coupled receptors is the largest and most ubiquitous of
the receptor mediated processes. We describe here the analysis in
real-time of the assembly and disassembly of soluble G protein-coupled
receptor-G protein complexes. A fluorometric method was utilized to
determine the dissociation of a fluorescent ligand from the receptor
solubilized in detergent. The ligand dissociation rate differs between
a receptor coupled to a G protein and the receptor alone. By observing
the sensitivity of the dissociation of a fluorescent ligand to the
presence of guanine nucleotide, we have shown a time- and
concentration-dependent reconstitution of the
N-formyl peptide receptor with endogenous G proteins.
Furthermore, after the clearing of endogenous G proteins, purified G GTP-binding regulatory protein-coupled receptors
(GPCR)1 represent the largest
class of cell surface receptors. A broad variety of physiological
processes depend on this family of seven transmembrane proteins, making
them prime targets for drug discovery (1). Several complementary
approaches are being taken in this therapeutic effort. One approach is
to define novel therapeutic agents including both agonists and
antagonists for specific receptors, as well as molecules that block
interactions between receptors and their G protein transduction
partners. In addition, the interactions between ligands and receptors
are beginning to be thoroughly mapped through studies incorporating
receptor mutagenesis as well as analysis of the binding and activity of
ligand analogs (2). Comparable efforts are being made to describe the
molecular mechanisms that influence the specificity of coupling
interactions between GPCR and their cognate heterotrimeric G proteins.
Several experimental systems for reconstitution provide alternatives
for studying receptors and G proteins, such as adding G protein to
stripped membranes, pairing receptors and G proteins in phospholipid
vesicles (3), and examining solubilized receptor-G protein interactions
using gradient centrifugation (4).
The kinetics of the interactions between ligand (L), receptor (R), and
G protein (G) are described by the ternary complex model (5). The
formation of LRG is required for the activation of the G protein and
typically involves the formation of a high affinity receptor, release
of GDP, and activation of the G protein through GTP binding (1).
Extended ternary complex models have been developed to describe the
transition of receptors from inactive to activated states (6). The
assembly kinetics of these systems are not completely understood as the
available methods do not provide the time resolution required to
evaluate all of the steps in the activation process. Moreover, analysis
of receptor-G protein interactions in cells is complicated by the
difficulty in elucidating the G protein numbers and concentrations
within the microdomains of the receptors and the transient association
between receptors and G proteins.
To further the understanding of GPCR-mediated processes, we have
utilized the N-formyl peptide receptor (FPR), which couples to a pertussis toxin-sensitive G protein and is expressed predominantly on leukocytes (7). This receptor recognizes the bacterially generated
N-formyl peptides that act as potent chemoattractants for
human phagocytes. The FPR is one of the better characterized receptors
in the chemoattractant/chemokine subclass of GPCR (8). It modulates
several cell functions including chemotaxis, superoxide formation, and
degranulation, as well as influencing nuclear regulation via activation
of MAPK cascades (7). It has been assumed that the FPR binds
preferentially to a G We have previously described a number of real-time assays of
ligand-receptor interactions using flow cytometry and fluorescence that
have been primarily directed toward viable cells or
detergent-permeabilized cells (12-14). These studies characterized
wild type and mutant receptors leading to a model of receptor-G protein
coupling for the FPR (13). More recently, we were able to assess the
efficiency of solubilization of the FPR using fluorescence methods, and
have shown that these receptors were able to reconstitute with G
proteins in a non-cellular format (15). In this report we have expanded our investigation of the detergent-solubilized FPR with emphasis on the
receptor-G protein interaction. Using reconstitution assays, we
demonstrate the ability of the receptor to couple with endogenous G
proteins, as well as with exogenous G proteins containing specific G Reagents and Cell Culture--
The generation of U937 cells
transfected with the FPR was previously described (16). Plasticware was
obtained from VWR Scientific Co. Chemicals and reagents were obtained
from Sigma except where otherwise noted. Cells were grown in tissue
culture-treated flasks (Corning) in RPMI 1640 (Hyclone) containing 10%
fetal bovine serum (Hyclone), 2 mM
L-glutamine, 10 mM HEPES, with 10 units/ml
penicillin and 10 µg/ml streptomycin. Cultures were grown in standard
tissue culture incubators at 37 °C with 5% CO2, and
passaged from subconfluent cultures every 3-4 days by reseeding at
2 × 105 cells/ml. Purified G protein Membrane Preparation by N2Cavitation--
U937 FPR
cells were harvested, centrifuged at 200 × g for 5 min, and resuspended in cavitation buffer (10 nM PIPES, 100 mM KCl, 3 mM NaCl, 3.5 mM
MgCl2, 600 µg/ml ATP) at a density of 107
cells/ml at 4 °C. The cell suspension was placed in a nitrogen bomb
and pressurized to 450 psi using N2 gas for 20 min at
4 °C. Nuclei and cytoplasmic material were separated by
centrifugation at 1000 × g for 5 min at 4 °C. The
supernatant, containing the membranes, was washed 2 times by
centrifugation at 135,000 × g for 30 min at
4 °C, then resuspended in HEPES sucrose buffer (200 mM
sucrose, 25 mM HEPES, pH 7), aliquoted, and stored until
use at Solubilization of the FPR--
Membranes were thawed and diluted
to 1-2 × 108 membrane cell equivalents/ml (CEQ/ml)
in binding buffer (30 mM HEPES, 100 mM KCl, 20 mM NaCl, 1 mM EGTA, 0.1% (w/v) bovine serum
albumin, 0.5 mM MgCl2). Preparations were
maintained at 4 °C throughout the extraction process. Membranes were
centrifuged at 135,000 × g for 30 min and resuspended
in 150 µl of binding buffer containing protease inhibitor mixture I
and 1% n-dodecyl Velocity Sedimentation--
Membranes (1 × 108
cell equivalents) were solubilized and applied to 1 ml of a 5-20%
linear sucrose gradient prepared in binding buffer plus 1%
n-dodecyl Reconstitution of Receptors with G Proteins--
Purified
individual
The activity and concentration of the G Spectrofluorometric Analysis--
Fluorescence associated with
fMLFK-FITC was measured by a SLM 8000 spectrofluorometer (SLM
Instruments, Inc.) using the photon counting mode in acquisition. The
sample holder was fitted with a cylindrical cuvette adapter to permit
measurements in stirred volumes of 200 µl using small cylindrical
cuvettes (Sienco) and 2 × 5-mm stir bars (Bel-Art). Excitation
was fixed at 490 nm, and stray light was reduced with a 490 nm, 10-nm
band pass filter (Corion). FITC fluorescence emission was monitored
using a 520 nm, 10-nm band pass interference filter (Corion) and a
3-70 orange glass, 500-nm long pass filter (Kopp). Additions to
samples during kinetic measurements were performed using 10-µl glass
syringes (Hamilton) adding reagents through an injection port on the
SLM 8000 above the sample cuvette.
Following preparation at 4 °C, samples were brought up to a volume
of 200 µl with binding buffer plus 0.1% n-dodecyl
Statistical Analysis--
Data were analyzed and graphed using
Prism software (Graph Pad Software Inc.). To determine the dissociation
characteristics of the receptor preparations, the fluorescence over
time in blocked control samples was subtracted point by point from the
fluorescence over time of ligand binding samples as described
previously (14). The data were then normalized and the relative
fluorescence was fit by nonlinear regression using single (Equation 1)
or double (Equation 2) exponential decay equations.
Clearing Sample of Endogenous G Protein--
Membrane
preparations were solubilized in detergent as outlined above. The
sample was then incubated with 20 µl of anti-G Western Blot Analysis--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene difluoride membranes (Gelman) with a semi-dry transfer
apparatus (Owl Scientific). Membranes were blotted with antibody
(rabbit) against either G Fluorometric Assay Development--
Receptor-G protein
interactions have been characterized in intact and permeabilized cells,
membranes, phospholipid vesicles, and in detergent using gradients (3,
21-23). We have extended the use of detergent-solubilized FPR and G
proteins, adding fluorescence detection, to study receptor-G protein
complexes in real-time. We have taken advantage of the fact that the
ligand dissociation rate of the LR complex of the FPR (in the absence
of G protein or in the presence of GTP
The assay is based upon a fluorescein-conjugated ligand used along with
an anti-fluorescein antibody that rapidly quenches the fluorescein of
the free ligand upon binding. The antibody is able to interact only
with the fluorescein on the free ligand, as FPR-ligand complexes
sterically inhibit the antibody from binding the fluorescein (14). In
this way we were able to determine the quantity of FPR-bound ligand
(Fig. 1) immediately following the
addition of the anti-fluorescein antibody. We were also able to follow
the ligand dissociation kinetics as the excess antibody further
quenches ligand released from the receptor. These methods were based on
assays that have been fully characterized using permeablized whole
cells (12-14). The dissociation data was analyzed as described under
"Experimental Procedures." The data were fit to one or two rates,
the slow rate apparently representing L dissociation from RG and the
fast rate apparently representing L dissociation from R.
Endogenous G Protein Reconstitution with a Solubilized
Receptor--
The human FPR was previously stably transfected into the
undifferentiated human myeloid cell-line, U937 (16). The U937 cells expressing the wild type FPR react specifically to formylated peptides
with calcium and chemotactic responses (26). Membrane fractions were
prepared using N2 cavitation and the work presented is data
from both intact membranes or membranes that have been solubilized with
1.0% detergent solution. The extent of ligand binding to the FPR is
similar for membranes and the solubilized membrane extract (15).
However, in the membrane samples it appeared that G proteins were
either pre-coupled to, or interacted almost immediately with, the
receptor after the addition of ligand, but once the membranes were
solubilized, no G protein interaction was observed over the same time
frame (Fig. 1A). In earlier studies we demonstrated the
ability of the solubilized receptor to interact with an exogenous G
protein, if the receptor was incubated in the presence of ligand for
2 h (15). Thus it was possible that the solubilized receptor would
reconstitute with endogenous G proteins over time if ligand was
present. As shown in Fig. 1B, there was indeed a time
dependence to the interaction. Additionally, when assays were prepared
in larger volumes, that diluted the concentration of both receptor and
G protein, reconstitution was not observed (data not shown). This
dependence on receptor and G protein concentration in solution suggests
that while within the membranes, the receptor-G protein interactions
take place rapidly in two dimensions, the solubilized proteins exist in
three dimensions requiring extra time for the proteins to associate.
To confirm that our receptor preparations were solubilized in the
presence of 1% detergent (n-dodecyl
Clearance of Endogenous G Proteins from Solubilized Membrane
Preparations--
In order to generate a system in which the
affinities of specific exogenous G protein subunits could be determined
for the FPR, we first needed to remove the endogenous G protein from
our solubilized receptor solution. This was accomplished by incubating the sample with an anti-G Reconstitution of the Solubilized FPR with G Proteins Containing
Specific G Requirement for the G Protein Heterotrimer to Induce Binding to the
Receptor--
In the previous set of experiments we combined the G
protein Inhibition of the FPR-G Protein Interaction--
The G
Further investigation of the receptor-G protein interface was
accomplished by incorporating G
The same experiments were also performed using membrane preparations in
place of solubilized receptors. Interestingly, none of the peptides or
the antibodies was able to inhibit the FPR-G protein interaction under
the same conditions (data not shown).
FPR-Arrestin Interactions--
The desensitization of activated
GPCR by arrestin molecules has been well documented (28). It has been
presumed that the arrestin-receptor interaction physically blocks the
inactivated G proteins from associating with the receptor (29). The
data obtained in the above series of experiments demonstrated that our
fluorometric assay was able to quantitate G protein coupled verses
uncoupled receptor. This suggested that our methods would be amenable
to a receptor-arrestin study. The FPR colocalizes with arrestin-2 and
arrestin-3 in U937 cells transfected to express the FPR and in
neutrophils (30).2 The
addition of wild-type arrestin-3 had no effect on the ligand dissociation rate or sensitivity to guanine nucleotide (Fig.
9). This was expected as desensitization
of GPCR by arrestin is dependent on the phosphorylation of the
activated receptor. The use of solubilized receptors in our system
precluded receptor phosphorylation. A truncated version of
arrestin-3-(1-393), that has been shown to bind to GPCR in a
phosphorylation independent manner (31), was then tested. When samples
were incubated with the truncated arrestin-3, the result was a loss of
the slow dissociation component and guanine nucleotide sensitivity
indicating that the FPR-G protein interaction was indeed inhibited
(Fig. 9). This demonstrated that the truncated arrestin-3 was able to
compete with G protein in binding to the receptor.
We have presented here a series of reconstitution assays that have
analyzed the ability of the FPR to interact with several G proteins and
arrestins. The reconstitution assays depended on the detergent micelles
supporting the structural conformation of the FPR adequately for it to
retain its ability to bind both ligand and the various proteins that
interact with the intracellular domain of the receptor. It was
demonstrated that the solubilized FPR was present as a monodisperse,
uncoupled protein (Fig. 2). This work has led to the development of
fluorometric methods that can systematically evaluate GPCR interactions
in real-time. As this was done in a non-cellular format, a controlled
environment could be maintained in which specific interactions were studied.
Fluorometric methods that analyze solubilized receptors by real-time
ligand binding were utilized. The kinetic data of ligand dissociation
was statistically analyzed to determine the off-rate. Bound ligand
fluorescence was fit by non-linear regression using single or double
exponential decay equations. Receptors that did not associate with G
proteins fit best to a single exponential decay. Conversely, when
ligand dissociation displayed an initial slow rate, the curve was best
fit to a double exponential decay equation, indicating that some
fraction of the receptors were bound to a G protein. Moreover, when
GTP When the FPR was examined in membrane preparations, the
ligand-stimulated receptor rapidly formed a G protein-receptor complex, leading to speculation of a receptor-G protein precoupled state. However, with the solubilized receptor, this interaction was highly time- and concentration-dependent (Fig. 1). If precoupling
was present in the membrane assays, it is possible the solubilization disrupted the interaction. Additionally, even without precoupling, within the membrane environment the interactions occur over two dimensions, possibly in microdomains. This is in contrast to the three
dimensions available in the solubilized sample where LRG assembly is
expected to depend on time and [R][G]. Thus in a previous study performed with a lower concentration of solubilized membrane than
used here, FPR reconstitution with endogenous G protein was not
observed (15). It was fortuitous that the receptor was not precoupled
to a G protein in the solubilized system as this allowed for the
depletion of the endogenous G proteins (Fig. 3). By isolating all the
components of our system, we were able to add specificity to the
assembly process.
The next step was to examine FPR interactions with several purified G
protein subunits. The G protein containing the G The addition of individual This system does not permit definitive conclusions about which G
proteins are coupled to receptors under physiological conditions. Considering the FPR, most cells expressing this receptor contain relatively high levels of G The ability to detect the interaction of truncated arrestin-3 with the
receptor indicates the potential generality of the assay for the study
of many types of molecular assemblies (Fig. 9). Moreover, the effect of
arrestin on ligand affinities can be
determined.3 This approach
could also be extended to multistep interactions, such as GPCR
desensitization, that requires the interaction of the receptor with
kinases, arrestins, and cytoskeletal components (28, 32). Another
opportunity may be the evaluation of the assemblies of signaling and
scaffolding components amenable to the suspension analysis suggested
here or particle-based analysis by flow cytometry as suggested
previously (15).
subunits premixed with bovine brain G
subunits were also able to
reconstitute with the solubilized receptors. The solubilized
N-formyl peptide receptor and G
i3 protein
interacted with an affinity of ~10
6 M with
other
subunits exhibiting lower affinities (G
i3 > G
i2 > G
i1
G
o). The
N-formyl peptide receptor-G protein interactions were
inhibited by peptides corresponding to the G
i C-terminal regions, by G
i mAbs, and by a truncated form of
arrestin-3. This system should prove useful for the analysis of the
specificity of receptor-G protein interactions, as well as for the
elucidation and characterization of receptor molecular assemblies and
signal transduction complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i2 protein as this Gi
isoform is highly expressed in neutrophils, while G
i3 is
expressed at low levels and there is no expression of
G
i1 (9, 10). A recent report using chimeric proteins
containing the FPR fused to G
i1, G
i2, or
G
i3, expressed in Sf9 cells, suggested that the
FPR couples to each of the Gi isoforms with similar
efficiency (11). However, several aspects of this interaction still
remain unresolved.
subunits. We were able to measure the affinities of the complexes and
found that the FPR binds to a G protein heterotrimer containing the
G
i3 subunit with somewhat higher affinity than to
heterotrimers containing G
i2 or
G
i1 proteins. The individual
subunits and the
complex alone were unable to induce a change in the dissociation of
ligand from the receptor indicating a necessity for the G protein heterotrimer. We were able to inhibit the G protein-receptor
interaction with peptides derived from the G
i subunits
as well as with anti-G
i antibodies. Finally, we have
expanded the system to include analysis of GPCR-arrestin interactions.
The methods described here provide an approach to study the mechanism
of GPCR interactions with G proteins, arrestins, and other potential
targets and at the same time provide a platform for identifying and
characterizing novel therapeutic agents.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits
G
i1, G
i2, G
i3, and G
o (functional, myristoylated, rat recombinant) were
purchased from Calbiochem. The bovine brain
complex was isolated
and purified as previously reported (17). Arrestins were expressed in
Escherichia coli (strain BL21) and purified by sequential
heparin-Sepharose and Q-Sepharose chromatography essentially as
described (18). Reagents for inhibition of reconstitution include three
G protein
subunit blocking peptides composed of the last 10 amino
acids of G
i1,2, G
i3, and
G
s, and anti-G
antibodies recognizing the C terminus
of G
i1,2, G
i3, G
o, and an
internal G
i3 antibody (Calbiochem).
80 °C.
-D-maltoside (Calbiochem). Preparations were incubated 60 min at 4 °C with agitation. The insoluble fraction was separated by centrifugation at 70,000 × g for 5 min in a Beckman Airfuge. The supernatant,
containing the solubilized fraction, was removed and this extract was
used for experimentation within 6 h.
-D-maltoside. Gradients were
centrifuged at 40,000 rpm in a TLS-55 rotor (Beckman) for 14 h and
fractionated into 20 × 50-µl fractions. To establish the
distribution of receptor, 25 µl of each fraction was incubated with
10 nM formyl-Met-Leu-Phe-Lys-fluorescein 5-isothiocyanate
(fMLFK-FITC, Peninsula Laboratories) for 2 h on ice. Fractions
were subjected to spectrofluorometric analysis, as outlined below.
Gradients containing 5 µg of bovine serum albumin (4.4 S) and rabbit
immunoglobulin (7.7 S) were centrifuged in parallel. These fractions
were analyzed by SDS-polyacrylamide gel electrophoresis, followed by
Coomassie staining. Based on the sedimentation of these standard
proteins, the migration of 4 S and 7 S proteins was calculated to peak
at fractions 9 and 15, respectively.
subunits were mixed in an equimolar ratio with the
complex and incubated on ice for 15 min to form the
heterotrimeric complex (17). Solubilized FPR (5-10 µl of ~10
nM receptor in 1% n-dodecyl
-D-maltoside prepared as above), was then incubated with
the specific G-protein heterotrimers or with a mixture of bovine brain
Gi/Go heterotrimer (Calbiochem) at a
concentration of up to 3 µM for up to 2 h on ice in
the presence of 10 nM fMLFK-FITC. Blocked samples were
incubated with 1 µM formyl-Met-Leu-Phe-Phe (fMLFF, CBI)
for 15 min prior to the addition of fMLFK-FITC. Samples were prepared
in small volumes (typically 15 µl) to maximize receptor
concentration. Control samples were prepared in the presence of
appropriate buffer(s). The G
subunit buffer contained 100 mM NaCl, 20 mM HEPES, 3 mM
MgCl2, 1 mM EDTA, pH 8.0. The G
buffer
contained 50 mM HEPES, 1 mM EDTA, 1 mM dithiothreitol, 0.5% sodium cholate, pH 8.0, The
arrestin buffer contained 10 mM Tris, 100 mM
NaCl, 2 mM EDTA, 2 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 0.7 µg/ml pepstatin A, 10 µM chymostatin, pH 7.5. Reconstitution incubations were carried out in the presence of 0.8 to
0.85% detergent (final concentration).
o subunit was
verified using a BODIPY FL GTP
S binding assay titrating the subunit (1 nM to 300 nM) against 50 nM
nucleotide and recording the resulting changes in fluorescence on the
spectrofluorometer (19, 20). The derived value for the
G
o subunit concentration was consistent with the value
reported by the supplier (data not shown).
-D-maltoside, equilibrated to 22 °C, and placed into
the spectrofluorometer with constant stirring. Data were acquired for
180-210 s in 1-s intervals. Typically, total fluorescence was obtained
for the first 20 s, then, an anti-fluorescein antibody was added
to the sample. The antibody binds free fMLFK-FITC with high affinity and results in essentially complete quenching of the fluorescence associated with free ligand (14). Thus, the remaining fluorescence represents the receptor-bound. In G protein experiments, GTP
S (100 µM) sensitivity was used to assess the coupling between
receptors and G proteins based on characteristic ligand dissociation
rates. Experiments were performed using a detergent concentration
slightly above the critical micelle concentration.
(Eq. 1)
Where I = fluorescence intensity in arbitrary
units, kx is the off rate of the receptor state in
s
(Eq. 2)
1, t is time in seconds, plateau is the
fluorescence when all peptide has dissociated and Ax
is the fraction of receptor in the state with the rate of
kx. Rates are given as mean ± S.E.
i1,2,3 antibody (Calbiochem) for 45 min on ice. To remove antibody-substrate conjugates, 100 µg of Protein A-agarose was added to the sample. After a 30-min incubation, the sample was centrifuged at 14,000 × g for 30 s and the supernatant was removed. The process
was then repeated.
i1,2,3 or
(Calbiochem)
followed by an horseradish peroxidase-conjugated goat anti-rabbit
secondary antibody (Sigma). The blots were developed using ECL Plus
(Amersham Pharmacia Biotech) and imaged using a PhosphorImager
(Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S) is many times faster than
ligand dissociation from LRG (12, 24, 25). Thus, the presence of the G
protein imparts a higher affinity for ligand to the receptor. When GTP
is not available, the G protein remains bound to the FPR. The addition
of GTP, or its non-hydrolyzable analog GTP
S, putatively induces the
dissociation of G protein from the receptor and results in a decrease
in the affinity of the ligand, increasing its dissociation rate. The
change in the ligand dissociation rate is likely to represent a switch
from the slowly dissociating LRG to the rapidly dissociating LR as the
activated G protein uncouples from the FPR.
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Fig. 1.
Receptor-G protein coupling in membranes and
solubilized receptor. A, a membrane sample from U937
cells stably transfected to express the FPR was washed once,
resuspended into 150 µl of binding buffer, then divided into two
aliquots. One of these was solubilized by incubating for 1 h at
4 °C in the presence of 1.0% dodecyl maltoside then centrifuged to
remove the insoluble fraction. The supernatant was collected,
containing the solubilized fraction. To compare ligand binding and
dissociation of the membrane preparation verses the
solubilized sample, 10 µl of each was added to a glass cuvette along
with 1 nM ligand (fMLFK-FITC) in a final volume of 200 µl
and equilibrated to room temperature for 2 min with stirring. Samples
were placed in the fluorometer, at 20 s an anti-FITC-Ab was added
and then at 120 s GTP S was added. Graph displays plot of
fluorometric analysis of the dissociation of the fluorescent ligand.
B, approximately 10 nM solubilized receptor (10 µl) was incubated at 4 °C in the presence of 10 nM
fMLFK-FITC for the indicated times. Prior to analysis, samples were
equilibrated to room temperature with constant stirring. The
anti-FITC-Ab was added at 20 s and GTP
S at 120 s. Plots
are representative of three experiments each done in duplicate.
-D-maltoside), a sucrose velocity sedimentation assay
was performed. This assay determines the extent of solubilization and
complex formation of proteins. Briefly, the solubilized receptor
fraction is layered onto a 5-20% sucrose gradient. Following
centrifugation, the gradients are fractionated, and the position of the
FPR within the gradient was determined by spectrofluorometric analysis.
The FPR was found in fractions 8-10, sedimenting as a 4 S species, as
expected for a monodisperse receptor (Fig.
2). Also, this further confirmed that the
solubilized FPR was not initially coupled to a G protein.
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Fig. 2.
Fluorescence analysis of FPR in
gradient. Membrane preparations were incubated in the presence of
1% n-dodecyl -D-maltoside for 1 h. The
solubilized fraction was collected and layered on a 5-20% sucrose
gradient as outlined under "Experimental Procedures." Twenty-50
µl fractions were collected, and each was tested for the presence of
FPR using spectrofluorometric analysis. Graph displays the
percent of the total fluorescence of bound ligand present in each
fraction. The positions of 4 S and 7 S standards are indicated.
Experiment was performed twice in triplicate.
i1,2,3 antibody then adding
protein A-agarose to clear the antibody-substrate conjugates. Our
solubilized receptor sample, when treated in this manner, lost all
sensitivity to GTP
S and the non-linear regression fit of the data
indicated only a single exponential decay corresponding to LR. This
suggested that the endogenous G proteins had indeed been removed from
the sample (Fig. 3A). Western
blot analysis of the sample prior to, and after antibody treatment,
confirmed that this procedure effectively cleared the endogenous
Gi proteins from the system. It appeared that the
Gi proteins were present in the heterotrimeric form as the
anti-G
i antibody pulled down both the
subunits and
the
complex (Fig. 3B). A receptor preparation treated
in this way would be appropriate for the analysis of specific
receptor-G protein interactions.
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Fig. 3.
Clearing samples of endogenous G
protein. After the solubilized fraction was collected, 10 µM of an anti-G i1,2,3-Ab was added and
incubated with gentle agitation for 45 min at 4 °C. To remove the
antibody-substrate conjugates, 15 µg of protein A-agarose was added
and sample was incubated again for 30 min at 4 °C. Sample was
microfuged and the supernatant removed. A control sample that
was not treated with antibody (
) and a treated sample
(
) were incubated in the presence of 10 nM fMLFK-FITC
for 2 h at 4 °C. Samples were equilibrated to room temperature
for 2 min with constant stirring prior to fluorometric analysis.
Anti-FITC-Ab was added at 20 s and GTP
S at 70 s. Plot
representative of three experiments, each in duplicate.
i Subunits--
It has been shown that the
FPR interacts with pertussis toxin-sensitive Gi proteins in
neutrophils and U937 cells (9, 10). The predominantly expressed
Gi protein in neutrophils has a G
i2 subunit.
Thus it has been assumed that the FPR preferentially binds to the
G
i2 proteins. Until now it has been difficult to directly assess the affinity between G proteins and receptors. We have
previously demonstrated the ability of solubilized receptors to
reconstitute with a bovine brain G protein mixture with an ED50 of ~10
6 M under conditions
where the ligand and receptor concentrations are ~10 nM
(15). Expanding this study, we examined this interaction using
specific, purified G protein
subunits (G
i1,
G
i2, G
i3, and G
o). The
individual
subunits were first mixed in an equal molar ratio with
bovine brain
to form a G protein heterotrimeric complex (17).
Solubilized receptor, that had been depleted of endogenous G protein,
was incubated with the individually prepared G protein heterotrimers in
the presence of fluorescein-conjugated ligand for 2 h. The
resulting plots, as seen in Fig. 4,
A-D, demonstrate the preference of the FPR for the
G
i subunits as the G
o protein does not
appear to induce the high ligand affinity state of the receptor.
Non-linear regression of the first phase (t = 20-50 s)
of the G
i curves (*) fit to a double exponential (after
subtraction of the free component), indicating that two different rates
were present. The slower rate was 0.0065 ± 0.0020 s
1, representing ligand dissociation from RG (high
affinity state), and the second rate was 0.0309 ± 0.0069 s
1, ligand dissociation from R (low affinity state). The
amplitudes were used to determine the percentage of the receptors in
the sample that were initially coupled to the G protein (Fig.
5). The data indicates that the FPR
interacts with the G
i3-specific G protein with an
apparent Kd of 1 µM with the other
subunits displaying lower affinities (Kd
G
i3
1 µM < Kd
G
i2 < Kd G
i1
Kd G
o). As G protein concentrations
of 3 µM were the highest we could obtain, the
Kd for the other G protein subunits could only be estimated. The G
i1 and G
i2 G proteins
have a Kd greater or equal to 3 µM and
the G
o G protein Kd is likely to be
greater than 10 µM given the levels of receptor-G protein
coupling observed. The G
o data as well as the lower
concentrations of G
i1 and G
i2
(t = 20-120 s) was best fit to a single exponential equation with a rate of 0.0315 ± 0.0052 s
1. The
ligand dissociation after the addition of GTP
S in all the curves
(t = 51-120 s) was also fit by a single exponential
decay equation, exhibiting a rate of 0.0325 ± 0.0063 s
1. These rates represent the mean ± S.E. of the
values obtained in Fig. 4. There was not a systematic difference
between the rates of ligand dissociation from LRG or from LR for any of
the G
subunits as represented in Table
I.
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Fig. 4.
Reconstitution of the FPR with exogenous G
proteins. Purified G subunits G
i1,
G
i2, G
i3, and G
o were
individually combined with bovine brain
in an equimolar ratio
(10 µM) for 15 min on ice. Solubilized protein that had
been cleared of endogenous G proteins was mixed the G
-specific G
proteins and with 10 nM fMLFK-FITC in a volume of 12 µl
and incubated for 2 h at 4 °C. Prior to analysis, sample were
expanded to 200 µl and equilibrated to room temperature for 2 min.
Anti-FITC-Ab was added at 20 s and GTP
S added at 50 s.
A, G
i1 subunit specific G protein;
B, G
i2 subunit specific G protein;
C, G
i3 subunit specific G protein;
D, G
o subunit specific G protein. Experiment
was repeated three times in duplicate with the same results. Data is
representative plots from one experiment.
View larger version (11K):
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Fig. 5.
Statistical analysis of receptor-G protein
coupling. GraphPad Prism software was used to fit the data from
Fig. 3 to single or double exponential decay equations. Samples that
displayed no GTP S sensitivity fit best to a single exponential decay
rate of 0.0315 ± 0.0052 s
1. Runs that display
nucleotide sensitivity were analyzed in two segments. Data collected
from 21 to 50 s fit to double exponential decay equations,
indicating two off-rates. The slower rate was 0.0065 ± 0.0020 s
1 and the second rate was 0.0309 ± 0.0069 s
1. The data from 51 to 120 s fit best to a single
rate of 0.0325 ± 0.0063 s
1. The open
bars represent the percentage of receptors displaying the slow
dissociation rate in the presence of 1.0 µM G protein,
the filled bars are at 3.0 µM G protein.
Asterisk (*), entire curve (21-120 s) was best fit by a
single exponential decay (fast) rate, thus a slow dissociation rate was
not observed. Bars display the mean ± S.E. for three
experiments each done in duplicate.
The fluorometric data from Fig. 4 were analyzed to determine ligand
dissociation rates
S were modeled in two parts, from t = 20-50
seconds and from t = 51-120 s. In these instances, the
data in t = 20-50 s fit to a double exponential decay
equation giving a "slow" rate (k1) and a
"fast" rate (k2). We define the slow rate as L
dissociation from LRG and the fast rate as L dissociation from LR. The
data in the t = 51-120 second time frame fit to a
single exponential decay equation (k). Curves that were
continuous, displaying no sensitivity to GTP
S also fit to a single
exponential decay (t = 20-120 s). Rates are given as
mean ± S.E. in s
1.
subunit and the
complex prior to mixing with
receptor. Mixing the subunits in this way results in them combining to
form an
heterotrimer (17). After reconstituting receptor-G
protein interactions with these heterotrimeric G proteins we next
investigated whether the
subunits or the
complex alone could
generate a high affinity state in the FPR. The solubilized receptor was incubated for 2 h with either a specific
subunit alone or
bovine brain
alone. As it appeared that G proteins containing
the G
i3 subunit bound to the receptor with higher
affinity, the G
i3 subunit was tried first. The
spectrofluorometric analysis showed that neither the G
i3
subunit nor the
complex on their own were able to induce the
slow ligand dissociation rate or any sensitivity to guanine nucleotide
(Fig. 6, A and B).
However, if the subunits are combined as described above, receptor-G
protein coupling was restored (Fig. 6C). This suggests that
the G protein heterotrimer is necessary for generating the high
affinity state of the receptor.
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Fig. 6.
Requirement for the G protein
heterotrimer. Solubilized receptor was cleared of endogenous
Gi protein. The receptor was combined with: A,
the G i3 subunit; B, the
complex; or
C, a previously combined G
i3 +
heterotrimer. Ligand was added (10 nM) and samples were
incubated for 2 h at 4 °C, then were equilibrated to room
temperature for 2 min prior to fluorometric analysis. Anti-FITC-Ab was
added at 20 s and GTP
S at 50 s. Plots are representative
of two experiments done with triplicate samples.
surface
that interacts with the receptor has been well defined in several
systems especially for the visual GPCR rhodopsin and its interaction
with transducin (27). As these studies implicated the C-terminal region
of the subunit as important in binding to the receptor, we next tested
the ability of a series of anti-G
antibodies to interfere with
endogenous G protein coupling to the FPR. The antibodies were added to
the reconstitution assays at the start of the 2-h incubation. Two
anti-G
i antibodies, anti-G
i1,2, and
anti-G
i3 each recognizing the C terminus of the
subunits, was able to inhibit the receptor-G protein interaction (Fig.
7). Conversely, an anti-G
o
C-terminal antibody had no effect and an anti-G
i3
antibody, that recognized an internal sequence, likewise had no effect
(data not shown).
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Fig. 7.
Reconstitution inhibited by
anti-G i-Ab. Reconstitution
assays were performed in the presence of either: A, an
anti-G
i1,2 or B, an anti-G
i3
antibody (both C-terminal). Solubilized receptor samples were not
cleared of G protein, contained receptor at a concentration of ~1
nM and fMLFK-FITC at 10 nM and were incubated
for 2 h on ice. Control samples (
) and samples incubated in the
presence of 3.0 µM antibody (
) were compared.
Spectrofluorometric analysis was done after expanding samples to 200 µl and warming to room temperature for 2 min. Anti-FITC-Ab was added
at 20 s and GTP
S added at 70 s. Data representative of
three experiments, each in duplicate.
C-terminal peptides into the assays.
Three peptides were tested, the first was a G
i1,2
peptide (the last 10 C-terminal amino acids of G
i1 and
G
i2 are identical), the second was the last 10 C-terminal residues of G
i3, and the third was from the
G
s subunit C terminus. Incubation of the solubilized receptor with a 1 µM concentration of the
G
i peptides inhibited reconstitution with endogenous G
proteins (data not shown) and with exogenous G
i proteins
(Fig. 8). In both cases the slow ligand dissociation and guanine nucleotide sensitivity was inhibited in the
presence of the peptide. While both peptides were effective in
disrupting the association of the receptor with the G protein, the
G
i3 peptide EC50 was ~0.1 mM
compared with an EC50 of 1 mM for the
G
i1,2 peptide. The G
s peptide had no
effect on the ligand dissociation kinetics and did not interfere with
the guanine nucleotide sensitivity (data not shown).
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Fig. 8.
Reconstitution inhibited by
G i blocking peptides.
Reconstitution assays were performed in the presence of either:
A, G
i1,2 C-terminal blocking peptide
(H(Cys)-Lys-Asn-Asn-Leu-Lys-Asp-Cys-Gly-Leu-Phe-OH; or B,
G
i3 C-terminal blocking peptide
(H(Cys)-Lys-Asn-Asn-Leu-Lys-Glu-Cys-Gly-Leu-Tyr-OH; reconstitution
assays were prepared in the standard format, solubilized receptor was
combined with 1 µM bovine brain G protein, 10 nM fMLFK-FITC and peptides at the indicated quantities at
the start of the 2-h incubation. Data are representative of three
experiments, each in duplicate.
View larger version (20K):
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Fig. 9.
FPR-arrestin interactions. Solubilized
receptor was combined with 1 µM bovine brain G protein,
10 nM fMLFK-FITC, and control buffer ( -), 3 µM wild type arrestin-3 (
), or 3 µM
truncated arrestin-3-(1-393) (
). Spectrofluorometric analysis was
done adding anti-FITC-Ab at 20 s and GTP
S at 100 s.
Experiment was performed three times, each in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S was added to these samples, a rapid dissociation rate was
induced that fit to a fast single exponential decay rate (the same rate
as k2 and k in Table I) indicating
the conversion of receptors in the RG state to R. This allowed for a
quantitative measure of the ability of the solubilized receptors to
interact with G proteins.
i3 subunit had the highest affinity for the solubilized receptor with a
Kd of ~1 µM (Fig. 4). Obtaining an
accurate Kd for the G
i1 and
G
i2 subunit G proteins was limited by the quantity of
available G proteins. However, given the level of receptor-G protein
coupling at the 3 µM concentration of G protein, the
Kd for G
i2 would be approximately
equal to 3 µM and the Kd for
G
i1 would be greater than 3 µM (Fig. 5).
We had previously reported that the solubilized FPR had an apparent
affinity for bovine brain G protein, which is predominantly
G
o, similar to what we report here for
G
i3 (15). In this prior work, we had not taken into
account the significant contribution endogenous G proteins were having
on the system. Consequently, the bovine brain G proteins were in
addition to the endogenous G proteins, resulting in a higher total G
protein concentration than was used to compute the
Kd. With this knowledge, the current work was
performed with samples in which the endogenous G proteins were cleared
prior to reconstitution with specific subunits.
subunits or the
complex, to
samples precleared of endogenous G proteins, did not induce the high
affinity state indicative of G protein binding (Fig. 6). While it is
not known if these subunits were able to bind to the receptor or not,
they could not induce the resultant state change observed with the
addition of the heterotrimer. This also confirmed that the precleared
samples were adequately depleted of both
and
subunits as the
exogenous addition of either component could not induce the high
affinity state of the FPR. Inhibition of the receptor-G protein
interaction was accomplished through the use of both
anti-G
i antibodies and by G
i C-terminal
peptides. Only antibodies to the G
i subunits were
effective in disrupting the receptor-G protein interaction. However,
the anti-G
i1,2 and the anti G
i3
antibodies acted equally (Fig. 7). The similarity of the two
G
i antibody preparations could result from the
relatively common peptide sequences which serve as antigens, even
though the antibodies have been reported as non-cross-reactive in
Western blots. The antibody behavior was in contrast to the results
obtained with the G
i peptides. Concentration curves
demonstrated that the G
i3 peptide EC50 for
inhibition was about 10-fold lower than that of the
G
i1,2 peptide (Fig. 8). This correlated well with our
previous results showing that G proteins containing the
G
i3 subunit bound to the FPR with higher affinity. The
ability of antibodies and especially small peptides to specifically
inhibit the receptor-G protein interaction could prove useful in the
development of drugs to modify cellular responses by interfering with G
protein activation.
i2 and only small amounts of
G
i3 (9, 10). Physiologically, it appears as though the
FPR may transduce most of its signal through G
i2
activation even though it may bind preferentially to
G
i3. There may be other determinants that operate at the
receptor-G protein interface that alter signaling specificity or
efficiency. Cell architecture or accessory proteins may differ between
cell types that influence potential interactions. A previous report
analyzed FPR interactions with the three G
i isoforms and
found that the receptor coupled with each with similar efficiency (11).
The assay fused the FPR to G
i1, G
i2, or
G
i3 and expressed the fusion proteins in Sf9
cells. The use of the fusion proteins creates a high G protein
concentration within the microdomain of the receptor, making it
difficult to discern difference in binding affinities that are
evidenced in the detergent soluble complexes.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM60799 and the New Mexico Cancer Research Fund RR01315 (to L. A. S.), National Institutes of Health Grants NIAID36357 (to E. R. P.) and EY11500 (to V. V. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cancer Center, 2325 Camino de Salud, CRF219A, University of New Mexico, Albuquerque, NM
87131. E-mail: lsklar@salud.unm.edu.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M009679200
2 T. A. Bennett and E. R. Prossnitz, unpublished observation.
3 T. A. Key, T. A. Bennett, T. D. Foutz, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
G protein, guanine nucleotide-binding
regulatory proteins;
L, ligand;
R, receptor;
G, G protein;
FPR, N-formyl peptide receptor;
FITC, fluorescein
5-isothiocyanate;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
Ab, antibody;
PIPES, 1,4-piperazinediethanesulfonic acid.
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