From the
G protein-coupled seven-transmembrane-containing receptors, such
as the N-formyl peptide receptor (FPR) of neutrophils, likely
undergo a conformational change upon binding of ligand, which enables
the receptor to transmit a signal to G proteins. We have examined the
functional significance of numerous conserved charged amino acid
residues proposed to be located within or near the transmembrane
domains. Whereas the wild type FPR exhibits a K
The activation of cellular processes through the binding of
agonists to cell surface membrane receptors is of central importance to
numerous aspects of biology. The recent explosion in the cloning of
cell surface receptors has demonstrated that a large number of these
signal-transducing receptors are coupled to GTP-binding regulatory
proteins (G proteins)
The ternary complex model (TCM) is the most accepted model
for describing the activation of G protein-coupled receptors
(3) . It describes the active form of the receptor as a ternary
complex among ligand, receptor, and G protein formed as a result of the
sequential association of ligand and G protein, in either order, with
receptor. The more favorable sequence is traditionally thought to
involve ligand binding to receptor followed by G protein binding. This
simple form of the TCM has been found to be inadequate for the
evaluation of recently described experiments
(4, 5) .
Characterization of constitutively active ( i.e. ligand-independent) mutants of
The FPR of neutrophils is representative of the class of G
protein-coupled receptors. Neutrophils respond to a large number of
structurally diverse ligands with functions such as chemotaxis,
superoxide production, and degranulation. Many of these chemotactic
receptors have recently been cloned, including the receptors for
N-formyl peptides
(8) , complement component C
G protein-coupled receptors may represent the largest class
of signal-transducing molecules, and as such they bind a vast array of
ligands and couple to many distinct intracellular effectors via a large
collection of G proteins. Although all members of this class contain
the canonical seven-transmembrane domain motif, they possess
considerable sequence diversity to accommodate the multitude of
functions they perform. Despite this diversity, G protein-coupled
receptors contain certain amino acid residues that are highly conserved
throughout the entire class. Perhaps the most distinct of these is the
Asp-Arg-Tyr triplet at the boundary between transmembrane domain 3
(TM3) and the second intracellular loop. Another almost invariant
residue, an Asp, is found in the middle of TM2. Highly conserved among
the muscarinic, adrenergic, dopaminergic, serotonin, and a number of
other G protein-coupled receptors is an Asp in the middle of TM3. In
some instances, this and the other conserved Asp residues are replaced
by the similarly charged acidic amino acid, Glu. We have also noted a
weak homology at the boundary between the TM7 and the carboxyl
terminus. This consensus consists of a Phe preceded and/or followed by
one or more basic amino acids. Examples of this include FR ( e.g. opsins), FRR ( e.g.
To examine the functional capabilities of the mutant forms
of the FPR, we measured the ability of the transfected cells to
mobilize calcium. As we have previously reported, addition of fMLP to L
cells transfected with the wild type FPR resulted in a dose-dependent
increase in intracellular levels of calcium with an EC
In this study, we have utilized site-directed mutants of a
well characterized G protein-coupled receptor, the FPR, to investigate
mechanisms of G protein coupling. We hypothesized that amino acid
residues strictly conserved between virtually all G protein-coupled
receptors would be critical to receptor function. Of the six mutants
generated, two appeared not to be functionally expressed at the cell
surface (D106A and D122A), whereas the remaining four were not only
expressed at the cell surface but were capable of binding ligand.
Detailed ligand binding analyses revealed that only one of these four
mutants (R163F) demonstrated high affinity, GTP
These results indicated that the D71A, R123G, and
R309G/E310A/R311G mutant FPRs were incapable of a productive
interaction with G protein. The data, however, did not address the
nature of the defect. To test whether the mutant forms of the FPR were
capable of physically interacting with G protein, the formation of a
receptor-G protein complex was determined. Formation of a physical
complex correlated well with the functional abilities of the mutant
receptors. These results suggest that either 1) the mutations occur at
the receptor-G protein interface, preventing their interaction, and as
a result the receptor exists in a low affinity state or 2) the
mutations prevent the receptor from switching to the high affinity
conformation and that this prevents the interaction with G protein. The
locations of the mutations provide some insight into the interpretation
of the results. The R123G and R309G/E310A/R311G mutations exist at the
membrane-cytoplasm interface of the receptor. It is therefore possible
that either of the two mechanisms is responsible for the uncoupling
observed with these mutants. However, the D71A mutation occurs in the
middle of the second transmembrane domain, making it unlikely that this
site interacts directly with G protein. As a result, the most probable
mechanism of uncoupling for this mutant is the stabilization of the low
affinity state (or conversely destabilization of the high affinity
state) of the FPR.
Our results suggest that the unliganded form of
the wild type FPR may be similarly inefficient at binding G protein. We
propose that only upon ligand binding, with its subsequent
conformational change, can the receptor bind G protein with high
affinity. These results require an additional intermediate to be
introduced into the ternary complex model (Fig. 8). The receptor
(R) exists in an inactive state (R
Sklar and co-workers
(6, 7) have proposed a model based on kinetic measurements in which
the N-formyl peptide receptor population exists in two states:
an uncoupled or slowly coupling state and a G protein precoupled or
rapidly coupling state representing up to 50% of the total receptor. In
these studies, comparable rates for the formation of the LR binary
complexes and LRG ternary complexes were observed. Fitting of the
experimental data to the TCM was significantly improved by allowing
precoupling of the G protein to the receptor. The results however could
not distinguish rapid coupling (half-times of less than 1 s) from
precoupling. Our data suggest that the inactive receptor
(LR
In this study, we have shown
that inactive receptor mutants are unable to bind G protein in a manner
similar to that of the liganded wild type receptor, suggesting the low
affinity inactive state of the wild type receptor has a significantly
reduced affinity for G protein. Furthermore, we suggest that the
ligand-induced change in the wild type receptor conformation is
critical for the receptor-G protein binding interaction to take place.
Constitutively inactive receptor mutants should provide a valuable tool
for studying the molecular properties of both the inactive and the
active receptor state.
Ligand binding assays were performed as described
under ``Experimental Procedures'' in either the presence or
absence of 2 µM GTP
We acknowledge Larry Sklar for helpful discussions and
Stacey Cavanagh and Daniel Cheng for excellent technical support.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
for an agonist of 1-3 nM, which is reduced to
40 nM in the presence of guanosine
5`-3- O-(thio)triphosphate (GTP
S), substitution of either
Asp
or Arg
resulted in mutant receptors that
bound ligand with only low affinity ( K
= 30-50 nM) independent of GTP
S. In
contrast, substitution of Arg
, predicted to be located at
a similar depth within the membrane as Asp
, had no effect
on ligand binding. Replacement of residues
Arg
-Glu
-Arg
resulted in an
FPR with intermediate ligand binding characteristics. Functional
analysis of the mutant receptors revealed that substitution of either
Asp
or Arg
resulted in a mutant receptor
that was unable to mediate calcium mobilization, whereas replacement of
residues Arg
-Glu
-Arg
yielded
a receptor with an EC
of 50 nM, compared with 0.5
nM for the wild type FPR. In order to determine the point of
the defect in signal transduction, we performed reconstitution of the
solubilized receptors with purified G proteins. The wild type FPR
displayed a K
for G protein of
0.6
µM compared with the
Arg
/Glu
/Arg
mutant with a
K
of approximately 30 µM. A
significant physical interaction between the Asp
or
Arg
mutants and G protein was not observed. The
implications of these findings for signal transduction mechanisms are
discussed.
(
)
(for a review, see Refs.
1 and 2). The class of G protein-coupled receptors is typified by its
unique membrane topology of seven-transmembrane-spanning domains. Under
physiological conditions, transmembrane signaling is initiated by the
binding of ligand to the extracellular surface of the receptor. This is
proposed to induce a conformational change in the receptor resulting in
the activation of a G protein and the subsequent activation of
downstream effectors. Despite recent advances in the study of G
protein-coupled receptors, little is known regarding the structural and
functional differences between the various states of the receptor, such
as the liganded, activated receptor and the unliganded, inactive
receptor.
- and
-adrenergic
receptors has led to the conclusion that the unliganded form of
receptors exists in an equilibrium between active and inactive states
and that the binding of ligand serves to shift the equilibrium from the
inactive to the active state. Only this active state is capable of
initiating signal transduction. The TCM of signal transduction has also
been investigated by studies of conformational changes in the
N-formyl peptide receptor (FPR) through rapid kinetic
spectrofluorometric methods using fluorescent chemotactic peptides as
ligands
(6, 7) . This technique takes advantage of the
large difference (approximately 2 orders of magnitude) in dissociation
rates between the high and low affinity states of the FPR. Analysis of
such kinetic data has shown that the assembly of the ternary complex is
rapid, occurring with a half-time of less than 1 s, suggesting that a
portion of the receptor population may be precoupled to G protein.
(9) , platelet-activating factor
(10) , and
interleukin-8
(11, 12) . These receptors are all members
of the class of G protein-coupled receptors, appearing to couple
through the pertussis toxin-sensitive G
subtype of G
protein. Recent work has suggested that the FPR couples to G proteins
via its second intracellular loop and carboxyl-terminal domain but that
the third intracellular loop is of only minor significance
(13, 14) . The carboxyl-terminal domain has also been
shown to be a substrate for G protein-coupled receptor kinase 2,
suggesting this kinase is involved in phosphorylation and
desensitization of the FPR
(15) . In this study, we describe the
characterization of FPR mutants in the second transmembrane domain, the
second intracellular loop, and the carboxyl-terminal domain that are
unable to bind ligand with high affinity and are also incapable of
signal transduction, suggesting a defect in receptor-G protein
interactions. Since it is known that the high affinity, liganded state
of the wild type FPR forms a stable physical complex with G protein
(16) , we used these mutants to test whether the low affinity
state of the FPR is similarly capable of interacting with G protein.
The results will provide important mechanistic information regarding
the dynamics of G protein-coupled receptors.
Materials
The cDNA that encodes the FPR was
obtained from a human HL-60 granulocyte library as described previously
(17) . Restriction enzymes, T4 DNA ligase, lipofectamine, G418,
and trypsin-free dissociation buffer were from Life Technologies, Inc.
Oligonucleotides were obtained from Operon. Mutagenesis was carried out
using the Amersham Corp. oligonucleotide-directed mutagenesis system.
Sequencing was carried out using Sequenase version 2.0 (U. S.
Biochemical Corp.). Bacterial cells were grown in Circlegrow medium
(BIO 101). fML[H]P (specific activity, 56
Ci/mmol) was obtained from DuPont NEN, and unlabeled fMLP was purchased
from Sigma. Carrier-free sodium [
I]iodide was
obtained from Amersham Corp.
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and indo-1/AM
were obtained from Molecular Probes. Mouse L cell fibroblasts (L2071)
were obtained from ATCC. Dulbecco's modified Eagle's medium
was from Whittaker Bioproducts; fetal bovine serum was from HyClone.
Enzymobeads were from Bio-Rad, and
sulfosuccinimidyl-2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionate
was from Pierce.
Construction and Expression of Site-directed
Mutants
The FPR gene was subcloned into the EcoRI site
in the polylinker of M13 mp18
(13, 17) . Single-stranded
M13 DNA in conjunction with mutant oligonucleotides (containing the
altered nucleotides necessary to generate the desired mutations) was
used to introduce mutations into the FPR gene. Plaques were amplified,
and the mutations were confirmed by dideoxy sequencing. For expression,
the mutated FPR genes were subcloned into the EcoRI site of
the vector pSFFV.neo, which contains the selectable marker
aminoglycoside transferase. Mutations were reconfirmed by dideoxy
sequencing prior to transfection. Mouse L cell fibroblasts were
transfected as follows. Approximately 10 cells were plated
out in a 25-cm
flask 20 h prior to transfection with 10
µg of linearized vectors by lipofectamine. Transfectants were
selected by their resistance to G418 sulfate at an active drug
concentration of 0.35 mg/ml. For each mutant, approximately 20-50
individual colonies from transfected cells were pooled for analysis.
Flow Cytometry
Mouse L cell fibroblasts were
detached from the culture flask with protease-free dissociation buffer,
harvested by centrifugation, washed once with phosphate-buffered
saline, and resuspended to 10 cells/ml in
phosphate-buffered saline. Binding was carried out in 0.5 ml with
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein at 20
nM. Following incubation for at least 15 min on ice, the cells
were analyzed on a FACScan flow cytometer (Becton Dickinson) for
fluorescent intensity. Debris and dead cells were excluded with a gate
on forward and side scatter. Nonspecific binding was determined in the
presence of 1 µM N-formyl-Met-Leu-Phe.
Membrane Preparation
Cells were detached from
culture flasks with protease-free dissociation buffer, collected at 500
g, and resuspended at 10
cells/ml in 10
mM PIPES (pH 7.3), containing KCl (100 mM), NaCl (3
mM), MgCl
(3.5 mM), ATP (0.6 mg/ml), and
the protease inhibitors chymostatin, phenylmethylsulfonyl fluoride and
diisopropyl fluorophosphate (Sigma). The cells were disrupted by
N
cavitation (500 p.s.i., 15 min, 4 °C), and the cell
nuclei and unbroken cells were removed by centrifugation at 1000
g for 5 min. The membranes were collected by
sedimentation at 130,000
g for 45 min, resuspended at
a protein concentration of 5 mg/ml in 25 mM HEPES (pH 7.0),
200 mM sucrose, and stored at -80 °C until use.
Photoaffinity Labeling of the FPR
Membranes were
photoaffinity-labeled with 20 nM N-formyl-Met-Leu-Phe- N-(2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionyl)-Lys
(fMLPK-[
I]SASD) as described previously
(18, 19) . Briefly, N-formyl-Met-Leu-Phe-Lys,
dissolved in dry dimethylformamide, was added to equimolar amounts of
sulfosuccinimidyl-2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionate
and triethylamine and incubated overnight in the dark at room
temperature. fMLPK-SASD was radiolabeled using 5 mCi of
Na
I and the Enzymobead reagent as described by the
manufacturer. The mixture was then chromatographed on a Bio-Gel P-2
(extra fine) equilibrated in 20 mM NaOH to obtain the
fMLPK-[
I]SASD, which eluted in the void volume.
FPR-expressing fibroblasts and their respective membrane preparations
were covalently labeled as follows. Cells (0.5
10
)
were harvested and resuspended in 200 µl of HEPES-buffered saline.
Membranes (50 µg) were diluted to 200 µl of HEPES-buffered
saline. fMLPK-[
I]SASD (5-10 µl of the
eluted sample, representing a final concentration of 10-30
nM) was added in the absence or presence of 1 µM
unlabeled fMLP to assess nonspecific labeling. Following a 10-min
incubation on ice, the radiolabel was covalently incorporated by
exposure on ice to UV light in a Rayonet ultraviolet light reactor
irradiating at 370 nm for 5-10 min. Labeled samples were
centrifuged and washed two times with HEPES-buffered saline prior to
further analysis.
Quantitative Radioligand Binding
Ligand binding
assays were performed on membrane preparations in a final volume of 0.2
ml. Membranes (30 µg of protein) were suspended in the binding
buffer (pH 7.4) consisting of 140 mM NaCl, 1.0 mM
KHPO
, 5 mM
Na
HPO
, 1.5 mM CaCl
, 0.3
mM MgSO
, 1 mM MgCl
, and 0.2%
bovine serum albumin. Binding was started by the addition of various
amounts of [
H]fMLP. Equilibrium binding was
carried out at 23 °C for 45 min and terminated by rapid filtration
through Whatman GF/C filters followed by three washes with 0.75 ml of
ice-cold binding buffer. Specific binding was calculated as total
binding minus nonspecific binding, which was determined in the presence
of 50 µM unlabeled fMLP. Each determination was done in
duplicate. The amount of bound ligand was estimated by scintillation
counting, and the binding data were analyzed by fitting to a double
rectangular hyperbola with the nonlinear curve fitting program,
SigmaPlot (Jandel Scientific). Measurement of
[Ca
]
-Cells were
harvested in dissociation buffer, washed once with phosphate-buffered
saline, and resuspended at 5
10
cells/ml in RPMI
1640 medium containing 10% fetal bovine serum. The cells were incubated
with 5 µM indo-1/AM for 25 min at 37 °C, washed once
with medium, and resuspended to a concentration of approximately
10
cells/ml. The elevation of intracellular Ca
by various amounts of fMLP was monitored by continuous
fluorescent measurement using an SLM 8000 photon-counting
spectrofluorometer (SLM-Aminco) detecting at 400 and 490 nm,
respectively, as described
(17) . The concentration of
intracellular Ca
was calculated as described
(20) .
Purification of G Proteins
G (containing subtypes G
and G
) was
purified from bovine brain essentially as described
(21) . After
the heptylamine-Sepharose purification step, resolution of G
from G
was achieved by purification on a 20-ml
DEAE-Sephacel column, which was equilibrated with TENL (25 mM
Tris-HCl, pH 8.0, 1 mM EDTA, 0.6% Lubrol) and eluted with a
200-ml linear gradient of 0-250 mM NaCl in TENL.
G
-enriched fractions were pooled and judged by silver stain
following SDS-polyacrylamide gel electrophoresis to contain
approximately 80% G
and 20% G
.
FPR/G Protein Reconstitution and
Analysis
Reconstitution was performed as described previously
(22) . Briefly, membranes, photoaffinity-labeled with
fMLPK-[I]SASD, were extracted with 1%
octylglucoside in 10 mM HEPES, pH 7.4, 100 mM KCl, 10
mM NaCl, 0.1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml chymostatin for 1 h on
ice. Extracts were incubated with or without G
for 2 h at 4
°C and then applied to 700 µl of 5-20% linear sucrose
gradients prepared in the extraction buffer. Gradients were centrifuged
at 45,000 rpm in an SW 50.1 rotor (Beckman) for 13.3 h and fractionated
into 14
50-µl fractions. Fractions were subjected to
SDS-polyacrylamide gel electrophoresis, followed by autoradiography to
confirm the specific labeling and distribution of the receptor.
Gradients containing 20 µg of bovine serum albumin (4.4 S) and
rabbit immunoglobulin (7.7 S) were centrifuged in parallel with
FPR-containing gradients. Based on the sedimentation of these standard
proteins, the migration of 4 S and 7 S proteins was calculated to peak
in fractions 6 and 10, respectively.
-adrenergic), FKK ( e.g. m4 muscarinic), FRK (D2 dopamine), KKFRKH ( e.g. platelet-activating factor), and KFRH ( e.g. interleukin-8). As a model G protein-coupled receptor to study the
role of these conserved residues, we have utilized the FPR, which
possesses all of these consensus residues. Instead of a Tyr at the
third position of the Asp-Arg-Tyr triplet, the FPR contains a Cys,
found in a small number of G protein-coupled receptors. The consensus
at the TM7/cytoplasm boundary of the FPR consists of the sequence FRER,
which is consistent with the pattern of sequences outlined above,
although the presence of an acidic residue near the Phe residue is
unusual. For the purposes of this study, we altered the conserved amino
acids described above as illustrated in Fig. 1. Arg
was also mutated because it represents a charged residue with a
predicted position in the fourth transmembrane domain at a similar
depth to Asp
.
Figure 1:
Schematic
representation of the structure of the FPR and the locations of
site-directed mutants. Opencircles represent
individual amino acids of the FPR; solidcircles,
with the indicated amino acid changes, show the residues that were
mutated. Potential N-linked glycosylation sites are also
indicated ( CHO). RER/GAG,
R309G/E310A/R311G.
Following the generation of the desired
mutations by site-directed mutagenesis, the mutant recombinant FPR
constructs in the expression vector pSFFV.neo were stably transfected
into mouse L cell fibroblasts. We have previously shown that these
cells are capable of expressing functional FPR upon transfection with
the wild type gene. After selection with G418, the stable transfectants
were analyzed for surface expression of the FPR. This was accomplished
in two ways: 1) by flow cytometry using a fluorescein-conjugated
hexapeptide ligand and 2) by covalent photoaffinity labeling using an
iodinated photoactivatable tetrapeptide ligand followed by
SDS-polyacrylamide gel electrophoresis. The results are shown in
Fig. 2
. Whereas mock-transfected cells show no specific binding
of either of the two ligands ( panelA), wild type
FPR-transfected cells show binding of both the fluoresceinated ligand,
as indicated by the increase in cell-associated fluorescence ( panelB), and the iodinated ligand, as evidenced by the
presence of a band of molecular mass 55,000-70,000 kDa upon
SDS-polyacrylamide gel electrophoresis ( panelB,
inset). In both cases, the binding of the ligand was
demonstrated to be specific by the addition of excess fMLP, which
competed for the binding of the labeled ligand. The addition of excess
unlabeled fMLP to the mock-transfected cells had no effect on the
nonspecific background binding of either of the labeled ligands.
Analysis of the mutant forms of the FPR revealed that mutants D71A,
R123G, R163F, and R309G/E310A/R311G were capable of binding
formyl peptide ligands to a similar extent as the wild type receptor.
However, the mutants D106A and D122A displayed no specific binding of
either ligand, suggesting that either these two mutants were not
expressed at the cell surface or that they were expressed at the cell
surface but were incapable of binding the ligand.
Figure 2:
Cell surface expression of the wild type
and mutant forms of the FPR. Expression was evaluated for cells
transfected with vector only, the wild type FPR, and each mutant FPR by
flow cytometry, plotting cell number versus fluorescent
intensity ( panelsA-H). Binding was determined
with N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein at 20
nM. The inset within each panel shows the
profile of membrane proteins photoaffinity-labeled with approximately
20 nM fMLPK-[I]SASD analyzed on a 12%
SDS-polyacrylamide gel. The major labeled protein, representing the
FPR, has a molecular mass of 55,000-70,000 kDa. In each assay,
binding was determined in the absence (-) and presence (+)
of 1 µM N-formyl-Met-Leu-Phe. Data are
representative of two to four experiments. RER/GAG,
R309G/E310A/R311G.
To examine the
ligand binding characteristics of the mutants in greater detail, we
performed fML[H]P binding assays using membranes
prepared from the transfected cells. Analysis of the wild type FPR
revealed the expected properties, high affinity binding representing
2.3 pmol/mg protein with a K
of 2.7
nM, which could be converted to low affinity binding by the
addition of 2 µM GTP
S, yielding a
K
of approximately 40 nM with a
receptor density of 1.6 pmol/mg of protein (Fig. 3 A,
). The low affinity state of the receptor generated in the
presence of GTP
S is presumed to be the result of the irreversible
activation and dissociation of G proteins from receptors and results in
an apparent reduction in the receptor density as determined by
filtration binding. Scatchard analysis as well as nonlinear curve
fitting of the binding data revealed the presence of a single site in
both the presence and absence of GTP
S (). Of the
mutant receptors observed to bind the fluoresceinated ligand, only the
R163F mutant displayed binding affinities characteristic of the wild
type receptor, although in the absence of GTP
S both the high and
low affinity sites were observed (approximately 1 nM and 20
nM, respectively). Binding of fMLP to the wild type FPR of
human neutrophils and HL60 cells routinely displays both high and low
affinity sites in the absence of GTP
S, with the receptor
population in the high affinity state being sensitive to GTP
S, as
observed here with the R163F mutant.
Figure 3:
Ligand binding characteristics of the FPR
mutants. Specific fML[H]P binding to isolated
membranes from cells expressing the wild type ( panelA) and the following mutant forms of the FPR: D71A
( panelB), R123G ( panelC), R163F
( panelD), and R309G/E310A/R311G ( RER/GAG)
( panelE). Binding was performed in the absence
(
) or presence (
) of 2 µM GTP
S. Binding
data were fit with a one-site model, except for the R163F mutant in the
absence of GTP
S, which was better fit with a two-site model. Data
are representative of three to five binding
assays.
Two of the mutants, D71A and
R123G, exhibited fMLP binding with a Kof
approximately 30-50 nM. This binding was unaffected by
the presence of GTP
S, suggesting that even in the absence of
GTP
S the FPR was unable to exist in the high affinity state. The
receptor densities for the D71A and R123G mutants were 0.4 and 0.9
pmol/mg protein, respectively. Although these values represent a small
percentage of the number of sites determined for the wild type FPR in
the absence of GTP
S, they represent up to 60% of the sites
determined for the wild type FPR in the presence of GTP
S, a more
valid comparison since this state of the FPR represents the uncoupled
form of the receptor. Analysis of the R309G/E310A/R311G mutant revealed
that its binding properties were intermediate between that of the wild
type FPR and mutants D71A and R123G. In the absence of GTP
S, this
mutant predominantly displayed a K
of 40
nM with a receptor density of 1.9 pmol/mg of protein
(representing 97-100% of the receptor sites). Scatchard analysis
did reveal the presence of a very small population of receptors with
high affinity (1.2 ± 0.9 nM) representing only
2-3% of the receptor sites. In the presence of GTP
S, no high
affinity binding was observed, but the number of low affinity sites was
reduced to 1.2 pmol/mg of protein with an unaltered
K
. This level of reduction in the
apparent number of fML[
H]P binding sites assayed
in the presence of GTP
S, is similar to that seen with the wild
type FPR.
of
0.5 nM (Fig. 4). Similar results were obtained with
cells expressing the R163F mutant. Cells expressing the
R309G/E310A/R311G mutant were also able to initiate calcium
mobilization upon the addition of fMLP; however, higher concentrations
of ligand were required (EC
50 nM), and the
maximal response was less than that observed for the wild type
receptor. Mutant D71A did not display any calcium mobilization up to
fMLP concentrations of 10 µM; however, at 100
µM fMLP a small but reproducible amount of calcium
mobilization was observed. The remaining mutant capable of binding
fMLP, R123G, was unable to mobilize calcium even at the highest
concentration of fMLP, 100 µM. Higher concentrations of
fMLP could not be used due to solubility limits of this hydrophobic
molecule. The remaining two mutants created in this study, namely D106A
and D122A, were also tested for calcium mobilization. Since our
filtration binding assays can determine binding only up to
approximately 500 nM, we considered the possibility that
either or both of these mutants might not display detectable binding
but, if functional, might exhibit signal transduction at the very high
fMLP concentration possible in our functional assay. This was, however,
not the case since both mutants displayed no calcium mobilization at
100 µM fMLP.
Figure 4:
Calcium mobilization by the wild type and
mutant forms of the FPR. Cells expressing the wild type () and
mutant forms of the FPR (D71A (
), D106A (
), D122A (
),
R123G (
), R163F (
) and R309G/E310A/R311G (RER/GAG)
(
)) were analyzed for fMLP-stimulated elevation of intracellular
calcium. The mutants D106A, D122A, and R123G exhibited no measurable
calcium mobilization even at 100 µM fMLP. The extent of
the increase in intracellular calcium is expressed as a percentage of
the maximal response for each cell type at saturating concentrations of
the heterologous ligand, ATP. Data are representative of three
experiments.
The data presented to this point suggest
that the mutants D71A and R123G are functionally uncoupled from G
proteins. The nature of this uncoupling cannot be determined from the
experiments already described. A number of possible mechanisms exist to
explain this phenomenon: 1) G protein can bind to the FPR, but this
binding neither results in conversion of the FPR to the high affinity
state nor signal transduction; 2) G protein cannot bind to the receptor
because the mutants created exist at the interaction site(s) between
the FPR and G protein, and 3) the mutant forms of the FPR are trapped
in the low affinity state, which is inherently incapable of binding G
protein. In order to gain more insight into the mechanism of uncoupling
we performed quantitative physical reconstitution between the wild type
and mutant forms of the FPR and purified G protein. This assay
determines the extent of complex formation between the FPR and G protein by sucrose density sedimentation. Briefly, FPR is
photoaffinity-labeled with fMLPK-[
I]SASD and
extracted from membranes with octylglucoside. The extracted receptor is
incubated with varying amounts of purified G protein and layered onto a
5-20% sucrose gradient. Following centrifugation, the gradients
are fractionated, and the position of the FPR within the gradient is
determined by analyzing the fractions on SDS-polyacrylamide gel
electrophoresis. In the absence of G protein, the FPR is found in
fractions 5-7, sedimenting as a 4 S species (Fig. 5). With
a saturating amount of G protein, the FPR is found in fractions
9-11, sedimenting as a 7 S species. At intermediate amounts of G
protein the sedimentation profile appears to represent a combination of
the complexed and uncomplexed forms of the FPR. The EC
for
formation of the 7 S complex was determined to be
0.6
µM. Previous reports of this value have ranged from
0.2-4 µM, the variation possibly being due to
the preparation of G protein. That the shift of the FPR from 4 S to 7 S
results from a specific binding interaction between the FPR and G
protein was confirmed by the addition of GTP
S, which activates and
dissociates G protein from the receptor, resulting in the conversion of
the 7 S species to the 4 S species despite the presence of G protein.
Figure 5:
Conversion of the wild type FPR from the 4
S to the 7 S form by reconstitution with G protein. Fractions from
gradients containing photoaffinity-labeled wild type FPR reconstituted
with increasing amounts of G protein as indicated were
solubilized in sample buffer and applied to SDS-12% polyacrylamide
gels. GTP
S was added to a final concentration of 2
µM. The positions of 4 S and 7 S standards are
indicated.
Mutant forms of the FPR capable of binding ligand were similarly
analyzed for their ability to complex with G protein (Fig. 6).
All of the mutants sedimented as 4 S species in the absence of added G
protein. In contrast to the wild type FPR (Fig. 6 A), the
mutants D71A (Fig. 6 B) and R123G
(Fig. 6 C) failed to form a complex with 4
µM G protein, a quantity sufficient to convert essentially
all of the wild type FPR to the 7 S species. At very high
concentrations of G protein (30 µM), the mutants underwent
only a minimal shift in their sedimentation profiles, similar to the
shift observed with the wild type FPR at a concentration of G protein
of about 0.1 µM. However, the addition of GTPS to the
samples containing 30 µM G protein resulted in no change
of the sedimentation profile, suggesting that this receptor species was
not a result of an active G protein complex. The mutant R163F, which
displayed high affinity ligand binding and almost wild type signal
transducing properties, formed a GTP
S-sensitive complex with
purified G protein with an EC
similar to that of the wild
type FPR,
1 µM G protein (Fig. 6 D).
The R309G/E310A/R311G mutant, like mutants D71A and R123G, also
displayed little change in the sedimentation profile at 4
µM G protein; however, at higher G protein concentrations,
this mutant displayed a partial shift in its sedimentation profile
(Fig. 6 E). The profile at 30 µM G protein
appeared to represent an equal distribution between complexed and
uncomplexed receptor, similar to that seen with the wild type FPR at G
protein concentrations of 0.4-0.8 µM (Fig. 7).
That the complex of the R309G/E310A/R311G mutant with 30
µM G protein represented a wild type-like complex was
assessed by determining the guanine nucleotide sensitivity of the
complex. Addition of GTP
S to the sedimentation assay converted the
complexed portion of the R309G/E310A/R311G mutant to an uncomplexed 4 S
species, suggesting the complexed species is a functional complex.
Figure 6:
Physical reconstitution of the mutant
forms of the FPR with G protein. Sedimentation profiles were determined
for photoaffinity-labeled membranes from cells expressing the wild type
FPR ( panelA) and the following mutant forms of the
FPR: D71A ( panelB), R123G ( panelC), R163F ( panelD), and
R309G/E310A/R311G ( panel E). The G protein concentration used
for each reconstitution is indicated.
Figure 7:
Quantitative analysis of complexed wild
type and mutant R309G/E310A/R311G FPR as a function of G protein
concentration. The fraction of complexed () and uncomplexed
(
) receptor was evaluated as a function of G protein concentration
for the wild type FPR ( panelA) and the
R309G/E310A/R311G mutant ( panelB). Uncomplexed
receptor was taken as that receptor in fractions 5, 6, and 7, whereas
complexed receptor was assessed in fractions 9, 10, and 11. In the
presence of 2 µM GTP
S, the fraction of complexed
(
) and uncomplexed (
) receptor was also
determined.
S-sensitive fMLP
binding as seen with the wild type receptor. The remaining three
mutants (D71A, R123G, and R309G/E310A/R311G) did not exhibit high
affinity, GTP
S-sensitive fMLP binding. Functional analysis showed
that of these three mutants, only the R309G/E310A/R311G mutant was
capable of signal transduction, although at fMLP concentrations
approximately 100-fold higher than that required for the wild type
receptor.
), which has a low
affinity for both ligand and G protein. In this model, the binding of
ligand follows a two-step process, first forming an inactive complex
(LR
), which can proceed to the activated
ligand-receptor complex (LR
). Through the cross-linking of
a photoactivatable ligand to the receptor, we are able to generate a
stable form of the wild type LR
state, or LR
state in the case of the mutant receptors. This wild type state
of the receptor exhibits a high binding affinity for G protein, which
binds to form the active ternary complex (LR
G). It is our
contention that mutants, such as the D71A FPR, prevent the conversion
of the LR
to the LR
state, preventing
association with G protein. Previous mutagenesis studies of other G
protein-coupled receptors have characterized nonfunctional receptors
capable of ligand binding; however, these studies could not identify
the nature of the receptor coupling defect (reviewed in Ref. 23).
Figure 8:
Revised model for the interaction of
seven-transmembrane receptors with ligand and G protein. In this model,
inactive receptor (R) binds ligand (L), which leads
to the formation of an inactive intermediate (LR
)
with its subsequent conversion to an active ligand-receptor complex
(LR
). This form then interacts with and activates G
proteins (G), leading to cellular activation. Also shown is the
equilibrium between inactive (R
) and spontaneously
active receptor (R
).
Mutations of the -adrenergic receptor that result in its
ligand-independent ( i.e. constitutive) activation have
recently been described
(4, 5) . To accommodate these
findings, the basic TCM was modified in such a way as to introduce a
step representing the spontaneous conversion of R
to
R
, resulting in ligand-independent activation of G proteins
(see Fig. 8). The assumption was made that only the activated
form of the receptor, R
, could activate G protein. In the
case of the wild type receptor, a small fraction of receptor would
exist in the active state, resulting in basal activity. Although the
energy of activation of the receptor should be sufficiently low that
the binding of a small ligand can provide sufficient energy to alter
the conformation of the receptor into its active state, the barrier to
activation should also be sufficiently high so that in the absence of
ligand the receptor spends little time in the spontaneously active
R
state. It was concluded that the effect of the mutations
was to mimic the effect of ligand binding and to convert the receptor
to an active state. It has since been shown that this constitutively
active mutant is also recognized by receptor kinases, further
suggesting that the conformation of the mutant receptor is that of the
active state
(24) . Despite these advances, little is known
regarding the nature of receptor activation and the differences between
the liganded and unliganded receptor and how these forms of the
receptor interact with G protein.
and presumably R
) has a low
affinity for G protein, at least 2 orders of magnitude lower than that
of the ligand-activated receptor (LR
). This makes the
existence of precoupled high affinity receptor questionable and
supports the alternative conclusion that two forms of G protein may
exist, one rapidly coupling and the other slowly coupling. One possible
explanation is that a fraction of G protein is maintained in close
proximity to receptors, by the
subunits for example, but
that only upon ligand binding to the receptor does the
subunit of
the G protein interact with the receptor to generate the high affinity
ligand binding state. Such a mechanism could provide for extremely
rapid formation of the ternary complex.
Table: Ligand binding parameters of wild type and
mutant FPRs
S as indicated. The data were
analyzed using a two-site model. Where one of the sites represented
less than 1% of the total number of sites (-), it was taken to
indicate that only a single site existed. Site 1 is arbitrarily taken
as the high affinity site and site 2 as the low affinity site. The
K
values are given in nM,
whereas the B
values are given in fmol/mg of
protein.
I]SASD,
N-formyl-Met-Leu-Phe- N
-(2-( p-azidosalicylamido)ethyl-1,3`-dithiopropionyl)-Lys;
TM, transmembrane domain; GTP
S, guanosine
5`-3- O-(thio)triphosphate; R, receptor; LR, ligand-receptor;
G, G protein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.