From the Molecular Cardiology Unit, Victor Chang
Cardiac Research Institute, St. Vincent's Hospital, Sydney 2010, Australia and the § School of Biochemistry and Molecular
Genetics, University of New South Wales,
Kensington, New South Wales 2033, Australia
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ABSTRACT |
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Pharmacophore mapping of adrenergic receptors
indicates that the phenyl ring of catecholamine agonists is involved in
receptor binding and activation. Here we evaluated
Phe310, Phe311, and Phe303 in
transmembrane VI (TMVI), as well as Tyr348 in TMVII of the
Binding of catecholamines by both For the This altered catechol ring orientation may also contribute to other
agonist docking differences between Previous studies of the hamster 1B-adrenergic receptor (
1B-AR), which
have been implicated in a catechol-ring interaction. Neither
catecholamine docking studies nor mutagenesis studies of
Phe311, Phe303, or Tyr348 supported
a role for these residues in catechol-ring binding. By contrast,
docking studies indicated that the Phe310 side chain is
well positioned to interact with the catechol-ring, and substituted
cysteine accessibility method studies revealed that the side chain of
the 310, but not 311 residue, is both solvent accessible and directed
into the agonist-binding pocket. Also, saturation mutagenesis of both
Phe310 and Phe311 revealed for the former, but
not for the latter, a direct relationship between side chain volume and
agonist affinity, and that aromaticity is essential for wild-type
agonist binding, and for both wild-type agonist potency and efficacy.
Moreover, studies of Phe310 mutants combined with a
previously described constitutively active
1B-AR mutant,
A293E, indicated that although not required for spontaneous receptor
isomerization from the basal state, R, to a partially
activated conformation R', interaction of
Phe310 with catecholamine agonists is essential for
isomerization from R' to the fully activated state,
R*.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-Adrenergic receptors
(
1-AR)1 are
members of the heptahelical superfamily that share a common structural
motif of seven putative
-helical transmembrane spanning regions
linked by three extra- and three intracellular loops, an extracellular
N terminus and intracellular C-terminal tail. Transmembrane signaling
by all
1-AR subtypes (
1A,
1B, and
1D) in response to the natural catecholamine agonists, norepinephrine and epinephrine, is mediated by
G-proteins of the Gq/11 family or in some
instances, the Gh family of tissue transglutaminases (1, 2). Based on certain key structural features,
1-ARs are more
closely related to rhodopsin or the type A subfamily of GPCRs that
includes
-ARs, than to the calcitonin (type B) or metabotropic (type
C) subfamilies.
1- and
-ARs
involves the formation of a salt bridge between the basic aliphatic
nitrogen atom common to all sympathomimetic amines and an aspartate
(Asp125 in the hamster
1B-AR;
Asp113 in the hamster
2-AR) in the third
transmembrane segment (TMIII) (3, 4). With rhodopsin, light induced
isomerization of the retinal chromophore leads to deprotonation of a
Schiff base linking the chromophore to Lys296 in TMVII (5).
In the ground state the protonated Schiff base is ionically bonded to a
TMIII acidic residue (Glu113) that is equivalent to
Asp125 and Asp113 in the
1B and
2-ARs, respectively (5). With the
1B-AR
there is evidence that receptor activation also is due to disruption of
an ionic interaction between the TMIII aspartate and a TMVII lysine
(Lys331), due to competition between the catechol
protonated amine and the TMVII lysine, for binding to the TMIII
aspartate (3). The TMIII aspartate thus serves as a counterion and most
likely is an important residue for agonist binding and activation of
all adrenergic receptors.
2-AR, two serine residues, Ser204
and Ser207, which are conserved in most adrenergic
receptors, have been proposed to hydrogen bond with the
meta- and para-hydroxyls, respectively, of the
catechol ring (6). Mutation of either serine to an alanine results in a
30-fold decrease in affinity for catecholamine agonists, and each
serine contributes about 50% to receptor activation. Thus, binding of
both catechol hydroxyls is required for full agonist activity. By
contrast, agonist binding to the
1A-AR involves an
interaction between the meta-hydroxyl and Ser188
(equivalent to Ser203, not Ser204 in the
2-AR) that plays a major role in receptor activation, being responsible for 70-90% of the wild-type response. An
interaction between the para-hydroxyl and Ser192
(equivalent to Ser207 in the
2-AR), on the
other hand, contributes minimally to receptor activation (7). Moreover,
since the interacting serines in the
1A-AR are separated
by four residues, whereas those in the
2-AR are
separated by only three residues, docking of the catecholamine ring
is in a more planar orientation in the
1A-AR, and is
rotated by about 120° to that in the
2-AR.
1- and
-ARs. For example, stereoselectivity of binding and activation has been attributed, in part, to a hydrogen bond interaction between the chiral
benzylic hydroxyl group attached to the
-carbon atom of catecholamines, and Asn293 in TMVI of the
2-AR (8). Although stereoselectivity of catecholamine binding and activation is preserved with
1-ARs, the
determinants of stereoselectivity have not been defined, and the
Asn293 equivalent is replaced by a residue (leucine or
methionine) lacking hydrogen-bonding potential. This finding again
provides evidence that some of the catecholamine-binding and activation
residues in
1-ARs are distinct from those in
-ARs.
2-AR suggested that a
phenylalanine in TMVI (Phe290, equivalent to
Phe311 in the
1B-AR, see Fig.
1), which is conserved only in biogenic amine-binding GPCRs, is involved in forming an aromatic-aromatic interaction with the phenyl ring of catecholamines (9). This conclusion
was based on the finding of a 10-fold decrease in agonist, but not
antagonist, binding with mutation of Phe290 to methionine.
However, no additional studies were performed to exclude a nonspecific
global or local change in receptor structure with this
Phe290 mutation, or to evaluate the role of the potential
aromatic-aromatic interaction in receptor activation. In addition,
substitution of an adjacent TMVI phenylalanine (Phe289;
equivalent to Phe310 in the
1B-AR) to
alanine, resulted in a 1000-fold decrease in agonist affinity with no
change in antagonist binding. Finally, Tyr326 in TMVII was
also suggested to potentially be involved in a catechol ring
interaction, since substitution of this residue with leucine decreased
agonist, but not antagonist binding by 10-fold (9).
View larger version (39K):
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Fig. 1.
Sequence alignment of TM VI residues of
hamster 1B and other G
protein-coupled receptors. Sequences were aligned to maximize
homology within this region using the GCG program
"Pileup." The conserved phenylalanines corresponding to
Phe310 and Phe311 in the hamster
1B-AR are shaded and in bold type,
while Phe303, which is highly conserved among all G
protein-coupled receptors is boxed. The dashed
line at the top delineates the transmembrane residues
of helix VI.
Here we show, based on macromolecular modeling studies, in which the
planar orientation of the phenyl ring in 1-ARs was taken into consideration when docking catecholamines, that interaction with
the phenyl ring involves Phe310 in TMVI and not
Phe311 in TMVI, or Tyr348 in TMVII (equivalent
to Tyr326 in the
2-AR). Furthermore, based
on mutagenesis studies coupled with the evaluation of group-specific
catecholamine analogs, as well as SCAM (substituted cysteine
accessibility method) studies, we provide evidence that
Phe310 is critically involved both in forming an
aromatic-aromatic interaction with the catecholamine phenyl ring and in
receptor activation.
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EXPERIMENTAL PROCEDURES |
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Materials--
()-Epinephrine, (
)-norepinephrine,
(±)-synephrine, (±)-halostachine, dopamine, phenethylamine, prazosin,
phentolamine hydrochloride, lithium chloride, and dl-propranolol were
purchased from Sigma. 5-Methylurapidil was from Research Biochemicals
International. AG 1-X8 (100-200 mesh, formate form) was from Bio-Rad,
and fetal calf serum and culture media from Trace Biosciences,
Australia. myo-[3H]Inositol (80 Ci/mmol) was
from Amersham. [125I]HEAT (2200 Ci/mmol) was from Du
Pont. Methanethiosulfonate ethylammonium (MTSEA) was from Toronto
Research Chemicals Inc. Other chemicals were of the highest grade
available commercially.
Site-directed Mutagenesis--
The construct used was the
hamster 1B-AR cDNA with an octapeptide tag (1D4) at
the end of coding region in the modified eukaryotic expressing vector,
pMT2' (10). The presence of the 1D4 epitope at the C-terminal of
1B-AR does not affect its expression and function (data
not shown). Site-directed mutagenesis was performed as described
previously (2, 11). Briefly, two primers, one carrying the nucleotide
change(s) to produce the desired amino acid mutation in the
1B-AR sequence, the other carrying the nucleotide changes to convert a unique restriction site (ClaI) to
another restriction site (NarI) in a non-essential region of
the vector, pMT2', were simultaneously annealed to the denatured
template and a new second strand DNA containing both primers was
synthesized by treatment with T4 polymerase. The DNA was then digested
with ClaI to linearize re-annealed parental plasmid and the
reaction mixture used to transform Escherichia coli cells
(BMH 71-18 mutS cells). Transformants were grown en
mass in liquid media and used to isolate plasmid DNA. The
resulting DNA was digested with ClaI again to linearize
remaining parental plasmid, and transformed into DH5
-cells.
Transformants were plated and plasmid DNA prepared and sequenced to
confirm the presence of the desired mutation.
Cell Culture and Transfection--
COS-1 cells (American Type
Culture Collection, Manassas, VA) were cultured and transiently
transfected with the indicated constructs using the DEAE-dextran
method, as described previously (2, 12, 13). The transfection
efficiency was 30-40%, as determined by in situ staining
of cells transfected with pSVLacZ, a plasmid encoding the reporter,
-galactosidase, and treatment of the cells with 0.2%
5-bromo-4-chloro-3-indoyl-
-D-galactoside. Cells were
harvested 72 h post-transfection.
Membrane Preparation--
Membranes were prepared from
transfected COS-1 cells, as described previously (2, 10). The membranes
were resuspended in HEM buffer (20 mM HEPES, pH 7.5, 1.5 mM EGTA, 12.5 mM MgCl2) containing
10% (v/v) glycerol, and stored at 70 °C. Protein concentration was determined by the Bradford method (14).
Western Blotting--
Membranes (50 µg of protein) were
dissolved in 1% CHAPS and SDS sample buffer overnight at 4 °C, and
then subjected to SDS-polyacrylamide gel electrophoresis, as described
previously (2, 12). The resolved proteins were electroblotted onto
Immobilon-P membranes and then immunostained for detection using the
ECL chemiluminescence system (Amersham), as described previously (2).
1B-AR was detected using a monoclonal antibody against
the 1D4 epitope (12).
Ligand Binding--
The ligand binding characteristics of the
membrane expressed receptors were determined in a series of radioligand
binding studies performed exactly as described previously (2, 10), using [125I]HEAT, an 1-specific
antagonist, as the radioligand. For saturation binding studies, the
concentrations of [125I]HEAT used ranged from 10-fold
below to 10-fold above the Kd value, whereas for
competition studies, a concentration near the Kd of
the radioligand value was used. Ki values were
determined using the Cheng-Prusoff equation (15). The membrane concentration used in these studies was selected to allow binding of
less than 10% of the total radioligand added. To avoid interassay differences replicate studies with the wild-type
1B-AR
were performed with the analysis of each mutant. Binding data were
analyzed using the iterative, nonlinear, curve-fitting program, Prism.
For comparison of the fit to a one-site or two-site model, the F test
was used, p < 0.05 was considered to be statistically significant.
Reaction with MTSEA and Binding Assays in Intact Cells-- Transfected COS-1 cells were harvested by trypsinization. After washing with phosphate-buffered saline, cells were resuspended in 1.4 ml of HEPES buffer (140 mM NaCl, 5.4 mM KCl, 1 mM EDTA, 0.006% bovine serum albumin, 25 mM HEPES, pH 7.4) as described previously (16). Aliquots (80 µl) of the cell suspension were incubated with 20 µl of freshly prepared MTSEA at the stated concentrations at room temperature for 2 min. Cell suspensions were then diluted 20-fold, and 100-µl aliquots were used to assay for [125I]HEAT (600 pM) binding in the presence or absence of 0.1 mM phentolamine in a total volume of 250 µl in triplicate. The result was analyzed as described above.
Construction of an 1B-AR Molecular Model and
Catecholamine Docking--
The coordinates of the
-carbon positions
were determined by overlay of putative
1-AR
transmembrane residues with the transmembrane coordinates of
bacteriorhodopsin (17), with data files generated using the Insight II
molecular modeling software from Biosym Technologies. The boundaries of
the putative transmembrane domains were determined using an algorithm
based on the weighted pairwise comparisons of adjacent residues (18).
The positioning of each helix with respect to the adjacent helices was
based where possible on data from
1B-AR mutagenesis
studies (10, 18, 19). The projections of the helices proposed by
Baldwin (21) were used to determine the tilt of each helix. The model
was minimized and conflicts adjusted to remove steric clashes based on
dynamic runs, as described previously (22).
Phosphatidylinositol Hydrolysis in Intact
Cells--
Phosphatidylinositol (PI) hydrolysis in intact, transfected
COS-1 cells was determined exactly as described previously (2), except
that for determining basal activation of PI hydrolysis, the cells were
seeded onto 6-well plates 1 day after transfection. Results are
expressed as the mean ± S.E. (error bars). An analysis of
variance and the Student's t test were used to determine
significant differences (p < 0.05).
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RESULTS AND DISCUSSION |
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A model of the 1B-AR developed previously (22) was
refined to accommodate the findings of recent mutagenesis studies and used to dock the agonist, (
)-epinephrine, into the binding pocket. In
particular the refined model takes into account an interhelical stacking interaction we have identified between Ala204 in
TMV and Leu314 in TMVI (10, 20), as well as the planar
orientation of the catechol ring. As shown in Fig.
2A, from this model it is
evident that the TMVI Phe310 side chain is well positioned
to interact with the catechol ring in a parallel stacked and displaced
conformation, which is an energetically favored structure for benzene
dimers (23). The TMVI Phe311 side chain, on the other hand,
is directed toward TMV, or is projecting into the lipid bilayer, and
Tyr348 is located toward the intracellular end of TMVII,
well below the plane of the catecholamine ligand.
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To more directly evaluate the involvement of Phe310 in
catechol ring binding, it was mutated to alanine or leucine, and the
resulting mutants (F310A and F310L), as well as the wild-type
1B-AR, were then evaluated in terms of membrane
expression, ligand binding, and stimulation of PI hydrolysis. As
controls for the potential Phe310-catechol ring
interaction, alanine and leucine mutants of two other TMVI
phenylalanines, Phe311 and Phe303, and a
leucine mutant of Tyr348 in TMVII, were also constructed
and similarly evaluated (Fig. 2B). Saturation binding and
immunoblotting studies indicated that the F303A, F303L, and Y348L
mutants were expressed in the plasma membrane and processed
(glycosylation) at levels almost equal to the wild-type
1B-AR (Table I and Fig.
3). All three mutants showed only small
decreases (1.7-2.2-fold) in affinity for the antagonist radioligand,
[125I]HEAT (Table I) and no change (Y348L) or an increase
(5-20-fold, F303A and F303L) in affinity for the agonists,
norepinephrine, epinephrine, and phenylephrine (Fig.
4). As will be reported in a subsequent
paper,2 the increased agonist
affinities observed with the Phe303 mutants are due to the
fact that these substitutions result in constitutive receptor
activation. Taken together, therefore, these findings do not support
involvement of Phe303 or Tyr348 in an
interaction with the catechol ring.
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As shown in Table II, the Phe310 mutants showed binding affinities for the antagonists, phentolamine, prazosin, and 5-methylurapidil that were 1.4-44-fold less than observed with the wild-type receptor. However, their affinity for the radioligand [125I]HEAT was largely unchanged (Table I) and their plasma membrane expression was equivalent to that of wild-type receptor (Table I and Fig. 3).
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The Phe311 mutants also displayed decreased antagonist binding (5-94-fold, Table II). However, in contrast to the Phe310 mutants, plasma membrane expression was markedly impaired with both F311A and F311L, and could not be enhanced by increasing the amounts of plasmid transfected (Table I and Fig. 3). These findings suggest that whereas substitution of Phe310 with alanine or leucine results in the loss of receptor interactions involved in the binding of some but not all antagonists, mutation of Phe311 produces a global change in receptor conformation that impairs not only ligand binding, but also protein folding and membrane expression.
To further characterize the effects of alanine or leucine substitution
at the Phe310 and Phe311 positions, the binding
of group-specific agonists was evaluated. The agonists evaluated
included the natural catecholamines, ()-norepinephrine, and
(
)-epinephrine, as well as (
)-phenylephrine, which is also a full
agonist but lacks the para-hydroxyl at the 4-position of the
catechol ring, and a group of partial agonists: (±)-synephrine, (±)-halostachine, phenethylamine, and dopamine, which lack a
meta-hydroxyl at the 3-position of the catechol ring; both
catechol ring hydroxyls; both catechol ring hydroxyls and the chiral
hydroxyl on the
-carbon; or only the chiral
-hydroxyl,
respectively. As shown in Fig. 4A, the F310A mutant showed
affinity losses of up to 1000-fold for the full agonists, but smaller
losses (10-50-fold) for the partial agonists. Consistent with the
involvement of Phe310 in ligand binding, the F310L mutant
in which the hydrophobicity but not the aromaticity of the wild-type
phenylalanine residue is preserved, displayed lesser decreases in
binding of both full agonists (30-40-fold) and partial agonists
(3-10-fold), than the F310A mutant (Fig. 4B).
As shown in Table III, the free energy
change (G) observed for the full agonists, with
substitution of Phe310 with alanine, was close to 4 kcal/mol, but only about 2 kcal/mol for the partial agonists. For the
leucine substitution the free energy change in agonist binding was
approximately 2 kcal/mol for the full agonists, and <1 kcal/mol for
the partial agonists. Given that the theoretical bond energy of an
aromatic-aromatic interaction is approximately 2 kcal/mol (23), one
interpretation of these findings is that the leucine substitution,
because of the hydrophobic character of its side chain, but not the
alanine substitution, which lacks both hydrophobicity and aromaticity, partially compensates for the wild-type phenylalanine. The greater free
energy loss observed for full agonists with the alanine substitution suggests that the aromatic component of the Phe310
interaction with the catechol ring is not only essential for ligand
binding, but for critical positioning of the catechol ring to allow
optimal interaction of other ligand moieties with the receptor, such as
the catecholamine meta-hydroxyl and Ser207. With
partial agonists, which lack at least one of the critical moieties of
full agonists and, thus, most likely have a less constrained binding
geometry, positioning of the catechol ring by an aromatic interaction
with the Phe310 side chain may be less critical. As a
result, the free energy losses for partial agonists are less than those
for full agonists, and almost exactly those anticipated (2 kcal/mol)
for the loss of a single aromatic-aromatic interaction.
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Like the Phe310 mutants, the Phe311 mutants also displayed decreased affinity for both full and partial agonists (Fig. 4). However, unlike the Phe310 mutants, the loss of agonist affinity with leucine substitution of Phe311 was greater than with alanine for the full agonists (up to 1000-fold versus 50-100-fold), but the same or less for partial agonists (5-10-fold versus 8-25-fold). Thus, the loss of both aromaticity and hydrophobicity with alanine substitution of Phe311 could not be compensated by a residue with a hydrophobic side chain (leucine) and, in fact, such a residue caused a greater impairment of agonist binding. Together with the markedly decreased membrane expression observed for the Phe311 mutants, these findings indicate a strict requirement for the phenylalanine side chain to allow proper folding into the wild-type receptor conformation, perhaps due to a critical interhelical stacking interaction between Phe311 and residues in the TMV helix.
To further evaluate the postulate that Phe310 forms a
critical aromatic-aromatic interaction with the catechol ring, whereas Phe311 is not directly involved in ligand binding but
rather in global receptor structure, additional Phe310 and
Phe311 mutants were constructed and their agonist-binding
properties evaluated. As seen in Fig. 5,
the Phe310 mutants produced graded decreases in agonist
affinity. Whereas the affinity for epinephrine, phenylephrine, and
halostachine was up to 1000-fold lower with the F310A mutant than with
the wild-type receptor, substitution of Phe310 with a
tryptophan, which although slightly larger than the native phenylalanine, preserves both its hydrophobicity and aromaticity, resulted in a receptor protein with near wild-type agonist affinities. Substitution with a tyrosine, which also has an aromatic side chain,
however, resulted in decreases in agonist affinity (25-30-fold) that
were comparable to those observed with the F310L mutation. This may be
due to the fact that the tyrosine ring contains an hydroxyl moiety. As
a result, its side chain, unlike those of phenylalanine or tryptophan,
is unlikely to be planar, and also has H-bonding potential. Thus, a
tyrosine at the 310 position may perturb optimal positioning of the
catechol ring due either to a steric clash or to loss of a favorable
planar stacking interaction. Other substitutions at the 310 position
with smaller hydrophobic or -branched residues (valine and
isoleucine) or polar residues (asparagine) resulted in significantly
greater reductions in agonist affinity than did tyrosine, leucine, or
phenylalanine. Not surprisingly, therefore, a positive and highly
significant correlation was evident between the volume of the
substituent side chain and agonist affinity (Fig. 5). This correlation
indicates a van der Waals component to the bonding between the catechol
ring and the side chain at the 310 position. This is not inconsistent
with an aromatic-aromatic interaction, which involves both a
dipole-dipole and a van der Waals component (23). In keeping with an
aromatic-aromatic interaction is the finding that the wild-type
receptor and the F310W mutant, which both have a planar aromatic side
chain at the 310 position, bind with considerably higher affinity than
the F310L mutant. In the case of this latter mutant the 310 side chain
is of similar volume to phenylalanine, but rather than being aromatic
and planar, is aliphatic and bulky.
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In contrast to the Phe310 mutations, no correlation was observed between the volume of the substituent side chain at the 311 position and agonist affinity (Fig. 5). Thus, small polar residues, such as asparagine, produced lesser decreases in affinity than larger hydrophobic residues, such as leucine or isoleucine.
If Phe310 indeed interacts with the catechol ring, as suggested by the above findings, it should be solvent accessible with its side chain projecting into the agonist-binding pocket. By contrast, Phe311, which we speculate projects toward TMV, should be much less solvent accessible. To directly evaluate the solvent accessibility of Phe310 and Phe311, we constructed the cysteine mutants, F310C and F311C, and used the SCAM (16, 24) to test their sensitivity to derivatization with the polar cysteine-modifying reagent, MTSEA. This compound is 2500 times more soluble in water than in n-octanol, fits into a cylinder of about 0.6 nm in diameter by 1 nm in length, and specifically adds its -SCH2CH2NH3+ moiety to reduced sulfhydryls to form mixed disulfides (24). Given the size of the -SCH2CH2NH3+ moiety, derivitization of a residue projecting into the binding pocket should sterically hinder ligand binding, whereas derivitization of a residue projecting toward an adjacent helix or into the lipid bilayer will occur much more slowly, and will either not influence ligand binding or will produce a more global change in receptor structure.
To evaluate the accessibility of Phe310 and
Phe311 to MTSEA derivitization, we initially evaluated the
accessibility of the 16 native cysteines (Fig. 2B) in the
wild-type 1B-AR. As shown in Fig. 6, MTSEA modification of intact COS-1
cells expressing the wild-type
1B-AR irreversibly
inhibited binding of the radioligand [125I]HEAT. This
inhibition could be rescued by treatment with the reducing agent,
dithiothreitol, and could be prevented by pretreatment with the
1-AR antagonist, phentolamine (data not shown). This indicates that MTSEA specifically modified cysteine residues rather than producing a nonspecific disruption of receptor structure. As will
be detailed in a subsequent paper,2 mutagenesis of three
native cysteines (Cys128, Cys129, and
Cys137) to serines, significantly reduced both the
sensitivity and reactivity of the
1B-AR to MTSEA
modification (Fig. 6, A and B). Importantly, the
triple mutant (C128S/C129S/C137S) displayed similar binding affinities
for various
1-AR antagonists ([125I]HEAT
and prazosin) and agonists (epinephrine and cirazoline), and similar
activity in stimulating PI hydrolysis, as the wild-type receptor (Table
IV). This triple mutant was thus used as
a template for SCAM studies of F310C and F311C.
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Substitution of Phe310 or Phe311 with cysteine
produced only a small decrease (3-15-fold) in [125I]HEAT
affinity, which was not further impaired when either F310C or F311C was
combined with the cysteine triple mutant to produce the quadruple
mutants F310C/C128S/C129S/C137S and F311C/C128S/C129S/C137S (Table
V). By contrast with the cysteine triple
mutant (IC50 for MTSEA-induced inhibition of
[125I]HEAT binding, >10 mM) additional
substitution of Phe310 to cysteine in the mutant,
F310C/C128S/C129S/C137S, restored sensitivity to MTSEA
(IC50 0.98 ± 0.23 mM, p < 0.001 as compared with the triple mutant) toward that observed with
the wild-type receptor (0.33 ± 0.06 mM) (Fig.
6A). However, sensitivity to MTSEA was not restored with
additional substitution of Phe311 to cysteine in the
mutant, F311C/C128S/C129S/C137S (IC50 > 10 mM)
(Fig. 6A). In agreement with these findings, the reactivity of Phe310 to modification with MTSEA in the
F310C/C128S/C129S/C137S mutant was rapid and identical to that observed
with the wild-type receptor (t1/2 = 1-3 s; Fig.
6B). By contrast, the reaction kinetics for MTSEA
modification of Phe311 in the F311C/C128S/C129S/C137S
mutant were extremely slow and identical to those observed with the
triple mutant, C128S/C1295/C137S (t1/2 > 600 s) (Fig. 6B). Nevertheless, given that MTSEA-induced
receptor inactivation was evaluated with the antagonist,
[125I]HEAT, and given that the residues involved in
antagonist and agonist binding may not be identical, these findings do
not exclude the possibility that Phe310 forms part of the
antagonist, but not agonist-binding pocket. To address this issue,
receptor-protection studies were performed to evaluate if agonist could
protect against MTSEA inactivation. As shown in Fig. 6C,
()-epinephrine fully protected both the wild-type receptor and the
F310C/C128S/C129S/C137S mutant. In contrast, the small amount of
inhibition of [125I]HEAT binding observed with MTSEA
treatment of both the F311C/C128S/C129S/C137S and C128S/C129S/C137S
mutants was unaltered by (
)-epinephrine pretreatment (Fig.
6C). Thus, the effect of MTSEA on [125I]HEAT
binding to the F311C/C128S/C129S/137S mutant is not due to
derivatization of Phe311. Moreover, in keeping with this
contention is the finding that, in contrast to the effect of MTSEA on
the F311C quadruple mutant, substituting Phe311 with a
variety of other residues that have considerably smaller side chain
volumes (
200 Å, Fig. 5, D-F) than that of the charged MTSEA moiety,
-SCH2CH2NH3+
(volume = 283 Å3), markedly perturbs
[125I]HEAT binding (Fig. 5, D-F). Taken
together therefore, these data indicate that the side chain of
Phe310, but not Phe311, is both solvent
accessible and directed toward the agonist-binding pocket.
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To determine the role of Phe310 and Phe311 in
receptor activation, the ability of substitution mutants to mediate
agonist-stimulated PI hydrolysis was compared with that of the
wild-type 1B-AR. With the F310L mutant, the maximal
response was unaltered but the potency of the epinephrine-stimulated PI
response was decreased by about 10-fold (EC50 = 0.57 µM versus 0.08 µM for the
wild-type
1B-AR), which is comparable to its decrease in
epinephrine binding affinity (Fig.
7A). However, the F310A mutant
showed a significant decrease not only in potency (EC50 = 120 µM versus 0.08 µM for the
wild-type
1B-AR), but also in efficacy of
epinephrine-stimulated PI hydrolysis (Emax = 60% of the wild-type response) (Fig. 7A). This decrease in
agonist efficacy was also observed with two other catecholamine
analogues, halostachine and phenethylamine, which lack either the
catechol para-hydroxyl, or both catechol hydroxyls and the
chiral hydroxyl on the
-carbon, respectively (Fig. 7, B and
C). Like the Phe310 mutants, alanine and leucine
substitutions at the 311 position also affected
(
)-epinephrine-stimulated PI hydrolysis. However, in this case the
leucine substituent produced a greater pertubation of receptor
signaling (decreased potency, EC50 = 219 µM
versus 0.08 µM for the wild-type
1B-AR, and efficacy, Emax = 40%
of the wild-type response) than the alanine substituent (decreased potency only, EC50 = 4.9 µM). Together with
the alterations in agonist binding observed above with these
Phe310 and Phe311 mutants, the data can be
interpreted to indicate that at the 310 position, substitution of the
native phenylalanine with a hydrophobic residue, such as leucine, can
partially compensate for the loss of both aromaticity and
hydrophobicity associated with an alanine substitution. At the 311 position, however, the greater perturbation of receptor signaling with
leucine than with alanine may be due to the bulky, non-planar leucine
side chain disrupting interhelical packing. In keeping with the
requirement of an aromatic side chain at the 310 position for both
agonist binding and receptor activation, is the finding that a mutant in which Phe310 was replaced with another aromatic residue,
tryptophan, displayed not only near wild-type agonist affinities, but
also wild-type PI hydrolysis (Fig. 7, A-C).
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Finally, to confirm that the effects of the F310A and F310L mutations
on PI signaling are not due to a global change in receptor structure,
we combined them with a previously described constitutively active
mutant, A293E (25, 26), to produce the double mutants, F310A/A293E and
F310L/A293E. Since Ala293 is about four helical turns below
Phe310, it is possible that a structural change caused by
the alanine or leucine substitutions will be transmitted to
Ala293, thus altering its structure and constitutive
signaling activity. As shown in Fig. 8,
signaling in the absence of agonist by the F310A or F310L mutants was
not different from that observed with the wild-type
1B-AR. Thus, F310A and F310L are not constitutively active. Moreover, increased basal signaling by the A293E mutant, which
is dependent on its level of expression, was not altered with the
double mutants, F310A/A293E or F310L/A293E (Fig. 8, inset). As seen in Fig. 9A, compared
with the wild-type
1B-AR, the A293E mutant showed all
the hallmarks of a constitutively active receptor, viz,
increased basal PI signaling, and a decreased EC50 and
increased Emax for epinephrine-stimulated PI
hydrolysis. Both double mutants showed similar increases in basal PI
signaling to that observed with the A293E mutant, alone. However, like
the single mutant, F310L, the F310L/A293E double mutant displayed a
decreased potency for epinephrine-stimulated PI hydrolysis
(EC50 = 113 nM versus 0.81 nM for A293E) without a change in efficacy
(Emax). Similarly, like the F310A single mutant,
the F310A/A293E double mutant displayed a greater decrease in agonist
potency (EC50 = 8020 nM) than F310L/A293E, as
well as a decrease in efficacy (Fig. 9A).
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Consistent with these changes in PI signaling, the increased
epinephrine affinity observed with the A293E mutant
(Ki = 0.017 µM) compared with the
wild-type 1B-AR (Ki = 0.75 µM), was perturbed by additional substitution of
Phe310 with leucine in the F310L/A293E double mutant
(Ki = 1.84 µM), and further decreased
in the F310A/A293E double mutant (Ki = 64 µM) (Fig. 9B). Thus, the effects of alanine or
leucine substitution of Phe310 on PI signaling do not
suggest distortion of global receptor structure, but are entirely
consistent with the involvement of Phe310 as a key switch
residue involved in ligand binding and receptor activation.
Pharmacophore mapping of adrenergic agonists suggests that the catechol
ring of ligands is important both for binding and receptor activity
(27). In this study, we provide several lines of evidence to support
the contention that Phe310 in TMVI of the
1B-AR mediates such effects through the formation of an
aromatic-aromatic interaction between its side chain and the catechol
ring. First, computerized modeling of the
1B-AR structure, and docking of catecholamines into the ligand-binding pocket, indicate that the Phe310 side chain is well
positioned to interact with the catechol ring. Second, as required for
a residue directly involved in ligand binding, SCAM studies reveal that
the Phe310 side chain is not only solvent accessible but is
directed into the agonist-binding pocket. Third, since substitution of
Phe310 with a variety of other amino acids does not perturb
membrane expression or post-translational processing (glycosylation) of the receptor, its effects on ligand binding and receptor activation are
due to local interactions, and not to alterations in global receptor
structure. In addition, even loss of all bonding potential with alanine
substitution of Phe310 does not impair spontaneous
isomerization of the receptor to a partially activated conformation, as
is evident with the F310A/A293E double mutant. Fourth, and most
importantly, the aromatic character of the Phe310 side
chain is essential for wild-type agonist binding and, in terms of
receptor signaling, for both wild-type agonist potency and efficacy.
Thus, although substitution of Phe310 with a residue that
has a hydrophobic side chain, such as leucine, partially compensates
for the loss of phenylalanine at the 310 position, only an aromatic
residue, e.g. tryptophan, can fully restore ligand-binding
and -receptor activation.
Our results do not support the direct involvement of Phe311
in agonist binding as proposed by Strader et al. (9) for the
equivalent residue (Phe290) in the 2-AR, or
alternatively, indicate that the residues interacting with the
catecholamine ring in the
1B and
2-AR,
differ. Thus, substitution of Phe311 with a variety of
different residues markedly impaired receptor expression and
post-translation processing, indicating that phenylalanine at the 311 position is essential for proper folding of the receptor protein. In
addition, neither the volume nor the hydrophobicity of
Phe311 substituent side chains could be directly related to
agonist binding or receptor activation. Finally, and most importantly, SCAM studies indicated that the Phe311 side chain is
neither solvent accessible nor directed into the ligand-binding pocket.
Thus, effects of Phe311 substitutions on ligand binding and
receptor activation are likely secondary to global changes in receptor
structure, rather than to loss of an interaction directly involved in
ligand binding.
Based on SCAM studies of the dopamine D2 receptor, it has been suggested recently that all of the aromatic residues in TMVI of biogenic amine receptors are solvent accessible (28). Furthermore, it was postulated that these aromatic residues relayed information from the site of agonist-binding in the extracellular half of the TMVI helix to produce a conformational change at the intracellular end of the helix, resulting in receptor activation. While an attractive hypothesis, the SCAM conclusions were based entirely on antagonist-inactivation data, and were not confirmed by evaluating an effect on agonist binding.
Apart from defining a critical residue (Phe310) involved in
ligand binding and signaling by the 1B-AR, the findings
of this study have potentially important ramifications for our
understanding of the mechanisms of GPCR activation. For rhodopsin,
activation involves initial disruption of an ionic bond between the
protonated Schiff base formed by the interaction of
11-cis-retinal with Lys296 in TMVII and
Glu113 in TMIII (5). Similarly, activation of the
1B-AR involves disruption of an ionic bond linking
Asp125 in TMIII and Lys331 in TMVII (3), and
partial receptor activation can be induced by a moiety mimic
(triethylamine) of the catecholamine protonated amine (29). For
rhodopsin, activation has been shown to result in rigid body movement
of the TMVI helix (30), and movement of this helix is also a feature of
2-AR (31) and
1B-AR
activation.2 Thus, it is likely that movement of TMIII, due
to disruption of a constraining interaction with TMVII, and movement of
TMVI, are common features of GPCR receptor activation. Movement of
these helices, which are contiguous with the second and third
intracellular loops, is also consistent with the known involvement of
these loops in G-protein activation (12).
Given these considerations, and based on the findings of this study
that implicate Phe310 in TMVI as a key switch residue in
1B-AR activation, it seems reasonable to postulate that
activation of adrenergic receptors involves initial disruption of the
Asp125/Lys331 ionic interaction followed by
Phe310/catechol ring-induced movement of TMVI. Indeed, the
findings of our studies with the F310A or L/A293E double mutants
support this model based on the following considerations: (i) central to the recently revised ternary complex model of GPCR activation is the
finding that mutant receptors can exist in a constitutively active
state that allows signaling in the absence of agonist; (ii) in support
of this model is the finding that overexpression of wild-type receptors
can also initiate biochemical responses in the absence of agonist;
(iii) accordingly, it has been proposed that receptors spontaneously
resonate between a basal state, R, and an active state,
R*, with only the latter being able to productively interact
with G-protein, and (iv) as a corollary, constitutively active mutants,
which partially mimic the active state, represent an intermediate
receptor conformation, R' (26). Based on these considerations and on the finding that agonists bind to constitutively active receptors with higher affinity than to wild-type receptors, it
has been suggested that rather than inducing the active conformation upon binding, agonists merely select or "trap" the active
conformation that results from spontaneous isomerization of
R to R* (32). Nevertheless, recent
structure-function studies of the angiotensin II (AT1)
receptor provide compelling evidence for an R' conformation that is a distinct intermediate between R and R*,
and that isomerization from the R' conformation to the
active state involves an inductive step that requires agonist binding
(33).
In the present study we demonstrate that little, if any, PI hydrolysis
is observed with the wild-type 1B-AR in the absence of
agonist. Moreover, signaling in the absence of agonist was similar with
the F310A and F310L mutants, and, importantly, was not reduced below
that observed with the unliganded wild-type
1B-AR, even
though these mutants markedly impaired agonist-induced signaling. Thus
the unliganded wild-type
1B-AR receptor likely represents the true basal or R conformation, whereas the
receptor maximally activated by high agonist concentrations represents the R* state. By contrast, and consistent with an
R' conformation, basal signaling was readily apparent with
the unliganded A293E mutant. Since the double mutants F310A/A293E and
F310L/A293E also showed similar basal signaling in the absence of
agonist, but impaired agonist-induced signaling, the
Phe310-catechol ring interaction is likely not required for
isomerization between R and R', but is critical
for full receptor activation. Consistent with the above considerations,
it seems reasonable to speculate that some other activation process,
e.g. disruption of the Asp125/Lys331
ionic interaction, is required for transition from R to
R', whereas the Phe310-catechol ring interaction
mediates isomerization from R' to R*. As with the
AT1 receptor (33), this latter requirement of a specific
agonist-receptor interaction also supports the notion that
isomerization from R' to R* is an
agonist-dependent inductive process. Clearly additional
studies, particularly aimed at directly evaluating receptor
conformational states, based, for example, on electron spin resonance
(30), NMR (34), fluorescence spectroscopy studies (35), Fourier
transform infrared resonance spectroscopy (36), and surface plasmon
resonance spectroscopy (37), will be required to more fully define the
activation process at atomic resolution.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Siiri Iismaa and Professor Ahsan Husain for critical reading of the manuscript and Elaine Martin for expert secretarial assistance.
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Note Added in Proof |
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While the paper was in review, additional
1B-AR mutants were constructed and evaluated in which
Phe310 or Phe311 in both the wild-type and
C128S/C129S/C137S receptors were substitute with lysine. Since the side
chain of this residue mimics the MTSEA moiety that binds to cysteine,
it should produce similar effects to MTSEA derivitization of F310C- or
F311C-substituted wild-type or C128S/C129S/C137S receptors. All
lysine-substituted mutants showed wild-type membrane expression
(Western blotting and in situ immunofluorescence), and as
observed with MTSEA derivitization of F310C-substituted receptors, the
F310K mutants displayed loss of [125I]HEAT binding. However,
unlike MTSEA derivitization of F311C-substituted mutants, which did not
perturb [125I]HEAT binding (Fig. 6B), the F311K
mutants displayed loss of radioligand binding. These findings further
indicate the Phe311 is not directed into the ligand-binding pocket.
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FOOTNOTES |
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* This work was supported by a grant from the National Health and Medical Research Council, Australia.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 requests for reprints should be addressed: Victor Chang Cardiac Research Institute, 384 Victoria St., Darlinghurst, NSW 2010, Australia. Fax: 612-9295-8501; E-mail: b.graham{at}victorchang.unsw.edu.au.
2 S. Chen, M. Xu, F. Lin, and R. M. Graham, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
1-AR,
1-adrenergic receptor;
SCAM, substituted cysteine
accessability method;
[125I]HEAT, 2-[
-(4-hydroxyl-3-[125I]iodophenyl)ethylaminomethyl]tetralone;
Emax, the maximal effector response;
TM, transmembrane;
MTSEA, methanethiosulfonate ethylammonium;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PI, phosphatidylinositol;
GPCR, G-protein coupled receptor.
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REFERENCES |
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