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J. Biol. Chem., Vol. 275, Issue 48, 37779-37788, December 1, 2000
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From the
Received for publication, March 13, 2000, and in revised form, August 6, 2000
Previous work in the
The Curiously, Using the substituted cysteine accessibility method (14, 15), we found
that in the dopamine D2 receptor each of the three aligned
serines (Ser-1935.42, Ser-1945.43,
Ser-1975.46) are accessible in the binding-site crevice
(16). This is consistent with experimental results in the
D2 receptor implicating each of the three serines in the
binding of various ligands, although different serines were found to be
more or less critical with different ligands (11, 12). More recently we
have applied the substituted cysteine accessibility method to the
aligned portion of TM5 in the human
To test this hypothesis, we investigated the functional
interaction between the Numbering of Residues--
Residues are numbered according to
their positions in the human Cell Culture and Transfection--
Human embryonic kidney cells
(HEK 293) were grown in Dulbecco's modified Eagle's medium/F-12 (1:1)
containing 3.15 g/liter glucose and 10% bovine calf serum at 37 °C
and 5% CO2. Transfection with wild type (WT) or mutant
Membrane Preparation--
Cells suspensions were centrifuged at
1000 × g for 5 min at 4 °C, and the pellets were
homogenized in 1 ml of binding buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 0.006%
bovine serum albumin) using an OMNI 1000 Polytron homogenizer at
setting 26-30 for 10-15 s at 4 °C. The homogenates were
centrifuged at 13,500 × g for 10 min at 4 °C, and
the membrane pellets (from a confluent 100-mm dish) were resuspended by
homogenization, as described above, in 1 ml of binding buffer. The
membrane suspensions were diluted (typically 1:20-1:40) in binding
buffer and used for radioligand binding studies.
[3H]CGP-12177 Binding--
Aliquots of diluted
membrane suspension (200 µl) were incubated in binding buffer either
with six different concentrations of the antagonist
[3H]CGP-12177 between 40 and 1100 pM (in
saturation binding experiments) or with increasing concentrations of
agonists or antagonists in the presence of 0.7 nM
[3H]CGP-12177 (in competition binding experiments). The
total volume was adjusted to 0.5 ml, and the binding experiments were
performed as described previously (20). The amount of membrane used was adjusted to ensure that the specific binding was always equal to or
less than 10% of the total concentration of the added radioligand. Specific [3H]CGP-12177 binding was defined as total
binding less nonspecific binding in the presence of 1 µM
alprenolol (Research Biochemicals). Data for saturation and competition
binding were analyzed by nonlinear regression analysis using GraphPad
Prism 3.0 (GraphPad Software, San Diego, CA). IC50 values
were obtained by fitting the data from the competition studies to a
one-site competition model. Ki values were
determined using the equation Ki = IC50/(1 + L/KD), where
L is the concentration of radioligand (21).
Reactions with Methanethiosulfonate (MTS) Reagents--
The
experiments for the reaction of methanethiosulfonate ethylammonium
(MTSEA) with the WT cAMP Accumulation Assays--
HEK 293 cells stably expressing
the WT Antagonist Binding to Mutant and WT Receptor
In competition experiments with [3H]CGP-12177 (Table I),
the affinity of pindolol was reduced 24-, 67-, and 65-fold by mutation of Ser-2035.42 to Ala, Val, and Cys, respectively. In
contrast, substitution of Thr for Ser-2035.42 only
decreased the affinity of pindolol 5-fold. These mutations did not
lower the affinities of alprenolol and propranolol for the
Probing the Accessibility of Ser-2035.42--
As
described above, mutation of Ser-2035.42 affected the
affinities of particular antagonists. This effect might have resulted from either a direct effect of an alteration in the interaction of
Ser-2035.42 with ligand or from an indirect effect through
a propagated conformational alteration. For the effect to be direct,
Ser-203 must be accessible on the surface of the binding-site crevice.
In ongoing studies we are using the substituted cysteine accessibility
method in TM5 of the Agonist Binding--
We examined the affinities of a series of
phenethylamine derivatives that only differed in the presence or
absence of the mOH and the pOH on the aromatic ring. Thus, the
derivative of phenethylamine containing a
The affinity of HAL-mOHpOH was reduced 71-, 25-, and 40-fold by
mutation of Ser-2035.42 to Cys, Ala, or Val, respectively,
whereas its affinity was reduced less than 3-fold by mutation of
Ser-2035.42 to Thr, which preserved a OH at the 203 position (Table II, Fig. 3). Furthermore, in contrast to the 96-fold
higher affinity of HAL-mOHpOH than HAL in WT, HAL-mOHpOH had an
affinity nearly identical to that of HAL after mutation of Ser-203 to
Cys, Ala, or Val. The affinity of HAL-mOHpOH, however, was 37-fold
higher than that of HAL in S203T, in which a OH at position 203 was preserved.
HAL-mOH had a 3-fold higher affinity than HAL in S203T, nearly the same
as the 4-fold difference in WT. In contrast, HAL-mOH had a slightly
lower affinity than HAL after substitution of Ser-2035.42
by Cys, Ala, or Val, suggesting that the presence of a OH at position
203 is critical for the positive effect of the mOH on binding affinity.
The affinities of HAL-pOH and HAL were essentially unaffected by
mutation of Ser-2035.42 to Thr and Cys and slightly
increased by mutation to Ala or Val.
The affinity of S204A for epinephrine was decreased 34-fold relative to
WT. If removal of the OH side chain at the Ser-204 position eliminated
interaction with the mOH, we would have expected that removal of the
mOH from epinephrine (synephrine) would not lower its affinity for
S204A. However, we observed a higher affinity of epinephrine than of
synephrine in this mutant (Table II), consistent with a preserved
impact of the mOH despite the absence of Ser-2045.43.
Moreover the affinity of HAL-mOH was higher than that of HAL in S204A,
also consistent with a preserved interaction of the mOH.
Simultaneous mutation of Ser-2035.42 and
Ser-2045.43 to Ala decreased the affinity for epinephrine
by 2 orders of magnitude (Table III, Fig.
3). Thus the affinity of epinephrine for S203A/S204A was similar to
that of HAL for WT. Moreover, removal of the mOH from epinephrine
(synephrine) did not further decrease its affinity for S203A/S204A,
consistent with the absence of a H bond with the mOH. Likewise,
addition of the mOH to HAL did not increase the affinity in the
simultaneous absence of Ser-2035.42 and
Ser-2045.43. Simultaneous mutation of
Ser-2035.42 and Ser-2075.46 to Ala also
decreased the affinity for epinephrine by nearly 2 orders of magnitude
(Table III, Fig. 3). Simultaneous mutation of Ser-2045.43
and Ser-2075.46 to Ala decreased the affinity of each of
the four phenethylamine derivatives tested (Table III, Fig. 3),
consistent with an effect of the mutations on the isomerization of the
receptor (24, 25).
Activation of Adenylyl Cyclase--
High levels of expression of
the WT
The effects of the mutations on the potencies of the agonists were
similar to those on their binding affinities. The addition of the mOH
to the aromatic ring of HAL (i.e. phenylephrine) increased its potency for WT only 2-fold (Ser-2035.42), whereas the
addition of the pOH alone (i.e. synephrine) slightly lowered
its potency for WT (Fig. 4, Table IV).
Thus, although the mOH appeared somewhat more important for receptor
activation than the pOH, the addition of both ring OHs to HAL,
resulting in epinephrine, dramatically and synergistically increased
its potency for WT and for S203T 176- and 32-fold, respectively. This synergism was preserved as well in S204A. In contrast, removal of the
OH from the side chain at position 203 by mutation to Ala, Val, or Cys
either completely abolished or greatly diminished the synergistic
effect of the catechol OHs in the potency of HAL-mOHpOH (<3-fold).
This synergism was also lost or greatly diminished in each of the three
double mutants.
HAL, which has no ring hydroxyls, cannot H bond with serines in TM5,
and thus, we anticipated that its ability to stimulate cAMP
accumulation would be unaffected by the presence of the TM5 serines.
Instead, the presence of a hydrophobic side chain at position
2035.42 in S203V led to a 6-fold greater potency for HAL
than for epinephrine. In addition, in S203V HAL produced a similar or
slightly higher maximal stimulation of cAMP than did epinephrine (Fig.
4). It is conceivable that the aromatic ring of HAL interacted
favorably with the hydrophobic Val side chain.
The addition of the mOH alone (phenylephrine) produced an increase in
intrinsic activity relative to HAL in the constructs that contain a
hydroxyl at position 2035.42, WT, S203T, S204A, and
S204A/S207A (Fig. 4). In contrast, the addition of the mOH did not
increase intrinsic activity beyond that of HAL in S203A, S203V, S203C,
S203A/S204A, or S203A/S207A.
Thermodynamic Mutant Cycle--
A method that has been used to
assess for the presence of direct interactions between a residue or
functional group in a ligand and a residue in a channel or receptor is
analysis of binding data based on the thermodynamic mutant cycle in
which each of the putatively interacting residues is mutated separately
and then together (24, 25). In this case, the "mutation" of the ligand was achieved by removal of one or both ring OHs from
epinephrine, and the mutations of the receptor were achieved by
substituting Ser-2035.42 with Thr, Ala, Val, or Cys. If a
mutated functional group of a ligand and a mutated residue in the
receptor do not interact, then the calculated coupling coefficient,
1/ We found that Cys substituted for Ser-2035.42 in
Each of the mutations that removed the OH from the side chain of the
residue at position 2035.42 resulted in a substantial
reduction of the binding affinity and adenylyl cyclase-activating
potency of HAL-mOHpOH and HAL-mOH, which both have mOHs. In contrast,
removal of the OH at the 2035.42 position increased or had
no effect on the affinity of HAL-pOH, which has a pOH but no mOH. The
increase in affinity, potency, and intrinsic activity of HAL-pOH and
HAL in S203A and S203V is consistent with a more favorable interaction
between the aromatic ring itself with the engineered hydrophobic side
chain introduced at the 2035.42 position in these mutants.
Preservation of the OH at position 2035.42 in S203T
maintained the affinities of HAL-mOHpOH and HAL-mOH, suggesting that
the mOH of the agonist is likely to H bond with the OH of
Ser-2035.42. Although the potencies of HAL-mOHpOH and
HAL-mOH were much less affected by mutation of Ser-2035.42
to Thr than they were by removal of the OH at position
2035.42, their potencies were nonetheless decreased
relative to WT. Thus, Thr is a good but not perfect substitute for Ser,
suggesting that the restricted side chain rotamer conformation of Thr
relative to that of Ser (17) and/or the increased side chain volume due to the presence of the extra methyl group exerted a negative influence on receptor activation despite the fact that the interaction of the
side chain OH with the mOH was preserved.
Analysis based on the thermodynamic mutant cycle was also consistent
with a direct interaction of the mOH with Ser-2035.42 (Fig.
5). Removal of Ser-2045.43 by mutation to Ala led to a
reduced affinity and potency for both HAL-mOHpOH and HAL-mOH, both of
which contain a mOH. These results suggest that Ser-2045.43
interacts with the mOH, in agreement with previous work (4). In S204A,
however, HAL-mOHpOH was more potent and vastly more efficacious than
HAL-pOH, demonstrating a persistent role of the mOH even in the absence
of Ser-2045.43. Based on the results of mutation of
Ser-2035.42 described above, we infer that the persistent
effect of the mOH in S204A comes from an interaction with
Ser-2035.42. Whereas in S203A/S204A and in S203A/S207A,
HAL-mOH was no better than HAL at activating, in S204A/S207A HAL-mOH
was better able to activate the receptor than was HAL, consistent with
a crucial role for an interaction of the mOH with
Ser-2035.42 in activation.
Thus we infer that in the WT receptor, the mOH interacts with both
Ser-2035.42 and Ser-2045.43 through a
bifurcated H-bonding network. Such a potential interaction is
illustrated in Fig. 6. Such three-center
bifurcated H bonds are common in protein structures, composing 20-25%
of H bounds in the crystal structures of small biological molecules
(28). Similarly, Ho et al. (29) suggested a simultaneous
H-bonding interaction of the hydroxyl group on the indole ring of
5-hydroxytryptamine with the OHs of both Ser-1985.42
and Thr1995.43 in TM5 of
5-hydroxytryptamine1A receptor (29), and Woodward et al. (10) also propose the existence of a H-bonding
network between the catechol OHs of dopamine and the analogous serines in the D2 dopamine receptor.
The Forgotten Serine
A CRITICAL ROLE FOR Ser-2035.42 IN LIGAND
BINDING TO AND ACTIVATION OF THE 
2-ADRENERGIC
RECEPTOR*
§,
,§,,, and**§§
Center for Molecular Recognition and the
** Departments of Psychiatry and Pharmacology, Columbia University
College of Physicians and Surgeons and the New York
State Psychiatric Institute, New York, New York 10032 and the
¶ Department of Physiology and Biophysics, Mount Sinai School of
Medicine, New York, New York 10029
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor demonstrated critical
interactions between Ser-204 and Ser-207 in the fifth membrane-spanning segment and the meta-OH and para-OH,
respectively, of catecholamine agonists (Strader, C. D.,
Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon,
R. A. (1989) J. Biol. Chem. 264, 13572-13578). Using the substituted cysteine accessibility method in the
2-adrenergic receptor, we have found that in addition to
Ser-204 and Ser-207, Ser-203 is also accessible on the surface of the
binding-site crevice and is occluded by bound agonist. Mutation of
Ser-203 to Ala, Val, or Cys reduced the binding affinity and adenylyl cyclase-activating potency of agonists containing a
meta-OH, whereas their affinities and potencies were
largely preserved by mutation of Ser-203 to Thr, which maintained an OH
at this position. Thus both Ser-203 and Ser-204 appear to interact with
the meta-OH of catecholamines, perhaps through a bifurcated
H bond. Furthermore, the removal of the OH at position 203 led to a
significant loss of affinity of antagonists with nitrogen in their
heterocyclic ring structure. The greatest effect was seen with
pindolol, a partial agonist, suggesting that a H bond between the
heterocyclic ring and Ser-203 may play a role in partial agonism. In
contrast, the affinities of antagonists such as propranolol or
alprenolol, which have cyclic structures without H-bonding capability,
were unaltered after mutation of Ser-203.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor
(2AR)1 has
been extensively studied and, along with rhodopsin, has served as a
prototype for our understanding of the structure and function of
related G-protein-coupled receptors (1, 2). In these receptors, the
binding site is formed among the seven-transmembrane segments (TMs) (2)
in a water-accessible binding-site crevice. A number of residues inferred to contact bound agonist in the 2AR have been
identified. Asp1133.32 (see "Experimental Procedures"
for a description of the indexing of residues) in the third TM (TM3) is
thought to interact with the protonated amine of catecholamines (3) and
is completely conserved in all amine receptors. A cluster of aromatic
residues in TM6 that is thought to interact with the aromatic ring of
catecholamines is highly conserved as well (2). A classical study by
Strader et al. (4) in the 2AR demonstrated
that two serines in TM5 directly interact with the catechol hydroxyls
(OHs) of agonists. Specifically, Ser-2045.43 interacts with
the meta-hydroxyl (mOH) and Ser-2075.46 with the
para-hydroxyl (pOH) (4). These interactions were shown to
contribute to the affinity, potency, and efficacy of various
catecholamine agonists. A potential role of Ser-2035.42 in
binding and activation, however, was obscured by the lack of expression
of the mutant 2AR in which Ser-2035.42 was
mutated to Ala (4). Nonetheless, because the catechol ring contains two
hydroxyls and because two serines were inferred to hydrogen bond to
these two hydroxyls, Ser-2035.42 has been generally assumed
not to play a significant role in agonist binding to and activation of
the wild-type 2AR.
2AR Ser-2035.42 is completely
conserved in all catecholamine receptors (Fig.
1), and this residue has been shown to play a role in ligand binding and receptor activation in the
1A, 2A, and 1B adrenergic
(5-8) and the dopamine D1, D2, and
D3 receptors (9-13). In the rat 1AAR, in
which a Ser is absent at 5.43, Ser-1885.42 (aligned with
2AR Ser-2035.42) interacts with the mOH of
catecholamines, and it is this H bond that is critical for ligand
binding and receptor activation (5).
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Fig. 1.
Sequence alignment of the regions in TM5 of
adrenergic and dopamine receptors containing the conserved
serines. Sequence alignment of the human receptor subtypes. The
conserved serines are shown in bold and are
highlighted. The numbers at the top signify the
positions of the amino acids in the primary sequence of each receptor.
The numbering uses as reference the highly conserved proline in TM5,
which is referred to as 5.50, in accordance with the nomenclature of
Ballesteros and Weinstein (17).
2AR,2 and we
were surprised to observe that substitution of Ser-2035.42
by Cys, unlike the published mutation of this Ser to Ala in the hamster
2AR (4), did not impair expression of the receptor but
instead lowered its affinity for both isoproterenol and for the
radiolabeled antagonist CGP-12177. Similar to our results in the
D2 receptor, we found that Cys substituted for each of the
three serines in TM5 of the 2AR reacted with charged
sulfhydryl reagents and that bound isoproterenol retarded the reaction
of the sulfhydryl reagents with the substituted cysteines. This
suggests both that Ser-2035.42 is on the water-accessible
surface of the binding-site crevice and that bound ligand sterically
obstructs access to this position. This observation prompted us to
reconsider the role of this Ser in ligand binding and receptor
activation. We hypothesized that in the 2AR each of the
serines interacts with catecholamine agonists and that the mode of
binding might not be dramatically different in the 2AR
as compared with related catecholamine receptors that also contain
three serines in TM5.
2AR and various agonists after
the mutation of Ser-2035.42 to a number of other residues,
of Ser-2045.43 to Ala, and of various combinations of two
of the three conserved Ser residues (Ser-2035.42,
Ser-2045.43, and/or Ser-2075.46) simultaneously
to Ala. Our results are consistent with an important role for Ser-203
in the binding of catecholamines and the resulting activation of the
2AR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR sequence. We also index
residues relative to the most conserved residue in the TM in which it
is located (17). By definition, the most conserved residue is assigned
the position index 50, e.g. Pro-2115.50, and
therefore Val-2105.49 and Leu-2125.51. This
indexing simplifies the identification of aligned residues in different
G-protein-coupled receptors.
2AR Plasmids and Site-directed
Mutagenesis--
The cDNA sequence encoding the human
2-adrenergic receptor, epitope-tagged at the amino
terminus with a cleavable influenza-hemagglutinin signal sequence
followed by the FLAG epitope (IBI, New Haven, CT) and tagged with six
histidines at the carboxyl terminus, was a gift from Dr. B. Kobilka
(Stanford University) (18). This cDNA was subcloned into the
bicistronic expression vector pcin4 (19), a gift from Dr. S. Rees
(Glaxo Wellcome), thereby creating the vector
pcin4-SF2AR6His. Mutations were generated by the
polymerase chain reaction-mediated mutagenesis using Pfu
polymerase (Stratagene). The polymerase chain reaction-generated DNA
fragments containing the mutations were subcloned into the
pcin4-SF2AR6His plasmid, and the mutations were
confirmed by DNA sequencing. Mutants are named as (wild-type
residue)(residue number)(mutant residue), where the residues are given
in the single-letter code.
2AR and selection for generation of stably transfected
pools of cells expressing the receptors were performed as described
previously (20).
2AR and the S203C mutant as well as
the experiments for the protection of this reaction by isoproterenol were performed as described previously (20).
2AR or the mutants were plated in 96-well cell
culture plates (pretreated with poly-L-lysine, 0.1 mg/ml)
at a density of 40,000-60,000 cells/well. After incubation overnight
at 37 °C in 5% CO2, the cells were 95-100% confluent. The medium was removed, and 100 µl of OPTIMEM (Life Technologies, Inc.) with or without (control) 60-120 µM
N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was
added. The cells were incubated for 1 h at 37 °C. The medium
was removed, and 100 µl of assay buffer (25 mM HEPES, pH 7.4, 2 mM choline, 288 mM sucrose, 0.9 mM CaCl2, 0.5 mM MgCl2, and 1 mM 3-isobutyl-1-methylxanthine) was added. After a
1-h incubation at 37 °C, more assay buffer without (basal levels) or
with 10 µM forskolin or with increasing concentrations of
agonists was added to a total volume of 200 µl, and the incubation
was continued for 10 min at 37 °C. At the end of the incubation the
assay buffer was removed. The cells were placed on ice and lysed with
3% trichloroacetic acid. Lysates were incubated on ice for 30-60 min
and stored at 
20 °C. After 1-5 days, frozen lysates were thawed
and centrifuged at 1,800 × g for 10 min at 4 °C,
and the supernatants were neutralized with 2 N NaOH.
Quantification of cAMP in the neutralized supernatants was performed
using a competitive binding assay as described previously by Watts
et al. (22) and Nordstedt and Fredholm (23) with minor
modifications. Supernatants were transferred to polypropylene mini-tubes (20 µl/tube) containing buffer B (100 mM
Tris-HCl, pH 7.4, 100 mM NaCl and 5 mM EDTA)
with 1-1.1 nM [2,8-3H]adenosine 3',5'-cyclic
phosphate (Amersham Pharmacia Biotech). Subsequently,
cAMP-binding protein (~100 mg of crude bovine adrenal cortex extract
in 500 µl of buffer B) was added to each tube. After incubation on
ice for 2.5-3 h, the mixtures were filtered through GF/B glass fiber
filters as described for radioligand binding assays. The amount of cAMP
in each sample (one-tenth of a well) was determined by comparison with
a standard curve of known concentrations of unlabeled cAMP (0.3-100
pmol/tube). EC50 values were obtained by fitting the data
to a one-site sigmoidal model using nonlinear regression analysis
(GraphPad Prism 3.0).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR
Expressed in HEK 293 Cells--
Using site-directed mutagenesis, we
constructed eight different 2AR mutants. The Ser at
position 2035.42 was mutated to Ala (S203A), Val (S203V),
Thr (S203T), or Cys (S203C); the Ser at position 2045.43
was mutated to Ala (S204A). In three other mutants, two of
Ser-2035.42, Ser-2045.43, and
Ser-2075.46 were simultaneously mutated to Ala
(S204A/S207A, S203A/S204A, S203A/S207A) to create a series of
constructs in which each Ser was the only residue in this region
capable of H-bonding to a catechol hydroxyl. Saturation analysis of
[3H]CGP-12177 binding to membranes from HEK 293 cells
stably expressing WT or the mutants showed that the receptor density
(Bmax) of the mutants was 31-370% that of WT
2AR (data not shown). The affinity (KD) of the antagonist [3H]CGP-12177
was reduced by mutation of
Ser-2035.42 to Ala (3-fold), Val (6-fold), and Cys
(10-fold) (Table I). In contrast, the substitution of Thr for
Ser-2035.42 (preserving the presence of a OH at this
position) did not lower the affinity of [3H]CGP-12177
(Table I).
Antagonist binding to WT 2AR and the serine mutants
) or increase (
) in ligand affinity after mutation of
2AR. For S204A, S203A/S204A, and S203A/S207A, the
Kd values (pM) for
[3H]CGP-12177 binding were 189 ± 10, 562 ± 112, and 237 ± 48, respectively.
2AR. Indeed, removal of the OH at position 203 (S203V,
S203C, S203A) increased the affinity of propranolol 2-, 5-, and
11-fold, respectively.
2AR to determine the residues that
form the surface of the binding-site crevice.2
Treatment of intact HEK 293 cells stably expressing WT
2AR with the hydrophilic, positively charged,
sulfhydryl-specific reagent, MTSEA, did not affect
[3H]CGP-12177-specific binding, suggesting that none of
the endogenous Cys of the receptor was accessible for reaction (or that
reaction took place but was without a functional effect) (Fig.
2). MTSEA, however, potently inhibited
[3H]CGP-12177 binding to the S203C mutant (Fig. 2),
suggesting that the wild-type Ser-2035.42 side chain is on
the water-accessible surface of the receptor. Isoproterenol (1 µM), a catecholamine with a structure similar to that of
epinephrine but with a propyl moiety instead of a methyl attached to
the protonated amine, substantially retarded the reaction of MTSEA with
the S203C mutant (data not shown), suggesting that the residue is on
the surface of the binding-site crevice and is sterically protected by
bound agonist.
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Fig. 2.
Effects of MTSEA on
[3H]CGP-12177 binding to WT and S203C mutant.
Specific binding was assayed as described under "Experimental
Procedures" after a 2-min incubation with the indicated
concentrations of MTSEA. The means and S.E. are shown for triplicate
determinations from a representative experiment; the experiment was
repeated three times with similar results. The fraction of initial
binding, Y, was fit to (1 plateau) × e
kct + plateau, where plateau
is the fraction of residual binding at saturating concentrations of
MTSEA, k is the second-order rate constant (in
M1 s1),
c is the concentration of MTSEA (M), and
t is the time (120 s). For this experiment,
k = 19.2 M1
s1 and plateau = 0.01.
-OH and a
N-CH3, halostachine (HAL), contains no ring hydroxyls.
Phenylephrine (HAL-mOH) is identical to HAL except for the addition of
a mOH, synephrine (HAL-pOH) is identical to HAL except for the addition
of a pOH, and epinephrine (HAL-mOHpOH) contains both the mOH and the
pOH simultaneously (Table II). In the
simplest scenario, if direct contact exists and disregarding solvation
effects, the effect of removing a OH from a drug is expected to be
similar to that of removing the associated side chain OH contact from
the receptor. Agonist competition of [3H]CGP-12177
binding (Table II, Fig. 3) in membranes
from HEK 293 cells expressing WT showed that HAL-mOHpOH had an affinity
96-fold higher than that of HAL, which contains no OHs on its aromatic ring. HAL-mOH had a 4-fold higher affinity than HAL, whereas HAL-pOH had an affinity nearly identical to HAL. Thus, the presence of both
ring OHs synergistically increased the affinity of HAL-mOHpOH for
WT.
Agonist binding to WT 2AR and serine mutants
) or increase
() in the affinity after the modification of the 2AR
(parentheses) or epinephrine
(brackets/italics).
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Fig. 3.
Competition binding of adrenergic agonists to
wild type, 2AR, and serine
mutants. Competition of [3H]CGP-12177 binding by
HAL-mOHpOH (
), HAL-mOH (
), HAL-pOH (×) and HAL (
) was
performed as described under "Experimental Procedures" on membranes
from HEK 293 cells stably expressing the WT 2AR or the
Ser mutants (indicated in each panel). The means and S.E.
(duplicate or triplicate determination) are shown from a representative
experiment repeated 2-8 times with similar results. The data were fit
to a one-site competition model by nonlinear regression. The log
Ki values determined from the resulting log IC50
values are given in Table II with the chemical structures of the
agonists.
Agonist binding to WT 2AR and double serine mutants
) or increase
() in the affinity after the modification of the 2AR
(parentheses) or epinephrine (brackets/italics).
2AR as well as the mutants in HEK 293 cells
resulted in masking of the partial agonist activity of the drugs tested
in this study. Thus, the maximal stimulation of cAMP accumulation by
the partial agonists, HAL-mOH, HAL-pOH, and HAL, was similar to that of
the full agonist, HAL-mOHpOH (data not shown). To overcome this problem
we measured agonist-stimulated cAMP accumulation after inactivating
95-99% of the receptors by pretreatment with the alkylating reagent
EEDQ (26). The concentration of EEDQ was chosen (a) to
equalize the number of cell surface receptors in each of the mutants
and (b) to achieve the minimal receptor density that still
gave maximal stimulation of cAMP by epinephrine as compared with
untreated cells.3 In the
resulting low receptor reserve environment we found that HAL-mOH,
HAL-pOH, and HAL were indeed partial agonists, being 40 ± 8, 32 ± 12, and 28 ± 5% (mean ± S.E.; n = 7-9) as effective as the full agonist, HAL-mOHpOH, in maximally
stimulating cAMP accumulation in WT receptor (Fig.
4). The maximal elevation of cAMP
produced by epinephrine in untransfected HEK 293 cells (in the absence
of EEDQ treatment) was less than 5% that seen in WT receptor after
treatment with EEDQ (data not shown). Since EEDQ treatment inactivates
both endogenous and heterologously expressed receptor, the contribution
of residual endogenous receptor in these assays is negligible.
View larger version (31K):
[in a new window]
Fig. 4.
Stimulation of cAMP accumulation by
adrenergic agonists. Stimulation of cAMP accumulation by the
indicated concentrations of HAL-mOHpOH ( ), HAL-mOH (),
HAL-pOH (×),y and HAL () was performed as described under
"Experimental Procedures" in intact HEK 293 cells stably expressing
the WT 2AR or the Ser mutants (indicated in each
panel). The means and S.E. (duplicate determination) are
shown from a representative experiment repeated 2-3 times with similar
results. The data were fit to a one-site sigmoidal dose-response model
by nonlinear regression.
Stimulation of cAMP accumulation by the WT 2AR and the
serine mutants
) or increase () in the affinity
after the modification of the 2AR (parentheses) or
epinephrine (brackets/italics).
, will be close to unity. In contrast, in the case of a direct
interaction, 1/ will be much greater than unity (an exception to
this will be a case in which the receptor mutation maintains the direct
interaction with the unmutated ligand, as in the case of S203T with
HAL-mOHpOH, and in this scenario 1/ is expected to be close to
unity). As depicted in Fig. 5, 1/ is
indeed close to unity for S203T but is 100-200 for S203A, S203V, and
S203C in their mutant cycles with HAL-pOH and HAL, the compounds in
which the mOH or the mOH and the pOH are absent. This strongly suggests
the presence of a direct interaction between Ser-2035.42
and the mOH of HAL-mOHpOH. In contrast, 1/ is much lower in the
cycles of these mutants with HAL-mOH in which the pOH is removed, consistent with the lack of a direct interaction between
Ser-2035.42 and the pOH. The slightly elevated 1/ values
for S203V and S203C in their cycles with HAL-mOH are consistent with
either additional interactions of the substituted side chain or an
indirect conformational alteration in binding due to the effects of the
mutant side chains.
View larger version (32K):
[in a new window]
Fig. 5.
Assessment of the interaction between the
residue at position 2035.42 of
2AR and the catechol OHs of epinephrine
using thermodynamic double mutant cycles. A, the
corners of a thermodynamic double-mutant cycle are formed by the
affinities (Ki) of HAL-mOHpOH before (epinephrine
(EPI)) and after removal (DRUG) of one or both
OHs from its aromatic ring to 2AR before (WT) and after
the mutation of Ser-2035.42 to Thr, Ala, Val, or Cys
(S203X). For example,
Ki(EPI/WT) and
Ki(EPI/S203X) represent the affinities of
EPI for the WT and for the S203X mutant, respectively,
whereas Ki(DRUG/WT) and
Ki(DRUG/S203X) represent the affinities
of EPI after removal of either the mOH, the pOH, or both for the WT and
for the S203X mutant, respectively. X1 = Ki(EPI/WT)/Ki(DRUG/WT), X2 = Ki(EPI/S203X)/Ki(DRUG/S203X),
Y1 = Ki(DRUG/WT)/Ki(DRUG/S203X),
Y2 = Ki(EPI/WT)/Ki(EPI/S203X).
The affinity change from the EPI/WT interaction to
DRUG/S203X interaction must be the same regardless of the
pathway followed. Thus, X1 × Y1= X2 × Y2. The factor,
= X1/X2= Y2/Y1 = [ Ki(EPI/WT)/Ki(DRUG/WT) ]/[
Ki(EPI/S203X)/Ki(DRUG/S203X)
], the coupling coefficient, reflects the extent of interaction
between the residue at position 2035.42 and the catechol
OHs (for details see "Results"). B,
three-dimensional plot of 1/ values, calculated according to the
above equation, for the pairs of S203X mutants (X
is Thr, Ala, Val, or Cys) with epinephrine before and after the removal
of mOH (SYN, synephrine), pOH (PHE,
phenylephrine), or both ring OHs (HALO, halostachine).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR was accessible to reaction with the charged
sulfhydryl-specific reagent MTSEA. Because MTSEA reacts
>109 times faster with the thiolate than with the thiol
(27) and only water-accessible Cys residues are likely to ionize to a
significant extent, we infer that Ser-2035.42 faces the
water-accessible binding-site crevice. The ability of the catecholamine
isoproterenol to protect the substituted Cys at position 203 from
reaction with MTSEA is consistent with the possibility of a direct
interaction between the drug and the OH side chain at position
2035.42 in the WT receptor.
View larger version (31K):
[in a new window]
Fig. 6.
The feasibility of the proposed H-bonding
interactions between the mOH of catecholamine ligands to
Ser-2035.42 is shown in a molecular model of epinephrine
(EPI) bound to the
2AR. A, a view from the
extracellular side of the seven TMs of the receptor showing the
proposed H-bonding interactions between epinephrine and the
receptor based on previous mutagenesis studies. The protonated amine of
epinephrine H bonds Asp-1133.32 in TM3 and also the
CO moiety of the Asn-2936.55 side chain in TM6. The NH
moiety of Asn6.55 in TM6 H bonds the -OH of epinephrine.
The catechol OHs of the phenyl ring of EPI participate in H-bonding
interactions with a cluster of Ser residues in TM5, shown in more
detail in panel B. The Ser-2035.42 studied here
and the conserved Pro residue at position 5.50, which bend TM5 away
from the ligand, are marked for reference. B, proposed
H-bonding between epinephrine (EPI, left) and the
Ser in TM5 (right). The side chain OH of
Ser-2035.42 H bonds the mOH of EPI in addition to the
previously proposed H-bonding of this mOH to Ser-2045.43
(4). The pOH moiety of EPI H bonds Ser-2075.46, as
previously proposed (4). The side chain OHs of Ser-2045.43
and Ser-2075.46 are capable of H-bonding simultaneously to
the backbone carbonyls of the preceding turn, which maintains their
-helical H-bonding pattern to the backbone NH moieties of the i+4
residue.
Ser and Thr residues in -helical environments have a tendency to
establish H bonds between the side chain OH and the backbone carbonyl
of the preceding turn of the -helix (30), and these interactions can
induce long range conformational changes in the -helix (31). This
interaction is illustrated in Fig. 6B, in which the side
chain OH of Ser-2045.43 H bonds to the backbone carbonyl of
residue 5.39, which in turn maintains the standard helical H-bonding to
the NH of the residue i+4. A similar pattern is shown for
Ser-2075.46, the side chain OH of which H bonds the
backbone carbonyl of residue 5.43. The OH moieties of Ser residues are
capable of participating in up to three H-bonding interactions
simultaneously, two as H-bonding acceptors on the oxygen atom and one
as a H-bonding donor through the hydrogen atom. Fig. 6B
illustrates that these H-bonding interactions most likely represent a
complex network of H bonds involving the catechol OHs, the Ser side
chain OHs, and the backbone carbonyls. The pattern of H-bonding
interactions between the Ser residues at positions 2035.42,
2045.43, and 2075.46 of the 2AR
and HAL-mOHpOH and the backbone is one of several possible
conformations, and alternative H-bonding networks are also possible
that would result in a different set of H-bonding interactions between
the ligand, the Ser side chains, and the backbone carbonyls. Because
alternative ligand-receptor H-bonding interactions may result in
different backbone conformations, such a mechanism may play a role in
the conformational changes that accompany receptor activation.
In the receptors with a OH at position 2035.42, namely WT and S203T, the simultaneous addition of the mOH and the pOH to HAL resulted in large synergistic increases in affinity and potency, in contrast to the relatively small effects of the addition of the mOH or the pOH alone. The synergism of the mOH and pOH on potency was abolished when the OH on the side chain at position 2035.42 was removed by mutating Ser-2035.42 to Ala, Cys, or Val. Notably the synergistic effect of the catechol OHs on the potency of and maximal activation of HAL-mOHpOH was preserved after the substitution of Ala for Ser-2045.43. Thus, although the effect of the presence of the mOH alone on potency and maximal activation was essentially abolished by mutation of Ser-2045.43 to Ala, consistent with the conclusion of an interaction between the mOH and Ser-2045.43, the effect of the simultaneous addition of the mOH (which was ineffective alone) and the pOH (which was also ineffective alone) was substantial in S204A. Thus this finding is consistent with an important role for the mOH in receptor activation even in the absence of Ser-2045.43. The synergistic effects of the mOH and pOH were absent in each of the double mutants, suggesting that the presence of Ser-2035.42 by itself is not sufficient for synergism.4 A similar synergistic effect of the catechol OHs has been observed on the binding affinity of dopamine (12).
Mutation of Ser-2035.42 to Ala, Cys, or Val also reduced
the affinities of the antagonists
pindolol5 and CGP-12177,
which contain an indole or imidazole ring, but not of alprenolol or
propranolol, which have cyclic structures lacking nitrogen (Table I).
In contrast, preservation of the OH by mutation of
Ser-2035.42 to Thr generally maintained the affinities of
CGP-12177 and pindolol for the 2AR (Table I). Both
ligands contain a nitrogen in their heterocyclic ring that is able to
participate in H-bond formation, and the OH of Ser-2035.42
may directly H bond to the nitrogen of these antagonists. Simultaneous replacement of Ser-2045.43 and Ser-2075.46 by
Ala did not significantly alter the affinities of the antagonists, suggesting that the nitrogen of the heterocyclic antagonists interacts only with the OH of Ser-2035.42. This is consistent with
the restrictive H-bonding capacity of the N-H moiety of pindolol, which
can only interact with a single H-bonding acceptor. In intact cells
expressing the 2AR, pindolol has been shown to be a weak
partial agonist (32, 33), whereas alprenolol and propranolol are
neutral or weak negative antagonists (32). Thus, the interaction
between the nitrogen of the heterocyclic ring of pindolol with the
hydroxyl of Ser-2035.42 may play a role in producing
partial agonism. Curiously, we observed an increase of the affinity of
the S203A, S203V, and S203C mutants for propranolol, suggesting that
the drug binds better when the side chain at position
2035.42 is hydrophobic and/or favorable for interaction
with the aromatic ring structure of propranolol.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Drs. Brian Kobilka and
Stephen Rees for gifts of the epitope-tagged 2
receptor DNA and the pcin4 plasmid, respectively. We thank Thomas
Livelli for the HEK 293 cells and for valuable advice and Myles Akabas,
Arthur Karlin, Lei Shi, Irache Visiers, and Harel Weinstein for
valuable discussion and comments on this manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institute of Mental Health Grants MH57324 and MH54137, by the G. Harold and Leila Y. Mathers Charitable Trust, and by the Lebovitz Trust.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: Dept. of Pharmacology, Medical School, University of Crete, Heraklion 71110, Greece.
Present address: Novasite Pharmaceuticals, Inc., 3520 Dunhill
St., San Diego, CA 92121.
§§ To whom correspondence should be addressed: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 West 168th St., P&S 11-401, New York, NY 10032. Tel.: 212-305-7308; Fax: 212-305-5594; E-mail: jaj2@columbia.edu.
Published, JBC Papers in Press, August 29, 2000, DOI 10.1074/jbc.M002092200
1
2AR, 2-adrenergic
receptor; WT, wild type; TM, transmembrane segment; mOH,
meta-hydroxyl; pOH, para-hydroxyl; H bond, hydrogen bond;
HEK, human embryonic kidney; MTS; methanethiosulfonate; MTSEA, MTS
ethylammonium; EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2 -dihydroquinoline; HAL, halostachine; HAL-mOH, phenylephrine; HAL-pOH,
synephrine; HAL-mOHpOH, epinephrine (EPI).
2 G. Liapakis, D. Fu, and J. A. Javitch, manuscript in preparation.
3
Similar treatment of L6 myoblasts and C6 glioma
cells with EEDQ decreased the maximal density of the -adrenergic
receptors (Bmax) without altering the affinity
(KD) of the residual receptor for radioligand or the
cell viability (34).
4 The energy changes associated with removal of H bonds are somewhat variable, and multiple complex factors such as solvation energies or the local rearrangement of water may also play a role. Stereoelectronic effects of the presence of the ring hydroxyls might also change the overall properties of the various agonists and thereby further alter the interactions.
5 The 24-fold lower affinity of S203A for pindolol may be partially responsible for the lack of binding seen in the original study of this mutant with radiolabeled iodocyanopindolol (4).
| |
REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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L. Shi, G. Liapakis, R. Xu, F. Guarnieri, J. A. Ballesteros, and J. A. Javitch beta 2 Adrenergic Receptor Activation. MODULATION OF THE PROLINE KINK IN TRANSMEMBRANE 6 BY A ROTAMER TOGGLE SWITCH J. Biol. Chem., October 18, 2002; 277(43): 40989 - 40996. [Abstract] [Full Text] [PDF]
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M. L. Lopez-Rodriguez, B. Vicente, X. Deupi, S. Barrondo, M. Olivella, M. J. Morcillo, B. Behamu, J. A. Ballesteros, J. Salles, and L. Pardo Design, Synthesis and Pharmacological Evaluation of 5-Hydroxytryptamine1a Receptor Ligands to Explore the Three-Dimensional Structure of the Receptor Mol. Pharmacol., July 1, 2002; 62(1): 15 - 21. [Abstract] [Full Text] [PDF]
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J. R. Bunzow, M. S. Sonders, S. Arttamangkul, L. M. Harrison, G. Zhang, D. I. Quigley, T. Darland, K. L. Suchland, S. Pasumamula, J. L. Kennedy, et al. Amphetamine, 3,4-Methylenedioxymethamphetamine, Lysergic Acid Diethylamide, and Metabolites of the Catecholamine Neurotransmitters Are Agonists of a Rat Trace Amine Receptor Mol. Pharmacol., December 1, 2001; 60(6): 1181 - 1188. [Abstract] [Full Text] [PDF]
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J. A. Ballesteros, L. Shi, and J. A. Javitch Structural Mimicry in G Protein-Coupled Receptors: Implications of the High-Resolution Structure of Rhodopsin for Structure-Function Analysis of Rhodopsin-Like Receptors Mol. Pharmacol., July 1, 2001; 60(1): 1 - 19. [Abstract] [Full Text]
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