From the Department of Molecular Cardiology NB5, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
Received for publication, April 9, 2001, and in revised form, April 27, 2001
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ABSTRACT |
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Although agonist binding in adrenergic receptors
is fairly well understood and involves residues located in
transmembrane domains 3 through 6, there are few residues
reported that are involved in antagonist binding. In fact, a major
docking site for antagonists has never been reported in any G-protein
coupled receptor. It has been speculated that antagonist binding is
quite diverse depending upon the chemical structure of the
antagonist, which can be quite different from agonists. We now report
the identification of two phenylalanine residues in transmembrane domain 7 of the Adrenergic receptors
(ARs)1 ( However, our knowledge of how antagonists bind to the adrenergic
receptor family is limited. Mutagenesis studies in our laboratory have
identified that the subtype selectivity of two Materials--
Drugs were obtained from the following
manufacturers: (a) alprenolol, ( Site-directed Mutagenesis in the
Cell Culture and Transfection--
COS-1 cells (American Type
Culture Collection, Manassas, VA) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 1%
(v/v) penicillin, and streptomycin. Cells were maintained and passaged
upon reaching confluence by standard cell culture techniques.
Experiments were conducted on cells between passages 10 and 25. Cells
were transiently transfected with either wild type or mutated
Membrane Preparation--
Membranes were prepared as described
previously (6). Briefly, transfected COS-1 cells were scraped 72 h
after transfection, collected, washed in Hank's balanced salt
solution, and then pelleted under low-speed centrifugation. The cell
pellet was resuspended in a 0.25 M sucrose solution and
centrifuged. The pellet was resuspended in water containing a mixture
of protease inhibitors and frozen at Measurement of Ligand Binding Affinities--
Saturation binding
experiments to measure the affinity of 125I-HEAT at
Data Analysis--
Competition binding data were analyzed using
the nonlinear regression functions of the noniterative curve fitting
program GraphPad Prism. Binding affinities (Ki) were
determined by transformation of the program-calculated IC50
value using the Cheng-Prusoff equation. The binding data for each
ligand were modeled to one- or two-site binding. The most suitable
model was determined by performing an F test comparison of the least
sum of squares fit of the data to these equations. Statistically
significant differences in the affinities of agonists and antagonists
were determined by t test analysis.
Molecular Modeling--
Models depicting the binding
interactions of antagonists to the Fig. 1 shows the comparison of
residues located in TM7 for the 1a-adrenergic receptor
(Phe-312 and Phe-308) that are a major site of antagonist affinity.
Mutation of either Phe-308 or Phe-312 resulted in significant losses of
affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378,
(+) niguldipine, and 5-methylurapidil, with no changes in affinity for
phenethylamine-type agonists such as epinephrine, methoxamine, or
phenylephrine. Interestingly, both residues are involved in the binding
of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and
may be more antagonist-like, which may explain their partial agonist
properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the
pocket closer to the extracellular surface than agonist binding
and appears skewed toward transmembrane domain 7.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1a,
1b,
1d,
2a,
2b,
2c,
1,
2, and
3) are members of the G-protein
coupled receptor superfamily of membrane proteins that mediate the
actions of the endogenous catecholamines, the neurotransmitter
norepinephrine, and the hormone epinephrine. Similar to rhodopsin,
these proteins are proposed to traverse the plasma membrane in a series
of seven transmembrane-spanning
-helical domains linked by three
intracellular and three extracellular loops (1). In accordance with the
observation that the greatest structural conservation is
localized to the transmembrane helical domains of the receptor, the
catecholamine binding pocket is also localized to these regions.
Mutagenesis studies in our laboratory and others have identified that
the endogenous agonist in biogenic amine receptors is stabilized in the
binding pocket by ionic, hydrogen bond, and aromatic/hydrophobic
interactions involving residues on TM3, TM5, and TM6, although there
are various modulations of these interactions between the families
(2-5). We have also recently shown that
1-ARs and
perhaps some other biogenic amine receptors have additional
aromatic/hydrophobic interactions with the endogenous agonist to
residues in TM4 and TM5 (6).
1a-AR
antagonists, phentolamine and WB4101, is conferred by interactions with
three consecutive residues of the second extracellular loop (7). Similar results were also obtained in the 5-HT1D
receptor (8) and the opioid receptor (9). This observation indicates
that in contrast to agonist binding, which is localized to the interior core of the receptor, antagonists interact with residues closer to the
extracellular surface of adrenergic receptors, above the plane of the
agonist binding pocket. Further point contacts between
1-antagonists and extracellular residues of this
receptor could not be identified using a series of
1a-/
2-AR chimeras. Therefore, the
high-affinity binding of these drugs to the receptor must involve
additional interactions with residues within the transmembrane domains
of the receptor (10). Other previous mutagenesis studies have indicated
the importance of phenylalanine residues located close to the
extracellular surface of the receptor in antagonist binding at
adrenergic receptors. A single phenylalanine residue about two turns
into the TM7 domain of the
2-AR (Phe-412) was shown to
promote high-affinity binding of yohimbine (11). Mutagenesis of a
phenylalanine residue (Phe-86) at the surface of TM2 in the
1a-AR accounts for the
1a
versus
1d selectivity of dihydropyridine antagonists such as niguldipine (12). Furthermore, a phenylalanine residue (Phe-310 of the
1b-AR) in TM6 has been
identified as being important not only for agonist binding but also for
the binding of certain
1-antagonists (13). In light of
these studies and given that the
1-antagonists contain
high aromatic/hydrophobic character, we continued our investigation
into the role of aromaticity/hydrophobicity in antagonist binding at
1-ARs. We now report the importance of two conserved
phenylalanine residues near the extracellular surface of TM7 involved
in nonselective binding for several
1-antagonists. Our
studies suggest that
1-antagonists bind the receptor in
an elevated pocket from agonist binding that is also skewed toward TM7.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) epinephrine,
oxymetazoline, phenylephrine, methoxamine, phentolamine, prazosin,
clonidine, yohimbine, alprenolol, and propranolol, Sigma;
(b) 125I-HEAT, PerkinElmer Life Sciences; and
(c) 5'-methylurapidil, BMY7378, (+) niguldipine, cirazoline,
and WB4101, Research Biochemicals Inc. (Natick, MA). Chemicals for
buffered solutions (HEPES (free acid), EGTA, and MgCl2)
were obtained from Mallinckrodt Baker (Phillipsburg, NJ).
1a-AR--
Site-directed mutagenesis of the
pMT2' rat
1a-AR plasmid (14) was performed
using polymerase chain reaction technology and commercially synthesized
oligonucleotides (Life Technologies, Inc.) specifically designed to
code for the desired mutation. A fragment of cDNA encoding either
the F312A or F312N mutation of the rat
1a-AR was
generated using sense primers containing the unique EcoRI
cloning site before the start site of translation and an antisense
primer targeted to the unique AhaIII site of the rat
1a-AR that also encoded the mutation. The F308A or F308L mutations were generated using a sense primer containing the mutation targeted to the endogenous MseI restriction site and an
antisense primer containing the NotI restriction site after
the stop codon. Using the Expand High Fidelity polymerase chain
reaction protocol (Roche Molecular Biochemicals), these specific
fragments of the rat
1a-AR were generated using 1 µg
of pMT2' rat
1a-AR plasmid, 300 nM sense and antisense primers, 200 nM each
deoxynucleotide triphosphate, and 2.6 units of Taq and Pwo
DNA polymerase in a 20 mM Tris-HCl, pH 7.5, buffer
containing 100 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 0.05% (w/v) Tween 20, and a final
MgCl2 concentration of 1.5 mM. All polymerase
chain reactions were conducted in 10% (v/v) glycerol. The
amplification reactions, which were repeated for 40 cycles, consisted
of denaturation at 95 °C for 3 min and an annealing and elongation
phase at 72 °C for an additional 3 min. The polymerase chain
reaction-generated fragments resulting from each of the reactions were
isolated and purified, followed by either
EcoRI/AhaIII (Phe-312 mutations) or
MaeI/NotI (Phe-308 mutations) restriction enzyme
digestion. The polymerase chain reaction products were ligated
with their respective WT fragment and subcloned into the pMT2'
expression vector to yield the full-length rat
1a-AR
construct containing single mutations. Mutations were confirmed by
full-length sequence analysis of the construct by the dideoxy method
(Cleveland Clinic Sequencing Core Facility).
1a-AR subcloned into eukaryotic expression plasmid pMT2'
using the DEAE-dextran method as described previously (15).
70 °C for 30 min. Pellets
were dounced from a B glass Dounce homogenizer. Nuclear debris was
removed by a low-speed centrifugation step. Membranes in the
supernatant were washed with HEM buffer and pelleted by high-speed
centrifugation. The membrane pellet was washed twice in HEM buffer, and
the final pellet was reconstituted in HEM buffer containing 10% (v/v)
glycerol and stored at
70 °C until use. The protein concentration
was determined by a Bradford assay, using bovine serum albumin as the
known standard.
1a-ARs were performed as described previously (6). The
binding affinities of various adrenergic receptor agonists and
antagonists were determined in a series of competition binding experiments performed as described previously (6). All binding assays
were performed in duplicate in HEM buffer, in a total assay volume of
250 µl. Nonspecific binding was defined as the amount of
radioactivity that remained bound to the filters in the presence of
10
4 M phentolamine.
1a-AR were generated
as described previously using Insight II molecular modeling software
from Biosym Technologies (4, 7). The coordinates of the
-carbon
positions were determined by an overlay of the
1a-AR
residues with the TM coordinates of rhodopsin (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-ARs as well as all
other adrenergic receptors. The biogenic amine family has a consensus
domain of WXGXNSXXNPXXY
that is thought to be involved in signal transduction and sequestration
but not directly involved in binding per se. (17).
Therefore, we focused our attention on aromatic residues located closer
to the extracellular border. TM7 has two aromatic residues; both are
absolutely conserved in both the
1- and
2-AR subtypes, but not in the
-AR subtypes. These two
residues, Phe-308 and Phe-312 in the
1a-AR subtype, are
predicted to be near the extracellular surface of the receptor (Fig.
2), with Phe-308 located in the first
helical turn, and Phe-312 located in the second turn on TM7. The
analogous residue to Phe-312 in the
2-AR is also thought
to confer
2-AR versus
-AR selectivity for
yohimbine (11). To study the importance of these residues in
1-AR binding, we mutated Phe-308 to either Ala or Leu
(to examine hydrophobicity versus aromaticity,
respectively), and we mutated Phe-312 to either Asn or Ala (to examine
-AR to
-AR selectivity and
1-AR hydrophobicity,
respectively).
View larger version (18K):
[in a new window]
Fig. 1.
Sequence alignment of TM7 among cloned
adrenergic family members. Residues mutated in the rat
1a (Phe-308 and Phe-312) are shown in bold
along with residues conserved in the corresponding position in other
adrenergic receptors. The numbering is in reference to the rat
1a sequence (14). Amino acids are represented by the
one-letter code.
View larger version (15K):
[in a new window]
Fig. 2.
Secondary structure model of the TM7 domain
of the rat 1a-AR. Mutated
residues (Phe-308 and Phe-312) are shown in gray. Phe-308 is
predicted to be in the first helical turn of TM7, whereas Phe-312 is in
the second helical turn. Amino acids are represented by the one-letter
code.
Binding Phenotype at Phe-308 Mutations--
Binding results for a
series of 1-AR,
2-AR, and
-AR
antagonists are presented in Table I. All
binding curves resulted in one-site fits. In saturation binding
studies, the radiolabeled antagonist 125I-HEAT bound to
each of the mutated receptors with comparable affinity and density as
determined for the wild type
1a-AR. To determine changes
in
1-antagonist affinity, a number of ligands were
tested at the Phe-308 mutants. There were no changes in affinity at
either mutant for phentolamine, prazosin, or 5-methylurapidil. However,
WB4101 (10-25-fold), BMY7378 (3-4-fold), and (+) niguldipine (8-30-fold) had lower affinity at either substitution. We then tested
both
2-AR and
-AR antagonists to address
cross-family selectivity issues. All ligands tested but
alprenolol showed no changes from WT
1a-AR
affinity; alprenolol had a 36-fold increased affinity, but only at
the F308L mutation.
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In agonist binding at the Phe-308 mutations (Table II), all phenethylamine-type agonists such as epinephrine, phenylephrine, and methoxamine displayed no changes in affinity from the WT receptor. However, imidazoline-type agonists such as oxymetazoline and clonidine displayed a 13-fold and a 4-fold decrease in affinity from the WT, respectively.
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Binding Phenotype at Phe-312 Mutations--
Binding results for a
series of 1-AR,
2-AR, and
-AR
antagonists are presented in Table I. In saturation binding studies, results of Phe-312 mutations were similar to those of the Phe-308 mutations. The radiolabeled antagonist 125I-HEAT bound to
each of the mutated receptors with wild type
1a-AR affinity. Receptor density of the Phe-312 substitutions was also similar to that of the WT and the Phe-308 mutations.
1-Antagonist affinities were decreased for prazosin
(85-fold), 5-methylurapidil (21-fold), WB4101 (5-fold), and (+)
niguldipine (1000-fold). These changes were all at the F312A mutation,
with only niguldipine showing additional changes at the F312N mutation.
Affinities for the
2-AR and
-AR antagonists were also
unchanged from the WT, as seen for the Phe-308 mutations.
In agonist binding at the Phe-312 mutations (Table II), the results
were similar to those of the Phe-308 mutations in that phenethylamine-type agonists such as epinephrine, phenylephrine, and methoxamine were unchanged from WT. Again, imidazoline-type agonists showed decreased affinity with oxymetazoline (150-fold), cirazoline (8-fold), and clonidine (25-fold).
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DISCUSSION |
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Aromaticity/hydrophobicity has been shown to be important in
the binding of agonists and antagonists to the various subtypes of
adrenergic receptors. Recently, we have identified two novel aromatic
interactions promoting agonist binding that involve two phenylalanine
residues located at the extracellular surface of TM4 and TM5 of the
1a-AR (6). These residues were modeled to form contacts
from above the plane of the agonist pocket. Until recently,
determination of residues involved in antagonist binding at
1-ARs was limited to two domains. One is located on the
second extracellular loop of the receptor. These consecutive residues (Gln-177, Ile-178, and Asn-179) are involved only in
1a-AR versus
1b-AR selectivity
issues and then for only three ligands, phentolamine, WB4101, and
partial effects on 5-methylurapidil (7). Another group has shown that
mutagenesis of a phenylalanine residue (F86M) at the surface of TM2 in
the
1a-AR accounts for the
1a
versus
1d selectivity of dihydropyridine
antagonists such as niguldipine (12). This aromatic residue is also
modeled to be in the first helical turn of TM2. Nevertheless, both
these previous interactions involve selectivity issues and separate
domains of the receptor and involved only certain antagonists. A major
antagonist docking site has never been reported in any adrenergic
receptor family member, let alone any G protein-coupled receptor; thus,
it has been thought that these ligands are very diverse in their
binding parameters because of the diversity of their chemical structures.
Aromaticity/hydrophobicity should be more important in antagonist
binding than in agonist binding, given the higher degree of these
contacts contained in adrenergic antagonists and their decreased
aqueous solubility. Agonists typically have one aromatic ring and
hydrophilic substitutents, whereas 1-AR antagonists are
larger molecules with multiple aromatic/hydrophobic ring components. However, little is known of specific residues involved in antagonist binding, even among other adrenergic receptor family members. Chimeras
between the
1- and
2-AR failed to reveal
discrete residues (18, 19). However, a single residue in TM7
(either Phe-412 in the
2 or Asn-385 in the
5-HT1a) seemed responsible for certain
-AR antagonist
selectivity (11, 20). Therefore, to explore additional residues
involved in antagonist binding, we postulated that in addition to the
antagonist pocket being closer to the extracellular surface than
agonists, the pocket must also be skewed but sufficient in overlap to
maintain competitive interactions. Because we and others have found
agonist interactions in TM3 through TM6 for the
1-AR
(4-6, 13, 21) and an antagonist-binding residue in TM7 in
2-/
2-ARs (11) and in TM6 for a few
1-antagonists (6), the antagonist pocket must also be
skewed toward TM6 and TM7. We therefore focused our attention on TM7 of
the
1-AR for mutagenesis studies.
As shown in the sequence alignment (Fig. 1), the phenylalanine residue
at position 308 of the 1a-AR is conserved across all the
cloned adrenergic receptor subtypes except the
2-AR,
where the aromaticity is conserved as a tyrosine residue. Two mutations of this residue were made: (a) a F308A mutation in which the
side chain packing and the potential for Van Der Waal's interactions were minimized, and (b) a F308L mutation in which the
aromaticity of the side chain was removed, but hydrophobicity was
maintained. The phenylalanine at position 312 was mutated in
expectation of cross-family selectivity issues such as those found in
the
2-AR,
2-AR, and 5-HT1a
receptors (11, 20). Therefore, to reproduce this same system, we
mutated Phe-312 to Asn, the same corresponding residue found in the
-ARs and the 5-HT1A. This mutation would also test
hydrophobicity. We also made the F312A substitution to explore
potential packing interactions.
Mutation of either Phe-308 or Phe-312 did not change the affinity of
125I-HEAT, the radiolabeled antagonist (Table I). This
supports the position that the overall global conformation of the
receptor was maintained, and the changes in affinity we do see with
other ligands are specific. Likewise, there are no significant
differences in receptor expression as measured by changes in
Bmax. Interestingly, there are no published
reports of any residue that specifically changes the affinity of
125I-HEAT, which leads to speculation regarding how this
antagonist actually binds in the pocket to accommodate this larger
iodinated species. Inclusive of this report, all
1-antagonists except for 125I-HEAT had at
least one point binding contact identified in the receptor.
Nevertheless, because mutation of either 308 or 312 does produce
specific affinity changes, but only in antagonists and imidazolines, it
is reasonable that the mutations are involved in direct contacts and
not in global or nonspecific changes in receptor structure.
Neither residue changed the affinity of the endogenous agonist,
epinephrine, or of other phenethylamine-type agonists such as
phenylephrine or methoxamine, suggesting that the changes in affinity
were antagonist-specific (Table II). In 1-antagonist binding, either Phe-308 or Phe-312 substitutions or sometimes both
residues displayed lower binding affinities for all
1-AR antagonists tested; the only exception was phentolamine (Table I). The
degree of change varied (3-1200-fold), but this is likely due to the
variances in structure. Both prazosin (85-fold) and niguldipine
(1200-fold) had the greatest decrease in affinity, which coincidently
correlates to these ligands having the highest binding affinities for
1-ARs (subnanomolar). It is likely that Phe-308 is
involved in mostly aromatic binding because both the Ala and Leu
substitutions resulted in roughly equal losses in affinity. In
contrast, it appears that Phe-312 is most likely involved in packing
interactions because most of the changes in affinity were at the Ala
substitution and not at the Asn substitution.
In modeling these interactions and using the current coordinates of
rhodopsin (16), the binding orientation of WB4101 is illustrated in
Fig. 3A. This model
incorporates the residues involved in subtype-selective affinity
changes in WB4101 (30-fold) that we described previously that are
located in the second extracellular loop (7). According to the
structure of rhodopsin, the second extracellular loop folds down into
the binding pocket of retinal and likewise in our model folds down
sufficiently to allow interactions with WB4101. The portion of the loop
near TM5 was previously found to contain charged interactions with
WB4101, and thus these moities are orientated toward the charged
portions of the antagonist. The extracellular loop interactions still
allow aromatic interactions with Phe-308 and Phe-312 that are likely
due to the phenyl ring. This docking of WB4101 is consistent
with its placement above the plane of the agonist pocket (note the
orientation of Ser-188 and Ser-192, which are involved in
phenethylamine agonist binding, (4)). Interaction of Asp-106 and the
amine group of the antagonist is still possible but would be a weak
interaction due to a greater separation than agonist binding. However,
this result is consistent with previous mutagenesis of Asp-125 in the
1b-AR, in which antagonist binding was not affected as
greatly as agonist binding (5). Modeling of the other antagonists
produced similar results, with one end of the molecule interacting with
Phe-308 and Phe-312, whereas the other end was orientated more toward
TM6 than TM4-5.
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The model is also consistent with the reason why phentolamine did not change affinity with either TM7 mutation. All of the ligands that changed affinity in this report have long and extended structures, whereas phentolamine is more compact and is likely confined to TM5-6 and the loop regions and not extended to TM7. Consistent with this interpretation, phentolamine displayed an 8-fold loss of affinity at the second extracellular loop mutations (7) and a 10-fold loss at the Phe-310 mutation in TM6 (13) but did not do so with either TM7 mutation.
Interestingly, we found that all imidazoline-type agonists also
decreased affinity with Phe-308 and Phe-312 substitutions. The affinity
of oxymetazoline, a weak partial agonist, was decreased at F308L
(13-fold) and F312A (150-fold). Decreases in affinity were also found
for other imidazolines such as cirazoline (8-fold) and clonidine
(25-fold). Imidazolines have been proposed to bind differently in the
agonist pocket than phenethylamine agonists such as epinephrine because
many mutants that affect phenylethylamine binding do not change
imidazoline binding (6, 22). In fact, in earlier studies, the
Eason-Stedman hypothesis predicted that imidazolines bind differently
than phenethylamines (23). This is also supported by signaling studies
in which imidazolines lack cross-desensitization with phenethylamine
agonists, suggesting different activation and signaling parameters
(24). Because the imidazoline ring has protonated nitrogens that are
involved in binding to Asp-106 in TM3 and is required for agonism (5), modeling of this ligand (Fig. 3B) requires that the
imidazoline ring must be pointed toward TM3, but the second aromatic
ring analogous to epinephrine must then orient toward TM7. This is in
sharp contrast to epinephrine binding, in which the aromatic ring is
orientated toward TM5 (4). Therefore, the binding of imidazolines is
also skewed toward TM7 and located higher in the pocket, much like
antagonist binding. Mutation of Phe-412 to Asn (analogous to Phe-312 in
the 1a-AR) in TM7 of the
2-AR also resulted in lower binding affinity for p-aminoclonidine,
another imidazoline agonist (11), suggesting perhaps a conserved
binding paradigm for imidazolines in both
1- and
2-ARs. However, our current results suggest that they
also have some antagonist-like binding character due to their
involvement of TM7, which may orient imidazolines differently than
phenethylamines and account for their generally poor agonism at
1-ARs (25).
Interestingly, mutation of Phe-312 to Asn in the 1a-AR
in mimicry of the
2-/
2-AR or
5-HT1a studies (11, 20) did not display any changes in
affinity for
-AR antagonists (alprenolol and propranolol) or the
2-AR antagonist (yohimbine) (Table I). Although Asn is
believed to be involved in a high-affinity point contact for
propranolol and alprenolol and other aryloxyalkylamine
-AR
antagonists (20) and is possibly discriminatory between
2- and
-AR antagonists (11) due to losses in
yohimbine binding, it does not appear that the corresponding residue
(Phe-312) in the
1-AR is involved in analogous
paradigms. However, F308L in the
1a-AR displayed
altered non-
1-AR binding only for alprenolol (36-fold
higher affinity) and could not reproduce it with propranolol. It is
possible for the propyl group of alprenolol to pack with Phe-308, and
perhaps the extended structure of the leucine substitution enhances
this interaction. Although it may be argued that propranolol contains
an extra aromatic ring and may pack differently in the pocket with
Phe-308, these results utilizing
- and
2-antagonists merely emphasize that there are significant differences in the binding
pockets among adrenergic family members, although they may conserve key residues.
In conclusion, we have demonstrated that two Phe residues in TM7 are
involved in high-affinity binding for a large number of
1-antagonists. These interactions are not involved in
subtype selectivity differences because both Phe residues are strictly conserved among all three
1-AR subtypes but represent a
major docking site for
1-AR antagonists. Imidazoline
agonists, which were recognized previously to bind differently than
epinephrine-like agonists, also bind to these two Phe residues in TM7,
suggesting that imidazolines bind with some antagonist characteristics.
This antagonist-like binding may explain their generally poorer agonism at
1-ARs. A consensus of the results of numerous
mutagenesis studies suggests that the agonist pocket lies deeper in the
hydrophobic core of the receptor and involves TMs 3 through 6, whereas
antagonist binding is located above the plane of agonist binding but is
skewed toward TM7. This study represents the first report of a major docking site for
1-AR antagonists and for a G
protein-coupled receptor in general. These studies suggest that whereas
antagonists generally have diverse structures, there are conserved
pharmacophores that recognize a common site on the receptor. These
studies may be helpful in the synthesis of selective ligands for these
receptors and may represent conserved paradigms for antagonist binding
in other adrenergic receptors or other nonpeptide G protein-coupled receptors.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1HL61438 (to D. M. P.), an American Heart Established Investigator Award (to D. M. P.), and an unrestricted grant from Glaxo Wellcome, Inc. (to D. M. P.).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.
Recipient of fellowship support from the American Heart
Association, Ohio Valley Affiliate.
§ Present address: Dept. of Oncology, Queens University, Belfast, Northern Ireland BT9 7AB.
¶ To whom correspondence should be addressed: Dept. of Molecular Cardiology, NB5, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-2058; Fax: 216-444-9263; E-mail: perezd@ccf.org.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M103152200
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ABBREVIATIONS |
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The abbreviations used are:
AR, adrenergic
receptor;
TM, transmembrane domain;
125I-HEAT, 2-[-(4-hydroxy-3-[125I]iodophenyl)ethylaminomethyl]tetralone;
WT, wild type;
HEM, 20 mM HEPES, pH 7.4, 1.4 mM
EGTA, and 12.5 mM MgCl2;
HT, hydroxy
trypamine.
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REFERENCES |
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Strader, C. D.,
Candelore, M. R.,
Hill, W. S.,
Sigal, I. S.,
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