(Received for publication, August 8, 1995; and in revised form, September 18, 1995)
From the
The purpose of the present study was to develop an approach to
directly monitor structural changes in a G protein-coupled receptor in
response to drug binding. Purified human adrenergic
receptor was covalently labeled with the cysteine-reactive, fluorescent
probe N,N`-dimethyl-N-(iodoacetyl)-N` -
(7 - nitrobenz -2 - oxa -1,3diazol-4-yl)ethylenediamine (IANBD). IANBD
is characterized by a fluorescence which is highly sensitive to the
polarity of its environment. We found that the full agonist,
isoproterenol, elicited a stereoselective and dose-dependent decrease
in fluorescence from IANBD-labeled
receptor. The
change in fluorescence could be plotted against the concentration of
isoproterenol as a simple hyperbolic binding isotherm demonstrating
interaction with a single binding site in the receptor. The ability of
several adrenergic antagonists to reverse the response confirmed that
this binding site is identical to the well described binding site in
the
receptor. Comparison of the response to
isoproterenol with a series of adrenergic agonists, having different
biological efficacies, revealed a linear correlation between biological
efficacy and the change in fluorescence. This suggests that the
agonist-mediated decrease in fluorescence from IANBD-labeled
receptor is due to the same conformational change as
involved in receptor activation and G protein coupling. In contrast to
agonists, negative antagonists induced a small but significant increase
in base-line fluorescence. Despite the small amplitude of this
response, it supports the notion that antagonists by themselves may
alter receptor structure. In conclusion, our data provide the first
direct evidence for ligand-specific conformational changes occurring in
a G protein-coupled receptor. Furthermore, the data demonstrate the
potential of fluorescence spectroscopy as a tool for further
delineating the molecular mechanisms of drug action at G
protein-coupled receptors.
The adrenergic receptor is a prototype member
of the G protein-coupled receptor family(1) . The receptor
family constitutes the largest group of plasma membrane receptors,
which are characterized by a remarkable diversity in the chemical
structure of their endogenous
ligands(1, 2, 3) . The receptors are all
believed to share a common topology with seven
-helical,
transmembrane segments; however, the helical arrangement and actual
three-dimensional structure of the receptors remain
unknown(1, 2, 3) . Mutagenesis studies in the
adrenergic receptor as well as in many other G
protein-coupled receptors have been able to assign distinct receptor
functions, such as ligand binding and G protein coupling, to specific
receptor domains(1, 2, 3) . Several molecular
models of these receptors have also been generated based on the
structure of bacteriorhodopsin and rhodopsin for which more detailed
structural information is
available(3, 4, 5) . Nevertheless, very
little is known about the molecular events and structural changes in
the receptor that provide the important link between ligand binding and
transmission of the signal across the membrane.
Drugs acting at G protein-coupled receptors are traditionally classified in biological assays as either full agonists, which elicit the maximal response, as partial agonists, which only elicit a fractional response, or as antagonists, which block the response induced by agonists(6, 7) . In addition, recent data have suggested that antagonists should be subclassified into at least two categories: neutral antagonists, which have no effect on basal receptor activity, and negative antagonists (also referred to as inverse agonists), which inhibit basal receptor activity occurring in the absence of agonist (7, 8, 9, 10, 11, 12, 13) . Thus, in contrast to the conventional view, it has now become evident even in vivo(13) that many antagonists actively inhibit receptor function rather than just passively block access of the agonist to its binding site(8, 9, 10, 11, 12, 13) . However, the structural basis for the biological classification of drug action at G protein-coupled receptors is not yet known. Any direct evidence for the existence of discrete ligand-specific conformational states of G protein-coupled receptors has not been obtained. To date, the conformational state of G protein-coupled receptors has been assessed only by indirect methods, such as the effect of receptor conformation on G protein GTPase activity or on the activity of the effector enzymes.
In the present study we have developed a
fluorescence spectroscopy approach to directly monitor ligand-induced
conformational changes in a G protein-coupled receptor. Our approach
takes advantage of the sensitivity of many fluorescent molecules to the
polarity of their molecular
environment(14, 15, 16, 17) .
Fluorescent labels incorporated into proteins can therefore often be
used as sensitive indicators of conformational changes and of
protein-protein interactions that cause changes in polarity of the
environment surrounding the
probe(14, 15, 16, 17) . To
accomplish these experiments, we expressed the human adrenergic receptor in SF-9 insect cells and established a
purification procedure, which allowed us to obtain the required amount
of pure protein for the spectroscopy analysis. The purified receptor
was labeled with the cysteine-specific and environmentally sensitive
fluorescent probe N,N`-dimethyl-N-(iodoacetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine
(IANBD). (
)We found that binding of full agonists and strong
partial agonists to the fluorescently labeled
adrenergic receptor produced a stereospecific, dose-dependent,
and reversible decrease in fluorescence emission, which is not observed
in response to weaker partial agonists or in response to neutral or
negative antagonists. The magnitude of the response correlated with
agonist efficacy, suggesting that this change in fluorescence emission
is due to the same conformational change as involved in agonist
activation of the receptor.
Figure 1:
Fluorescence properties of IANBD and
IANBD-labeled receptor. a, emission spectra
of cysteine-reacted IANBD (0.3 µM) in solvents of
different polarity. Excitation was set at 481 nm. b, emission
spectrum of IANBD-labeled
receptor (0.15 µM receptor, 1.2 mol IANBD per mol receptor). Control is emission
spectrum of 0.15 µM
receptor
``labeled'' with IANBD prebound to free cysteine instead of
free IANBD to asses possible nonspecific attachment of the probe to the
receptor during labeling. Insert, 10% SDS-polyacrylamide gel
electrophoresis of IANBD-labeled
receptor. Lane
1, 150 pmol of IANBD-labeled
receptor; lanes
2 and 3, 150 pmol of
receptor
preincubated before exposure to IANBD with iodoacetamide (lane
2) and N-ethylmaleimide (lane 3). Inset:
left panel, Coomassie Blue staining of gel; right panel,
gel photographed under UV light. The weak band with an apparent
molecular mass of 32.5 kDa is a degradation product of the
receptor.
Figure 2:
Competition binding profiles of unlabeled
and IANBD-labeled, purified receptor. a and b, competition binding of
[
H]dihydroalprenolol (1.2 nM) with
isoproterenol (a) and alprenolol (b) to unlabeled,
purified
receptor. c and d,
competition binding of [
H]dihydroalprenolol (1.2
nM) with isoproterenol (a) and alprenolol (b) to IANBD-labeled, purified
receptor.
Data are expressed as percent of maximum bound
[
H]dihydroalprenolol (mean ± S.E., n = 3).
Figure 3:
Stereospecificity of isoproterenol induced
decrease in fluorescence from IANBD-labeled receptor. a, emission spectra of IANBD-labeled
receptor obtained immediately (t = 0) and after
15 min (t = 15) following addition of 30 µM of the less active (+)-isomer of the agonist, isoproterenol
((+)ISO). b, Emission spectra of IANBD-labeled
receptor obtained immediately (t = 0)
and after 15 min (t = 15) following addition of 30
µM of the active (-)-isomer of the agonist,
isoproterenol ((-)ISO). The experiments shown is
representative of three identical experiments. Fluorescence
measurements were done as described under ``Experimental
Procedures'' with excitation set at 481
nm.
Figure 4:
Time
course and dose-dependence of isoproterenol induced decrease in
fluorescence from IANBD-labeled receptor. a,
emission from IANBD-labeled
receptor measured over
time following stimulation with indicated concentrations of
isoproterenol. Excitation was 481 nm and emission measured at 523 nm.
Isoproterenol (ISO) was added at the time indicated by the arrow. Fluorescence in the individual traces was normalized to
the fluorescence observed immediately after addition of ligand. The
experiment shown is representative of four identical experiments. b, the percent change in fluorescence at t =
15 min following addition of isoproterenol plotted against the
isoproterenol concentration and fitted to a simple hyperbolic function (F = F
L/K
+ L; F, change in fluorescence at the ligand
concentration L; F
, maximum change in
fluorescence; K
, affinity constant). c, the relative change in fluorescence at t =
15 min following addition of isoproterenol plotted against the
logarithm of the isoproterenol concentration and fitted to a one-site
sigmoid curve. The percent change in fluorescence was calculated as the
change in fluorescence relative to the extrapolated base line at t = 15 min after addition of isoproterenol. Percent change is
given as mean ± S.E. of the following number of experiments,
1000 µM, n = 5; 300 µM, n = 4; 100 µM, n = 4; 30
µM, n = 4; 10 µM, n = 4; 3 µM, n = 4; 1
µM, n = 2; 0.1 µM, n = 2, H
O, n = 5. Curve fittings
were performed using Inplot 4.0 from GraphPad Software, San Diego,
CA.
Figure 5:
Reversibility of isoproterenol-induced
decrease in fluorescence from IANBD-labeled receptor. a, control addition of water (H
O). b and c, reversal
of the response to isoproterenol (ISO) by the
active(-)-isomer of the antagonist propranolol,(-)PROP (b), but not by the less active (+)-isomer,
(+)PROP (c). Dotted lines indicate
extrapolated base line. Excitation was 481 nm and emission measured at
523 nm. Fluorescence in all the individual traces shown was normalized
to the fluorescence observed immediately after addition of ligand. All
traces shown are representative of at least three identical
experiments.
Figure 7:
Effect of antagonists on fluorescence from
IANBD-labeled receptor. a, emission from
IANBD-labeled
receptor measured over time following
stimulation with 1 µM(-)-propranolol
((-)PROP), 1 µM (+)-propranolol
((+)PROP) and water (H
O). b, emission from IANBD-labeled
receptor
measured over time following stimulation with 10 µM ICI
118,551 (ICI), 10 µM pindolol (PIND),
and water (H
O). c, emission from
IANBD-labeled
receptor measured over time following
exposure to 10 µM dichloroisoproterenol (DCI),
alprenolol (ALP) and water (H
O). d, emission from guanidinium chloride denatured IANBD-labeled
receptor measured over time following exposure to 10
µM(-)-propranolol ((-)-PROP) and 10
µM ICI 118,551 (ICI). The compounds were added at
the time indicated by the arrows. Excitation was 481 nm, and
emission was measured at 523 nm. Fluorescence in the individual traces
was normalized to the fluorescence observed immediately after addition
of ligand. The experiment shown is representative of at least three
identical experiments.
The time
course analysis showed that the response to isoproterenol was
dose-dependent and reached a maximum amplitude below the extrapolated
base line after 10-15 min (Fig. 4a). The relative
change in fluorescence at t = 15 min following addition
of isoproterenol could be plotted against the isoproterenol
concentration and fitted to a simple hyperbolic function, describing a
single binding site with a K of 29 µM (Fig. 4b). Similarly, the fluorescent change could
be plotted against the logarithm of the isoproterenol concentration
showing the best fit to a one-site sigmoid curve with an EC
of 19 µM (Fig. 4c). The K
value of 29 µM and the EC
value of 19 µM for isoproterenol are higher than the
binding constants observed using conventional radioligand binding
techniques as shown in Fig. 2(
1 µM). One
explanation for this apparent discrepancy could be technical
differences in the methods by which the binding constants were obtained
(the fluorescence studies were done with more than 100-fold higher
receptor concentrations (100 nM receptor) for 15 min).
Although apparent maximal change in fluorescence was observed already
after 15 min, full equilibrium may not have been reached.
Unfortunately, the fluorescent change upon ligand binding cannot be
reliably determined after 1 h of incubation due to magnification of
base-line differences over a longer period of time. Another potential
explanation for the difference in apparent K
values could be the incomplete labeling of the cysteine in the
receptor that is responsible for the agonist-induced
response. The fraction of labeled receptors may exhibit a lowered
agonist affinity in contrast to the fraction of receptors labeled at
other sites. However, this explanation is inconsistent with our
radioligand binding data, which detect only one agonist affinity site (Fig. 2). Furthermore, we do not observe a change in the total
number of binding sites following labeling with IANBD.
We also investigated whether the response seen following stimulation with isoproterenol could be reversed by addition of antagonist. As shown in Fig. 5, the response to isoproterenol could be readily reversed by the active(-)-isomer of the antagonist propranolol but not by the less active (+)-isomer. The response to isoproterenol was similarly reversed by several other antagonists, including alprenolol, ICI 118,551, pindolol, and dichloroisoproterenol (data not shown).
Figure 6:
a, Effect of different adrenergic agonists
on fluorescence from IANBD-labeled receptor. The
percent change (mean ± S.E.) in fluorescence was calculated as
the change in fluorescence relative to the extrapolated base line at t = 15 min after addition of ligand. The ligands and
concentrations used were (number of experiments in parentheses); H
O, water (n = 5); EPH, 10
M ephedrine (n = 3); DOB, 10
M dobutamine (n = 4); SAL,
10
M salbutamol (n = 3); ISO, 10
M isoproterenol (n = 5); and EPI, 10
M epinephrine (n = 3). Responses significantly
different from water are indicated by *p < 0.000005
(unpaired t test). The ligand concentrations used were chosen
to ensure full saturation of the receptor with all ligands. This was
confirmed by the ability of all compounds (at the concentration used)
to fully displace [
H]dihydroalprenolol from the
receptor in a binding assay (data not shown). b, effect of
adrenergic agonists on adenylate cyclase activity in membranes from
SF-9 cells. Data are maximal response to indicated ligands (see above)
expressed as percent of basal activity (mean ± S.E., n = 2) in membranes from cells infected with
receptor baculovirus at a density of 1.2 pmol/mg of protein. The dotted bar shows basal activity in an equivalent amount of
membranes from non-infected cells in percent of basal activity in
membranes from the infected cells. c, plot of percent change
in fluorescence at t = 15 min against percent change in
adenylate cyclase activity. The squared correlation coefficient (r
) was 0.98.
The fluorescent changes were compared with the ability
of the antagonists to affect basal adenylate cyclase activity in SF-9
cell membranes. The effect of the antagonists on basal adenylate
cyclase activity was evaluated in SF-9 cells expressing a high level of
receptor (approximately 7 pmol/mg of protein) which
leads to an elevated receptor-mediated basal level of adenylate cyclase
activity (Fig. 8b). In contrast, partial agonists were
evaluated at a lower receptor density (approximately 1.2 pmol/mg of
protein) (Fig. 6b), as partial agonists are difficult
to differentiate from full agonists in the presence of a large receptor
reserve(25) . Alprenolol, pindolol, propranolol,
dichloroisoproterenol, or ICI 118,551 did not affect basal adenylate
cyclase activity at the low receptor density (data not shown). However,
ICI 118,551 and propranolol, which induced the largest increase in
fluorescence, potently decreased basal adenylate cyclase activity
40-60% at the high receptor density (Fig. 8b).
Alprenolol and pindolol induced an approximately 30% decrease in basal
activity, whereas dichloroisoproterenol revealed weak agonist activity
at this high receptor density by increasing activity about 30%.
Figure 8:
a, effect of different adrenergic
antagonists on fluorescence from IANBD-labeled receptor. The percent change (mean ± S.E.) in fluorescence
was calculated as the change in fluorescence relative to the
extrapolated base line at t = 15 min after addition of
ligand. The ligands and concentrations used were (number of experiments
in parentheses); H
O, water (n = 5); PIND, 10
M pindolol (n = 4); ALP, 10
M(-)alprenolol (n = 3); DCI, 10
M dichloroisoproterenol (n = 3); (-)PROP, 10
M(-)-propranolol (n = 3); and ICI, 10
M ICI 118,551 (n = 4). Responses significantly different from water are
indicated by *p < 0.0005 (unpaired t test). The
ligand concentrations used were chosen to ensure full saturation of the
receptor with all ligands. This was confirmed by the ability of all
compounds (at the concentration used) to fully displace
[
H]dihydroalprenolol from the receptor in a
binding assay (data not shown). b, effect of adrenergic
antagonists on adenylate cyclase activity in membranes from SF-9 cells.
Data are maximal response to indicated ligands (see above) expressed as
percent of basal activity (mean ± S.E., n = 2)
in membranes from cells infected with
receptor
baculovirus at a density of 7 pmol/mg protein. The dotted bar shows basal activity in an equivalent amount of membranes from
noninfected cells in percent of basal activity in membranes from the
infected cells.
Figure 9:
Stern-Volmer plots of quenching of
IANBD-labeled receptor. Increasing concentrations of
KI were added sequentially to labeled receptor (open square),
labeled receptor treated with 10 µM ICI 118,551 (open
circle) and 100 µM(-)-isoproterenol (closed
circle). Fluorescence was measured and plotted as described under
``Experimental Procedures.'' The quenching constant K
was 4.477 ± 0.074 (mean ± S.E., n = 3) in absence of any ligand, 4.436 ± 0.073
(mean ± S.E., n = 3) in presence of ICI 118,551
and 4.209 ± 0.036 (mean ± S.E., n = 3) in
presence of isoproterenol. The difference between isoproterenol and
buffer and between isoproterenol and ICI 118,551 was significant (p < 0.05) (unpaired t test).
In the present study we have been able to directly monitor
conformational changes in a G protein-coupled receptor. As a molecular
reporter we have used the cysteine-selective and environmentally
sensitive, fluorescent probe, IANBD, which can be covalently
incorporated into the purified, human adrenergic
receptor without perturbing the pharmacological properties of the
receptor. Using the IANBD-labeled protein we were able to detect an
agonist specific and reversible decrease in fluorescence emission from
the labeled receptor protein. The response to the full agonist,
isoproterenol, was shown to be dose-dependent and could be plotted as a
simple, hyperbolic binding isotherm demonstrating interaction with a
single binding site in the receptor. The ability of several adrenergic
antagonists to reverse the isoproterenol-induced response confirmed
that this binding site must be identical to the well described ligand
binding site in the
receptor. These data provide
structural evidence in a pure system for the existence of
ligand-specific conformational states of a G protein-coupled receptor.
The agonist induced change most likely represents changes in the polarity of the environment surrounding one of more labeled cysteines in the hydrophobic core of the protein. Two possible mechanisms that could account for these changes are outlined in Fig. 10. According to the first model, a change in the environment around the indicated fluorophore (F) could be the result of a ligand induced movement of a transmembrane segment perpendicular to the plane of the lipid bilayer (Fig. 10a). This could result in the exposure of the fluorophore to the solvent causing quenching of the fluorescence and thus a decrease in the net fluorescence from the labeled receptor. Alternatively, it could be imagined that the agonist could induce a rotation of the membrane spanning domain resulting in movement of the fluorophore into the core of the protein, which is predicted to be more polar (Fig. 10b)(3, 4, 5) . According to the first model, the fluorophore should be more accessible to an aqueous quencher like potassium iodide following agonist binding. However, we observed that the agonist isoproterenol caused a slight decrease in quenching of the fluorescence from the IANBD-labeled receptor (Fig. 9). This would favor the latter model in which the fluorophore is moved to a more hydrophilic pocket in the core of the protein following agonist binding. In this model aqueous quenchers might be expected to have a more limited access to the fluorophore. It should be noted that at this point we can only speculate on the molecular mechanism behind the ligand-induced changes in fluorescence. With a stoichiometry of labeling at 1.2 mol of IANBD per mol of receptor at least two sites are being labeled in the receptor. Thus, the isoproterenol-mediated decrease in fluorescence may involve a different labeled cysteine than that responsible for the isoproterenol-induced effect on KI quenching. The observed changes may therefore be due to a more complex mechanism than that illustrated in Fig. 10.
Figure 10: Illustration of two possible mechanisms that may cause changes in polarity of the environment around the IANBD fluorophore. a, transmembrane segments of the receptor viewed from within the lipid bilayer. A change in the environment around the indicated fluorophore could be the result of a ligand induced movement of a transmembrane segment perpendicular to the plane of the lipid bilayer. b, transmembrane segments viewed from the extracellular face of the membrane. In this model, ligands could induce a rotation of the membrane spanning domain resulting in either movement of the fluorophore into the core of the protein, which is predicted to be more polar, or into the more hydrophobic, membrane-embedded shell of the protein.
Our fluorescence data suggest that the
receptor can exist in at least two conformational
states, an unliganded state and an agonist-bound state. It is therefore
tempting to consider these data in the context of the prevailing
two-state model for activation of G protein-coupled receptors. This
model predicts that the receptors exist in a dynamic equilibrium
between two states, an inactive (R) and active conformation (R*); and that the biological response to a given ligand is
governed by its intrinsic ability to change the overall equilibrium
between the two
states(8, 9, 10, 13, 24, 26, 27) .
According to this model the fluorescence properties of the unoccupied
IANBD-labeled receptor would be expected to represent the average
fluorescence properties of the population of
receptors in the inactive state (R) and the active state (R*). The fluorescent properties of the R state alone
should be observed in the presence of a negative antagonist, while
those of the R* state alone should be observed in the presence
of a full
agonist(8, 9, 10, 13, 24, 26, 27) .
Using our fluorescence assay we compared a series of adrenergic
agonists, which exhibited distinct efficacies in a biological assay (Fig. 5b). We found an apparent linear correlation
between the functional efficacy of these compounds and their ability to
promote a change in fluorescence from the labeled receptor (Fig. 5c). These data can be interpreted in agreement
with the two-state model. Hence, the different functional efficacies
may result from differences in the ability of the different agonists to
pull the equilibrium toward R* and thus decrease the
fluorescence signal. Alternatively, the data also could support a
multistate model in which the biological efficacy of an agonist may be
a consequence of the magnitude of conformational change that it induces
in the receptor, rather than just affecting the equilibrium between
only two states. We should note, however, that without analyzing the
fluorescently labeled receptor in a reconstituted system together with
the corresponding G protein, we cannot exclude the possibility that the
agonist-mediated change in fluorescence does not describe the
structural changes of direct importance for G protein activation.
Nevertheless, the correlation between intrinsic activity of the
agonists and change in fluorescence suggests that the change is
actually describing the conformational change in the receptor that
leads to G protein activation.
It has been suggested, but never
structurally verified, that antagonists may stabilize a conformation of
the receptor that is distinct from unliganded receptor, and thus from
the R state in the two-state
model(11, 28, 29, 30, 31, 32) .
For example, this has been proposed to explain the unexpected
observation that non-peptide antagonists of G protein-coupled peptide
receptors can act as competitive antagonists for agonist peptides
without sharing apparent binding sites in the
receptor(28, 29, 30, 31) . In other
words, agonists and antagonists may be able to mutually exclude each
others binding to the receptor by stabilizing different receptor
conformations(28, 29, 30, 31) . It
was therefore intriguing to observe that a series of adrenergic
antagonists produced small but very reproducible increases in base-line
fluorescence from the IANBD-labeled receptor ( Fig. 7and Fig. 8). Except in the case of
dichloroisoproterenol, the changes showed a correlation with the
negative intrinsic activity of the tested compounds. Thus, propranolol
and ICI 188,551, which exhibited the strongest negative intrinsic
activity, caused the largest increase in base-line fluorescence,
whereas alprenolol and pindolol with a smaller negative intrinsic
activity also caused a smaller increase in fluorescence. However, given
the small size of responses observed following stimulation with this
group of compounds, it is difficult to assess the molecular
significance of the fluorescence changes at the present stage.
Nevertheless, the data are still of interest as they suggest that
antagonists by themselves may alter receptor structure. The findings
are also consistent with previous proteolysis studies on membrane bound
receptor which show that agonists and antagonists are equivalent in
protecting the
receptor from
proteolysis(33) . This would not be expected if antagonists
only changed the conformation of the small percentage of the receptor
population which, in the absence of ligand, would be predicted to be in R* according to the two-state model.
Summarized, our data
demonstrate the sensitivity of using fluorescence techniques for
studying ligand-receptor interactions and their potential for
delineating ligand-induced structural changes in G protein-coupled
receptors. Importantly, if site-specific fluorescent labeling of the
receptor can be achieved, the approach may be useful
for mapping conformational changes in the receptor structure to
specific subdomains. In this way it should be possible to more
precisely define the molecular mechanism of transmembrane signal
transduction in G protein-coupled receptors.