ACCELERATED PUBLICATION
Is the Isolated Ligand Binding Domain a Good Model of
the Domain in the Native Receptor?*
Dustin
Deming,
Qing
Cheng, and
Vasanthi
Jayaraman
From the Department of Integrative Biology and Pharmacology,
University of Texas Health Science Center, Houston, Texas 77030
Received for publication, March 11, 2003, and in revised form, March 24, 2003
 |
ABSTRACT |
Numerous studies have used the atomic level
structure of the isolated ligand binding domain of the glutamate
receptor to elucidate the agonist-induced activation and
desensitization processes in this group of proteins. However, no study
has demonstrated the structural equivalence of the isolated ligand
binding fragments and the protein in the native receptor. In this
report, using visible absorption spectroscopy we show that the
electronic environment of the antagonist
6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated
protein and the native glutamate receptors expressed in cells.
Our results hence establish that the local structure of the
ligand binding site is the same in the two proteins and validate the
detailed structure-function relationships that have been developed
based on a comparison of the structure of the isolated ligand binding
domain and electrophysiological consequences in the native receptor.
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INTRODUCTION |
Glutamate receptors are the predominant mediators of excitatory
synaptic signals in the central nervous system and play an important
role in the regulation of synaptic strength and in diverse neuropathologies, including epilepsy and ischemia (for reviews, see
Refs. 1 and 2). Based on agonist affinity profiles, they can be
subdivided into three subfamilies:
-amino-3-hydroxy-5-methyl-4-isoxazole propionate
(AMPA),1
N-methyl-D-aspartate, and kainate
receptors (1, 3). The AMPA subtype of the glutamate receptors are homo-
or hetero-oligomers composed of GluR1-GluR4 subunits. The recent
determination of the structure of the soluble ligand binding domain
(S1S2) for the GluR2 subunit (4-8) complemented with spectroscopic
investigations (7-9), in combination with the vast existing
electrophysiological data on the native receptor (1, 6, 10), has
provided the first direct structural insight into how the changes at
the ligand binding site lead to the sequence of conformational changes
that regulate ion flow in this class of important proteins (5, 6). The
validity of these inferences hinges on the assumption that the ligand
binding domain is the same in the model construct as in the native
receptor (Fig. 1). There is no direct structural (x-ray or
spectroscopy-based) comparison between the S1S2 protein and the native
receptor, which would clearly indicate whether the S1S2 protein is a
good model for the ligand binding domain in the full receptor. The
assumption that the S1S2 protein is a representative model of the
domain in the full glutamate receptor is currently based on equilibrium
ligand binding assays that suggest similar ligand binding properties
for the two proteins (11, 12). In the present report using absorption
spectroscopy, which is sensitive to changes in the ligand binding
domain, we establish that the electronic environment and therefore
structural environment of the ligand binding site is the same in the
S1S2 and in the native receptor. Perhaps the most noteworthy aspect of
this approach is that it is straightforward to conduct visible
absorption spectroscopy on cells in their native state without the need
to make model constructs or purify the full receptors expressed in cells.
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EXPERIMENTAL PROCEDURES |
S1S2 Protein Preparation--
The GluR2-S1S2 construct
containing the S1 segment (amino acids 390-506 in the GluR2 sequence)
and the S2 segment (amino acids 632-763 in the GluR2 sequence) with
the two domains being linked together via an amino acid linker (GT) was
kindly provided by Dr. Gouaux (Columbia University, New York, NY) (5).
The protein was expressed, purified, and characterized as described by
Chen et al. (13). The digestion and purity of the protein
was tested using SDS-PAGE, and the activity (ligand binding) was tested
by measuring the Kd values using fluorescence
spectroscopy. GluR4-S1S2 was kindly provided by Dr. Madden (Dartmouth
College, Hanover, NH).
Human Embryonic Kidney 293 (HEK-293) Cell Cultures--
HEK-293
(ATCC CRL 1573) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Invitrogen) and 2 mM glutamine, 50 µg/ml penicillin, and 50 µg/ml
streptomycin. One day before transfection, cells were replated in 35-mm
culture dishes coated with rat tail collagen (Sigma). Transfections
were performed using FuGENE 6 (Roche Applied Science) transfection reagent with 1-2 µg of glutamate receptor cDNA. The plasmid
encoding for the GluR4-flip subunit of the glutamate receptors used for the transfection was generously provided by Dr. Seeburg (Max Planck Institute, Heidelberg, Germany). Transfected cells were allowed to grow for 1-3 days before use.
Absorption Measurements--
The UV-visible absorption spectra
were obtained using Agilent 8453 or Shimadzu UV 2501 spectrometers
(photometric repeatability of 0.0003 absorbance units) and analyzed
using Gram's Spectral Notebase (Thermo Galactic, Salem, NH). The
spectra were collected in the range of 500-250 nm with a sampling
interval of 1 nm using a 1-cm quartz cuvette. The absorption spectra
for GluR2-S1S2 and GluR4-S1S2 were obtained using 10 µM
protein and 20 µM
6-cyano-7-nitro-2,3-dihydroxyquinoxaline (CNQX). For the absorption
experiments with HEK-293 cells the number of cells used was such that
the background due to scattering from cells was 0.7 absorbance units at
310 nm; this ensured that the total absorption was less than 1 absorption unit upon adding 20 µM CNQX.
When determining the difference absorption spectra for GluR4-S1S2 and
GluR2-S1S2 using competitive ligands, parallel control experiments were
performed by adding the corresponding concentrations of ligands to 20 µM CNQX in buffer to ensure that no spectral changes were
observed due to ligand-CNQX interactions. Similarly for the
HEK-293 cells the difference spectra have been corrected for any
differences arising due to nonspecific changes in the CNQX spectrum
that arise due to the addition of ionic ligands by performing parallel
control experiments using HEK-293 cells that have not been transfected.
Specifically difference spectra were obtained between the spectra for
homogenized non-transfected HEK-293 cells with 20 µM CNQX
in the presence and absence of competitive ligands. Small differences
were observed only at millimolar concentrations of competitive ligands,
and these were subtracted from the corresponding difference spectra
generated for transiently transfected cells.
Determination of Kd Values from the Dose-Response
Curves--
A logistic equation (Equation 1) was used to fit the
dose-response curves to obtain the IC50 values for the
agonists competing with CNQX.
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(Eq. 1)
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where min and max are the minimum and maximum y
values, respectively, in the dose-response curves. The IC50
values thus obtained were used to calculate the Kd
values for the various agonists using the corrected Cheng-Prusoff
equation (14) (Equation 2).
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(Eq. 2)
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where y is the ratio of the bound to free
concentrations of CNQX before the addition of competitive agonist,
p is the total initial concentration of CNQX (20 µM), Kd2 is the dissociation constant for CNQX, and Kd1 is the dissociation
constant for the agonist. The bound and free concentrations of CNQX
were calculated using Kd2 and the protein
concentration (10 µM for GluR2-S1S2).
For calculating the Kd value for glutamate binding
to GluR4 receptors expressed in HEK-293 cells, the Cheng-Prusoff equation (Equation 3) was used since the concentration of protein was
significantly lower, and therefore depletion of CNQX was small.
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(Eq. 3)
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RESULTS AND DISCUSSION |
A number of ligands bind with high
specificity to the glutamate family of receptors. Of particular
interest is CNQX, which exhibits electronic transitions in the visible
region as evidenced by its orange/red color. Moreover CNQX exists in a
unique electronic configuration when bound to the S1S2 protein as
established by the dramatic changes in its infrared vibrational modes
(9). The difference in the electronic configurations of the free and protein-bound forms of CNQX is expected to be reflected in its absorption spectra.

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Fig. 1.
The schematic representation of the topology
of the AMPA receptor (shown on the left) and the
isolated ligand binding domain (S1S2 protein, shown to the
right, contains the S1 and S2 domains joined by a
linker). AMPA receptors are thought to be tetramers, each subunit
having three transmembrane domains, a pore-loop sequence that lines the
channel, and an extracellular domain with two lobes (S1 and S2) forming
a ligand-binding cleft.
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The UV-visible absorption spectra of CNQX bound to GluR2-S1S2 and in
the free form are shown in Fig. 2. A
decrease in the absorption is observed at 330 and 343 nm upon CNQX
binding to the protein. Based on the wavelength as well as on the
structure of CNQX (extended conjugation and presence of a nitro group)
these absorbance bands are expected to arise from
-
* or
n-
* transitions. The change in the absorption
spectral features are highlighted by examining the difference
absorption spectrum (shown in Fig. 3).
These changes in the absorption and the underlying changes in the
electronic environment could arise due to specific and nonspecific
binding of CNQX. By conducting a series of competitive binding studies
we have identified that the changes in the absorption spectra are
mainly due to specific binding of CNQX to the GluR2-S1S2. Specifically
we have examined the competition with glutamate receptor agonists such
as glutamate (Fig. 3B), AMPA (Fig. 3C), kainate (Fig. 3D), and a non-binding compound,
-aminobutyric acid
(Fig. 3E). The difference spectrum between the bound and
unbound forms of CNQX is only observed upon selective displacement of
CNQX from the ligand binding site and not with a nonspecific binding
compound. Additionally we have investigated the changes in the
absorption spectrum of CNQX due to changes in pH and in the presence of
dimethylsulfoxide as a solvent (Fig. 4).
These studies clearly indicate that the differences in the absorbance
bands due to CNQX binding to S1S2 protein cannot be mimicked by changes
in the electronic environment of CNQX induced by changes in pH and
solvent, thus indicating that the differences in the absorbance bands
of CNQX upon binding to S1S2 is unique to the environment of the ligand
binding site in S1S2.

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Fig. 2.
The top panel shows the
structures of CNQX in the free (A) and S1S2-bound
(B) forms (9); the bottom panel shows
the absorption spectrum of free CNQX (A) (dashed
line), CNQX bound to S1S2 (B) (solid
line), and S1S2 (C) (solid
line).
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Fig. 3.
Difference absorption spectra between 20 µM CNQX in buffer and CNQX in the
presence of GluR2-S1S2 (A) and 20 µM CNQX bound to GluR2-S1S2 in the
presence and absence of 500 µM AMPA
(B), 500 µM
glutamate (C), 500 µM kainate (D), and
500 µM
-aminobutyric acid (E). The
difference spectra were obtained at pH 7.4 using a phosphate buffer and
at room temperature. a.u., absorbance units.
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Fig. 4.
Difference absorption spectra between CNQX in
buffer (pH 7.4) and CNQX in the presence of GluR2-S1S2
(A), CNQX at pH 7.4 and at pH 4 (B),
CNQX at pH 7.4 and at pH 9 (C), and CNQX in water
buffer at pH 7.4 and in dimethylsulfoxide
(D).
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Dose-Response Curves--
The absorbance bands shown in Fig.
3A are not only useful for qualitative investigations of
electronic environment of the ligand binding site, but they also allow
the quantitative determination of the fraction of bound CNQX.
Specifically, since the absorbance is directly proportional to the
concentration of the chromophore (CNQX), a quantitative measure of the
concentration of CNQX dissociated from the binding site can be obtained
from the integrated area of the difference spectral features (320-353
nm). Based on this, dose-response curves were obtained for the
displaced CNQX at various concentrations of competitive agonists, and
the dissociation constants of the competitive agonists were determined.
A plot of the integral areas as a function of the concentration of the
competitive ligands is shown in Fig. 5
for various ligands added to a solution containing CNQX bound to
GluR2-S1S2. Using a logistic fit for these dose-response curves the
IC50 values were determined to be 7 ± 2, 22 ± 5, and 115 ± 25 µM for AMPA, glutamate, and
kainate, respectively. Based on these IC50 values,
using the corrected Cheng-Prusoff equation (14) (corrected for ligand
depletion; see "Experimental Procedures" for details), the
Kd values were determined for AMPA, glutamate, and
kainate (shown in Table I). These values
are in good agreement with the values previously reported based
on radioactive ligand binding experiments (Table I) (13). The
ability to use the differences in the absorbance of CNQX to determine
the affinities of various ligands makes this an excellent assay to
screen for compounds that selectively bind to the this receptor.

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Fig. 5.
Dose-response curves for the competitive
displacement of CNQX by AMPA (filled circles),
glutamate (filled squares), and kainate
(triangles) from GluR2-S1S2 and displacement of CNQX
by glutamate from GluR4 homomeric receptors expressed in HEK-293 cells
(open squares). The dose-response curves were fit
to a logistic equation to obtain IC50 values. The
experiments were performed using 20 µM CNQX.
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Structure of S1S2 Versus the Ligand Binding Domain in the Full
Receptor--
Since the difference spectrum provides a fingerprint of
the local electronic structure of the ligand binding site, we used this
to compare the structure of the isolated ligand binding domain with the
same domain in the full receptor. For these investigations we have
compared the difference absorption spectra for CNQX binding to the
GluR4-S1S2 (isolated ligand binding domain of GluR4 subunit) and
homomeric GluR4 receptors expressed in HEK-293 cells (Fig. 6). The difference spectra are identical
for the isolated domain and the native receptor. Additionally no
spectral features were observed in displacement studies with a
non-binding compound
-aminobutyric acid, confirming that the
spectral features shown in Fig. 6 arise due to specific binding of
CNQX. Finally for a quantitative comparison a dose-response curve was
obtained for competitive displacement of CNQX by glutamate from
homomeric GluR4-flip receptors expressed in HEK-293 cells (Fig. 5,
open squares). The IC50 value determined using a
logistic fit was 20 ± 5 µM, similar to that
obtained for GluR2-S1S2, and the corresponding Kd
value for glutamate was 450 ± 110 nM assuming the
Kd for CNQX is 460 nM (15-17). This
value is in reasonably good agreement with previously published values
for glutamate binding based on radioactive ligand binding (15-17).

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Fig. 6.
Difference absorption spectra between 20 µM CNQX bound to GluR4-S1S2 in the
presence and absence of 500 µM
glutamate (A) and difference absorption spectra
between 20 µM CNQX in the presence
of homogenized HEK-293 cells transiently transfected with c-DNA for
GluR4-flip in the presence and absence of 100 µM glutamate (B) and
100 µM
-aminobutyric acid (C).
a.u., absorbance units.
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The results presented here provide definitive evidence that the
electronic environment of CNQX is the same in the homomeric GluR4
glutamate receptors transiently expressed in HEK-293 cells as that in
the isolated ligand binding domain, therefore clearly indicating that
the GluR2-S1S2 and GluR4-S1S2 are good models for the ligand binding
domain in the native receptor, thus providing additional validation
for the structure-function insights currently available based on the
atomic level structure of GluR2-S1S2.
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ACKNOWLEDGEMENTS |
We thank Dr. Eric J. Gouaux and Yu Sun for
the GluR2-S1S2 construct and for guidance with the purification
procedure and Dr. Dean R. Madden for providing us with GluR4-S1S2
protein. We also thank Dr. Eric J. Gouaux, Dr. George P. Hess, Dr.
Meyer B. Jackson, Dr. Dean R. Madden, and Dr. Thomas G. Spiro for
reading the manuscript and providing helpful comments.
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FOOTNOTES |
*
This work was supported by National Science Foundation Grant
NSF-0096635 (to V. J.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Integrative
Biology and Pharmacology, MSB 4.106, 6341 Fannin, University of Texas
Health Science Center, Houston, TX 77030. Tel.: 713-500-6236; Fax:
713-500-7444; E-mail: vasanthi.jayaraman@uth.tmc.edu.
Published, JBC Papers in Press, March 25, 2003, DOI 10.1074/jbc.C300105200
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ABBREVIATIONS |
The abbreviations used are:
AMPA,
-amino-3-hydroxy-5-methyl-4-isoxazole propionate;
GluR, glutamate
receptor;
CNQX, 6-cyano-7-nitro-2,3-dihydroxyquinoxaline;
HEK, human
embryonic kidney.
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Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.