(Received for publication, December 4, 1995; and in revised form, December 28, 1995)
From the
Activation of the - and
-adrenergic receptors (AR) involves hydrogen bonding
of serine residues in the fifth transmembrane segment (TMV) to the
catechol hydroxyls of the endogenous agonists, epinephrine and
norepinephrine. With the
-AR both Ser
and Ser
but not a third TMV serine
(Ser
) are required for binding and full agonist activity.
However, with the
-AR only one of two TMV serines
(Ser
, equivalent to Ser
in the
-AR)
appears to contribute partially to agonist-binding and activation.
Because the
-AR uniquely contains only two TMV
serines, this subtype was used to systematically evaluate the role of
hydrogen bonding in
-AR activation. Binding of
epinephrine or its monohydroxyl congeners, phenylephrine and
synephrine, was not decreased when tested with alanine-substitution
mutants that lacked either Ser
(Ser
Ala) or Ser
(Ser
Ala). With the
substitution of both serines in the double mutant, Ser
Ala, binding of all three ligands was significantly
reduced (10-100-fold) consistent with a single hydrogen bond
interaction. However, receptor-mediated inositol phosphate production
was markedly attenuated only with the Ser
Ala
mutation and not with Ser
Ala. In support of the
importance of Ser
, binding of phenylephrine (meta-hydroxyl only) by Ser
Ala increased
7-fold over that observed with either the wild type receptor or the
Ser
Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser
Ala mutation. In addition, when combined with a recently
described constitutively active
-AR mutation
(Met
Leu), only the Ser
Ala
mutation and not Ser
Ala relieved the high
affinity binding and increased agonist potency observed with the
Met
Leu mutation. A simple interpretation of these
findings is that the meta-hydroxyl of the endogenous agonists
preferentially binds to Ser
, and it is this hydrogen bond
interaction, and not that between the para-hydroxyl and
Ser
, that allows receptor activation. Furthermore, since
Ser
and Ser
are separated by three residues
on the TMV
-helix, whereas Ser
and Ser
of the
-AR are separated by only two residues,
the orientation of the catechol ring in the
-AR
binding pocket appears to be unique and rotated approximately 120°
to that in the
-AR.
Adrenergic receptors (ARs) ()are members of the
superfamily of receptors that exert their physiological effects through
coupling to guanine nucleotide-binding proteins (G-proteins). Strictly
conserved among this superfamily is the presence of seven
transmembrane-spanning domains connected by hydrophilic loops
alternately exposed to the extracellular and intracellular environment.
The intracellular loops bind and activate
G-proteins(1, 2) . The AR family (
,
,
,
,
,
,
,
,
) mediate the effects of the
sympathetic nervous system through the actions of epinephrine and
norepinephrine and control the homeostasis of the cardiovascular
system. However, the ligand binding pockets are distinct between the
receptor subtypes, as they can discriminate a wide variety of synthetic
agonists and antagonists(3) .
Previous studies with the
-AR have identified key residues involved in binding
epinephrine and norepinephrine. These interactions likely are conserved
in the other ARs since they all bind the natural hormones with similar
affinity. In particular, a highly conserved aspartic acid residue
(Asp
in the
-AR) in TMIII is involved in
forming a salt bridge with the protonated amine of the
catecholamine(4) . There are also two serine residues in TMV
(Ser
and Ser
in
-AR) that
are highly conserved among receptors that bind catecholamines, but not
in other G-protein-coupled receptors (Fig. 1). In the
-AR, one of these two serine residues, Ser
as well as a non-conserved serine (Ser
), have been
shown to be involved in hydrogen bond interactions with the hydroxyl
groups on the catechol ring(5) . Both residues are required for
high affinity binding of agonists and for full agonist activity. These
interactions were confirmed with the use of various agonists that lack meta- and/or para-hydroxyl groups of the catechol
ring. A model was therefore proposed in which Ser
hydrogen bonds with the meta-hydroxyl group of the
catechol ring while Ser
interacts with the para-hydroxyl group. Extrapolation of this model to the
-AR is unclear. Mutation of either of the equivalent
serines in the
-AR (Ser
and
Ser
) resulted in a 10-fold decrease in affinity for
epinephrine but no change in affinity for synephrine that lacks a meta-hydroxyl group (suggesting the para-hydroxyl is
unimportant)(6) . The Ser
mutant attenuated the
functional activity (65% active) but only with synephrine, implicating
a para-hydroxyl interaction. From these studies it was
concluded that the para-hydroxyl group of the catechol ring is
involved in a hydrogen bonding interaction with Ser
, as
has been described previously for the corresponding serine in the
-AR. Furthermore, in contrast to the situation with
the
-AR, it was suggested that the
-AR Ser
residue does not participate
directly in receptor-agonist interaction.
Figure 1: Sequence alignment of TMV residues of AR subtypes. Sequences were aligned to maximize homologies within this region(16) . The numbers at the top signify the positions of the amino acids in the primary sequence for each receptor subtype. The conserved serines are boxed. Serine residues shown in previous studies (5, 8) to be involved in binding and activation are bold. The single-letter amino acid code is used.
With the recent cloning of
the -AR, which uniquely contains only two TMV
serines, we explored the serine requirements for
-AR
agonist binding and activation. We also evaluated if the interaction
proposed for the
-AR was conserved.
Using site-directed mutagenesis and pharmacophore mapping of
catecholamine agonists, Strader et al.(5) proposed
that hydrogen bond interactions involving the hydroxyl groups on the
phenyl ring are important for ligand binding to the
-AR. A model was proposed in which two of three
serines in the TMV are involved in these hydrogen bond interactions
with the catechol hydroxyl groups of catecholamine agonists. In
particular, it was proposed that Ser
of the
-AR forms a hydrogen bond with the meta-hydroxyl group of the catechol ring while Ser
forms a hydrogen bond with the para-hydroxyl group. In
support of this model, substitution of alanines for either of these two
serines resulted in mutants that had a 30-fold decrease in their
affinity for several catecholamine agonists, with each serine
contributing about 50% to the activation of the receptor(5) .
Consistent with the importance of serine residues for catecholamine
binding, it is of interest that two of the three serines in TMV of the
-AR are highly conserved, although only one of these
two conserved residues is implicated in catecholamine binding (Fig. 1). The involvement of the serine residues (Ser
and Ser
) in the
-AR is less
clear(6) . Although both serine mutations in the
-AR did produce a decrease in affinity with
epinephrine and phenylephrine, only synephrine produced an attenuation
of function with Ser
Ala, the equivalent serine to
Ser
in the
-AR. Other adjacent potential
hydrogen-bonding residues (Cys
) were not mutated, so the
-AR paradigm is still incomplete.
With the cloning
of the -AR and the finding that this subtype contains
only two serine residues in TMV, we have been able to study the
interaction of the catechol hydroxyls of agonists with
-AR serine residues. In order to assess the
interactions between each of the conserved serine residues and the
catechol hydroxyls, site-directed mutagenesis was used to create three
different mutated
-ARs analogous to those created by
Strader et al.(5) . These receptors are denoted as
Ser
Ala, Ser
Ala, and
Ser
Ala and correspond to the substitution of
serine residues by alanines at the indicated amino acid number of the
-AR (Fig. 1). However, in the
-AR there is no equivalent serine residue at position
204 as in the
-AR. The N-terminal TMV
-AR serine is located one residue higher in the helix
at the analogous position 203 of the
-AR (Fig. 1).
The binding of epinephrine and norepinephrine to
the wild type receptor is consistent with a single population of
binding sites and both displayed similar K values (Table 1). There is no apparent high affinity component of
agonist binding, as is typically seen with other G-protein-coupled
receptors, and addition of GTP analogs does not produce a low affinity
shift of epinephrine binding. With the wild type
-AR,
binding of synephrine, a congener of epinephrine that lacks the meta-hydroxyl, was 15-fold lower than that of epinephrine.
However, binding of phenylephrine, a congener of epinephrine that lacks
the para-hydroxyl, was similar to that of epinephrine. This
suggests that the meta-hydroxyl group contributes
predominantly to determination of catecholamine affinity for
-ARs (Table 1, Fig. 2).
Figure 2:
Competition binding and IP stimulation
with substituted catechol hydroxyls to wild type, Ser
Ala, and Ser
Ala mutant
-ARs. Competition binding and IP stimulation was
performed on cells prepared from transfected COS-1 cells with the wild
type
-AR receptor (
), Ser
Ala (
), or Ser
Ala (
) as described
under ``Experimental Procedures,'' in the presence of
epinephrine (panel A), phenylephrine (panel B), and
synephrine (panel C). The experiment shown is the mean curve
(± S.E.) generated from at least three separate
experiments.
Replacement of
either Ser or Ser
in TMV of the
-AR with an alanine did not significantly reduce the
binding affinity for any of the agonists tested compared to the wild
type receptor (Table 1, Fig. 2). In fact, the binding
affinity for phenylephrine was significantly increased (7-fold) with the Ser
Ala mutant (Table 1, Fig. 2). There also was a noticeable higher
affinity for the other monohydroxyl agonist, synephrine with the
Ser
Ala mutant, but this increase in affinity was
not significant. These results are quite distinct from the
-and
-ARs paradigms, where either
serine mutation was able to reduce agonist-binding affinity. To confirm
a hydrogen bond interaction, the double mutant, Ser
Ala, was created and found to decrease the binding
affinity by 25-120-fold for epinephrine, norepinephrine and
phenylephrine, consistent with a decrease in binding energy upon
substitution of
G = 3-5 kcal/mol. The
decrease in synephrine binding affinity by this mutant was only 6-fold.
The free energy values are consistent with the disruption of a single
hydrogen bond (
G= 3-7 kcal/mol). Since
either serine residue is sufficient in itself in maintaining the wild
type binding affinity but the free energy values derived from the
double serine mutation indicates only one hydrogen bond is formed, the
data are consistent with both serines contributing a weak hydrogen bond
to the agonist when both catechol hydroxyls are present. However, it
seems that the meta-hydroxyl interaction with Ser
is the strongest since with the wild type receptor phenylephrine
has essentially the same binding affinity as epinephrine (Table 1, Fig. 2). The finding that binding is maintained
with removal of one or the other serine residue is explained by
competition of the catechol hydroxyls for the remaining serine residue.
The agonist will then dock to optimize its interaction (thus, higher
binding affinity than the wild type receptor) with the remaining serine
residue, suggesting promiscuity of ligand docking. The energy
difference between the possible ligand binding sites would be very
different to account for Hill coefficients of unity. Based on affinity
differences between phenylephrine and Ser
versus Ser
(14-fold difference, 0.9 versus 12.6
µM, respectively), and assuming an initially direct
relationship between affinity and distance (i.e. higher the
affinity, the smaller the distance), the meta-hydroxyl would
be closer to Ser
than Ser
. Likewise, from
the affinity differences between synephrine and either serine mutation
(4-fold difference, 28.2 versus 104.7 µM,
respectively), the para-hydroxyl group is also closer to
Ser
than Ser
. Therefore, it appears that
-ARs maintain their catechol hydroxyls not equal
distant from the two serine residues but in a parallel position
relative to the surface of the receptor (Fig. 4B) with
the meta-hydroxyl and Ser
forming the strongest
interaction. This model is not only valid for the mutated receptors but
is also consistent for the wild type receptor since the binding of
phenylephrine (6.2 µM) is similar to epinephrine (3.3
µM), while synephrine binding decreased by 10-fold for the
wild type receptor. These results demonstrate a strong binding
interaction and, therefore, a closer distance of the meta-hydroxyl position to a serine residue and a weaker and
more distant interaction of the para-hydroxyl. Also in support
of this model for the wild type receptor, synephrine's affinity
is minimally changed (6-fold) for the wild type receptor (52.5
µM) as compared to the double serine mutant (324
µM), while all other agonists tested displayed
25-120-fold affinity differences (Table 1). This also
suggests that the para-hydroxyl is only weakly bonding with a
serine residue.
Figure 4:
A
model of the agonist epinephrine in the ligand binding pocket of the
-AR (A) and the
-AR (B). Model was constructed as described under
``Experimental Procedures.'' TM helices V and VI are only
shown for clarity. Panel A, model of the
-AR
showing the docking of epinephrine (epi). The meta-hydroxyl group of epinephrine interacts with Ser
and is closer to TMVI, while the para-hydroxyl group of
epinephrine interacts with Ser
and is closer to TMIV. The
-AR catechol hydroxyls equally interact strongly with
its respective serine residue, resulting in a tilt of the catechol ring
with respect to the extracellular surface. Panel B, model of
the
-AR showing the docking of epinephrine. The meta-hydroxyl group of epinephrine interacts strongly with
Ser
of the
-AR and is closer to TMIV,
while the para-hydroxyl group of epinephrine interacts weakly
with Ser
closer to TMVI. This results in a rotation of
the catechol ring by about 120° compared with the
-AR model and a parallel orientation of the catechol
ring with respect to the extracellular
surface.
The activation requirements for
-ARs appear distinct from its binding interactions (Fig. 2). At equal receptor numbers of 0.3 pmol/mg protein, only
Ser
plays a major role in receptor activation,
contributing 70-90% of the wild type response. On the other hand,
the effect of Ser
on receptor activation is minimal. Both
full agonists, epinephrine and phenylephrine, produced similar effects
on activation with either serine mutant, consistent with the meta-hydroxyl of epinephrine being nearest to Ser
(Fig. 2, A and B). Synephrine produced
effects similar to those of epinephrine and phenylephrine on receptor
activation (Fig. 2C) with either serine mutation (but
with less efficacy). Only Ser
Ala (Ser
intact) allowed full receptor activation. Since synephrine can
activate the receptor, this indicates that either hydroxyl group on the
catechol ring is capable of activating the receptor but only an
interaction with Ser
will produce a wild type response.
This also supports the hypothesis of promiscuity of ligand docking,
since the para-hydroxyl would need to move to allow
interaction with Ser
. Since this would not be an optimal
interaction, it accounts for the partial agonist properties of
synephrine. Likewise, the full agonism of phenylephrine is due to its
close contact with Ser
, as has been postulated in earlier
studies (15) in which the greatest activity among monophenolic
analogs of phenethylamines always resides in the meta-substituted derivative, with the para-hydroxylated phenethylamines being significantly weaker.
We recently described a chimeric point mutation in the
-AR, Met
Leu, that was created
to explore the agonist binding pocket differences between
- and
-ARs. This mutant is
constitutively active as evidenced by increased basal signaling and
increased agonist binding and potency(16) . The mechanism by
which this mutant imparted constitutive activity appears to be by
preventing normal packing of an adjacent valine residue in TMV
(Val
), which, in turn, perturbs the helix resulting in
constitutive activity(16) . Since this
mutation, Met
Leu, appeared to be operating
through a conformational change of TMV, we explored the possibility
that the serine residues on TMV might also be involved in manifesting
its constitutive activity. Therefore, we combined the constitutively
active mutation, Met
Leu, with either serine
mutation, Ser
Ala or Ser
Ala,
in a single receptor and evaluated the resulting double mutant for
changes in potency, basal IP
release, and agonist binding
affinity. The Met
Leu mutation alone was
constitutively active, as evidenced by its higher IP
basal
activity (Fig. 3A), higher binding affinity (Fig. 3B), and increased potency (Fig. 3C) compared to the wild type receptor. However,
basal IP
signal transduction remained higher when combined
with either serine mutation (Met
Leu/Ser
Ala or Met
Leu/Ser
Ala), indicating an agonist-independent property of this
mutant. However, the high binding affinity for epinephrine as seen in
Met
Leu alone was abolished by combination with
either serine mutation. This is in contrast to the single serine
mutations and most likely is due to both serines in the Met
Leu mutant being moved closer to the agonist binding
pocket. However, the exact nature of the conformational change and
whether it truly mimics the native activated state are unknown.
Nevertheless, these results are still consistent with both serines
participating in binding affinity. Likewise, in dose-response studies
with epinephrine, the Met
Leu/Ser
Ala combination virtually abolished the signal
transduction and lowered the EC
back to wild type values,
essentially reversing the agonist-dependent manifestations of
constitutive activity. These results are also consistent with the
proposed
-AR paradigm that both serines contribute to
binding affinity but only Ser
is critical in receptor
activation.
Figure 3:
Basal IP release (A),
epinephrine binding affinity (B), and IP dose response by
epinephrine (C) for wild type, Met
Leu,
and mutant
-ARs. IP
production in the
absence of agonists (panel A), competition binding (panel
B), and IP stimulation (panel C) was measured in COS-1
cells expressing the constitutively active
-AR
mutation, Met
Leu alone (
), or in
combination with either Ser
Ala (
) or
Ser
Ala (
) relative to the wild type
(
) or to mock transfected cells (vector alone). Expression levels
for each receptor were adjusted to similar values (0.3 ± 0.1
pmol/mg membrane protein) by titrating the amount of DNA used in the
transfection. The results are the mean ± S.E. of at least three
separate experiments.** indicates significant differences from the wild
type (**, p < 0.01;***, p <
0.001).
The data presented here are consistent with the model of
the -AR ligand binding site presented in Fig. 4. Two serine residues in TMV of the
-AR,
Ser
and Ser
, are responsible for part of
the agonist binding affinity. Each catechol hydroxyl is not located
equal distant from its respective serine as in the
-AR. Both hydroxyls appear closer to Ser
with the meta-hydroxyl forming the closest interaction.
For receptor activation, however, only Ser
is necessary
for full agonism. This
-AR paradigm is likely
conserved to the other two
-AR subtypes, the
- and
-ARs. Although both these
-receptor subtypes have an extra serine located at the
analogous position of 189 in the
-AR (Ser
in the
-AR, Fig. 1), we previously
demonstrated that a Ser
Ala mutation in the
-AR had no effect on agonist binding affinity and
functional responsiveness(3) . Therefore, the two serine
residues in the
-ARs are located three residues apart
in the helix while the
-AR serines are located two
residues apart. Due to the helical nature of the TM domains, this
displacement by one residue can be predicted to result in a total
rearrangement of the
-AR catechol hydroxyls in the
ligand binding pocket compared to the
-AR. As shown in Fig. 4B, the meta-hydroxyl serine residue
(Ser
) of the
-AR would be closer to
TMIV and the para-hydroxyl serine residue (Ser
)
closer to TMVI with the extra serine residue in the
-
(Ser
) and
-subtypes (Ser
)
facing away from the ligand binding pocket. This arrangement of the
hydroxyls in the
-AR would be opposite to the
alignment in the
-AR where the meta-hydroxyl
serine residue (Ser
) would be closer to TMVI and the para-hydroxyl serine residue (Ser
) would be
closer to TMIV. Therefore, the orientation of the catechol ring in the
-AR binding pocket appears to be unique and rotated
approximately 120° to that in the
-AR. This
resulting difference between the
- and
-ARs has major implications for drug design and
possible mechanistic differences in receptor activation.