(Received for publication, September 26, 1995; and in revised form, January 24, 1996)
From the
We have identified previously two amino acids, one in each of
the fifth and sixth transmembrane segments of both the
-adrenergic receptor and the
-adrenergic receptor (AR), that account almost
entirely for the selectivity of agonist binding by these receptor
subtypes (Hwa, J., Graham, R. M., and Perez, D. M.(1995) J. Biol.
Chem. 270, 23189-23195). Thus reversal of these two
residues, from those found in the native receptor of one subtype to
those in the other subtype, produces complementary changes in subtype
selectivity of agonist binding. Here we show that mutating only one of
these residues in either the
-AR or the
-AR to the corresponding residue in the other subtype
(Ala
Val for the
; Met
Leu for the
-AR) results in chimeras
that are constitutively active for signaling by both the phospholipase
C and phospholipase A
pathways. This is evident by an
increased affinity for agonists, increased basal phospholipase C and
phospholipase A
activation, and increased agonist potency.
Although mutation of the other residue involved in agonist binding
selectivity, to the corresponding residue in the other subtype
(Leu
Met for the
-AR;
Val
Ala for the
-AR) does not
alter receptor binding or signaling, per se, when combined
with the corresponding constitutively activating mutations, the
resulting chimeras, Ala
Val/Leu
Met (
-AR) and Val
Ala/Met
Leu (
-AR), display wild
type ligand binding and signaling. A simple interpretation of these
results is that the
- and
-ARs
possess residues that critically modulate isomerization from the basal
state, R, to the active state R*, and that the native receptor
structures have evolved to select residues that repress active state
isomerization. It is likely that the residues identified here modulate
important interhelical interactions between the fifth and sixth
transmembrane segments that inhibit or promote receptor signaling.
-Adrenergic receptors are members of the
G-protein-coupled receptor superfamily. All three cloned subtypes
(
,
, and
)
activate multiple signal transduction pathways via distinct G-proteins
and effectors, including phospholipase C (PLC) (
)and A
(PLA
)(1, 2) .
Recently a number
of point mutations have been described in both the -AR
and other adrenergic receptors that result in partial activation in the
absence of agonist stimulation(3, 4) . Such mutations
are not unique to the adrenergic receptor superfamily, since
constitutively active somatic mutations of the human thyrotropin
receptor that result in adenomas and hyperthyroidism have also been
identified(5) . In addition, mutant hyperfunctioning
luteinizing hormone receptors have been reported that lead to
precocious puberty in young males(6) . In general, these
mutations are characterized by increased agonist binding, increased
agonist independent receptor activation, and increased agonist potency
(EC
). The increased agonist binding is independent of
G-protein binding and thus is an intrinsic property of the receptor.
Additionally, constitutive activity of these mutants can be reversed by
some antagonists. Based on these findings, as well as the observation
that overexpression of wild type receptors can also initiate
biochemical responses in the absence of agonist, it has been proposed
that receptors spontaneously isomerize between a basal state, R, and an
active state, R*. Furthermore, agonists bind with higher affinities to
the active state and ``trap'' the receptor in the R*
conformation.
With regard to the hamster -AR, two
noteworthy mutations have been identified. Substitution of Ala
in the third intracellular loop to any of the other amino acids
results in varying degrees of constitutive activity(3) . In
addition, in the third transmembrane domain, replacement of the natural
cysteine residue, Cys
, by phenylalanine also results in
constitutive activity(4) . However, by contrast with the
Ala
mutants, the increased basal effector activation and
increased agonist potency observed with the Cys
Phe mutation are pathway-specific, since they are confined almost
entirely to the PLC pathway. Here we show that when one of two residues
in either the
- or the
-ARs, which
we have shown previously are involved in determining subtype
selectivity for agonists (7) , is mutated to the corresponding
residue in the other subtype (Ala
Val,
-AR; Met
Leu, (
)
-AR), the resulting chimeras are
constitutively active for both the PLC and PLA
pathways.
Mutations in Val
in the
-AR (the
residue corresponding to Ala
in the
-AR) or Leu
in the
-AR (the residue corresponding to Met
in the
-AR) when replaced by the corresponding
residue in the other subtype, are not constitutively active. However,
when combined with the constitutively activating mutants, the resulting
double mutants, Ala
Val/Leu
Met and Val
Ala/Met
Leu, show
a restoration of native receptor signaling. This paper documents that
active state isomerization of receptors can be modulated by the
composition of specific amino acids in adjacent transmembrane domains.
Figure 1:
Schematic diagram of
the fifth and sixth transmembrane domains (TMV and TMVI) showing the locations of the targeted residues.
Ala (bold, italics) in the wild type
-AR is positionally equivalent to Val
(bold) in the wild type
-AR. Similarly
Leu
(bold, italics) in the wild type
-AR is positionally equivalent to Met
(bold) in the wild type
-AR. These
residues are postulated to be at equivalent positions on transmembrane
domains V and VI, both being one helical turn away from the
extracellular surface.
Leu
Met
alone exhibited no significant increase in the binding of either the
nonselective agonists,(-)-epinephrine and
(-)-norepinephrine (Table 1), or antagonists (data not
shown). There was, however, an increased binding affinity for
oxymetazoline and methoxamine, two
-selective
agonists, as described previously(7) . When Leu
Met was combined with Ala
Val in the
same
-AR construct, there was an overall decrease in
binding affinity for nonselective agonists as compared to the
Ala
Val mutant alone. The additional mutation of
Leu
to methionine, therefore, had an inhibitory effect on
the binding affinity for the two natural ligands. This same trend was
not apparent for the synthetic subtype-selective agonists.
Equilibrium binding studies revealed expression levels
(3.0-3.9 pmol/mg protein, respectively) for both the Ala
Val and the Leu
Met single mutants
which were similar to that observed with the wild type
-AR (Table 1). When combined, the expression
level of the double mutant (Ala
Val/Leu
Met) dropped to 1.2 pmol/mg protein, a value not
dissimilar to that observed for the wild type
-AR
(1.0 pmol/mg protein).
Figure 2:
Basal IP release by COS-1
cells mock-transfected (control) or transfected with the
-AR constructs. Panel A, basal IP
release by
-AR constructs was determined using
a radioreceptor assay as outlined under ``Experimental
Procedures.'' Values shown are the mean ± S.E.. for
10-20 separate 60-mm plates (one measurement per plate) from four
separate transfections, each containing an internal control of wild
type receptor and mock transfection. Mean receptor expression levels of
the various constructs were determined from equilibrium binding studies
and were 0.6 pmol/mg protein for the
-AR (WTb), 0.63 pmol/mg protein for the Ala
Val (A204V), 3.03 pmol/mg protein for the Leu
Met (L314M), and 1.22 pmol/mg protein for the
combination Ala
Val/Leu
Met (A204V/L314M). From each plate, 4 µl of cytosolic
supernatant from a total of 1 ml (per plate) was used to calculate
total IP
production. This represents the IP
production for approximately 1
10
cells.
Plates of cells subjected to mock transfection (no plasmid) were used
to measure background counts. The Ala
Val was the
only construct that resulted in a basal IP
release that was
statistically different from the
-AR (WTb)
(*, p < 0.05). Panel B, basal IP
release by the various
-AR constructs were
determined as described in panel A. Receptor expression was
determined as described above. In this experiment only the IP
released by Met
Leu differed significantly
from that of the wild type
-AR (WTa) (*, p < 0.05). Panel C, relationship of receptor
density to basal IP
release. Wild type (
) or the
Ala
Val mutant (
) were expressed in COS-1
cells at different receptor densities by varying the amount of cDNA
used in each transfection. The slope of the wild type receptor was 0.04
pmol of IP
/fmol of receptor, while that of the Ala
Val was 0.12 pmol of IP
/fmol of
receptor.
Figure 4:
Basal IP and arachidonic acid
release in the presence or absence of phentolamine for the wild type
and constitutively active mutations. Panel A, using the same
technique as detailed in Fig. 2, basal IP
by each of
the constitutively active mutants was determined in the presence (gray) and absence (black) of phentolamine (100
µM). Phentolamine decreased the basal IP
release for both the Ala
Val and Met
Leu mutations, although this achieved statistical
significance (*, p < 0.05) only with the Ala
Val mutant. Panel B, using the same technique as
detailed in Fig. 3, basal arachidonic acid release was
determined for both constitutively active mutations in the presence (gray) and absence (black) of phentolamine (100
µM). Both the Ala
Val and Met
Leu mutants showed a statistically significant decrease in
arachidonic acid production in the presence of phentolamine (**, p < 0.01; *, p < 0.05).
Figure 3:
Basal arachidonic acid release by COS-1
cells mock-transfected (control) or transfected with the various
-AR constructs shown. Panel A, arachidonic
acid release for the various
-AR constructs were
determined as detailed under ``Experimental Procedures.''
Values shown are the mean ± S.E. for 10-20 separate 60-mm
plates from four separate transfections, each containing an internal
control of wild type and mock transfection. One milliliter of cytosolic
supernatant from each plate was counted for radioactivity. The mock
transfection consisted of plates that were transfected with no plasmid.
This represented the background counts. Only the Ala
Val showed a significant difference from the wild type
-AR (*, p < 0.05). Receptor expression
levels were the same as for Fig. 2. Panel B, an
identical assay to the above. However, in this experiment the mutant
-AR constructs were used. The Met
Leu was the only mutant that showed a statistical
difference from the wild type
-AR (*, p <
0.05). Panel C, relationship of receptor density to basal
arachidonic acid release. Wild type (
) or the Ala
Val mutant (
) were expressed in COS-1 cells at
different receptor densities by varying the amount of cDNA used in each
transfection. The slope of the wild type receptor was 5.7 cpm/fmol
receptor, while that of the Ala
Val was 23.5
cpm/fmol receptor.
To determine agonist potency
for the PLC pathway, total inositol phosphates were determined by
adding [H]inositol 24 h before the assay and then
measuring epinephrine-stimulated inositol phosphate release. The
Ala
Val mutation showed a leftward shift in
the(-)-epinephrine concentration-response curve, as compared to
the wild type
-AR or the
-AR. The
EC
(concentration of agonist producing 50% of maximal
activation) values ± S.E. were 11 ± 1 nM, 38
± 4 nM, and 77 ± 8 nM, respectively (Fig. 5A). This change correlated with the
3-fold
decrease in the K
value for epinephrine and
norepinephrine with this mutant. Maximum
(-)-epinephrine-stimulated inositol phosphate turnover by the
Ala
Val mutant was not different from that of the
wild type receptor (Fig. 6C).
Figure 5:
Dose-response curves for IP and
arachidonic acid release using(-)-epinephrine as the agonist. Panel A, an IP assay was used to measure the potency of second
messenger production upon addition of varying concentrations
of(-)-epinephrine for the two wild type and Ala
Val mutant receptor. Five to 10 separate dose-response
experiments were performed to determine each response. Each point
represents the mean ± S.E. for each concentration. There was no
significant difference for the two wild type receptors. However, there
was a leftward shift for the mutation Ala
Val by
approximately 1 log fold. Panel B, an arachidonic acid assay
was used to measure the potency of second messenger production with
different concentrations of (-)-epinephrine, for the two wild
type receptors and for Ala
Val mutant. Five to ten
separate assays were performed to establish each response. Each point
represents the mean ± S.E. for each concentration. The two wild
type receptors did not differ significantly. However, there was a 1 log
fold leftward shift with the Ala
Val mutation. Panel C, dose-response curve for IP production using varying
concentrations of (-)-epinephrine for the wild type
-AR, the Ala
Val, and the
combined Ala
Val/Leu
Met
mutations. Each data point represents the mean ± S.E. derived
from eight separate experiments. With the Ala
Val
mutation, there was a shift to the left in the dose-response curve.
However, with the double mutation, Ala
Val/Leu
Met, the dose-response curve was now
similar to that observed for the wild type receptor. Panel D,
dose-response curve for arachidonic acid production using varying
concentrations of(-)-epinephrine for the wild type
-AR, the Ala
Val, and the
combined Ala
Val/Leu
Met
mutations. Each data point represents the mean ± S.E. derived
from eight separate experiments. Similar to panel C, with the
Ala
Val mutation there was a shift to the left in
the dose-response curve. However, with the double mutation, Ala
Val/Leu
Met, the dose-response curve
was now similar to that observed for the wild type
receptor.
Figure 6:
Dose-response curves of IP and arachidonic
release for the single and double -mutants using
(-)-epinephrine as the agonist. Panel A, dose-response
curve for IP production using varying concentrations of
(-)-epinephrine. Each data point represents the mean ±
S.E. derived from eight separate experiments. With the Met
Leu mutation, there was a shift to the left in the
dose-response curve. However, with the double mutation, Met
Leu/Val
Ala, the dose-response curve
was now similar to that observed for the wild type
-AR. Panel B, dose-response curve for
arachidonic acid production using varying concentrations
of(-)-epinephrine. Each data point represents the mean ±
S.E. derived from eight separate experiments. Similar to panel
A, with the Met
Leu mutation there was a
shift to the left in the dose-response curve. However, with the double
mutation, Met
Leu/Val
Ala,
the dose-response curve was now similar to that observed for the wild
type receptor. Panels C and D, maximal IP (C) and arachidonic acid (D) release observed
with(-)-epinephrine stimulation of the wild type
and
receptors and their corresponding single
and double mutants. Data were generated as described in panels A and B and in Fig. 5. The maximum responses for the
mutants were not significantly different (using an ANOVA) from that
observed with their respective wild type
receptors.
Like other G-protein-coupled receptors, the molecular
mechanism involved in -AR activation remains unclear.
Some insights into receptor activation have come recently from the
discovery of mutations that constitutively activate G-protein-coupled
receptors. These studies suggest that the native receptors
spontaneously isomerize between a basal (R) and activated (R*)
conformation, with only the R* conformation being able to productively
interact with the receptor-coupled G-protein and, thus, to result in
effector activation. Furthermore, agonists bind to the R* state with
higher affinity than to the R state and ``trap'' the R*
conformation. For wild type receptors, the equilibrium between the R
and R* states markedly favors the basal conformation, and, in the
absence of agonist, effector activation is minimal, unless the
receptors are highly overexpressed. With marked overexpression, the
number of receptor molecules in the R* state increases sufficiently to
allow effector activation in the absence of agonist, even though the
ratio of molecules in the R versus R* state is unchanged. By
contrast to the wild type receptors, with mutations that constitutively
activate the receptor, the energetic requirements for spontaneous
isomerization from the R to R* are reduced and thus at any instance
more receptors are in the R* conformation. This allows effector
activation in the absence of agonist and explains the other hallmarks
of constitutive activity, i.e. an increase in both agonist
binding and in agonist potency.
In this paper we have characterized
novel and
chimeras that involve
residues previously shown to be major determinants of the selectivity
of these subtypes for agonist binding. From our earlier
experiments(7) , we demonstrated that one of these chimeras,
which involves a change of Ala
in the
-AR to the equivalent residue in the
-AR (Val
), resulted in a mutant
-receptor that recognized a number of agonists
including the natural ligands,(-)-epinephrine and
(-)-norepinephrine, with higher affinity (Table 1). Because
both the wild type
- and
-ARs bind
both (-)-epinephrine and(-)-norepinephrine with similar
affinity, we questioned whether the higher agonist affinity of the
Ala
Val mutant signified that it was
constitutively active. The results show that in addition to increased
agonist binding, this mutant displayed all the properties of a
constitutively activated receptor, including agonist-independent
effector activation and increased agonist potency. What was also
intriguing was that when this Ala
Val mutation was
combined with a change in Leu
(the other residue
identified previously to be involved in subtype selectivity for
agonists) to methionine (the equivalent residue in the
-AR), all three parameters of constitutive activity
were suppressed. Thus, the phenotype of the double mutant was
indistinguishable from the wild type
-AR, except for
a reversal of its agonist-binding profile to that of the
-AR.
To confirm that this phenomenon was not
merely serendipitous or unique to the -AR structure,
we reversed the mutations in the
-AR.
Characterization of these chimeras revealed analogous but complementary
effects to those observed with the
chimeras.
Mutation at Met
to leucine (the equivalent residue in the
-AR) resulted in constitutive activity. Mutation of
Val
to alanine (the reverse of the Ala
Val mutation in the
-AR) did not change
receptor binding or signaling from that observed with the wild type
-AR. However, when combined with the Met
Leu mutation all parameters of constitutive activity were
suppressed, so that binding of (-)-epinephrine
and(-)-norepinephrine, and signaling by the double mutant did not
differ from the wild type
-AR.
To further
characterize the constitutively active chimeras, we
evaluated antagonist binding and the effects of antagonists on basal
effector activation. Phentolamine suppressed basal effector activation
by both the
-AR constitutively active mutant,
Ala
Val, and the
-AR
constitutively active mutant, Met
Leu, indicating
that phentolamine is a negative agonist. However, there was no change
in the binding affinity of the mutants for phentolamine or other
-antagonists, including the
-selective compounds, 5-methylurapidil and WB4101.
One would predict that these constitutively active mutants would
recognize these compounds with lower affinity, since fewer receptor
molecules would be in the R (basal) conformation that binds negative
agonists with higher affinity than the R* conformation. Failure to
detect a change in affinity, however, can be explained by the fact that
the Ala
Val (
-AR) and
Met
Leu (
-AR) mutations only
result in partial activation. Thus, with these mutations the proportion
of molecules in the R* state is presumably not large enough to detect a
change in affinity. In support of this notion, combination of the
Ala
Val mutation with two previously defined
constitutively active mutations, Cys
Phe and
Ala
Glu(4) , resulted in not only
increased constitutive activity, compared to either single mutation
alone, but also a lower affinity (
10-20-fold) for a number
of antagonists including phentolamine. (
)
Based on these
findings, the question arises as to the mechanism(s) involved in the
altered binding and signaling properties of these chimeras. Differences based simply on alterations in
agonist/receptor contacts due to altered properties of the substituting
residues, in terms of hydrophobicity or size, as proposed previously to
explain the changes in agonist binding alone(7) , cannot simply
explain the increased agonist-independent effector activation observed
with the Ala
Val and Met
Leu
mutations, or the restoration of wild type signaling with the addition
of the Leu
Met and Val
Ala
mutations, respectively. Rather, we speculate that the observed changes
in agonist binding and receptor signaling are due to perturbations in
the helical structure of the involved transmembrane segments. The amino
acid composition of an
-helix, for example, has been found to be
critical for determining
conformation(12, 13, 14, 15) . Also,
changing the amino acids in an
-helix can perturb interhelical
interactions(16) . In this regard, the complementarity of the
binding and signaling effects observed with the chimeras indicates
there are critical interactions between the fifth and the sixth
transmembrane segments. Thus the residues identified here may influence
helical conformation, either by direct intrahelical effects or
indirectly by an effect at the level of the side-chain packing between
adjacent helices.
Although structural resolution at the atomic level
will be required eventually to discern effects on helical conformation,
based on the following considerations, we propose a model involving
predominant effects on interhelical packing, which accounts for the
phenotypes observed with the chimeras, and provide insights into the
mechanism(s) of G-protein-coupled receptor activation. First, it is of
interest that the Ala
Val constitutively
activating mutation, as well as a previously characterized
constitutively activating
-AR mutation, Cys
Phe, involve transmembrane residues that are located
approximately one helical turn above or below residues putatively
involved in forming critical interactions with agonists. Thus, in the
fifth transmembrane domain the Ala
is one helical turn
above Ser
, which hydrogen-bonds with the catechol
hydroxyl of phenethylamines; and in the third transmembrane domain
Cys
is one helical turn below Asp
, which
forms a salt bridge with the protonated amine of agonists. This
suggests that these mutations alter the conformation of the third and
fifth transmembrane segments, or the relative positioning of the
helices, leading to an enhanced propensity for the receptor to
isomerize spontaneously to the R* state, as evidenced by increased
basal signal transduction, an agonist-independent manifestation of
constitutive activity. However, this conformational change may also
facilitate the bonding of critical residues such as Ser
and Asp
with agonist, thus leading to
agonist-dependent manifestations of constitutive activity such as high
affinity binding and increased potency. Second, the constitutive
activity of the Met
Leu
chimera, suggests that a conformational change in the sixth
transmembrane segment may also be involved in receptor signaling.
However, this may be indirect through an interaction with the fifth
helical segment, which then also influences agonist bonding with
Ser
, since mutation of Val
in the fifth
transmembrane segment to Ala relieves the constitutive activity of
Met
Leu.
Based on these considerations, we
propose that the constitutive activity of the Ala
Val is not due to the loss of alanine, which in the native receptor
structure may be postulated to act as a repressor of active state
isomerization. Rather, the valine, either because of its bulk or
-substituent, prevents normal side-chain packing between the fifth
and the sixth helical segments. As a result there is a conformational
distortion of the fifth helical segments that mimics the active state
conformation and allows enhanced interactions between Ser
and the agonist (Fig. 7). With the additional substitution
of Leu
by a methionine residue, this conformational
distortion is relieved and the receptor is no longer constitutively
active. Similarly, with the native
-AR there is
normal packing between the fifth and the sixth helical segments,
despite the presence of the wild type valine at position 185, since the
native methionine at position 292 allows the valine to be accommodated.
However, with a leucine at position 292, as occurs in the Met
Leu mutation, valine at position 185 now induces
constitutive activation, since normal interhelical packing is
prevented. Finally, this latter steric constraint can be relieved with
the substitution of Val
by alanine.
Figure 7:
A
schematic diagram outlining the concept of ``molecular repressors
and inducers.'' In the wild type -AR at position
204, there is an alanine that does not modulate activity. When replaced
to a valine, the valine either because of its bulk or
-substituent, prevents normal side-chain packing between the fifth
and the sixth helical segments. As a result there is a conformational
distortion of the fifth helical segments that mimics the active state
conformation. With the additional substitution of Leu
by
a methionine residue, this conformational distortion is relieved and
the receptor is no longer constitutively active. Similarly, with the
native
-AR there is normal packing between the fifth
and the sixth helical segments, despite the presence of the wild type
valine at position 185, since the native methionine at position 292
allows the valine to be accommodated. However, with a leucine at
position 292, as occurs in the Met
Leu mutation,
valine at position 185 now induces constitutive activation, since
normal interhelical packing is prevented. Finally, this later steric
constraint can be relieved with the substitution of Val
by alanine. Thus the valine at this position in both the
-AR and
-AR acts as a molecular
inducer by perturbing conformation, whereas a methionine in
transmembrane VI functions as a repressor, reverting the valine-induced
conformational change back to normal. TMV, transmembrane V; TMVI, transmembrane VI.
Recently,
mutational analysis of the tachykinin NK-1 receptor revealed that
replacement of two residues located at the extracellular surface of the
fifth and sixth transmembrane domains (Glu and
Tyr
, respectively) with histidine, enabled Zn
ions to be coordinated and to act as an antagonist (17) .
Although the replacement of the wild type residues with histidine may
have resulted in altered helical conformations allowing Zn
coordination, it is of interest that Glu
and
Tyr
are exactly one helical turn above Ala
(
) or Val
(
),
and Leu
(
) or Met
(
), respectively. Thus this finding with the
NK-1 receptor provides support for our contention of an important
interhelical interaction between the fifth and sixth transmembrane
domains. In addition, if the rotation of helix V is transfixed such
that Ser
of the
-AR (or Ser
of the
-AR) faces the ligand binding pocket to
allow coordination with agonist, then, as suggested by Liu et
al.(18) , the helices, including helix V and VI, have to
be arranged in a counterclockwise orientation, as viewed from the
extracellular surface. Arrangement in a clockwise orientation is
precluded, since if Ala
and Leu
(
-AR) or Val
or Met
(
-AR) have to interact, this would result in
Ser
facing away from the ligand-binding pocket toward the
lipid bilayer. If one now considers the interhelical connectivity
suggested by this study (helix V with VI), as well as the NK-1 receptor
study(17) , in addition to the connectivity between helices II
and VII, I and VII, and III and VII, demonstrated from studies of the
gonadotrophin-releasing hormone receptor(19) , the muscarinic
acetylcholine receptors(18) , and rhodopsin(20) ,
respectively, then it follows that the helices must be arranged, not
only in a counterclockwise direction, but also such that the helices
are juxtaposed in the order I, II, III, etc., to VII, rather than in
some other order, as suggested by Pardo et al.(21) and Zhang and Weinstein(22) .
At present the only available structural data on receptor activation (23) , which is of low resolution, comes from studies of bacteriorhodopsin. These data implicate conformational movement of the sixth and seventh transmembrane segments. However, this receptor is not G-protein-coupled. In support of our notion that the receptor activation involves conformational movement of the third, fifth, and possibly the sixth transmembrane domains is the finding that both the third and second intracellular loops, which are contiguous with these transmembrane segments, are involved in G-protein activation. In addition, recent spin labeling data from Khorana and Hubbell (24) indicate that photoexcitation of rhodopsin, a member of the G-protein-coupled receptor family, involves rigid movement of the third helix relative to the others in the ligand binding helical bundle.
The findings of this study also provide potential insights
into the evolution of receptor structure. This is evident from the fact
that the wild type receptors show little, if any, agonist-independent
effector activation, yet mutation of a single residue can result in
constitutive activation. This suggests that the native structures have
evolved to select residues that repress active state isomerization.
Furthermore, if a mutation did occur that introduced such a residue,
the resulting constitutively active receptor may have been deleterious
to the organism. Propagation of this receptor structure would then only
be possible if a simultaneous mutation occurred in the complementary
residue in the adjacent transmembrane segment, which could serve as a
surrogate ``molecular brake'' to suppress constitutive
activity. Although such a double mutant would have an altered agonist
binding profile when evaluated with a variety of synthetic ligands, as
observed here with the chimeras, binding of the
natural ligands,(-)-epinephrine and(-)-norepinephrine,
would be unchanged. This finding, as well as the fact that the
- and
-AR appear to activate all
effector pathways similarly, suggests that from the point of view of
the organism, such a double mutant would be phenotypically silent.
Indeed, based on such considerations, one can postulate that such an
evolutionary mechanism may underlie subtype diversity for some classes
of G-protein-coupled receptors, such as the
-AR. Our
ability to discriminate these subtypes may, thus, be due to the
development of synthetic ligands that can detect subtle differences
between the ligand recognition sites of these subtypes, which can be
exploited to develop subtype selective drugs, particularly as different
subtypes have been shown to possess different functions, even when
expressed in the same tissue. However, further studies will be
required, particularly with other classes of G-protein-coupled
receptors to confirm or refute this postulate.