(Received for publication, April 26, 1995; and in revised form, July 25, 1995)
From the
-Adrenergic receptor (AR) subtypes mediate many
effects of the sympathetic nervous system. The three cloned subtypes
(
-AR,
-AR,
-AR),
although structurally similar, bind a series of ligands with different
relative potencies. This is particularly true for the
-AR, which recognizes a number of agonists and
antagonists with 5-50-fold higher affinity than the
- or
- subtypes. Since ligands bind
to receptor-residues that are located in the transmembrane spanning
domains, we hypothesize that subtype differences in ligand recognition
are due to differences in the binding properties of nonconserved
transmembrane residues. Using site-directed mutagenesis, selected
putative ligand-binding residues in the
-AR were
converted, either individually or in combination, to the corresponding
residues in the
-AR. Mutation of two such residues
(of approximately 172 amino acids in the transmembrane domains)
converted the agonist binding profile entirely to that of the
-AR. Over 80% of this conversion was due to an
Ala
Val substitution; the remainder was due to the
additional substitution of Leu
Met. To confirm
that Ala
and Leu
are indeed critical for
agonist subtype-selectivity, the equivalent residues in the
-AR (Val
and Met
) were
reversed of that of the
-AR. Correspondingly, the
agonist-binding profile of this double
-AR mutant
reverted to that of the
-AR. From these data, in
conjunction with macromolecular modeling of the ligand-binding pocket,
a model has been developed, which indicates that the determinants of
these two residues for agonist subtype-selectivity are due not only to
interactions between their side chains and specific ligand moieties but
also to a critical interaction between these two amino acids.
-Adrenergic receptors
(
-ARs)
are members of the seven
transmembrane G-protein-coupled receptor family that are activated by
norepinephrine released from sympathetic nerve endings and by
epinephrine released from the adrenal medulla. They are part of a
larger subset of related but distinct adrenergic receptors, which
include the
- (
,
,
) and the
- (
,
,
) adrenergic receptors.
-ARs are expressed in a wide variety of tissues
including the brain, liver, myocardium, and vascular smooth
muscle(2, 3) . Thus far, three
-AR
subtypes have been cloned and pharmacologically characterized, the
-AR(4) , the
-AR(2, 5) , and the
-AR(6, 7) . The
-subtype has a 5-50-fold higher affinity for
the agonists oxymetazoline and methoxamine and for the antagonists
5-methylurapidil, (+)-niguldipine, and phentolamine when compared
with the
- or the
-subtype(2, 7) . However, there are
currently no good subtype-selective drugs readily available that can
discriminate between the subtypes, based on 100-1000-fold differences
in affinity.
Recent studies have indicated that
-subtypes can mediate different and sometimes opposing
physiological effects. In particular the
-AR appears
to promote automaticity and arrhythmias during myocardial ischemia,
whereas the
-AR subtype can activate a
Na
/K
pump leading to cell
hyperpolarization, thus decreasing the propensity for abnormal heart
rhythms(8) . In addition, it is now known that the
-subtype regulates the predominant dynamic component
involved in the development of benign prostatic
hypertrophy(9) . Hence, development of better
-subtype-selective drugs to either block or enhance
function may help in combating subtype-related disorders.
In order
to design selective drugs, an understanding of subtype differences in
the ligand binding pockets would be invaluable. Although the residues
forming the -AR binding-pocket have not been defined,
key interactions are likely to be similar to those of the
-adrenergic receptor, since both receptor families are activated
by the catecholamines, norepinephrine, and epinephrine. Based on
studies of the
-AR, key points of interaction are
believed to occur between Asp
in the third transmembrane
helix (TMIII) with that of the protonated amine of the agonists, and
between two serine residues (TMV; Ser
and
Ser
) that hydrogen-bond with the meta- and parahydroxyl
groups of the catechol ring. These critical binding contacts with the
natural ligands are well conserved between the subtypes. However, there
are 48 amino acid differences in the transmembrane domains between the
-AR and the
-AR. We hypothesize
that only a few of these nonconserved residues are critical for the
ligand binding specificity of each
-AR subtype since
only a subset of these nonconserved residues occur in the regions of
the transmembrane domain thought to be involved in ligand binding.
Therefore, rather than sequentially mutating all amino acids that
differ between the -subtypes, we have developed simple
criteria to select those residues that may be critical for ligand
binding and thus are involved in subtype differences in ligand binding
profiles. Eight
-AR residues were mutated
individually to the corresponding residue in the
-AR.
Two of these changes, when combined in a double mutant, conferred on
the
-AR the agonist binding properties of the
-AR. A single change, Ala
Val (
)in TMV, made the greatest contribution to the change in
ligand binding. Reversal of these two residues in the
-AR to those in the
-AR reversed
the agonist binding profile back to that of the
-AR.
The -AR and the
-AR,
although structurally similar in their transmembrane domains, have some
significant differences in their ligand-binding affinities for a number
of agonists and antagonists. Determining the critical amino acids
responsible for these differences in binding properties may assist in
the design of better subtype selective drugs. We hypothesized that
there are only a few nonconserved transmembrane amino acids that
distinguish the
-AR agonist-binding pocket from that
of the
-AR.
Candidate residues potentially involved in
subtype selectivity were selected for mutation from the 48 nonconserved
transmembrane amino acids, based on the following criteria: 1) location
in the upper half of the transmembrane domain, where previous studies
indicate that small ligands are bound; 2) exclusion of the first
transmembrane domain since, based on models of bacteriorhodopsin and
rhodopsin, this appears not to be directly involved in forming the
ligand binding pocket(11) ; 3) location on the transmembrane
-helices such that their side-chains are oriented toward the
putative binding pocket or adjacent transmembrane domains rather than
toward the surrounding phospholipid bilayer (orientation of the
side-chains was determined based on consideration of models of the
-AR developed in our laboratory(13) , and on
the model of rhodopsin developed by Baldwin (15) ); 4) lack of
conservation not due merely to interspecies differences, since there is
conservation of
-subtype ligand-binding profiles
across species.
Seven amino acids (Fig. 1) of the
-AR were identified that fulfilled the criteria, and
these residues were mutated to the corresponding residues on the
-AR. An eighth residue Leu
Phe
was also mutated. Although this residue was predicted to be facing the
hydrocarbon environment, it was selected because the corresponding
-AR residue represented a significant change in size
and functional group. Moreover, if our prediction of its orientation
was correct, mutation of this residue should not alter ligand binding
and, thus, this mutant would serve as a negative control.
Figure 1:
Schematic
representation of the -AR highlighting the eight
amino acid residues selected for mutagenesis and their transmembrane
location. TMI, -II, -III, -IV, -V, -VI, and -VII represent the seven
transmembrane domains. The mutations were as follows: TMII, Ser
Thr and Phe
Ser; TMIV, Thr
Leu and Leu
Phe; TMV, Ala
Val and Ser
Ala; Ala
Val and Leu
Met.
Figure 2:
A, the
effects of the -AR point mutations on the affinities
for agonists that have a hydrophobic ortho group on their phenyl ring (y axis); B, binding affinities for agonists without
a hydrophobic ortho group (y axis). For both panels, the x axis represents the
-AR (WTb) and the
-AR (WTa) along with the mutations that were
statistically different (by a Student's t test) from the
WTb. The z axis represents the difference in K
(-log) from the
WTb.
Figure 5:
A,
the reverse mutations of Leu
Met and Ala
Val (
-AR) was performed in the
-AR (Met
Leu and Val
Ala, respectively). Removal of the inhibitory effect of
methionine (Met
Leu) resulted in an increased
affinity for agonists that lacked an ortho hydrophobic group
(epinephrine, norepinephrine, and phenylephrine). This is equivalent to
the Ala
Val in the
-AR. B, the combination of Met
Leu
and Val
Ala in the
-AR revealed
an
-AR agonist pharmacology. Shown for comparison is
the
-AR double mutant Ala
Val/Leu
Met.
Our results indicate that
with agonist binding, individual amino acids, particularly in the 5th
and 6th transmembrane domains, are critical in defining the agonist
binding pocket between the -AR and the
-AR. Each agonist is similarly effected by changes in
critical residues but not to the same degree. The Ala
Val mutation appears to explain most of the higher
affinity for agonists as seen with the
-AR. The
increased affinity observed with the substitution of valine for alanine
may be related to an increased hydrophobic interaction between the
valine and the aromatic ring of the ligands. The increase in binding
affinity with the Leu
Met may be due to an
increased interaction as a result of the extended chain length of the
methionine residue with the ortho hydrophobic group found with many of
the synthetic agonists. Supporting this is the progressive increase in
affinity conferred by Leu
Met with a progressive
increase in size and hydrophobicity of the ortho side chain. The
descending order of binding affinities (Fig. 2A) are
cirazoline > methoxamine > oxymetazoline and their respective
side chains are a cyclopropyl, a methoxy, and a methyl group.
Figure 3:
Binding
affinities for the combination -AR mutations.
Ser
Thr/Phe
Ser, Leu
Phe, and Ser
Ala represents the
combination of four mutations that individually didn't
significantly differ from the WTb. The Ala
Val and
Leu
Met double mutant represents the combination
of the two mutations that did significantly differ in their agonist
binding affinity from that of the WTb toward that of the
WTa.
Figure 4:
A model
of the Ala
Val and Leu
Met
(
) mutations showing proposed interactions in the
agonist binding pocket. The graph represents the individual mutations
followed by the combination of Leu
Met and
Ala
Val. The y axis shows K
(-log) differences from the WTb.
Below each graph is a model that describes the data. Oxymetazoline is
used to represent an agonist with a hydrophobic ortho group and
phenylephrine, an agonist without such a group. Only transmembranes 5
(TMIV) and 6 (TMVI) are shown. Only the phenyl ring and the ortho group
of the ligand is represented. Arrows indicate the putative
sites of interactions. A, for oxymetazoline, Leu
Met interacts with the ortho methyl group, and Ala
Val interacts with the phenyl ring. In combination the 314
position is pulled away by the ortho substituent from the 204 position
allowing both residues to interact freely with the ligand, thus having
an additive effect. B, for phenylephrine, the lack of an ortho
group results in steric inhibition of the 204 position by the bulkier
methionine, leading to an inhibitory effect when they are
combined.
From our data we were able to model the subtype
differences between their agonist binding pockets (Fig. 6, A and B). It appears that when an agonist is docked into
the binding pocket, the presence of a hydrophobic ortho substituent on
the phenyl ring will interact with the bulkier methionine residue at
position 293 on the -AR (Leu
on the
-AR) thus increasing binding affinity significantly.
Similarly the larger hydrophobic valine residue in the
-AR at position 185 (Ala
in the
-AR) interacts directly with the phenyl ring. For
agonists with an ortho hydrophobic substituent as with cirazoline,
oxymetazoline, and methoxamine, the effect of these two residues is
additive conferring upon the
-AR increased binding
affinity. In the absence of the ortho group (phenylephrine,
norepinephrine, and epinephrine) Met
(
)
sterically hinders Val
(
) from
interacting with the phenyl ring, thus there is no increase in
affinity. These two residues in combination differentiate the agonist
binding pocket of these two
-AR subtypes. The
metahydroxyl group appears to interact with the Ser
as
there was no difference in binding affinity between the wild-type
-AR and Ser
(
) for
phenylephrine, which contains only a metahydroxyl group. Thus each
agonist docks into the ligand binding pocket in a tilted manner.
Figure 6:
A, model of the -AR
showing the docking of oxymetazoline (oxy) in the mutated
ligand binding pocket (Leu
Met and Ala
Val), which mimics that of the
-AR. The
ortho methyl group interacts with Met
, the phenyl ring
with Val
, and the metahydroxyl with Ser
. B, with phenylephrine (PH) the lack of an ortho
substituent allows steric interaction of Leu
Met
and Ala
Val and therefore no difference is seen in
agonist binding affinity. TMI, -II, -V, -VI, and -VII are shown; TMIII
and -IV have been removed to allow visualization of the binding
pocket.
From our data we have defined two important residues that are
critical in differentiating the ligand binding pocket between two
-AR receptor subtypes. We are able to model the
interaction between these two residues and in so doing have provided a
rational basis for the design of better subtype-selective agonists to
allow optimal binding and receptor activation.