From the Division of Cellular and Molecular
Physiology, Department of Medical Physiology, The Panum Institute,
Copenhagen University, DK-2200 Copenhagen N, Denmark, the
¶ Department of Physiology and Biophysics, Mount Sinai School of
Medicine, New York, New York 10029, and
Novasite Pharmaceuticals
Inc., San Diego, California 92121
Received for publication, June 6, 2000, and in revised form, November 14, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The environmentally sensitive,
sulfhydryl-reactive, fluorescent probe
N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of
agonist-induced conformational changes in the G protein-coupled receptors
(GPCRs),1 or
seven-transmembrane segment receptors, comprise the largest superfamily
of mammalian proteins with now more than 1000 different members (1, 2). The While converging on the involvement of TM 3 and 6 in receptor
activation, the spectroscopic studies on rhodopsin and the To clarify this issue and to obtain further insight into the molecular
mechanisms involved in GPCR activation, we wished to establish new
read-outs for agonist-induced conformational changes in the To explore the putative conformational changes in the predicted
cytoplasmic region of TM 6 of the Mutagenesis--
The template used for site-directed mutagenesis
was a cDNA named Expression in Sf-9 Insect Cells--
Membrane Preparation, Adenylyl Cyclase Assay, and GTPase
Assay--
Membranes were prepared as described from 30-ml Sf-9 cell
cultures in 125-ml disposable Erlenmeyer flasks, grown at a density of
3 × 106 cells/ml, and infected with baculovirus
encoding the different receptor constructs for 48 h before
harvesting (9). Adenylyl cyclase assays were preformed in the Sf-9 cell
membranes as described (18). GTPase assays were carried out on Sf-9
cell membranes expressing Binding Assays--
Saturation binding assays and competition
binding assays on membrane preparations from cells expressing the
different receptors were carried out as previously described using
[3H]dihydroalprenolol (DHA) (Amersham Pharmacia Biotech)
as radioligand (19). Binding assays on solubilized and purified Purification Procedure--
The IANBD Labeling of Purified Receptor and Fluorescence Spectroscopy
Analysis--
The purified receptor was labeled with IANBD (Molecular
Probes, Eugene, OR) according to described procedures (17). Briefly, 1-3 nmol receptor was bound to a 150-µl nickel column (Chelating Fast Flow Sepharose Resin, Amersham Pharmacia Biotech), equilibrated in
high salt buffer (20 mM Tris-HCl, pH 7.5, with 500 mM NaCl and 0.08% D Collisional Quenching Experiments--
Stock solutions (1.0 M) of the hydrophilic quencher potassium iodide containing
10 mM Na2S2O3 was
prepared freshly for each round of experiments. The experiments were
preformed with 10-20 pmol of IANBD-labeled receptor in 400 µl of
buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCl and 0.08% D Computational Methods--
TM 6 was modeled from residues
Ser-2626.24-Val-2976.59 as an Cysteine Substitutions at the Cytoplasmic Side of TM 6--
To
achieve the possibility of site-selective, covalent incorporation of
the sulfhydryl-reactive fluorophore IANBD into the purified
Four consecutive residues in the cytoplasmic region of TM 6, His-2696.31, Lys-2706.32,
Ala-2716.33, and Leu-2726.34, were mutated to
cysteines in the
Next we wished to assess whether IANBD labeling of cysteines introduced
in positions 2696.31, 2706.32,
2716.33, or 2726.34 would affect the ability of
the receptor to assume the activated state and transmit the signal to
the G protein. Because the GTPase activity of Gs Fluorescence Spectroscopy Analysis of Site-selectively Labeled
Mutant
We have previously shown that agonist binding to the purified and
IANBD-labeled wild type
The time-based spectra for all four mutants differed significantly from
those of
Agonist stimulation of both
The kinetics of the agonist-induced changes in fluorescence in
IANBD-labeled Collisional Quenching Experiments with Iodide--
To further
elaborate the character of the ISO-induced change in the molecular
environment surrounding Cys-2716.33 and
Cys-2726.34, a series of collisional quenching experiments
were carried out on IANBD-labeled
The quenching experiments were also carried out in the presence of ISO.
In Dose-response Analysis and Investigation of Correlation between
Agonist Efficacy and Fluorescence Response at IANBD-labeled
We have previously shown that the agonist-induced changes in
fluorescence measured on IANBD-labeled wild type Molecular Modeling--
TM 6 was modeled as a Pro-kinked
To further explore this, a sequence alignment of 56 GPCRs homologous to
the Computational Simulations--
Based on the TM 6 model described
above we attempted to define the preferred conformations of the IANBD
moiety attached to the inserted cysteines by carrying out a series of
computer simulations. In our previous study (10), the preferred
conformations of the IANBD moiety attached to Cys-1253.44
and Cys-2856.47 were also predicted from computational
simulations. Because both Cys-1253.44 and
Cys-2856.47 reside in a highly hydrophobic environment,
these simulations was carried out in vacuum. However, our TM 6 model
predicts that Cys-2696.31, Cys-2706.32,
Cys-2716.33, and Cys-2726.34 reside in a mixed
hydrophobic-hydrophilic environment. Reliable predictions about the
preferred IANBD conformations would obviously require that this complex
environment is taken into consideration. Therefore, a computational
method in which this mixed environment is approximated as a dielectric
gradient was developed (see "Experimental Procedures"). This method
is based on the data of White and Wimley (reviewed in Ref. 25), who
showed evidence that a membrane consists of an ~30-Å relatively
constant, low dielectric region, sandwiched between two complex regions
of variable dielectric behavior, both of which are ~15 Å (reviewed
in Ref. 25). The 30-Å-thick core region corresponds to the hydrophobic
lipid chains while the surrounding 15 Å thick region corresponds to
the phospholipid headgroup domain (Fig. 7A). Thus, the
transmembrane helical domains reside in a complex environment
consisting of three distinct phases: a hydrophobic core of the membrane
defined by the phospholipid chains, a mixed hydrophobic-hydrophilic
region comprised by the phospholipid headgroups, and the aqueous
cytoplasm (25). A similar complex environment can be envisioned for
detergent micelles in which the experiments in the present study were
carried out, because they would be expected to follow the same polarity
pattern, i.e. a hydrophobic core, a mixed
hydrophobic-hydrophilic region comprised by the polar headgroups of the
detergent, and the aqueous exterior. Notably, the spectral responses to
agonists in IANBD-labeled
The conformational memories simulations were performed on the four
cysteine mutants IANBD derivatized peptides of TM 6 (see "Experimental Procedures"). The technique of conformational
memories, which utilizes energy-based Monte Carlo simulations, was
applied to explore the preferred conformations of the IANBD-labeled
cysteines. The most likely conformations of the IANBD-moieties attached
to the substituted cysteine residues are those more populated in the
Monte Carlo simulation, analyzed in terms of their dihedral angles,
which can be classified in terms of rotamers. Rotamer configurations
populated more than 5% during the course of the simulation were
selected as possible conformations of each IANBD-labeled cysteine
residue. An important result of the new computational method was the
observation of multiple IANBD rotamer configurations populated in all
four IANBD-labeled constructs. This result contrasts the results of our
previous simulations on IANBD attached to Cys-2856.47,
which demonstrated a restricted conformational space for IANBD (10).
Hence, the mixed dielectric medium that corresponds to the mixed
hydrophobic-hydrophilic region allows apparently for considerable more
conformational freedom of IANBD than the same moiety attached to
Cys-2856.47, which resides in a hydrophobic environment.
The composite of the populated conformations defines the set of
conformations available to each IANBD-derivatized cysteine residue.
Figs. 8 and
9A show the most populated
conformations for each of the four IANBD-derivatized cysteine residues
at the bottom of TM 6 in the structural context provided by a model of
the TM domain of the receptor. TM 6 was oriented based on the
predictions and experimental data identifying which residues are
oriented toward the protein interior or facing the lipid membrane as
described above and shown in Fig. 7B.
The data from the present study provide structural evidence that
agonist binding to the To understand the fluorescence changes in a structural context,
we generated a molecular model of TM 6. Importantly, this model
corresponds well to the newly published high resolution structure of
rhodopsin (33). The TM 6 model proposed that the cysteine-substituted
residues (Cys-2696.31, Cys-2706.32,
Cys-2716.33, and Cys-2726.34) reside in an
A simple rotation of TM 6 was sufficient to explain our previous data
based on IANBD labeling of Cys-2856.47 (10). However, the
observation in the present study that IANBD at all four inserted
cysteines residues likely moves into a more hydrophobic environment
upon agonist binding suggests that the helical movement is more
complex. Of particular interest, EPR spectroscopy analysis of
spin-labeled rhodopsin mutants has provided strong evidence that light
activation of rhodopsin involves a rigid body movement of the
cytoplasmic part of TM 6 away from TM 3 (6). The present data could
support a similar movement of TM 6 in Except for the The biophysical analyses of conformational changes in rhodopsin and in
It is important to emphasize that our experiments have been carried out
in the absence of G protein and that it is very likely that the G
protein can affect the kinetics of the transition between the inactive
AR state and the active AR* state. Clearly, the slow kinetics of the
agonist-induced conformational change in the absence of G protein would
predict a high activation energy barrier for this transition (13). It
is conceivable, however, that the G protein is able to stabilize the
agonist-receptor complex and accordingly lower the activation energy
barrier substantially and cause receptor activation to occur
significantly faster. This may provide an explanation for the apparent
discrepancy between the slow kinetics of agonist-induced conformational
changes observed for the purified 2
adrenergic receptor, a prototype hormone-activated G
protein-coupled receptor. In the background of a mutant
2 adrenergic receptor, with a minimal number of
endogenous cysteine residues, new cysteines were introduced in
positions 2696.31, 2706.32,
2716.33, and 2726.34 at the cytoplasmic side of
transmembrane segment (TM) 6. The resulting mutant receptors were fully
functional and bound both agonists and antagonist with high affinities
also upon IANBD labeling. Fluorescence spectroscopy analysis of the
purified and site-selectively IANBD-labeled mutants suggested that the
covalently attached fluorophore was exposed to a less polar environment
at all four positions upon agonist binding. Whereas evidence for only a
minor change in the molecular environment was obtained for positions
2696.31 and 2706.32, the full agonist
isoproterenol caused clear dose-dependent and reversible increases in fluorescence emission at positions
2716.33 and 2726.34. The data suggest that
activation of G protein-coupled receptors, which are activated by
"diffusible" ligands, involves a structural rearrangement
corresponding to the cytoplasmic part of TM 6. The preferred
conformations of the IANBD moiety attached to the inserted cysteines
were predicted by employing a computational method that incorporated
the complex hydrophobic/hydrophilic environment in which the cysteines
reside. Based on these preferred conformations, it is suggested that
the spectral changes reflect an agonist-promoted movement of the
cytoplasmic part of TM 6 away from the receptor core and upwards toward
the membrane bilayer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (
2AR) was cloned more than
a decade ago and has since served as a prototypic member of the
receptor family (3, 4). A key issue in our understanding of GPCR function is the nature of the molecular mechanisms that couple agonist
binding to receptor activation and transmission of the signal across
the plasma membrane. Only recently spectroscopic techniques on purified
receptor preparations have been taken into use and permitted the first
direct insight into structural changes that occur during receptor
activation (4). The use of EPR spectroscopy by Hubbell, Khorana,
and co-workers (5-7) has showed evidence that activation of the
light-sensing receptor rhodopsin involves movements of transmembrane
segment (TM) 3 and 6 relative to one another. In addition, movements of
TM 6 in rhodopsin have been predicted from fluorescence spectroscopy
studies (8). Our application of fluorescence spectroscopy to the
2AR
has supported a role of TM 3 and 6 also for activation of GPCRs
activated by "diffusible" ligands. The sulfhydryl-reactive
fluorescent probe, IANBD, was covalently incorporated into the purified
2AR and used as a molecular reporter of the structural changes that
takes place following agonist binding to the receptor (9). Subsequent
mutagenesis identified two cysteines, Cys-1253.44 in the
middle of TM 3 and Cys-2856.47 in middle of TM 6, as
responsible for the observed agonist-induced fluorescence changes
(10).2
2AR also
indicated some possible important differences between the two
receptors. The EPR spectroscopic read-outs for rhodopsin demonstrated a
rapid formation of the active metarhodopsin II state (within microseconds) following light-induced conversion of the prebound cis-retinal to all-trans-retinal, whereas the
conversion of metarhodopsin back to the inactive metarhodopsin III
state was slow, with a t1/2 of about 6 min (12). In
contrast to the rapid activation and the slow inactivation kinetics
observed for rhodopsin, the spectroscopic analyses of the
2AR
indicated slow agonist-induced conformational changes
(t1/2 = ~2-3 min), significantly slower than the
predicted association rate of the agonist (9, 10). However, the
reversal of the agonist-induced conformational change was
relatively fast (t1/2 = ~30 s) (9, 13). These
differences in activation kinetics could either reflect inherent
differences between the two receptors or be a consequence of
differences in how the changes were detected.
2AR. We
decided to focus on the cytoplasmic end of TM 6 for two major reasons.
First, the evidence for TM 6 movements in response to light activation
of rhodopsin, was based on spectroscopic analysis of mutants that
contained cysteine residues in this particular region of TM 6, labeled
with either nitroxide spin labels or fluorescent probes (6-8).
Labeling of the
2AR in this region with a molecular reporter of
conformational changes would thus allow a more direct comparison
between rhodopsin and the
2AR. Second, many mutagenesis-based studies have indicated the importance of the cytoplasmic region of TM 6 in receptor activation and G protein coupling (3, 14); nonetheless, its
precise role is still not well understood, and conformational changes
at the cytoplasmic side of TM 6 have not been described for receptors
activated by diffusible ligands (4). Notably, our earlier evidence for
TM 6 movements in the
2AR was based on fluorescent labeling of a
naturally occurring cysteine (Cys-2856.47) situated in the
hydrophobic middle part of TM 6 (10). Labeling at this position did not
allow reliable predictions about the character of potential movements
at the cytoplasmic side of the helix.
2AR, four residues in this region
were substituted with cysteines. The cysteines were inserted in a
mutant
2AR containing a reduced number of endogenous cysteines (
2AR-Cys-min) (see Fig. 1). The spectroscopic analyses of the purified and fluorescently labeled
2AR cysteine mutants provided evidence that activation of the
2AR involves structural changes at
the cytoplasmic side of TM 6. Employment of a computational method,
which incorporated the complex hydrophobic/hydrophilic environment in
which the inserted cysteines reside, allowed a prediction of the
preferred conformations of the attached IANBD moiety. Analysis of these
preferred conformations in context of a receptor model supported the
possibility that TM 6 undergoes movements that are similar to those
predicted to take place in rhodopsin during photo-activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR-Cys-min, which encoded the human
2AR,
containing the mutations C77V, C265A, C327S, C378A, and C406A (10). In
addition, the construct was tagged at the N terminus with the cleavable influenza-hemagglutinin signal sequence followed by the FLAG epitope (Sigma), and at the C terminus with six histidines (15). Mutations were
all generated by polymerase chain reaction-mediated mutagenesis using
Pfu polymerase according the manufacturer's instructions (Stratagene, La Jolla, CA). The generated polymerase chain reaction fragments were digested with the appropriate enzymes, purified by
agarose gel electrophoresis, and cloned into the baculovirus expression
vector pVL1392 containing
2AR-Cys-min (10) or
2AR-Cys-min-Gs
(16). All mutations were confirmed by
restriction enzyme analysis and DNA sequencing.
2AR-Cys-min and mutant
constructs were expressed in Sf-9 insect cells and high titer virus
stocks generated as described using the the BaculoGold transfection kit
(Pharmingen, San Diego, CA) (17).
2AR-Gs
fusion constructs as
described (16). Protein was determined using the Bio-Rad DC protein
assay kit.
2AR
were also performed as described using [3H]DHA as
radioligand (9). Binding data were analyzed by nonlinear regression
analysis using Prism 2.0 from GraphPad Software (San Diego, CA).
2AR-Cys-min and mutant
receptors were solubilized in 0.8%
n-dodecyl-
-D-maltoside (D
M) (Anatrace) and
subsequently purified by nickel chromatography using Chelating
Sepharose (Amersham Pharmacia Biotech) followed by alprenolol affinity
chromatography as previously described (17, 20). Approximately 1-2
nmol of purified protein could generally be obtained from a 500-ml
culture. The specific activity of the purified receptors varied between
3 and 9 nmol/mg protein. Protein was determined using the
detergent-insensitive Bio-Rad DC protein assay kit. Purified receptors
were analyzed by 10% SDS-polyacrylamide gel electrophoresis and
visualized by standard Coomassie staining.
M), and labeling achieved by
recycling 1.0 ml of 0.5 mM IANBD in high salt buffer
several times over the nickel column for 20 min. Excess dye was removed
by extensive washing of the column with high salt buffer. The labeling
procedure resulted in incorporation of 1.1-1.5 mol of IANBD/mol of
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 mass of 50,000 Da for the
receptor. Fluorescence spectroscopy was performed at room temperature
on a SPEX Fluoromax-2 spectrofluorimeter connected to a PC equipped with the Datamax 2.2 software package (Jobin Yvon Inc., Edison, NJ) as
described (9, 17). In all experiments the excitation and emission
bandpass were set at. 4.2 nm. Both emission scans and time course
experiments were done with 10-20 pmol of IANBD-labeled receptor in 500 µl of buffer (20 mM Tris-buffer, pH 7.4, containing 100 mM NaCl and 0.08% D
M) under constant stirring. In
emission scan experiments the excitation wavelength was 481 nm, and
emission was measured from 490 to 625 nm. During time scan experiments the excitation wavelength was 481 nm, and emission was measured at a
wavelength of 530 nm. The volume of the added ligands was one
percentage of total volume, and fluorescence was corrected for this
dilution. The compounds tested in the fluorescence experiments had an
absorbance of less than 0.01 at 481 and 525 nm in the concentrations used excluding inner filter effects.
M). To correct for dilution/ionic
strength effects on fluorescence, measurements were performed in
parallel using a 1.0 M stock of KCl. 10 µl of quencher
(potassium iodide) or control solution (potassium chloride) were added
sequentially followed by thorough mixing after each addition and
subsequent recording of fluorescence using the Constant Wavelength
Analysis program in the Datamax software package (Jobin Yvon Inc.). The
excitation wavelength was 481 nm, and the emission wavelength was 530 nm. The data were plotted according to the Stern-Volmer equation,
Fo/F = 1 + KSV[Q], where
Fo/F is the ratio of fluorescence
intensity in the absence and presence of quencher ([Q]), and
KSV is the Stern-Volmer quenching constant
(21).
-helix as
described under "Results" and according to Ref. 11. The conformational memories method (22, 23) was used to explore the
preferred conformations of IANBD attached to the inserted cysteines (Cys-2696.31, Cys-2706.32,
Cys-2716.33, and Cys-2726.34) (24). The
simulations were performed on the isolated TM 6 peptide using a new
computational method that incorporated the mixed
hydrophobic/hydrophilic environment in which the cysteines are located.
To reduce the required computing time, only the dihedrals of residues
from Glu-2686.30 to Ile-2766.38 were varied.
The computational method is based on the work of White and Wimley (25),
who showed evidence that the membrane cannot be considered a homogenous
hydrophobic layer but consists of an ~30 Å relatively constant, low
dielectric region, sandwiched between two complex regions of variable
dielectric behavior. In the method, the interfacial regions are
approximated as a dielectric gradient. Because solutions of the
Poisson-Boltzmann equation are iterative, it is not possible to perform
such calculations within a statistical mechanics simulation-like
conformational memories. Thus, the following procedure was implemented:
1) the 15Å interfacial region was grid into 0.2Å spacing; 2) the
water-interface boundary was set to a dielectric constant of 78; 3) the
interface-lipid boundary was set to a dielectric constant of 5; 4) the
penetration dielectric constant was set to to
e(z) = 78(1
z) + 5z, z = 0.2n/15, n = 0,1,2,
,75; 5) the Poisson Boltzmann
equation for each dielectric slice was solved, and the potentials were
stored on the grids numerically in data tables; 6) potentials were
obtained over a coverage of at least the 60 Å region spanning the
membrane; and 7) a linear interpolation of the potentials between grids
was implemented. Still and co-workers (26) have developed a generalized Born surface area continuum solvation model for water and chloroform. Creation of the membrane interface was implemented as follows: 1) for
each new configuration of the peptide, either a conformational change
or a rigid body translation or rotation, both the chloroform and water
solvation energy was computed; 2) atoms that have a z
coordinate less than 15 were assigned the chloroform solvation energy;
3) atoms that have a z coordinate greater than 0 were assigned the water solvation energy; 4) atoms that have 15 < z < 0 are assigned a solvation energy
esolv = (x)(water) + (1
x)(chloroform), x = (15 + z)/15.
The starting spatial configuration of the peptide consisted of placing
the
-carbon of the IANBD-labeled cysteine mutant 0.5 Å into the
interface at the interface-lipid boundary (i.e. 0, 0,
14.5), and placing the
-carbon of the i+7 residue in
the chloroform environment such that the vector between i
and i+7 are perpendicular to the xy plane. The
i+7
-carbon residue resides at an approximate coordinate
of 0, 0,
24. The different Pro-kink conformations were selected from
our previous and published simulations on TM 6 of the
2AR (10). The
preferred IANBD conformations defined by the computational simulations
described above were analyzed in the context of a three-dimensional
model of the seven-helix
2AR transmembrane domain modeled after the rhodopsin template (27, 28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR, we
wanted to reduce the number of endogenous cysteines available for
chemical derivatization. All of the free cysteines could not be
substituted without severe decrease in receptor expression; however,
the expression was preserved in a mutant where Cys-772.52,
Cys-2656.27, Cys-3277.54, Cys-378, and Cys-406
were substituted with valine or serine (10). This mutant still contains
three cysteines available for chemical derivatization,
Cys-1163.35, Cys-1253.44, and
Cys-2856.47, but additional mutation of any of these
residues substantially reduced expression (data not shown and Ref. 10).
Therefore, we chose this construct, which we named
2AR-Cys-min, for
further studies. Importantly,
2AR-Cys-min binds agonists and
antagonists with the same affinities as the wild type
2AR and
couples to adenylyl cyclase with the same efficiency (10).
2AR-Cys-min background (Fig. 1). The substitutions had only minor
effects on the general pharmacological properties of the receptor
(Table I). Saturation binding experiments revealed unchanged affinities in the mutants for the antagonist [3H]DHA (Table I). Competition binding experiments with
the full agonist isoproteronol (ISO) showed Ki
values for inhibition of [3H]DHA binding to the mutant
receptors that were either similar or slightly smaller than that of
2AR-Cys-min (Table I). Following purification and labeling with
IANBD, the mutants and
2AR-Cys-min were analyzed again for their
capability to bind [3H]DHA and ISO. Both the purified
2AR-Cys-min and the purified mutants exhibited high affinity for
[3H]DHA, although a moderate decrease in apparent
[3H]DHA affinity was observed in
2AR-Cys-min-L272C
(Table I). However, the affinities for ISO were well preserved in all
constructs upon purification and IANBD labeling (Table I). The mutants
were finally analyzed for their ability to activate adenylyl cyclase in
Sf-9 cell membrane preparations. All mutants exhibited evidence for
efficient coupling to adenylyl cyclase, and the observed
EC50 values for ISO were comparable to that of
2AR-Cys-min (Table I). Of the four mutants, only
2AR-Cys-min-L272C showed evidence of increased agonist-independent
activity as indicated by a small increase in basal receptor activity in
comparison with the wild type (Table I).
View larger version (65K):
[in a new window]
Fig. 1.
Snake diagram of the
2AR. The residues at the cytoplasmic side of
TM 6 that were mutated to cysteines are indicated by white
letters in black circles. The positions of the four
mutated residues are indicated by their generic number in the
2AR
followed by their number according to the Ballesteros-Weinstein
nomenclature (11), which assigns the identifier 6.50 to the conserved
Pro-288 (Pro-2886.50) in TM 6. The cysteines in the
transmembrane segments that are present in
2AR-Cys-min
(Cys-1163.35, Cys-1253.44, and
Cys-2856.47) are indicated by black letters in
white circles.
Binding properties and functional coupling of 2AR-Cys-min and mutant
receptors
L where
L = concentration of [3H]DHA (40). The
IC50 values for inhibition of [3H]DHA binding by
isoproterenol were determined by nonlinear regression analysis of data
from competition binding experiments on Sf-9 cell membranes and on
purified IANBD-labeled receptors. The IC50 values were
subsequently converted to Ki values using the
equation Ki = IC50/(1 + L/Kd), where L = concentration of [3H]DHA. Data are the means of
n = 3-4 with the S.E. intervals calculated from
pIC50 ± S.E. shown in parentheses. Functional coupling to
adenylyl cyclase was measured in Sf-9 cell membranes as described under
"Experimental Procedures." Basal activity was calculated as a
percentage of basal
2AR-Cys-min activity (means ± S.E.,
n = 3-5). EC50 values (means of
n = 3-5 with S.E. intervals shown in parentheses and
percentages of maximum cyclase (means ± S.E., n = 3-5) were determined from isoproterenol dose-response curves.
shows
less sensitivity to cysteine modifications as compared with downstream
effectors such as adenylyl cyclase, we subcloned all four mutant into
the previously described fusion construct between the
2AR and
Gs
. This fusion construct allows direct and efficient
assessment of G protein coupling by measurement of GTPase activity in
membrane preparations (16, 29). IANBD labeling was achieved directly
in situ by treating Sf-9 cell membranes, expressing the
2AR-Gs
fusion constructs, with a large excess of
IANBD (0.5 mM) for 30 min. Following this labeling
procedure, ISO was found to cause clear increases in GTPase activity in
both
2AR-Cys-min-Gs
and in the cysteine-substituted
fusion constructs. The GTP hydrolysis was linear for at least 30 min,
and the relative increases in GTPase activity were similar to those
observed without IANBD labeling (Table
II). Furthermore, the EC50
values for ISO also were well preserved upon IANBD labeling. For
2AR-Cys-min-H269C,
2AR-Cys-min-A271C, and
2AR-Cys-min-L272C,
the values were essentially unchanged, whereas in
Cys-2706.32 a 4-5-fold increase in the EC50
value for ISO was found after IANBD derivatization. Conceivably, this
could reflect a partial steric inhibition of the receptor-G protein
interaction because of covalent modification of a cysteine inserted in
a region known to form part of the G protein binding site (30).
ISO stimulated GTPase activity of 2AR-Cys-min and cysteine mutants
fused to Gs
with and without IANBD labeling
constructs were
determined in Sf-9 cell membranes as described under "Experimental
Procedures." The EC50 values for ISO (means of
n = 3 with S.E. intervals shown in parentheses) were
determined from nonlinear regression analysis of ISO dose-response
curves. The maximum GTPase activities (means ± S.E.,
n = 3-5) are indicated as the specific GTPase activity
measured in presence of 10 µM ISO in percentages of the
basal GTPase activity measured in absence of ligand.
2AR--
The emission from the IANBD fluorophore is highly
dependent on the polarity of the surrounding solvent. Decreasing the
polarity of the solvent is accompanied by a concurrent increase in
fluorescence emission and a blue shift of the
max
(wavelength at which maximal fluorescence emission occurs). Although
the
max for IANBD in aqueous solvent is 538 nm,
max is reduced to around 532 nm in 90% dioxane, 529 in
95% dioxane, and 523 nm in 100% dioxane (Table III). The fluorescence emission
spectrum of purified
2AR-Cys-min labeled with IANBD showed a
max of 530 nm (Table III), consistent with our previous
data and the localization of the major labeling sites,
Cys-1253.44 and Cys-2856.47, in a highly
hydrophobic environment (10). The emission spectra of the purified and
IANBD-labeled mutants all showed an ~2-nm red shift in
max as compared with IANBD-labeled
2AR-Cys-min (Table
III). The red shift in
max suggests that IANBD attached to Cys-2696.31, Cys-2706.32,
Cys-2716.33, and Cys-2726.34 is located in a
slightly less hydrophobic environment as compared with IANBD bound to
the endogenous cysteines Cys-1163.35,
Cys-1253.44, and Cys-2856.47. In this context
it is important to emphasize that the pharmacological data described in
the previous section provided evidence that the general structure of
the receptor is preserved in the mutants. Accordingly, any new
fluorescent signal detected in the IANBD-labeled mutants can most
likely be attributed to labeling of the inserted cysteines rather than
to altered labeling of the endogenous cysteines present in the
background,
2AR-Cys-min.
max values for the fluorescence emission of IANBD in solvent
and bound to the different receptor constructs
max values were determined from emission scan experiments
using an excitation wavelength of 481 nm. The values for buffer and
dioxane were determined using 10 µM free IANBD. For the
different IANBD-labeled receptor constructs data are the means ± S.E. of n = 4-5.
2AR is accompanied by a reversible decrease
in fluorescence intensity and that the spectral changes reflect
conformational changes involved in receptor activation (9).
Cys-1253.44 in TM 3 and Cys-2856.47 in TM 6 were found to be responsible for these spectral changes (10). Both
residues are still present in
2AR-Cys-min, and accordingly stimulation of this receptor with ISO leads to a decrease in
fluorescence emission over time that is readily reversed by the
antagonist alprenolol (ALP) (Fig.
2A, left panel). A
similar response is not observed in response to addition of ALP alone
(Fig. 2A, right panel). Therefore, the
fluorescence spectra of the four mutant receptors should be interpreted
in the context of this background response. It should also be noted
that, as in our previous studies (9, 17), we observed a drift in
base-line fluorescence from all IANBD-labeled constructs prior to the
addition of any ligand (Fig. 2). The drift was unaffected by the
addition of 0.1% bovine serum albumin, 10% glycerol, or phospholipids
to the cuvette (9) and is most likely due to IANBD photobleaching and
minor loss of receptor (17). Importantly, the decline in fluorescence
over time was constant in each individual experiment allowing detection of even minor changes in base-line fluorescence over time. Moreover, no
correlation between the magnitude of the decline and the different mutations was observed.
View larger version (34K):
[in a new window]
Fig. 2.
Time course experiments with purified,
IANBD-labeled mutants of the 2AR.
A, emission from purified IANBD-labeled
2R-Cys-min
measured over time in response to 3 × 10
4
M (
)ISO followed by 10
4
M (
)ALP (left panel) and in response to
10
4 M (
)ALP followed by 3 × 10
4 M ISO (right panel). Please
note that the affinity for ALP is 50-100-fold higher than for ISO. The
dashed lines represent extrapolations of the base lines
before addition of ligand. B, left panels,
emission from IANBD-labeled
2R-Cys-min mutants (H269C, K270C, A271C,
and L272C) measured over time in response to either 1 × 10
3 M or 0.3 × 10
3
M (
)ISO followed by 10
4 M
(
)ALP. Right panels, emission from IANBD-labeled
2R-Cys-min mutants (H269C, K270C, A271C, and L272C) measured over
time in response to 10
4 M (
)ALP followed by
1 × 10
3 M (
)ISO. Excitation was set
at 481 nm, and emission was measured at 530 nm. Fluorescence in the
individual traces was normalized to the fluorescence observed just
before addition of ligand. The experiments shown are representative of
at least three identical experiments.
2AR-Cys-min (Fig. 2B). For both
2AR-Cys-min-H269C and
2AR-Cys-min-K270C, no change in base-line
fluorescence could be detected in response to either 1000 µM of the full agonist ISO or 100 µM of the
antagonist ALP (Fig. 2B). Also, no change was observed in
response to 300, 100, and 30 µM of ISO (data not shown).
A possible explanation is that agonist binding causes an increased
hydrophobicity of the environment surrounding IANBD bound to
Cys-2696.31and Cys-2706.32 and that the
resulting increase in emission counterbalances the decrease in emission
from IANBD bound to the background Cys-1253.44 and
Cys-2856.47. Alternatively, we cannot exclude the
possibility that the apparent absence of response could be due to a
structural perturbation of the receptor in the mutants that alters in
the labeling of (or the signal from) the background cysteines
Cys-1253.44 and Cys-2856.47. However, given the
preserved pharmacological function of both mutants, we would argue that
an environmental change around Cys-2696.31 and
Cys-2706.32 is a likely interpretation. Moreover, because
IANBD derivatization of Cys-2696.31and
Cys-2706.32 in membranes still permit receptor activation
and G protein coupling (see previous section), it is also reasonable to
assume that the lack of response is not due to inability of the
IANBD-labeled receptors to adopt the activated state.
2AR-Cys-min-A271C and
2AR-Cys-min-L272C lead to clear increases in fluorescence emission
relative to the extrapolated base line with the largest change observed for
2AR-Cys-min-L272C (Fig. 2B). Both responses could be
completely reversed by the antagonist ALP (Fig. 2). In addition, the
agonist-induced fluorescence changes for both mutant
2AR-Cys-min-A271C and
2AR-Cys-min-L272C could be blocked by
addition of ALP before addition of ISO (Fig. 2B). Please
notice that the affinity of ALP is ~50-100-fold higher than the
affinity for ISO, and therefore, 10
4 M ALP
should easily displace a 3- or 10-fold higher concentration of ISO. It
should also be noted that the ISO-induced increases in fluorescence
intensity observed for positions Cys-2716.33 and
Cys-2726.34 were not accompanied by detectable changes in
max (data not shown). In summary, the data suggest that
binding of the agonist ISO causes a conformational change in the
receptor that exposes IANBD bound to Cys-2716.33 and
Cys-2726.34 to a more hydrophobic or less hydrophilic environment.
2AR-Cys-min,
2AR-Cys-min-A271C, and
2AR-Cys-min-L272C were analyzed by nonlinear regression analysis
using a single exponential function to determine
t1/2 values for the observed changes. To carry out
this analysis, raw traces were transformed into the kinetic curves
(shown in Fig. 3) by subtracting all data
points from an extrapolated base line with a slope defined from the
fluorescence trace after the fluorescence intensity has stabilized upon
ISO stimulation (last 300 s before ALP is added). This was chosen
instead of a simple extrapolation of the base line prior to ISO
addition to eliminate potential nonspecific effects of ISO on the
base-line drift. In
2AR-Cys-min-A271C and
2AR-Cys-min-L272C, a
normalized
2AR-Cys-min trace was also subtracted to exclude the
influence of the background decrease on the kinetics of the ISO induced
increase in fluorescence. If the agonist-induced increase in
fluorescence observed in
2AR-Cys-min-A271C and
2AR-Cys-min-L272C
represent new read-outs of the same series of conformational changes
that are also reported by IANBD bound to Cys-1253.44 and
Cys-2856.47, it would be expected that the changes would
occur with similar kinetics. As shown in Fig. 3, the agonist-induced
changes in fluorescence in
2AR-Cys-min,
2AR-Cys-min-A271C, and
2AR-Cys-min-L272C did display similar kinetics with
t1/2 values of 197 ± 24 s (mean ± S.E., n = 3), 155 ± 6 s (mean ± S.E., n = 4), and 181 ± 11 s (mean ± S.E.,
n = 4), respectively (Fig. 3). The
t1/2 value for the response observed in the wild
type
2AR was 135 ± 7 s, (mean ± S.E.,
n = 2) (data not shown). Although not identical, these
values are within a similar range, in particular in comparison with the
microsecond time scale reported for conformational changes in
rhodopsin. It is, therefore, reasonable to assume that the fluorescence
changes represent different read-outs of the same sequence of
structural changes (Fig. 3).
View larger version (20K):
[in a new window]
Fig. 3.
The kinetics of the isoproterenol induced
changes in fluorescence. Representative kinetic curves for
IANBD-labeled 2AR-Cys-min (upper panel),
2AR-
2AR-Cys-min-A271C (middle panel), and Cys-min-L272C
(lower panel) are shown. The kinetic curves for ISO
stimulation were obtained from representative raw traces by subtracting
all data points from an extrapolated base line with a slope defined
from the fluorescence trace after the fluorescence intensity has
stabilized upon ISO stimulation (last 300 s before ALP is added).
The resulting curves were analyzed by nonlinear regression analysis
using a single exponential function to determine
t1/2 values for the fluorescent changes (means ± S.E. n = 4). The t1/2 values were
197 ± 24 s (mean ± S.E., n = 3),
155 ± 6 s (mean ± S.E., n = 4), and
181 ± 11 s (mean ± S.E., n = 4) for
2AR-Cys-min,
2AR-Cys-min-A271C, and
2AR-Cys-min-L272C,
respectively.
2AR-Cys-min,
2AR-Cys-min-A271C,
and
2AR-Cys-min-L272C. Collisional quenching requires a bimolecular
interaction between the quencher and the fluorophore and, accordingly,
such experiments can determine the accessibility of the fluorophore to
the surrounding solvent (21). The aqueous quencher iodide
(I
) is a strong quencher of the fluorescence from IANBD
as illustrated by the linear Stern-Volmer plot in Fig.
4, in which
Fo/F is plotted against the potassium
iodide concentration. The slope of the line represents the Stern-Volmer
constant (KSV), which was 5.9 M
1 for the free probe. The iodide quenching
of IANBD-labeled
2AR-Cys-min was substantially smaller but still
apparent with a linear Stern-Volmer plot and a
KSV value of 3.1 M
1
(Fig. 4A). This is consistent with the localization of the
major labeling sites, Cys-1253.44 and
Cys-2856.47, in a hydrophobic environment that is only
partially accessible to the aqueous solvent. Interestingly, the
KSV values for both
2AR-Cys-min-A271C and
2AR-Cys-min-L272C were markedly higher (4.6 and 4.5 M
1), suggesting that IANBD bound to
Cys-2716.33 and Cys-2726.34 are more exposed to
the solvent than Cys-1253.44 and Cys-2856.47
(Fig. 4, B and C). These data provide additional
direct support for the prediction that Cys-2716.33 and
Cys-2726.34 are situated close to the cytoplasmic
membrane-water interface (Fig. 1).
View larger version (16K):
[in a new window]
Fig. 4.
Stern-Volmer plots of iodide quenching
in 2AR-Cys-min,
2AR-Cys-min-A271C, and
2AR-Cys-min-L272C. A, quenching of
2AR-Cys-min with the aqueous quencher potassium iodide in presence
(open circles) and absence (closed triangles) of
300 µM ISO. B, quenching of
2AR-Cys-min-A271C with the aqueous quencher potassium iodide in
presence (open circles) and absence (closed
triangles) of 300 µM ISO. C, quenching of
2AR-Cys-min-L272C with the aqueous quencher potassium iodide in
presence (open circles) and absence (closed
triangles) of 300 µM ISO. The quenching experiments
were carried out and data plotted as described under "Experimental
Procedures." In absence of ISO the Stern-Volmer quenching constants
(KSV) were 3.13 ± 0.07 M
1 (n = 2), 4.6 ± 0.3 M
1 (n = 3) and 4.5 ± 0.12 M
1 (n = 3) (means ± S.E.) for
2AR-Cys-min,
2AR-Cys-min-A271C, and
2AR-Cys-min-L272, respectively. In presence of 300 µM
ISO the Stern-Volmer quenching constants (KSV)
were 3.09 ± 0.05 M
1 (n = 2), 4.0 ± 0.2 M
1 (n = 3), and 4.1 ± 0.23 M
1
(n = 3) (means ± S.E.), respectively. In all
experiments the excitation was 480 nm, and emission was recorded at 530 nm. The
2AR-Cys-min Stern-Volmer plot (A) is shown in
B and C as a dashed line for
comparison.
2AR-Cys-min, iodide quenching was identical in presence of ISO,
indicating that the change in environment surrounding Cys-1253.44 and Cys-2856.47 observed in the
time course experiments is not accompanied by a measurable change in
iodide accessibility (Fig. 4A). However, a minor decrease in
iodide accessibility was observed for both
2AR-Cys-min-A271C and
2AR-Cys-min-L272C (Fig. 4, B and C).
Importantly, this observation is consistent with the time course
experiments shown in Fig. 2, which indicated a movement of
IANBD-labeled Cys-2716.33 and Cys-2726.34 to a
more hydrophobic and expectably a less solvent-accessible environment.
2AR-Cys-min-A271C and
2AR-Cys-min-L272C--
The ISO-mediated
response at both
2AR-Cys-min-A271C and
2AR-Cys-min-L272C
displayed a clear dose dependence (Fig.
5). The maximum response at
2AR-Cys-min-L272C was twice as big as the response at
2AR-Cys-min-A271C. Fitting the data points to a single-site hyperbolic function showed EC50 values of 32 µM for
2AR-Cys-min-A271C and 3.9 µM for
2AR-Cys-min-L272C. These values should be compared with the
observed EC50 value of 29 µM for the
IANBD-mediated response caused by labeling of Cys-1253.44
and Cys-2856.47 (9). It must be emphasized that these
EC50 values are determined on purified protein in absence
of G protein and thus cannot be compared with the EC50
values determined in functional assays (adenylyl cyclase or GTPase
activity). As outlined under "Discussion" it can be expected that
the G protein can affect the kinetic parameters of agonist-promoted
structural changes substantially.
View larger version (15K):
[in a new window]
Fig. 5.
Dose dependence of isoproterenol induced
changes in fluorescence. The calculated percentages of change in
fluorescence for IANBD-labeled 2AR-Cys-min-A271C (upper
panel) and
2AR-Cys-min-L272C (lower panel) were
plotted against the used ISO concentration and fitted to a hyperbolic
function (F = FoL/EC50 + L;
F, change in fluorescence at the ligand concentration
(L); Fo, maximum change in
fluorescence; EC50, ligand concentration at which half
maximum response occurs). The ISO-induced changes in fluorescence were
calculated from the magnitude of the alprenolol reversal
(10
4 M). The changes are means ± S.E.
of n = 3. Curve fittings were done using Prism 2.0 (GraphPad Software).
2AR correlates directly with the intrinsic efficacy of the agonist (9). We inferred
from this correlation that IANBD attached to the receptor were
reporting conformational changes associated with receptor activation
(9). A similar experiment was carried out on IANBD-labeled
2AR-Cys-min-A271C and
2AR-Cys-min-L272C. In addition to the full
agonist ISO, we tested the partial agonists dobutamine (DOB) and
salbutamol (SAL). Before measuring the effect of these compounds on
fluorescence, we wanted to ensure that they bound with high affinity to
the receptor and were able to act as strong partial agonists.
Competition binding experiments demonstrated that both dobutamine and
salbutamol bind with high affinity to the purified and IANBD-labeled
2AR-Cys-min-A271C and
2AR-Cys-min-L272C. The Ki values for dobutamine and salbutamol were 2.2 (2.1-2.3) and 1.4 (1.2-1.5) µM, respectively, at
A271C and 3.1 (2.0-5.0) and 0.27 (0.25-0.30) µM,
respectively, at L272C. This should be compared with 1.3 (0.7-2.4) and
0.7 (0.3-1.3) µM, respectively, in
2AR-Cys-min (all
data are means of n = 3 with the S.E. intervals in
parentheses calculated from pIC50 ± S.E.). As shown in
Fig. 6A, dobutamine and
salbutamol also acted as strong partial agonist as determined from
adenylyl cyclase experiments on SF-9 cell membranes expressing
2AR-Cys-min-A271C and
2AR-Cys-min-L272C. The observed efficacies
correspond well to those reported previously for the wild type
2AR
(9). The apparent higher efficacy at
2AR-Cys-min-L272C is most
likely a reflection of the weak constitutive receptor activity observed
in this mutant (Table I) (31). In the fluorescence experiments we
observed a strong correlation between these efficacies of dobutamine,
salbutamol, and ISO and the observed increases in emission at both
2AR-Cys-min-A271C and
2AR-Cys-min-L272C (DOB < SAL < ISO) (Fig. 6). It should be noted that the absence of response to
dobutamine at
2AR-Cys-min-A271C does not necessarily reflect that
dobutamine is not causing a conformational change at
Cys-2716.3 but only that the expected increase in
fluorescence either counterbalances the decrease in fluorescence
because of labeling of Cys-1253.44 and
Cys-2856.47 or is below our detection limit. No change in
base-line fluorescence was observed in response to the inverse agonist,
ICI 118,551 (data not shown).
View larger version (20K):
[in a new window]
Fig. 6.
Comparison of agonist efficacy and changes in
fluorescence in IANBD-labeled
2AR-Cys-min-A271C and
2AR-Cys-min-L272C. A, efficacy of
DOB and SAL relative to ISO in adenylyl cyclase experiments on
membranes expressing
2AR-Cys-min-A271C or
2AR-Cys-min-L272. Data
are increases in adenylyl cyclase activity in presence of 100 µM DOB or SAL in percentage of the increase observed in
presence of 100 µM ISO (means ± S.E.,
n = 2). B, effect of DOB, SAL, and ISO on
fluorescence from IANBD-labeled
2AR-Cys-min-A271C and
2AR-Cys-min-L272C. The magnitude of the alprenolol reversal
(10
4 M) was used as a measure of the
ligand-induced changes in fluorescence. The percentages of change are
means ± S.E., n = 3. The concentrations of
ligands used were as follows: 3 × 10
4 M
ISO, 3 × 10
4 M salbutamol, and 3 × 10
4 M dobutamine.
-helix
from Ser-2626.24 to Val-2976.59 (Fig.
7, A and B). A
proline kink motif around the highly conserved Pro-2886.50
has been directly indicated from the electron density maps of rhodopsin
(27) and supported by application of the substituted cysteine
accessibility method to the dopamine D2 receptor (32). Moreover, this kink is apparent in the very recently published high
resolution structure of rhodopsin (33). The spin labeling studies in
rhodopsin have identified the N terminus of TM 6 at position 6.23-6.24
(7), whereas substituted cysteine accessibility method studies
identified the C terminus of TM 6 at position 6.59 (32) (Fig.
7B). These limits are also very close to those observed in
the high resolution structure of rhodopsin showing the N terminus at
position 6.27 and the C terminus at position 6.59 (33). The sequence
identity among these GPCRs suggests that similar helical segments
should be expected for the
2AR. However, because of the presence of
four positively charged residues (Lys-2636.25,
Lys-2676.29, Lys-2706.32, and
Lys2736.35), the hydrophobicity plot, on which most
common prediction methods for transmembrane segments rely, is not
predicting that the segment between Ser-2626.24 and
Lys-2736.35 is helical in the
2AR. (Fig. 7C).
The simplest interpretation would be that TM 6 differs substantially
from rhodopsin and that the TM 6 helix does not commence before
Lys-2736.35. We find it likely that this is not the correct
interpretation and, thus, that the hydrophobicity plot is misleading.
The same hydrophobicity criterion that considers the membrane as a
hydrophobic medium predicts that in a helical transmembrane segment,
polar residues would be expected to face the protein interior while lipid-facing residues are expected to be apolar. This is not the case
in the
2AR where the four lysines are predicted to face the lipids
if they are a part of the TM 6 helix (Fig. 7B).
Interestingly, it is known from structures of membrane proteins that
Arg and Lys residues tend to concentrate at the cytoplasmic boundary of the helices, where they face the lipids and their positive charge interact with the phospholipid headgroups (11, 34). This Arg/Lys cytoplasmic motif could explain why the hydrophobicity plot of TM 6 in
the
2AR is not reflecting the actual secondary structure of the
segment from Ser-2626.24 to Lys-2736.35.
View larger version (43K):
[in a new window]
Fig. 7.
A, molecular model of TM 6 as a
Pro-kinked -helix (yellow ribbon). The four residues
substituted with cysteines at the cytoplasmic boundary of TM 6 are
shown in purple (His-2696.31,
Lys-2706.32, Ala-2716.33, and
Leu-2726.34). The endogenous Cys-2856.47 is
also indicated in purple. The side chains of residues in TM
6 experimentally identified to face the protein interior are shown in
green. The same residues are shown in yellow in
the helical net representation in panel B. The three Lys
side chains at the cytoplasmic side of TM 6 are oriented toward the
phospholipid headgroups, where they can participate in ionic
interactions. Positions where nonconserved Arg or Lys residues appear
in any neurotransmitter GPCR aligned (not shown) are marked with a
thick circle. Note the consistency between the
yellow residues supposed to face one side of the helix
(right face) and the thick circle supposed to
face the lipid headgroups (left face). C,
hydrophobicity plot commonly used to predict the TM helix boundaries
predicts a TM 6 for the
2AR receptor that is three turns shorter
than the observed TM 6 helix end for rhodopsin and the D2 receptor (see
text for further details).
2AR was constructed, and the Arg and Lys residues within TM 6 of
any aligned GPCR were identified. The Arg/Lys cytoplasmic motif
predicts that Arg/Lys in any homologous receptor that belongs to the TM
6
-helix at the cytoplasmic interface should concentrate on one face
of the
-helix facing the phospholipid headgroups. Whenever an Arg or
Lys residue was found in any of the 56 neurotransmitter GPCRs aligned
(data not shown), the position is highlighted by a thicker
circle in Fig. 7B. Interestingly, we observed that the arginines and lysines are clustering in a pattern consistent with the
presence of an
-helix from Ser-2626.24 to
Lys-2736.35. In contrast, all positions prior to
Ser-2626.24 contain arginines and lysines in a given
receptor, and thus this segment lacks any
-helical periodicity.
Accordingly, it is our prediction that the cytoplasmic N terminus of
the TM 6 helix is around position 2626.24, or at least at
position 6.27 as revealed in the rhodopsin crystal structure (33), and
that the four cysteine substituted residues in TM 6 reside in an
-helical region. Furthermore, we predict that the residues are
situated in a mixed hydrophobic-hydrophilic environment at the
lipid-water interface consistent with the spin labeling data in
rhodopsin, indicating the presence of a hydrophilic/hydrophobic boundary around residues 6.33-6.37 (7).
2AR-Cys-min-A271C were similar following
reconstitution into phospholipid vesicles as compared with detergent
micelles (data not shown). For the purpose of defining the approximate
conformational space for the IANBD moiety, we therefore allowed the
assumption that the mixed hydrophobic-hydrophilic region is at least
roughly the same in detergent micelles as in phospholipid membranes.
View larger version (64K):
[in a new window]
Fig. 8.
Extracellular view of the TM domain of
the 2AR. An illustrative set of the
preferred conformations for the IANBD side chain covalently attached to
the four substituted cysteines at the cytoplasmic side of TM 6 are
shown color-coded. Note that IANBD attached to Cys-2716.33
and Cys-2726.34 are facing the interior of the TM helix
bundle, whereas IANBD attached to Cys-2696.31 and
Cys-2706.32 are oriented toward the lipid membrane.
View larger version (31K):
[in a new window]
Fig. 9.
Proposed conformations of the inactive and
active states of the 2AR. A,
inactive conformation of the receptor characterized by a highly kink TM
6 helix (blue) with the cytoplasmic end in close proximity
to TM 3 and the helix bundle. An illustrative set of the preferred
conformations of the IANBD moiety covalently attached to the four
substituted cysteines at the cytoplasmic side of TM 6 is shown. IANBD
attached to Cys-2696.31 (blue) and
Cys-2706.32 (red) are facing the hydrophobic
milieu, whereas IANBD attached to Cys-2716.33
(yellow) and Cys-2726.34 (purple) are
facing the protein interior. B, a hypothetical active
conformation of the receptor in which the cytoplasmic side of TM 6 is
moved away arbitrarily from the helix bundle and upwards toward the
hydrophobic region, marked by straight lines. This putative
rearrangement of TM 6 moves all four IANBD-labeled residues upwards and
outwards allowing them to penetrate further into the more hydrophobic
region of the membrane/detergent micelles and away from the more
hydrophilic polar headgroups as well as from the predicted more
hydrophilic interior of the receptor protein. The movement can explain
the observed shift for all four IANBD-labeled cysteines toward a less
polar environment upon receptor activation. Because residues
Cys-2696.31 and Cys-2706.32 are facing the
hydrophobic aliphatic chains in both the inactive and active states,
whereas Cys-2716.33 and Cys-2726.34 transfer
from the protein interior to being exposed to the hydrophobic aliphatic
chains upon activation, the latter residues could be expected to
display a larger change in fluorescence emission upon agonist binding,
in agreement with the experimental observations. Notably, the movement
of the cytoplasmic part of TM 6 is shown to occur around the conserved
proline kink but could as well involve a rigid body movement of the
entire helix. However, our previous simulation of the TM 6 helix
indicated the possibility that the kink in the TM 6 helix induced by
Pro-2876.50 could behave as a flexible hinge, which can
modulate the movement of the cytoplasmic side of TM 6 helix relative to
the extracellular region (10). Whereas the more kinked conformation
would correspond to the inactive conformation (A), the
active state may be represented by a less kinked conformation,
resulting in the cytoplasmic portion of TM 6 moving away from the
receptor core (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR leads to a conformational rearrangement at the cytoplasmic side of TM 6. Cysteines were introduced in four
positions at the cytoplasmic side of TM 6 and site-selectively labeled
with the conformationally sensitive fluorophore IANBD. The
pharmacological properties of the cysteine mutants were largely unchanged, supporting that the overall structure of the receptor is
preserved and, thus, that the new fluorescence signal detected in the
IANBD-labeled mutants can be attributed to labeling of the inserted
cysteines rather than to altered labeling of the endogenous cysteines
present in the background,
2AR-Cys-min. The spectroscopic analysis
showed that binding of the full agonist ISO caused clear increases in
the fluorescence quantum yields in positions Cys-2716.33
and Cys-2726.34. The increases were reversible and
dose-dependent. Moreover, the magnitude of the responses
correlated with the efficacy of the used agonist, suggesting that the
observed changes are relevant for receptor activation (Fig. 6). The
increase in quantum yield is consistent with a decrease in the polarity
of the environment surrounding the IANBD-labeled
Cys-2716.33 and Cys-2726.34, which was also
indicated by the observation that agonist stimulation lead to a minor
decrease in accessibility to the aqueous quencher iodide (Fig. 4). The
spectroscopy analysis was also consistent with movement of
Cys-2696.31 and Cys-2706.32 to a more
hydrophilic environment, although the data were not as clear as for
Cys-2716.33 and Cys-2726.34 (Fig. 2). The
changes observed at
2AR-Cys-min-H269C and
2AR-Cys-min-K270C were
very discrete, essentially only counterbalancing the decrease derived
from Cys-1253.44 and Cys-2856.47, which are
still present in
2AR-Cys-min. We cannot exclude that the apparent
absence of response could be due to changes in the labeling of the
background cysteines Cys-1253.44 and
Cys-2856.47, but given the preserved function of both
mutants we would argue that an environmental change around
Cys-2696.31 and Cys-2706.32 is a likely
interpretation. It should be noted that the amplitude of the change in
quantum yield only indicates the relative change in the polarity of the
molecular environment and not the actual magnitude of a conformational
change. Therefore, we cannot say whether the magnitude of movement is
larger at Cys-2716.33 and Cys-2726.34, as
compared with Cys-2696.31 and Cys-2706.32, but
only that the change in the polarity of the environment must be
significantly larger.
-helical region whose environment corresponds to a mixed
hydrophobic-hydrophilic region (Fig. 7). In agreement with this,
collisional quenching experiments on
2AR-Cys-min-A271C and
2AR-Cys-min-L272C revealed a higher accessibility to the hydrophilic
quencher iodide consistent with the localization of both
Cys-2716.33 and Cys-2726.34 in a more
water-accessible environment (Fig. 4). Obviously, to interpret our data
correctly, it is highly important to predict the conformations of the
bound cysteine IANBD moiety as precisely as possible in this mixed
hydrophobic-hydrophilic region. In this context, it should be taken
into consideration, for example, that cysteine substitutions and
subsequent IANBD labeling in this part of the receptor could produce
local structural changes that are not affecting the overall
conformation of the receptor. Therefore, to obtain the most optimal
framework for our data interpretation, a new computational method was
developed. Notably, current computational simulation methods do not
incorporate the complexity of the mixed hydrophobic-hydrophilic region.
Only recently, a bi-phasic lipid-water solvent continuum model was
developed (24). In this study, this solvent model was extended by
developing a computational method in which the mixed
hydrophobic-hydrophilic regions are approximated as a dielectric
gradient. The results of the simulations, illustrated by the most
preferred conformations for each of the four IANBD-derivatized cysteine
residues, are shown in context of a molecular model in Fig. 8 (viewed
from the extracellular side) and Fig. 9A (viewed perpendicular to the plane of the membrane).
2AR in response to agonist
binding. As seen from the side (Fig. 9A), TM 6 is predicted
to form a kinked
-helix because of the presence of a highly
conserved proline (Pro-2876.50) (11). The projection maps
of rhodopsin, which are believed to represent the inactive state of the
receptor, indicates that the cytoplasmic part of TM 6 below the proline
kink is almost perpendicular to the plane of the membrane, whereas the
part above the proline kink is tilted ~25° (27). A rigid body
movement of the cytoplasmic part of TM 6 away from TM 3, and thus the
receptor core will result in large changes in the axial positioning of all four IANBD-labeled substituted cysteines. In the inactive conformation the IANBD moieties would be predicted to reside in the
polar headgroup region (Fig. 9A). However, if the
cytoplasmic part of TM 6 is moved away from the receptor core all four
IANBD-labeled residues are brought upwards and outwards, allowing them
to penetrate further into the more hydrophobic region of the
membrane/detergent micelles and away from the more hydrophilic polar
headgroups as well as from the predicted more hydrophilic interior of
the receptor protein (illustrated by the hypothetical active structure
in Fig. 9B where the cytoplasmic part of TM 6 with the IANBD
moieties attached is tilted arbitrarily away from the receptor core).
This movement could explain the observed shift for all four
IANBD-labeled cysteines toward a less polar environment upon receptor
activation. Because residues Cys-2696.31 and
Cys-2706.32 are facing the hydrophobic aliphatic chains in
both the inactive and active states, whereas Cys-2716.33
and Cys-2726.34 transfer from the protein interior to being
exposed to the hydrophobic aliphatic chains upon activation, the latter
residues could be expected to display a larger change in fluorescence
emission upon agonist binding, in agreement with the experimental
observations. This interpretation of our data does not contradict
previous data suggesting a counterclockwise rotation of TM 6 (19, 35).
In fact, it is highly conceivable that the outward movement of the cytoplasmic part of TM 6 could be accompanied by a helical rotation because it has also been predicted for rhodopsin (6). Of notable interest, the movement of the cytoplasmic part of TM 6 is shown to
occur around the conserved proline kink but could as well involve a
rigid body movement of the entire helix (Fig. 9B).
However, our previous simulation of the TM 6 helix indicated the
possibility that the kink in the TM 6 helix induced by
Pro-2876.50 could behave as a flexible hinge that can
modulate the movement of the cytoplasmic side of TM 6 helix relative to
the extracellular region (10). Whereas the more kinked conformation
would correspond to the inactive conformation (Fig. 9A), the
active state may be represented by a less kinked conformation,
resulting in the cytoplasmic portion of TM 6 moving away from the
receptor core (Fig. 9B). Evidently, further studies are
required to investigate such a role of the TM 6 proline kink in
receptor activation.
2AR, rhodopsin is the only other member of the GPCR
family that has been subject to biophysical analyses allowing direct
insight into conformational changes accompanying receptor activation
(6, 8). Even though we have shown evidence before that TM 6 of the
2AR may move during receptor activation, the current data provide
the first structural evidence that the actual character of these
movements may be conserved between the highly specialized light
receptor rhodopsin and the
2AR, a prototypic receptor activated by
the much more common diffusible ligands. Of interest, a role of TM 6 in
receptor activation has also been supported by several other studies
(4, 14). For example, the possibility of inhibiting transducin
activation by rhodopsin using disulfide cross-linking (6) or
engineering of bis-histidine metal ion binding sites between the
cytoplasmic extensions of TMs 6 and 3 indicated the possibility that
these two domains move relative to one another during receptor
activation (36, 37). Indirect evidence for a rotation or tilting of TM
6 in the activated state of the receptor has also been obtained in the
2AR by observing that constitutive activation of the receptor
results in an increased accessibility of the endogenous
Cys-2856.47 in TM 6 to the charged sulfhydryl-reactive
reagent methane thiosulfonateethyl ammonium (19, 35). Moreover,
the results of insertion mutagenesis in the muscarinic M2
receptor were consistent with a conformational change in the
cytoplasmic part of TM 6 upon receptor activation (38).
2AR raise new interesting questions about molecular modes of
agonist-induced receptor activation. Most significantly, the present
data substantiate differences between the kinetics of rhodopsin and
2AR activation. In rhodopsin, formation of the activated
metarhodopsin II state occurs essentially within microseconds. Interestingly, metarhodopsin II subsequently undergoes a slow (t1/2 = ~6 min) transition to the inactive
metarhodopsin III (12). The rapid activation kinetics for rhodopsin is
likely directly facilitated by the fact that its ligand,
cis-retinal, is covalently bound to the receptor as an
inverse agonist and upon absorption of a photon isomerizes to an
agonist (trans-retinal) within the binding pocket (reviewed in Ref. 39). Thus, ligand binding is not part of the activation process. This specialized mechanism of activation may be necessary to
facilitate the very rapid response of rhodopsin to light. In contrast
to the rapid activation and the slow inactivation kinetics observed for
rhodopsin, the agonist-induced conformational changes in the
2AR are
slow (t1/2 = ~2-3 min) (9, 13). It cannot be
excluded, of course, that this difference between rhodopsin and the
2AR is caused by a difference in the methodological approach.
However, because the measurements were performed under similar
conditions they may reflect differences between rhodopsin and a
receptor activated by a diffusible ligand. In the present study we find
that the agonist-induced spectral changes observed following IANBD
labeling of cysteines introduced at the cytoplasmic side of TM 6 occur
with kinetics similar to those observed following labeling of the
endogenous cysteines (Cys-1253.44 and
Cys-2856.47). This consistency for the different read-outs
and the clear correlation between change in fluorescence and biological
efficacy (9) support the contention that the slow kinetics is an
intrinsic property of the receptor, at least when conformational
changes are being probed using the IANBD fluorophore.
2AR with the rapid responses to
agonist stimulation of GPCRs in cells, such as for example activation
of ion channels. Unfortunately, it has not yet been possible to test
the kinetic importance of the G protein because of technical
difficulties. Credible spectroscopic analysis of the fluorescently
labeled receptor reconstituted with Gs
requires
essentially 100% coupling efficiency between the receptor and
Gs
and thus a huge excess of Gs
. At the
current stage, we have not been able to establish these experimental conditions because of instability of the Gs
protein.
![]() |
ACKNOWLEDGEMENTS |
---|
Dorte Frederiksen and Lis Soerinsen are thanked for excellent technical assistance. Dr. Jonathan Javitch is thanked for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
§ Present address: Maxygen, DK-2970 Hørsholm, Denmark.
** Recipient of an Ole Rømer Associate Research Professorship from the Danish Natural Science Research Council. To whom correspondence should be addressed: Div. of Cellular and Molecular Physiology, Dept. of Medical Physiology 12-5-22, The Panum Inst., University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45-3532-7548; Fax: 45-3532-7555; E-mail: gether@mfi.ku.dk.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M004871200
2 The numbers of the residues according to the Ballesteros-Weinstein numbering scheme are indicated in superscript (see Ref. 11).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein coupled receptor;
2AR,
2-adrenergic receptor;
TM, transmembrane segment;
IANBD, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
ethylene-diamine;
ISO, isoproterenol;
ALP, alprenolol;
DHA, dihydroalprenolol;
D
M, n-dodecyl-
-D-maltoside;
DOB, dobutamine;
SAL, salbutamol.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kolakowski, L. F. (1994) Receptors Channels 2, 1-7[Medline] [Order article via Infotrieve] |
2. |
Ji, T. H.,
Grossmann, M.,
and Ji, I.
(1998)
J. Biol. Chem.
273,
17299-17302 |
3. | Strader, C. D., Fong, T. M., Tota, M. R., Underwood, D., and Dixon, R. A. F. (1994) Annu. Rev. Biochem. 132, 63101-63132 |
4. |
Gether, U.
(2000)
Endocr. Rev.
21,
90-113 |
5. | Farahbakhsh, Z. T., Ridge, K. D., Khorana, H. G., and Hubbell, W. L. (1995) Biochemistry 34, 8812-8819[Medline] [Order article via Infotrieve] |
6. |
Farrens, D. L.,
Altenbach, C.,
Yang, K.,
Hubbell, W. L.,
and Khorana, H. G.
(1996)
Science
274,
768-770 |
7. | Altenbach, C., Yang, K., Farrens, D. L., Farahbakhsh, Z. T., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470-12478[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Dunham, T. D.,
and Farrens, D. L.
(1999)
J. Biol. Chem.
274,
1683-1690 |
9. |
Gether, U.,
Lin, S.,
and Kobilka, B. K.
(1995)
J. Biol. Chem.
270,
28268-28275 |
10. |
Gether, U.,
Lin, S.,
Ghanouni, P.,
Ballesteros, J. A.,
Weinstein, H.,
and Kobilka, B. K.
(1997)
EMBO J.
16,
6737-6747 |
11. | Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25, 366-428 |
12. | Farahbakhsh, Z. T., Hideg, K., and Hubbell, W. L. (1993) Science 262, 1416-1419[Medline] [Order article via Infotrieve] |
13. |
Gether, U.,
Ballesteros, J. A.,
Seifert, R.,
Sanders-Bush, E.,
Weinstein, H.,
and Kobilka, B. K.
(1997)
J. Biol. Chem.
272,
2587-2590 |
14. | Wess, J. (1998) Pharmacol. Ther. 80, 231-264[CrossRef][Medline] [Order article via Infotrieve] |
15. | Guan, X., Peroutka, S. J., and Kobilka, B. K. (1992) Mol. Pharmacol. 41, 695-698[Abstract] |
16. | Seifert, R., Lee, T. W., Lam, V. T., and Kobilka, B. K. (1998) Eur. J. Biochem. 255, 369-382[Abstract] |
17. | Jensen, A. D., and Gether, U. (2000) Methods Mol. Biol. 126, 345-361[Medline] [Order article via Infotrieve] |
18. |
Suryanarayana, S.,
Daunt, D. A.,
Von Zastrow, M.,
and Kobilka, B. K.
(1991)
J. Biol. Chem.
266,
15488-15492 |
19. |
Rasmussen, S. G.,
Jensen, A. D.,
Liapakis, G.,
Ghanouni, P.,
Javitch, J. A.,
and Gether, U.
(1999)
Mol. Pharmacol.
56,
175-184 |
20. | Kobilka, B. K. (1995) Anal. Biochem. 231, 269-271[CrossRef][Medline] [Order article via Infotrieve] |
21. | Lakovicz, J. R. (1999) Principles of Fluorescence Spectroscopy , 2nd Ed. , pp. 238-318, Kluwer Academic/Plenum Publishers, New York |
22. | Guarnieri, F., and Weinstein, H. (1996) J. Am. Chem. Soc. 118, 5580-5589[CrossRef] |
23. | Guarnieri, F., and Wilson, S. R. (1995) J. Comput. Chem. 16, 648-653 |
24. |
Ballesteros, J.,
Kitanovic, S.,
Guarnieri, F.,
Davies, P.,
Fromme, B. J.,
Konvicka, K.,
Chi, L.,
Millar, R. P.,
Davidson, J. S.,
Weinstein, H.,
and Sealfon, S. C.
(1998)
J. Biol. Chem.
273,
10445-10453 |
25. | White, S. H., and Wimley, W. C. (1999) Annu. Rev. Biophys. Biomol. Struct. 28, 319-365[CrossRef][Medline] [Order article via Infotrieve] |
26. | Mohamadi, F., Richards, N. G. J., Guida, W. C., Liskamp, R., Lipton, M., Caulfield, C., Chang, G., Hendrickson, T., and Still, W. C. (1990) J. Comput. Chem. 11, 440-467 |
27. | Unger, V. M., Hargrave, P. A., Baldwin, J. M., and Schertler, G. F. (1997) Nature 389, 203-206[CrossRef][Medline] [Order article via Infotrieve] |
28. | Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144-164[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Seifert, R.,
Wenzel-Seifert, K.,
Lee, T. W.,
Gether, U.,
Sanders-Bush, E.,
and Kobilka, B. K.
(1998)
J. Biol. Chem.
273,
5109-5116 |
30. |
Wess, J.
(1997)
FASEB J.
11,
346-354 |
31. |
Samama, P.,
Cotecchia, S.,
Costa, T.,
and Lefkowitz, R. J.
(1993)
J. Biol. Chem.
268,
4625-4636 |
32. | Javitch, J. A., Ballesteros, J. A., Weinstein, H., and Chen, J. (1998) Biochemistry 37, 998-1006[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Palczewski, K.,
Kumasaka, T.,
Hori, T.,
Behnke, C. A.,
Motoshima, H.,
Fox, B. A.,
Le T.rong, I.,
Teller, D. C.,
Okada, T.,
Stenkamp, R. E.,
Yamamoto, M.,
and Miyano, M.
(2000)
Science
289,
739-745 |
34. | Ballesteros, J. A., and Weinstein, H. (1992) Biophys. J. 62, 107-109[Abstract] |
35. |
Javitch, J. A.,
Fu, D.,
Liapakis, G.,
and Chen, J.
(1997)
J. Biol. Chem.
272,
18546-18549 |
36. | Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996) Nature 383, 347-350[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Sheikh, S. P.,
Vilardarga, J. P.,
Baranski, T. J.,
Lichtarge, O.,
Iiri, T.,
Meng, E. C.,
Nissenson, R. A.,
and Bourne, H. R.
(1999)
J. Biol. Chem.
274,
17033-17041 |
38. |
Liu, J.,
Blin, N.,
Conklin, B. R.,
and Wess, J.
(1996)
J. Biol. Chem.
271,
6172-6178 |
39. | Sakmar, T. P. (1998) Prog. Nucleic Acids Res. Mol. Biol. 59, 1-34[Medline] [Order article via Infotrieve] |
40. | DeBlasi, A., O'Reilly, K., and Motulsky, H. J. (1989) Trends Pharmacol. Sci. 10, 227-229[CrossRef][Medline] [Order article via Infotrieve] |