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J Biol Chem, Vol. 274, Issue 31, 21867-21872, July 30, 1999
From the The substituted cysteine-accessibility
method and two sulfhydryl-specific reagents, the methane-thiosulfonate
derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the
Recently, a technique utilizing charged methanethiosulfonate
derivatives to probe the accessibility of substituted cysteine residues
has been used to study the structure of the dopamine D2 and the
We recently used the agonist chloroethylclonidine (CEC) to map the
structure of TM5 of the human In our previous study, TM5 of H To explore such a conformational change in the H Materials--
[3H]RX821002
(2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline) was obtained
from Amersham Pharmacia Biotech; specific activity, 56 Ci/mmol).
Phentolamine, UK 14,304 (5-bromo-N-(4,
5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine), and CEC were supplied
by Research Biochemicals (Natick, MA). MTSEA was purchased from Toronto
Research Chemicals Inc. (North York, Canada). Cell culture reagents
were supplied by Life Technologies, Inc. (Gaithersburg, MD). Other
chemicals were of analytical grade and were purchased from commercial suppliers.
Mutagenesis and Expression Vectors--
Site-directed
mutagenesis was performed utilizing the Altered Sites II in
vitro mutagenesis system (Promega, Madison, WI) as described
previously (9). The wild type H Cell Culture and Transfections--
Adherent CHO cells (American
Type Culture Collection, Manassas, VA) were cultured as reported
previously (9). The pREP4-based expression constructs were transfected
into cells using the Lipofectin reagent kit (Life Technologies, Inc.)
(9). Hygromycin B (Roche Molecular Biochemicals)-resistant (550 µg/ml) cell cultures were examined for their ability to bind the
Ligand Binding--
Ligand binding assays were performed in 50 mM K+-phosphate buffer (pH 7.4 at 21 °C) as
described previously (9, 12).
Reactions with CEC and MTSEA--
Six large culture flasks (175 cm2) of CHO cells were harvested into chilled
phosphate-buffered saline, pelleted, washed, suspended in 2.5 ml of
ice-cold 50 mM K+-phosphate buffer (pH 7.4 at
21 °C), and homogenized with an Ultra-Turrax homogenizer (model T25,
Janke & Kunkel, Staufen, Germany; setting 9500 rpm, twice for 10 s). Aliquots (45 µl) of cell homogenate were incubated for 10 min at
room temperature. Five µl of freshly prepared CEC or MTSEA dilutions
were added to each vial to reach the appropriate final concentration.
Reactions took place at room temperature for 15 min (CEC) or 2 min
(MTSEA); the reaction mixtures were then diluted 20-fold with 50 mM K+-phosphate buffer (pH 7.4 at 21 °C).
Residual
The second-order rate constant (k) for the reaction of CEC
or MTSEA with wild type H Measurement of Change in Extracellular Acidification
Rate--
Extracellular acidification rates were measured using the
Cytosensor® Microphysiometer (Molecular Devices Corp., Menlo Park, CA)
(13). CHO cells were seeded into 12-mm capsule cups at 3 × 105 cells/cup and incubated in 5% CO2 at
37 °C for 20 h. The capsule cups were loaded into the sensor
chambers and perfused with running medium (bicarbonate-free Molecular Modeling--
The H
CEC was docked manually in the binding-site crevice into a position
that allows hydrogen bonding between the protonated nitrogen atom in
the imidazoline ring of CEC and Asp-113 in TM3 of the receptor.
Simultaneously, a covalent bond was created between CEC and the
cysteine residue in TM5. Each receptor-CEC complex was then
energy-minimized using CHARMm (17) (Molecular Simulations Inc., San
Diego, CA) using harmonic constraints for backbone atoms to maintain
the helical structures of the TMs. Energy minimizations were also
performed without constraints to completely relax the structure of the
complex. MTSEA was docked into the same region of the binding-site
crevice as CEC, and a disulfide bond between the sulfur atom of MTSEA
and the cysteine residue of the receptor was created.
Site-directed Mutagenesis and Transfections--
The introduced
mutations were confirmed, and the absence of secondary mutations was
verified by dideoxy sequencing of double-stranded DNA. Mutated and wild
type receptors were expressed in CHO cells. Hygromycin B-resistant cell
cultures were examined for their ability to bind the
Ligand Binding Assays--
Saturation isotherms of
[3H]RX821002 binding, and LIGAND (18)-derived
Kd (receptor affinity), and
Bmax (receptor density) values were determined
in three separate experiments performed in triplicate for each cell
line. The expression levels of two point-mutated receptors,
H Reactions with CEC--
Fig.
1A illustrates the inhibition
of antagonist binding produced by CEC. Binding to the mutants Cys-197,
Cys-200, and Cys-204 is very sensitive to CEC, whereas the mutants
Ser-201, Cys-202, and Cys-203 are relatively resistant to CEC, and the
wild type receptor Cys-201 shows intermediate sensitivity. The rate and extent of receptor inactivation by CEC with cysteine substitutions for
Ile-202 and Gly-203 was comparable with that of H
To quantitate the susceptibility of the engineered cysteines to CEC,
the second-order rate constants for reaction with these compounds were
determined as described under "Experimental Procedures" (Table
II). It is apparent from these data (Fig.
1) that H Reactions with MTSEA--
MTSEA inhibited binding to all of the
mutants (Fig. 1B), with Cys-202 and Cys-203 being the most
resistant, consistent with the relative inaccessibility of these
cysteines. The results for the Ser-201 mutant were not accurately
described by an exponential decay function; this receptor was
relatively resistant to MTSEA up to a concentration of 125 µM but showed quite significant inhibition at higher
MTSEA concentrations. Consequently, no estimate for the second-order
rate constant of inactivation of this receptor with MTSEA could be
computed. The reasons for this unexpected inhibitory pattern remain
unclear but may reflect the reactivity of other cysteines present in
the Ser-201 background. In Fig. 1B the MTSEA results for the
Ser-201, Cys-202, and Cys-203 mutants are presented without
curve-fitting. Reasonable single exponential decay fits were obtained
for Cys-197, Cys-201, and Cys-204. For Cys-200, the data also show a
"lag" at low concentrations, similar but less pronounced than that
seen with Ser-201 (see above), and thus the fit is less satisfactory
than that seen with the other three reactive residues. This results
in an overestimation of the rate of reaction of Cys-200 with MTSEA,
although it is impossible to determine the exact rate of reaction of
this substituted cysteine in the presence of the Ser-201 background.
Receptor-mediated Changes in Extracellular Acidification
Rate--
CEC increased the extracellular acidification rate in CHO
cells expressing H Receptor Modeling--
Our current receptor model is based on an
CEC was docked manually into the binding-site crevice of the wild type
receptor and the mutants in a position that allows hydrogen bonding
between Asp-113 in TM3 and the protonated nitrogen atom of the
imidazoline ring in CEC. At the same time, the other end of the CEC
molecule was able to form a covalent bond between a reactive carbon
atom and the sulfhydryl group of the cysteine residue in TM5 of the
receptor. The models were then energy-minimized (Fig.
4B). In the H
The starting position of MTSEA was similar to that of CEC. Fig.
4A shows the energy-minimized models of the receptor-MTSEA complexes. MTSEA forms ideal disulfide bonds with sulfhydryl residues in the H Using the substituted cysteine-accessibility method and
irreversible binding of the agonist CEC, we have previously mapped the
residues accessible in the binding-site crevice in TM5 of human
In the current study, the rate of reaction of engineered cysteines in
TM5 of human The concept that G-protein-coupled receptors may possess some degree of
spontaneous or constitutive activity and thus activate their cognate
G-proteins in the absence of receptor agonist has recently become
widely accepted (25, 26). G-protein-coupled receptors are thought to
exist in an equilibrium between two (or more) interconvertible
allosteric states, active (R*) and inactive (R) conformations. Binding
of a receptor agonist causes an increase in the ratio of R* to R (5,
25). It has also been suggested that the R state can isomerize, not to
a single active conformation, but to multiple different ligand-specific
active conformations (27, 28). This multistate model suggests that the
biological efficacy of an agonist may be a consequence of the
qualitative and quantitative preference of the ligand for
conformational variant forms of the receptor rather than just affecting
the equilibrium between only two alternative states (28). MTSEA has no
significant affinity for the receptor and, thus, reacts with cysteines
accessible in the predominant resting receptor form (R). The activated
state of a receptor can only be investigated with this reagent by using constitutively active mutant receptors (7). When the agonist CEC binds
to TM5 of H There are several other factors that might also contribute to a
difference in the rate of reaction of a cysteine with CEC and with
MTSEA. Local steric factors might favor the reaction of one reagent.
Although this is difficult to rule out, MTSEA is somewhat smaller than
CEC, and such a selective interference with the reaction of MTSEA would
be surprising. Because the methanethiosulfonate derivatives react
vastly faster with the thiolate than with the thiol form of their
substrates (29), changes in the ionization state of the sulfhydryl
would have a significant impact on the rate of reaction of MTSEA. It is
significant to note, however, that the relative difference in rates
results in significant part from the very rapid reaction of CEC at the
Cys-200 position. The simplest explanation for this rapid reaction is
the proximity of Cys-200 to the reactive moiety of bound CEC.
We have previously used a H These results suggest that CEC and MTSEA may recognize different
conformations of the receptor, with CEC accessing the cysteine at
position 200 more readily than MTSEA. This may be because of agonist-dependent clockwise rotation of TM5 of the
activated human Dr. Joyce Baldwin (MRC
Laboratory of Molecular Biology, Medical Research Council Centre,
Cambridge, UK) is acknowledged for kindly providing us the *
This work was financially supported by the Academy of
Finland and the Technology Development Centre of Finland.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. Tel.: 358-2-333 7021;
Fax: 358-2-333 7000; E-mail: anmarja@utu.fi.
2
J. M. Peltonen, unpublished observations.
The abbreviations used are:
AR, adrenergic
receptor;
CEC, chloroethylclonidine;
CHO, Chinese hamster ovary;
H
Chloroethylclonidine and 2-Aminoethyl Methanethiosulfonate
Recognize Two Different Conformations of the Human
2A-Adrenergic Receptor*
§,,,
,,,
Department of Pharmacology and Clinical
Pharmacology, Department of Biochemistry and Pharmacy,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (2-AR) agonist chloroethylclonidine (CEC), were used to determine the relative
accessibility of engineered cysteines in the fifth transmembrane domain
of the human 2A-AR (H2A). The second-order rate
constants for the reaction of the receptor with MTSEA and CEC were
determined with the wild type H2A (cysteine at position 201) and
receptor mutants containing accessible cysteines at other positions
within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these
compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor,
likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the
active receptor conformation (R*). This hypothesis was consistent with
three-dimensional receptor-ligand models, which further suggest that
2A-AR activation may involve the clockwise rotation of
transmembrane domain 5.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-Adrenergic receptors
(2-ARs)1
belong to the family of G-protein-coupled receptors and are integral
cell membrane proteins with seven transmembrane (TM) domains (1). The
TM regions are predicted to be -helical and to form a
water-accessible crevice containing the binding site for receptor
ligands (2). Some of the amino acid residues forming the surface of
this crevice directly interact with 2-AR agonists and/or
antagonists, whereas some others in the TM domains may affect ligand
binding indirectly (3, 4). G-protein-coupled receptors are thought to
exist in an equilibrium between two (or more) conformations or
allosteric states, R and R* (5). In the absence of ligand, the inactive state R predominates. Agonist binding stabilizes the receptor protein
in its active state R*, promoting its coupling with G-proteins. The
resulting G-protein activation initiates a cascade of intracellular events leading to physiological responses.
2-adrenergic receptors (4, 6-8). This method identifies residues that form the surface of the binding-site crevice by replacing
consecutive amino acid residues in the membrane-spanning segments with
cysteine, one at a time. The sulfhydryl side chain of a cysteine can
face the water-accessible binding-site crevice, the interior of the
protein, or the lipid bilayer. Sulfhydryls facing the binding-site
crevice react rapidly with sulfhydryl-specific methanethiosulfonate
derivatives such as 2-aminoethyl methanethiosulfonate (MTSEA), with
formation of a covalent bond with the free sulfhydryl group of an
accessible cysteine residue (4, 8). MTSEA has no preference for
receptor conformations, and it reacts, therefore, with the predominant
(inactive) form of the receptor (R). In this method, agonists cannot be
used to stabilize and thereby to study the structure of the active
receptor species, because the presence of an agonist in the binding
pocket would interfere with the access of MTSEA to the engineered
cysteines. The activated state of the receptor (R*) can, however, be
detected with MTSEA by using a constitutively active receptor
mutant (7). This approach, combined with molecular modeling, has
suggested rotation and/or tilting of the TM6 segment of the
2-AR as a consequence of receptor activation (7).
2A-AR with a modified
substituted cysteine-accessibility method (9). The reactive aziridinium ion derivative of CEC forms a covalent bond with the sulfhydryl side
chain of a cysteine exposed in the binding-site crevice and accessible
to CEC. This method has the advantage of introducing recognition
specificity by using a thiol-reactive group incorporated into an
affinity ligand of the target receptor. In functional studies, CEC
irreversibly activates 2-AR, both prejunctionally in the
rat vas deferens and postjunctionally in the dog saphenous vein (10,
11), suggesting that the compound is covalently tethered to an
endogenous cysteine near the binding site.
2A was shown to be consistent with an
-helix with residues Val-197, Cys-201, and Ser-204, pointing into
the binding-site crevice, residues Ile-198, Ser-199, Ile-202, and
Gly-203, facing the lipid bilayer, and Ser-200, pointing partly toward
TM4 (9). Surprisingly, the pattern of accessibility to MTSEA in TM5 of
the dopamine D2 receptor was not consistent with a fixed -helical
structure for TM5 (8). A possible explanation for these results is that
this region changes conformation. Because this region is known to be
directly involved in agonist binding and because the intracellular end
of TM5 interacts with G-proteins, conformational changes in TM5 may be
critical to the activation mechanisms of these receptors.
2A receptor, we have
compared the rates of reaction of the agonist CEC and of MTSEA with
accessible cysteines at positions 197, 200, 201, and 204 in TM5 of
H2A. Nonaccessible cysteines at positions 202 and 203 and a
H2ASer201 mutant (containing no substituted cysteines) were used as
controls. MTSEA reacted with accessible cysteines with the following
rank order of reactivity: Cys-200 < Cys-201(wt) < Cys-197 = Cys-204. In contrast, for CEC, the cysteine at position 200 was found to be the most reactive (rank order of the rate constants: Cys-204 = Cys-201(wt) = Cys-197 < Cys-200).
Our findings suggest that the agonist CEC and MTSEA may recognize two
different receptor conformations, with MTSEA reacting with the
predominant inactive form and CEC reacting with the
agonist-dependent active conformation of TM5 of H2A.
Such a hypothesis is consistent with three-dimensional models of the
receptor-MTSEA and receptor-CEC complexes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A and the mutated receptor cDNAs
were subcloned into the KpnI/BamHI sites of the expression vector pREP4 (In Vitrogen, NV Leek, The Netherlands).
2-AR antagonist [3H]RX821002. The
transfected cells chosen for further experiments were subsequently
maintained in 200 µg/ml hygromycin B.
2-AR binding was assessed by incubating the
homogenate (30-60 µg of protein/assay tube) with 2.5 nM
[3H]RX821002. Nonspecific binding was determined by
including 10 µM phentolamine in parallel assays.
2A and each susceptible mutant was
estimated by determining the extent of the reaction after a fixed time, 15 min (CEC) or 2 min (MTSEA), with seven concentrations of CEC (0.5, 1, 5, 10, 50, 100, and 500 µM) or MTSEA (5, 20, 50, 100, 150, 200, and 250 µM) as previously reported (8). The
fraction remaining of the initial total binding, Y, was fit
to (span × e-kct) + plateau, where k is the
second-order rate constant (M
1
s1), c is the concentration of CEC or MTSEA
(M), t is the reaction time (s), and plateau is
the fraction of binding remaining at saturating concentrations of
reagent. For Cys-197, Cys-200, and Cys-204, the highest concentration
of MTSEA and of CEC produced nearly complete inhibition of ligand
binding (see below). Thus, for all of these fits, the plateau was set
to a constant value of 0, and the span was correspondingly fixed to 1 because binding in the absence of added reagent is by definition 100%.
The plateau was not fixed for the other CEC fits, because the data did
not show complete inhibition at the highest concentration of CEC
tested. For these fits the span was set equal to 1 
plateau. The
results were analyzed with GraphPad Prism (GraphPad Software, San
Diego, CA).
-minimum
essential medium supplemented with 2 mM glutamine, 26 mM NaCl, 50 units/ml penicillin, and 50 µg/ml
streptomycin) at a flow rate of 100 µl/min. CEC and UK 14,304 were
diluted with the running medium. Following base-line stabilization, cells were exposed to agonists for 15 min, and a 30-min wash period was
employed between successive agonist exposures. The alterations in the
extracellular acidification rate were calculated as the difference
between the maximum effect after agonist exposure and the average of
three measurements taken immediately before agonist addition.
2A receptor model contains seven
transmembrane helices, and it was built using the computer program
MODELLER 4.0 (14). The model is based on a template (15) generated
using electron density of the frog rhodopsin (16) and the analysis of
~500 sequences of G-protein-coupled receptors (15). Receptor models for each mutant were generated by replacing corresponding residues at
positions 197, 200, or 204, one at a time, with a cysteine, and by
replacing Cys-201 with a serine.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-AR antagonist radioligand [3H]RX821002.
Three clones from each transfection expressing the expected receptor
were isolated for the preliminary experiments, and one cell line from
each transfection was expanded for further experiments and subsequently
maintained in 200 µg/ml hygromycin B.
2ASer201Cys202 and H2ASer201Cys203, were 451 ± 10 and
2922 ± 91 fmol/mg of protein, respectively. The affinities of
these two mutants for the 2-AR antagonist
[3H]RX821002 were comparable with the H2Awt receptor
(Kd values for H2Awt, H2ASer201Cys202, and
H2ASer201Cys203 were 0.60 ± 0.02, 0.25 ± 0.02, and
0.68 ± 0.02 nM, respectively) (Table I). A similar characterization of CHO
cells expressing H2Awt(Cys201), H2Ser201Cys197,
H2ASer201Cys200, and H2ASer201Cys204 was reported previously (9)
and is also shown in Table I. In all investigated cell lines expressing
wt and mutant receptors, the addition of CEC to competition binding
assays inhibited specific binding of 2.5 nM
[3H]RX821002 with steep monophasic competition curves
(9). The concentration of CEC that inhibited specific
[3H]RX821002 binding by 50% (IC50) was used
to calculate apparent Ki values (inhibition
constant) according to the Cheng-Prusoff equation (19). Competition
binding analysis of the control mutants H2ASer201,
H2ASer201Cys202, and H2ASer201Cys203 containing no exposed
cysteines in the binding cavity in our receptor model, indicated that
the lack of irreversible inhibition of binding by CEC in these
receptors was not due to a lack of binding affinity but rather to the
absence of accessible cysteine residues on the surface of the
binding-site crevice (apparent Ki = 260 ± 32, 345 ± 38, and 808 ± 37 nM, respectively) (Table
I).
Pharmacological characterization of CHO cells expressing wild type and
mutated human 2A-ARs
2ASer201, which
contained no substituted cysteines, suggesting that residues 202 and
203 face the lipid bilayer or are closely packed against other
membrane-spanning segments.
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Fig. 1.
Inhibition of antagonist binding by CEC and
MTSEA. A, % of antagonist binding remaining after 15 min of incubation in the presence of different concentrations of CEC.
The Cys-200 ( 
) mutant is much more sensitive to CEC than is the wild
type receptor Cys-201 (
). 
, Cys-202; 
, Ser-201; 
, Cys-203;
, Cys-204; 
, Cys-197. B, % of antagonist binding
remaining after 2 min of incubation in the presence of different
concentrations of MTSEA. The Cys-200 mutant is somewhat less sensitive
to MTSEA than the wild type receptor. The lines are the
single exponential decay fits (see "Experimental Procedures"),
except in the case of and Ser-201 (), Cys-202 (), and Cys-203
() in B, where no curve fits are shown (see
"Results"). The data points represent means ±S.E. of 3-6 separate
experiments, each performed in triplicate. , Cys-200; , Cys-201;
, Cys-204; , Cys-197.
2ASer201 is not resistant to the effects of CEC and that it
contains, therefore, one or more relatively slowly reacting cysteines.
For all of the mutants except H2Awt(Cys201), however, the simple
single exponential decay function gave a reasonable fit of the CEC
data, especially at low concentrations of CEC (Fig. 1A).
This is likely the case because Cys-197, Cys-200, and Cys-204 are so
reactive and because reaction at these positions produces such a large
inhibition of binding that the relatively slowly reacting endogenous
cysteines are nearly irrelevant to the resulting pattern of inhibition. For the H2wt(Cys201) receptor, however, the plateau determined by
this fit was substantially greater than the experimentally determined
value, even though the fit is consistent with the inhibition seen at
low concentrations of CEC. If the plateau was fixed to 0, however, a
single exponential fit provided a gross underestimate of the rate of
reaction (data not shown), a conclusion based on the significant
inhibition of binding to this receptor observed at even very low
concentrations of CEC. The data for the wild type receptor are better
fit by a 2-site exponential decay function, but the variation in the
rates obtained for the very reactive component for this receptor (and
for the other mutants) was too great to make this a reliable method for
analyzing the data (data not shown). Thus the value shown in Table II
is an approximation of the rapid rate of reaction with Cys-201, and the
remaining inhibition is likely because of reaction with the other more
slowly reacting cysteines in H2ASer201.
Second-order rate constants (k) of inactivation of the wild type and
mutated human 2A-ARs by MTSEA and CEC
2ASer201) were obtained by dividing each k value by the k
determined for the wild type or H2ASer201 receptor. The ratio
CEC/MTSEA represents the relative reactivity of CEC and MTSEA at each
receptor. ND, not reliably
determined.
2Awt in a concentration-dependent and
irreversible manner (Fig. 2). After CEC
treatment followed by extensive washing, the reversible
2A-AR agonist UK 14,304 was unable to further increase
the acidification rate, consistent with the complete reaction of the
entire receptor population with the covalently tethered agonist CEC and
persistent activation. Exposure of CHO cells expressing H2Awt and
H2ASer201Cys200 receptors to UK 14,304 resulted in reversible
increases in the rate of extracellular acidification (Fig.
3). These results confirmed the efficacy
of the tethered agonist and the functionality of the mutant receptor. The functionality of this mutant receptor was also shown in a [35S]GTP
S binding
assay.2
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Fig. 2.
Effect of CEC on the extracellular
acidification rate in CHO cells expressing human
2A-ARs. Cells were exposed to CEC
for 15 min at the concentrations indicated, and a 30-min wash was
employed between CEC exposures. CEC irreversibly increased the
extracellular acidification rate in CHO-H2Awt cells. After CEC
treatment and a further 30-min wash, the 2A-AR agonist
UK 14,304 (1 µM) did not further increase the
acidification rate. Each point represents the measured rate of
acidification for one of the six chambers of cells (3-6 × 105 cells/chamber). CEC additions (
) did not have any
effect in nontransfected CHO cells (control (
)), in which the
acidification rate was similar to CHO-H2Awt with no CEC treatment
(basal ()). Each division of the abscissa represents a
45-min treatment cycle. Numerically, 1 µV s1 is close to 1 × 103 pH units per minute (see Ref. 13).
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Fig. 3.
Effect of
2A-AR agonist UK 14,304 on the
extracellular acidification rate in CHO-H
2Awt (A) and
CHO-H2ASer201Cys200 (B)
cells. Exposure of CHO-H2Awt and CHO-H2ASer201Cys200 cells
to the 2A-AR agonist UK 14,304 resulted in reversible
increases in rates of extracellular acidification. Cells were exposed
to UK 14,304 for 15 min at the concentrations indicated, and a 30-min
wash was employed between UK 14,304 exposures. Each point represents
the measured rate of acidification for one of the six chambers of cells
(3-6 × 105 cells/chamber). UK 14,304 additions ()
did not have any effect in nontransfected CHO cells (control ()).
Transfected CHO cells with no UK 14,304 treatment showed stable
acidification rates (basal ()). Each division of the abscissa
represents a 45-min treatment cycle. Numerically, 1 µV s1
is close to 1 × 103 pH units per minute (see Ref. 13).
-carbon template of the transmembrane helices in the rhodopsin
family of G-protein-coupled receptors (15). Mutant receptor models were
built by replacing the corresponding residues. Because only two
residues at a time were replaced, and the mutations were quite
conservative, such as Cys-201 to Ser and Ser-200 or Ser-204 to Cys or
Val-197 to Cys and because radioligand antagonist binding to the
mutants was similar to that of wt receptor, it is reasonable to assume that these mutations did not have major effects on the receptor structure; therefore, these residues were simply replaced in the wt model.
2Awt receptor,
in the H2ASer201Cys197 mutant, and in the H2ASer201Cys204 mutant,
cysteine residues are readily accessible in the binding-site crevice,
and an ideal covalent bond can be formed. In the mutant
H2ASer201Cys200, the cysteine residue is less accessible, and the
predicted covalent bonding distance is longer than in the other cases,
suggesting that a conformational change is necessary to shorten this
distance and thereby to boost the rate of reaction between CEC and this
cysteine. When the minimization is made without constraints, the
cysteine residue can move. This allows the orientation of the TM5 helix to change, and makes the cysteine at position 200 more accessible for
covalent bonding with CEC (Fig. 4C).
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Fig. 4.
Energy-minimized hypothetical model of the
H 2A binding-site crevice with MTSEA and
CEC. MTSEA (A) forms a covalent disulfide bond with a
cysteine residue in TM5 of H2A at position 200 blue, the
wild type 201 (red), and 204 (green). CEC
(B) forms a covalent carbon-sulfur bond with a cysteine
residue in TM5 of H2A at position 200 (blue), 201 (red), and 204 (green). An aspartate residue
(Asp-113) in TM3 of H2A, the wt, and mutants participates in
recognition of CEC; this is shown in blue, red,
and green, respectively. C, energy-minimized model
of the H2ASer201Cys200 mutant with CEC without constraints. Note
that Cys-200 rotates to point into the binding-site crevice.
2Awt receptor, the mutant H2ASer201Cys197, and the mutant H2ASer201Cys204. However, the disulfide bond in the mutant
H2ASer201Cys200 was slightly longer, and the cysteine residue was
less accessible in the binding-site crevice than it is in the
H2ASer201Cys197, H2ASer201Cys204, or H2Awt receptor models. As
MTSEA has no other points of attachment in the binding-site crevice,
energy minimization of the H2ASer201Cys200 mutant without
constraints did not make the Cys-200 residue more accessible, as was
the case with CEC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2A-AR (9). Surprisingly, this pattern of accessibility was quite different from that observed in TM5 of the homologous dopamine D2 receptor (8). We postulated that the differences in
accessibility might relate to the use of the agonist CEC. CEC has been
used to discriminate between 1-AR subtypes in functional assays (20, 21), and it has also been shown to irreversibly bind to
H2A and H2C but not H2B (9, 22). In functional studies with
tissue preparations, CEC has been shown to behave as an
2-AR agonist, both at prejunctional and postjunctional 2-ARs (10, 11, 23, 24). However, the agonist action of CEC has previously not been shown in recombinant cell lines expressing only one defined receptor subtype. In this work, the agonist effects of
CEC in recombinant CHO cells expressing H2A were confirmed with
Cytosensor® microphysiometry. These results suggest that CEC is
covalently tethered to the side chain of Cys-201 in such a manner that
it produces irreversible receptor activation.
2A-AR was determined using two different sulfhydryl-specific agents, CEC and MTSEA. The rates of reaction of CEC
and MTSEA were similar at the wt receptor (Cys-201). The rates of
reaction of the two reagents were likewise similar, with cysteine
substituted for residues 197 and 204. In contrast, the rate of reaction
with a cysteine at position 200 was more than 5-fold faster for CEC
than for MTSEA. The true difference in rate is even greater than
5-fold, because the fit obtained for the inhibition of Cys-200 by
MTSEA, as discussed above, significantly overestimates the
rate of reaction of this residue with MTSEA. At particular positions
including the reaction of Cys-200 with MTSEA, a simple
single-exponential decay function is clearly inadequate to deal with
the complex molecular interactions underlying the experimental results.
There must be other sites in the receptor protein in addition to the
investigated cysteines that are susceptible to inactivation by CEC and
MTSEA, and these sites may also interact with each other. In addition,
residual CEC and MTSEA present in the ligand binding assays
may further complicate the situation, although experiments in which the
reagents were removed by extensive washing gave similar results (data
not shown). Nonetheless, both the qualitative results and the derived
second-order rate constants support a similar reactivity of Cys-197,
Cys-201, and Cys-204 to both CEC and MTSEA, and this contrasts with the
substantially greater rate of reaction of Cys-200 with CEC than with
MTSEA.
2A, however, it likely stabilizes one or several of the
active conformations (R*) and reacts with engineered cysteines
accessible in the active conformation. Thus, the difference in the
reactivity of CEC and MTSEA toward the substituted cysteine in position
200 in the H2ASer201Cys200 mutant may result from a difference in
the accessibility of this residue in the active and the inactive forms
of the receptor.
2A receptor model based on the electron
diffraction structure of bacteriorhodopsin (30). The model was
consistent with the -helical structure and orientation of TM5 in
H2A-AR (9), and the observed helical structure is also consistent
with our current revised receptor model based on the -carbon
template for the TM helices in the rhodopsin family of
G-protein-coupled receptors (15). When CEC was docked into the receptor
model based on the bacteriorhodopsin structure, residues 200, 201, and
204 were equally accessible to form the covalent carbon-sulfur bond
with CEC or with MTSEA. In our current receptor model,
however, the TM3 helix is tilted into the binding-site crevice, thereby
limiting the size of the binding site and the access of ligands to the
residue at position 200. Energy minimizations without constraints
indicated that the presence of CEC may influence the orientation of
position 200, thereby optimizing the interactions between the receptor
and CEC, i.e. both hydrogen bonding of CEC with Asp-113 in
TM3 and covalent bonding between CEC and Cys-200 in TM5 are
simultaneously favored. Our results with MTSEA support the
prediction that Cys-200 in the H2ASer201Cys200 mutant is not
quite as readily accessible as Cys-201 in H2Awt, Cys-197 in the
H2ASer201Cys197 mutant, and Cys-204 in the H2ASer201Cys204 mutant. Removing constraints during the energy minimization does not
cause any conformational changes in TM5 in the H2ASer201Cys200-MTSEA complex.
2A-AR. The current receptor model thus
appears to more accurately predict the three-dimensional receptor
structure than the previous bacteriorhodopsin-based receptor model.
ACKNOWLEDGEMENTS
-carbon
coordinates for helical parts of rhodopsin-like receptors. Anna-Mari
Pekuri and Ulla Uoti are gratefully acknowledged for their skillful
technical assistance. We also thank Tuomas Lönnberg (Juvantia
Pharma Ltd, Turku, Finland) for his help with receptor models, Drs.
Juha M. Peltonen (Dept. of Pharmacology and Clinical Pharmacology,
University of Turku, Turku, Finland) and Siegfried Wurster (Juvantia
Pharma Ltd, Turku, Finland) for performing the
[35S]GTPS binding assays and Dr. J-M. Savola (Juvantia
Pharma Ltd, Turku, Finland) for valuable comments on this manuscript.
FOOTNOTES
ABBREVIATIONS
2A, human 2A-adrenergic receptor;
H2B, human
2B-adrenergic receptor;
H2C, human
2C-adrenergic receptor;
MTSEA, 2-aminoethyl
methanethiosulfonate;
TM, transmembrane domain;
wt, wild type;
GTPS, guanosine 5'-O(3-thiotriphosphate)..
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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