(Received for publication, October 9, 1996, and in revised form, November 18, 1996)
From the Howard Hughes Medical Institute and the
¶ Division of Cardiovascular Medicine, Stanford University Medical
School, Stanford, California 94305 and the § Department of
Physiology and Biophysics, Mount Sinai School of Medicine,
New York, New York 10029
Mutations in several domains can lead to
agonist-independent, constitutive activation of G protein-coupled
receptors. However, the nature of the structural and molecular changes
that constitutively turn on a G protein-coupled receptor remains
unknown. Here we show evidence that a constitutively activated mutant
of the 2 adrenergic receptor (CAM) is characterized by
structural instability and an exaggerated conformational response to
ligand binding. The structural instability of CAM could be demonstrated
by a 4-fold increase in the rate of denaturation of purified receptor
at 37 °C as compared with the wild type receptor. Spectroscopic
analysis of purified CAM labeled with the conformationally sensitive
and cysteine-reactive fluorophore,
N,N
dimethyl-N-(iodoacetyl)-N
-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We
propose that the mutation that confers constitutive activity to the
2 adrenergic receptor removes some stabilizing
conformational constraints, allowing CAM to more readily undergo
transitions between the inactive and the active states and making the
receptor more susceptible to denaturation.
In classical receptor theory, binding of agonist has been considered essential for receptor activation and transmission of the biological signal across the plasma membrane. However, expression of many members of the seven-transmembrane, G protein-coupled receptor family in transfected cell lines has revealed that activation of intracellular messenger systems can occur in the absence of agonists (1-6). Moreover, it has been found that mutations in several different regions of G protein-coupled receptors can dramatically enhance agonist-independent receptor activity and in some cases confer oncogenic properties to the receptor (1-3, 7-14). Nevertheless, the molecular and structural changes in the receptor that are responsible for constitutive, agonist-independent activation are poorly understood. According to the prevailing two-state model for activation of G protein-coupled receptors, constitutive activation has been explained as a disturbance of the normal equilibrium between the inactive (R)1 state and the active (R*) state leading to a higher proportion of receptor molecules in the active R* state (3, 15). However, the conformational state of a constitutively active receptor protein has so far only been deduced from its effects on intracellular second messenger systems; therefore, this hypothesis has not been substantiated by any direct structural analysis.
Recently, we have described the use of fluorescence spectroscopy to
directly analyze ligand-induced conformational changes in the purified
wild type 2 adrenergic receptor (16). The approach is
based on the sensitivity of many fluorescent molecules to the polarity
of their molecular environment (17). In this study, we use the same
techniques to directly study conformational changes associated with
constitutive activation. Our results reveal novel characteristics of a
constitutively active receptor that provide insight into the mechanism
of altered signaling behavior of this mutant
2
receptor.
The cDNA encoding CAM was generously provided by Dr. R. J. Lefkowitz (Duke University, Durham, NC) and cloned into the baculovirus expression vector pVL1392 (Invitrogen) as described for the wild type receptor (18). The resulting construct had the cleavable influenza-hemagglutinin signal-sequence followed by the FLAG epitope (IBI) at the amino terminus and a tail of six histidines at the carboxyl terminus. Baculovirus containing the tagged CAM sequence was generated using the BaculoGold kit (Pharmingen) and plaque-purified. Sf-9 cells were grown as described (16).
Assessment of Receptor Expression in the Presence and the Absence of LigandSuspension cultures of Sf-9 cells at a density of
3 × 106 cells/ml were infected in the presence or the
absence of ligands plus 2 mM ascorbate. After 48 h
1.0-ml samples of the cultures were collected, and ligands were added
to controls before centrifugation (10 min at 10,000 × g). The pellets were frozen at 70 °C, thawed, and
resuspended in 1.0 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, with 1 mM EDTA, 10 µg/ml leupeptin, 10 mg/ml
benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride). The
lysed cells were centrifuged (10 min at 10,000 × g),
and the membrane pellet was washed three times in 1.0 ml of binding
buffer (75 mM Tris-HCl, pH 7.4, with 12.5 mM
MgCl2, and 1 mM EDTA) plus 10 µg/ml leupeptin and 10 µg/ml benzamidine. For binding assay 50 µl of membrane suspension was incubated in binding buffer in a total volume of 0.5 ml
with 10 nM [3H]dihydroalprenolol (Amersham
Corp.) for 2 h as described (19).
For purification, Sf-9 cells were
grown in 600-ml cultures, infected with a 1:100 dilution of a high
titer virus stock at a density of 3 × 106 cells/ml
and harvested after 48 h. To avoid a complete loss of the unstable
CAM protein it was necessary to exclude the M1 anti-FLAG antibody
column from our previously described purification procedure allowing
purification in 1 day instead of over 2 days (16, 20). Briefly, lysed
cell membranes were solubilized in 1.0% (w/v)
n-dodecyl--D-maltoside (D
M) (CalBiochem)
followed by nickel column chromatography using Chelating Sepharose
(Pharmacia Biotech Inc.) and alprenolol affinity chromatography as
described (16, 20). 1-2 nmol of pure CAM protein generally could be
obtained from three 600-ml cultures. The specific activity of purified
CAM was ~3 nmol/mg protein and ~8 nmol/mg protein for the WT
purified by the same procedure. Purified protein was analyzed by 10%
SDS-polyacrylamide gel electrophoresis. The protein was either
visualized by Coomassie staining or by Western blotting using the M1
anti-FLAG antibody (IBI) and the ECL system (Amersham Corp.).
Nickel-purified
receptor (2 pmol) was incubated at 37 °C for up to 3 h in 100 µl of 100 mM Tris-HCl, pH 7.5, containing 0.08% (w/v)
DM, 200 mM NaCl plus 4 mg/ml bovine serum albumin. The amount of functional receptor was determined in binding assays using a
saturating concentration of [3H]dihydroalprenolol (10 nM) according to described methods (16). To investigate the
ability of ligands to protect the receptor from degradation, 2 pmol of
CAM was incubated for 15 min and 2 pmol of WT was incubated for 60 min
at 37 °C in the above mentioned buffer plus 2 mM
ascorbate with our without ligands. Ligands were removed by binding
receptors to 100-µl nickel columns (chelating Sepharose) that were
subsequently washed 8-10 times with 500 µl of buffer. Receptors were
eluted in 500 µl of buffer with 200 mM imidazol before
binding analysis.
Purified receptor
was bound to a 150-µl nickel column (chelating Sepharose). Labeling
with the cysteine-reactive fluorophore IANBD (Molecular Probes, Eugene,
OR) was achieved by recycling 1.0 ml of 0.5 mM IANBD in
buffer (Tris-HCl, pH 7.5, containing 500 mM NaCl, and
0.08% DM) with 10
6 M alprenolol over the
nickel column for 20 min. Excess dye was removed by extensive washing
of the column with approximately 50 column volumes of buffer. Labeled
receptor was eluted with 200 mM imidazol in buffer. The
labeling procedure resulted in incorporation of 1.0-1.5 mol of
IANBD/mol of WT or CAM receptor, as determined by measuring absorption
at 481 nm and using an extinction coefficient of 21,000 M
1 cm
1 for IANBD and a
molecular weight of 50,000 for the receptor. Protein concentration was
determined using the Bio-Rad DC protein assay kit. The
results with IANBD-labeled wild type receptor purified and labeled by
the procedures described here were indistinguishable from results
obtained using our previous procedures (16). Fluorescence spectroscopy
was performed at room temperature on a SPEX Fluoromax spectrofluorometer with photon counting as described previously (16).
Lefkowitz and co-workers previously showed that a discrete change
in the carboxyl-terminal part of the third intracellular loop leads to
constitutive activation of the 2 adrenoreceptor (CAM)
(3). We expressed an epitope-tagged version of this mutant in Sf-9
insect cells to obtain large quantities of receptor needed for
purification and subsequent structural characterization. The functional
properties of CAM in insect cell membranes were similar to those
observed for CAM in membranes from transfected mammalian cells (3). As
expected we observed increased agonist affinity, an elevated basal
level of adenylyl cyclase and GTPase activity, and a higher maximal
agonist-stimulated adenylyl cyclase and GTPase activity for CAM than
for the
2 WT receptor when expressed at a similar level
(data not shown). Together this confirms that CAM not only possesses
constitutive activity but also is "superactive" compared with the
wild type receptor.
The expression of functional CAM in Sf-9 insect cells was considerably
lower than for the wild type (3.4 ± 1.3 pmol/mg protein versus 8.7 ± 1.8 pmol/mg protein, mean ± S.E.,
n = 4) in agreement with earlier studies in mammalian
cells (3). However, incubation of the cells with either an agonist or
an inverse agonist (also referred to as negative antagonist) during the
48-h infection markedly increased the expression of CAM (Fig.
1). A similar increase was also observed for neutral
antagonists (data not shown). This surprising lack of correlation
between the increase in expression and the pharmacological properties
of the added ligands strongly argues that the lowered expression of CAM
in the insect cells cannot be explained by receptor down-regulation as
a consequence of constitutive activation. Rather, the increased
expression of CAM in the presence of ligand may be due to biochemical
stabilization of an inherently unstable protein. Of interest, an even
more dramatic up-regulation is observed in transgenic mice expressing
CAM in response to antagonist treatment (21). Most likely, these data also reflect ligand stabilization of an inherently labile protein.
The instability of CAM can be demonstrated by observing the rate of
denaturation at 37 °C as shown in Fig. 2A.
Assuming an exponential decay, t1/2 for degradation
of CAM was 12.3 min versus 49.9 min for the wild type
receptor (Fig. 2A). The decrease in binding activity for
both wild type and CAM could be partially prevented by both the
agonist, isoproterenol, and the inverse agonist, ICI 118,551 (22, 23)
with isoproterenol being slightly less effective than ICI 118,551 (Fig.
2B). Western blotting of partially purified receptor before
and after exposure to 37 °C for 3 h revealed essentially no
changes in the intensity of the receptor band and no evidence of
proteolysis (Fig. 2A, inset).
The cysteine-reactive fluorescent probe IANBD can be used as a
sensitive molecular reporter of ligand-induced conformational changes
in the 2 adrenoreceptor (16). Agonist stimulation of purified IANBD-labeled
2 receptor leads to a
dose-dependent, reversible decrease in fluorescence,
indicating that one or more cysteines labeled with IANBD are exposed to
a slightly more polar environment upon agonist binding. By systematic
mutation of cysteines we have localized the responsible residues to the
third and sixth transmembrane domain.2
There was a linear correlation between the magnitude of the
fluorescence change in response to a series of adrenergic agonists and
the intrinsic efficacy of the compounds in adenylyl cyclase assays implying that the agonist-mediated decrease in fluorescence ensues from
a conformational change involved in receptor activation and G protein
coupling (16). In contrast to agonists, we found that inverse agonists
caused a relative increase in fluorescence emission (16). Emission
scans of IANBD-labeled CAM and wild type receptor both showed maximal
fluorescence at an emission wavelength of 523-526 nm (data not shown).
Time course analysis revealed that stimulation of IANBD-labeled CAM
with the full agonist, isoproterenol, and the partial agonist,
salbutamol, elicited substantially greater decreases in fluorescence
emission than in the IANBD-labeled WT receptor (Fig. 3,
A-E). In addition, the ratio of the salbutamol response
relative to the isoproterenol response increased from 0.40 in the WT to
0.76 in CAM (Fig. 3E). This agrees with the well described
increase in the efficacy of partial agonists at constitutively
activated receptor mutants in biological assays (Refs. 3 and 4 and data
not shown). The changes were fully reversible by antagonist for both
receptors (Fig. 3, A-D). As with the WT receptor, the
inverse agonist ICI 118,551 induced an increase in fluorescence of the
IANBD-labeled CAM receptor; however, the magnitude of the response was
greater for the CAM receptor (Fig. 3E). The larger changes
in fluorescence induced by both agonists and inverse agonists in the
IANBD-labeled CAM receptor are even more impressive considering that
the specific activity of CAM was lower than that of the WT (CAM, 3 nmol/mg protein; WT, 8 nmol/mg protein).
Taken together, our findings delineate two novel properties of a
constitutively activated receptor: structural instability and an
exaggerated conformational response to drug binding. Our findings, in
particular the larger changes in fluorescence observed with CAM in
response to agonists, are unexpected. If we assume that the CAM
mutation simply causes the unliganded receptor to adopt a conformation
mimicking the agonist bound form of the WT receptor, then the effect of
agonist binding might have been expected to produce little additional
change in fluorescence. One likely explanation for this apparent
discrepancy could be that constitutive activation of the
2 receptor confers a higher degree of conformational flexibility to the receptor protein due to the disruption of
stabilizing conformational constraints. This higher degree of
conformational flexibility may allow CAM to more readily undergo
transitions between the R state and the R state* in response to ligand
binding, thus leading to larger fluorescence changes and structural
instability (as outlined below and in the model in Fig.
4). In this thermodynamic model, we propose that the R*
state in the prevailing two-state model for activation of G
protein-coupled receptors (3, 23-27) can be considered a high energy,
intermediate state that can be stabilized by the G protein and/or the
agonist (Fig. 4). However, in the absence of agonist or G protein, the
expected lifetime of the excited R* state for both CAM and the WT
receptor would be short due to its high energy. Receptor in R* will
either rapidly return to the ground state or denature. Therefore, at
any given time the fraction of both CAM and WT receptor in R* should be rather low in absence of agonist and G protein ([R*WT]
<<< [RWT] and [R*CAM]
[RCAM]) (Fig. 4). Our model proposes that agonists stabilize both R*WT and R*CAM; however, because
of the smaller energy difference between the R and R* state of CAM,
agonists would cause a higher proportion of CAM molecules than WT
molecules to undergo the transition from R to R*. For example, agonist
occupancy of the wild type receptor in the absence of G protein might
cause a change in the R*WT fraction from 5 to 20%, whereas
the change could be from 10 to 35% for CAM. This greater change in the
fraction of R* for CAM than for the wild type receptor in response to
agonists is consistent with the larger changes in fluorescence, because the change in fluorescence most likely reflects receptor molecules undergoing transition from R to R* (16). Moreover, it explains the
superactivity of agonist-activated CAM as compared with
agonist-activated WT measured in GTPase and adenylyl cyclase assays
(3). Finally, the model can explain the larger increase in fluorescence
over time in response to the inverse agonist ICI 118,551. ICI 118,551 stabilizes the receptor in the ground state R and, as discussed above,
the fraction of CAM molecules in R* would always be higher than for the
WT receptor in the absence of ligands; therefore, the population of
receptor going from R* to R upon the addition of the inverse agonist
would be greater, and a larger increase in fluorescence would be
expected.
An alternative explanation for our observations would be that CAM has an altered structure with an altered environment around the fluorophore, and the active state (R*CAM) is structurally distinct from the active state of the WT (R*WT). Thus, CAM may accommodate a superactivated state that interacts more efficiently with the G protein and causes the more dramatic changes in fluorescence. Our data cannot distinguish between these two explanations. However, the model we have outlined in Fig. 4 represents the simplest hypothesis, because it does not propose any changes in the tertiary structure of R* for CAM as compared with R* for the WT receptor. Thus it is compatible with the prevailing two-state model for activation of G protein-coupled receptors (3, 23-27). It is also highly unlikely that the environment around the fluorophore is markedly changed in CAM, because the mutant has the same stoichiometry of IANBD labeling (see "Experimental Procedures") and the emission maxima from IANBD-labeled CAM and WT are indistinguishable (data not shown).
The structural instability should also be expected according to the
model outlined in Fig. 4. Due to the higher energy, the excited R*
state is predicted to be structurally more unstable; therefore it
follows that CAM is more susceptible to denaturation. A higher
structural instability of the R* state as compared with R is supported
by molecular dynamics simulations of ligand-receptor complexes
(28-30). CAM may also be more susceptible to denaturation from the
ground state due to the proposed higher energy level of this state
(RCAM). Of interest, the association of structural instability with constitutive activation is not restricted to G
protein-coupled receptors. A constitutively active mutant of the Gs
protein has been shown to be thermolabile and to degrade rapidly at
37 °C (31). Analogous to the observed ligand stabilization of CAM,
the degradation of the constitutively active Gs 224 protein could be
prevented by the addition of nucleotides (31).
In conclusion, this study represents the first direct structural analysis of a constitutively active G protein-coupled receptor. Interestingly, the data can be predicted from an activation model of G protein-coupled receptors that involves formation of an unstable high energy intermediate, which can be considered analogous to the active R* state in the prevailing two-state model for activation of G protein-coupled receptors (3, 23-27). Hence, our results underscore the importance of constitutively active mutants as critical tools for understanding the molecular processes involved in activation of G protein-coupled receptors.
We thank Dr. Robert J. Lefkowitz for helpful suggestions. Drs. Hans T. Schambye, Sansan Lin, and Pejman Ghanouni are thanked for critical reading of the manuscript.