From the Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195
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ABSTRACT |
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A cysteine-to-phenylalanine mutation of residue
116 in the third transmembrane domain of the
2-adrenergic receptor caused selective
constitutive activation of Na+/H+ exchange
through a pathway not involving cAMP. This selectivity was identified
by comparing binding and signaling characteristics of wild-type (WT)
versus C116F mutant receptors transiently transfected into
COS-1 cells. Indicating constitutive activity, ligand binding to the
C116F mutant showed a 78-fold higher than WT affinity for isoproterenol
and a 40-fold lower than WT affinity for ICI 118551. Although
agonist-independent activation of cAMP production was not exhibited by
the C116F mutant, a constitutive stimulation of the
Na+/H+ exchanger (NHE1) was observed. This was
identified by measuring either basal intracellular pH (pHi) or
rate of pHi recovery from cellular acid load. Due to a higher
rate of H+ efflux through NHE1, C116F transfectants
exhibited a significantly higher pHi (7.42) than did WT
transfectants (7.1). Furthermore, the rate of pHi recovery from
acid load facilitated by NHE1 was 2.1-fold faster in mutant
transfectants than in WT transfectants. The lower rate seen in the WT
case was stimulated by epinephrine, and the higher rate seen in the
mutant case was inhibited by ICI 118551. These findings, which show
that a C116F mutation of the
2-adrenergic receptor
evokes selective constitutive coupling to NHE1 over cAMP, form the
basis of our prediction that multiple and distinct activation states
can exist in G protein-coupled receptors.
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INTRODUCTION |
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The
2AR 1 is one
member of a large family of G protein-coupled receptors that mediate
the effects of numerous peptidic and nonpeptidic hormones and
neurotransmitters. As with all G protein-coupled receptors, the common
structural feature of this family is a single polypeptide chain with
seven hydrophobic regions that constitute seven transmembrane-spanning
domains. Classification of adrenergic receptors (
1,
2, and
), which as a family mediate the physiological effects of the sympathetic nervous system, is based on homology of
amino acid sequences and pharmacology of various ligands (1). In the
case of the
2-subtype, dogma suggests that the
endogenous catecholamines epinephrine and norepinephrine promote
receptor coupling to G
s and downstream cAMP production
(2, 3). Since
2ARs are most abundant in smooth muscle,
the best characterized effects of cAMP activation include smooth muscle
relaxation in bronchial tubes and in vascular tissue (4).
The process of receptor activation and G protein coupling that evokes cellular effects is described by the widely accepted revised ternary complex model (5, 6). In this allosteric model, the active conformation of a native receptor is the cornerstone of the agonist-receptor-G protein complex that leads to signaling. Without agonist present, the model predicts spontaneous receptor isomerization between the inactive (R) and active (R*) conformations, with equilibrium under native conditions shifted toward R. At any given time, even though most receptors reside in R, a small population will reside in R*, permitting formation of the R*·G protein complex that causes effector activation. The model predicts that the addition of agonist does not directly convert the receptor from R to R*. Rather, the agonist will preferentially bind to receptors already in R*, thereby shifting isomerization equilibrium away from R. Receptor mutations that induce an agonist-independent shift in isomerization equilibrium toward the R* conformation are termed constitutively active and so by definition couple to and evoke second messenger responses in the absence of agonist.
The studies contributing to the development of the revised ternary
complex model were based primarily on the characterization of
2AR signaling, which was assumed to be through a single
effector pathway (adenylate cyclase via coupling to G
s).
However, recent studies indicate that besides coupling to
G
s,
2ARs also couple to an alternate
protein in the G
family (possibly G
13),
which leads to newly discovered cellular effects, including stimulation
of NHE1 (7-9), via Cdc42- and RhoA-dependent pathways (10). Therefore, in the present study,
2AR signaling via
both cAMP production and Na+/H+ exchange was
examined in a receptor that was engineered to have a single amino acid
mutation of Cys-116 in the third transmembrane domain. Cys-116 is
situated approximately one helical turn below Asp-113, which is the
putative counterion that binds the protonated amine of adrenergic
ligands (11, 12). Because of the important role of Asp-113 in ligand
binding, any perturbation of the third transmembrane helix could affect
both the ligand-binding pocket and second messenger coupling.
Interestingly, the C116F mutation induces a receptor conformation that
constitutively activates Na+/H+ exchange while
only maintaining competent coupling to cAMP. This finding is similar to
the result observed in an analogous C128F mutation of the
1bAR, which exhibits selective constitutive coupling to
inositol metabolism over the arachidonic acid pathway (13). Work in our
laboratory with C116F
2AR and C128F
1bAR
has formed the basis of an emerging hypothesis suggesting that
receptors are capable of forming multiple activation states that are G
protein-specific. An understanding of conformational differences
between these distinct states may eventually lead to the design of
signaling-specific therapeutic agents. At least in the case of
receptors that couple to more than one G protein, we suggest that the
current model of receptor activation be revised to include
isomerization from the R conformation to distinct active conformations
that exhibit G protein selectivity.
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EXPERIMENTAL PROCEDURES |
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Media-- HEM buffer was composed of 20 mM Hepes (pH 7.5), 1.4 mM EGTA, and 12.5 mM MgCl2. Fluorescence medium consisted of 125 mM NaCl, 5 mM KCl, 1.7 mM CaCl2, 0.7 mM NaH2PO4, 0.8 mM Na2SO4, 0.5 mM MgCl2, 15 mM Hepes (pH 7.4), 2 mM L-glutamine, and 10 mM D-glucose. Potassium-based fluorescence medium was identical to fluorescence medium except that it contained 20 mM NaCl and 110 mM KCl.
Site-directed Mutagenesis--
The cysteine-to-phenylalanine
mutation was made at residue 116 of the human 2AR using
cassette replacement as described previously (14). cDNAs were
sequenced by the dideoxy method (Sequenase kit, Amersham Corp.) to
confirm the mutation. The synthetic human
2AR was then
subcloned into the eukaryotic expression plasmid pMT2
(15) and
purified.
Cell Culture and Transient Transfection--
COS-1 cells
(American Type Culture Collection), known to be AR-negative based on
the lack of epinephrine-evoked cAMP responses (16), were grown in
Dulbecco's modified Eagle's medium (Sigma) + 10% fetal bovine serum
(Life Technologies, Inc.). Cells were plated onto either 150- or 60-mm
tissue plates for membrane preparations or second messenger studies,
respectively, or onto UV-clear
T dishes (Bioptechs) for fluorescence
assay. Transient transfection was achieved by the DEAE-dextran method
(17). Efficiency of transfection was typically near 20%. Cells were
harvested or assayed 60 h after transfection.
Membrane Preparation and Radioligand Binding--
COS-1 cell
membranes were prepared as described previously (15). Membrane protein
concentration was determined using the Bradford method (18).
Competition binding experiments with the antagonist radioligand
[125I]CYP were carried out in a final volume of 0.25 ml
containing HEM buffer + 0.1% bovine serum albumin, 150 pM
[125I]CYP, COS-1 membranes, and varying concentrations of
unlabeled ligand. Nonspecific binding was determined in the presence of 105 M propanolol. Reactions were run at room
temperature for 1 h, after which time the mixtures were filtered
onto Whatman GF/C glass fiber filters using a Brandel cell harvester.
Bound radioactivity was quantitated with a Packard Auto-gamma 500 counter. Binding data were analyzed with the iterative curve-fitting
software package GraphPad Prism. Saturation binding studies were
performed with increasing concentrations of [125I]CYP
(5-600 pM) in the same buffer as used in competition
binding experiments. To minimize variation, WT and C116F
2AR binding experiments were always performed
simultaneously. Statistical significance in both binding and functional
assays was identified by the two-tailed Student's t
test.
cAMP Determination--
Accumulation of cAMP in WT and C116F
2AR transfectants was measured using a commercially
available cAMP assay system (Amersham Corp.) according to the
directions supplied by the manufacturer. Cell extracts were derived
from cultures in 60-mm dishes that were preincubated for 30 min with 5 mM theophylline and then for 30 min with both theophylline
and increasing concentrations of agonist.
Measurement of Intracellular pH Using BCECF--
Transfected
COS-1 cells, cultured in T dishes, were incubated for 15 min at
37 °C with 0.3 µM BCECF/AM (Calbiochem).
Unincorporated dye was washed away by perfusion with fluorescence
medium until extracellular fluorescence was undetectable.
Intracellularly trapped BCECF fluorescence was measured with a Delta
Scan dual wavelength spectrofluorometer (Photon Technology
International) functionally linked to an Olympus inverted fluorescence
microscope. Cells were alternately excited with 440- and 500-nm light
while the intensity of the 530 nm emission was measured with a
photomultiplier tube. Emission intensities at the two exciting
wavelengths were collected every 0.2 s during continuous perfusion
of the cells with medium. Using an adjustable shudder, the emission
from an area containing only 20-30 cells was collected for analysis.
Cellular autofluorescence and contaminating fluorescence from leaked
dye were found to be negligible, but background corrections were made
to remove contributions from ambient light. Excitation ratios (500/440
nm) were calibrated for pHi with 10 µM nigericin
in potassium-based fluorescence medium as described previously (19).
Fig. 1 shows the averaged results of
seven separate calibrations that indicate a linear relationship between
the 500/440 nm ratio and pHi in the physiological range (pH
6.3-7.8). For each experimental ratio collected, a pHi value
was extrapolated from this calibration plot. For certain experiments,
cells were acid-loaded via a 6-min transient exposure to 20 mM NH4Cl (20). Rate of recovery of pHi
from this induced acidosis was determined by calculating the derivative
of the slope of the pHi time course tracing for 10-s intervals
through the first 120 s of recovery.
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RESULTS |
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Pharmacological Comparison of WT and Mutant
Receptors--
Receptor density and [125I]CYP binding
affinity were determined in COS-1 cells transfected with either the WT
2AR cDNA or the C116F mutant.
[125I]CYP labeled an apparently homogeneous population of
binding sites with similar affinity in membranes prepared from both
transfected cell populations. Binding of the radioligand was
statistically best fit to a one-site model with mean
KD values for the WT and C116F receptors of 97.3 and
125.1 pM, respectively (data not shown). When transfecting
cells cultured in 150-mm plates with 8 µg of plasmid DNA, the mean WT
and mutant receptor densities were 0.398 and 0.058 pmol/mg,
respectively.
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Stimulation of cAMP Formation--
Functional coupling of WT and
C116F 2ARs to the cAMP pathway was examined by measuring
accumulation of cAMP. For this experiment, the transfection protocol
was modified to assure similar receptor expression levels in WT and
C116F transfectants. This was accomplished by titering down the amount
of WT cDNA introduced to the cells until
Bmax was approximately equal to values seen in
the mutant case. Mean receptor expression in titered WT transfectants
was 0.063 pmol/mg compared with 0.056 pmol/mg in mutant transfectants. Fig. 2 compares the isoproterenol
concentration dependence of cAMP production in cells expressing similar
levels of either WT or C116F receptors. Arguing against constitutive
coupling to G
s and cAMP production, the C116F mutant
receptor did not evoke a response in the absence of isoproterenol, and
it did not affect the potency of isoproterenol to evoke a response
compared with the WT receptor. The agonist concentration curves seen in
the two transfected cell populations were nearly identical, with mean EC50 values of 90.8 and 79.3 nM, respectively.
Like isoproterenol, epinephrine concentration-response curves and
EC50 values seen in WT and C116F transfectants were not
different (data not shown).
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Stimulation of Na+/H+ Exchange--
A
possible alternative constitutive coupling of C116F 2ARs
to G
13 and downstream NHE1 was initially examined by
studying the effect of this hypothetical constitutive activity on basal pHi. Continuous stimulation of H+ extrusion through
the Na+/H+ exchanger by a constitutively active
C116F mutant receptor would likely cause cytosolic alkalosis.
Therefore, using basal pHi as a marker of constitutive
activity, resting pHi levels in COS-1 cells were determined.
Fig. 3 illustrates that C116F receptor-transfected cells had a significantly more alkaline
pHi (7.42) than either WT transfectants (7.1) or untransfected
cells (7.04). This was despite 6.8-fold more receptors on WT than on mutant cell membranes. As expected, intracellular alkalosis in the
C116F transfectants was inhibited by inactivating the mutant receptors
with the inverse agonist ICI 188551 (10
5
M).
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DISCUSSION |
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In the current revised ternary complex model describing the
activation of G protein-coupled receptors (5, 6), agonists do not
directly drive the inactive receptor to assume the active conformation.
Rather, the model predicts that agonists will preferentially bind to a
receptor's R* conformation, thus stabilizing R* and shifting the
isomerization equilibrium away from R. Conversely, inverse agonists,
which preferentially bind to R, promote receptor inactivation by
shifting the equilibrium away from R* (23, 24). Constitutively active
mutant receptors that exhibit an increase in agonist affinity and/or a
decrease in inverse agonist affinity do so because a shift in
equilibrium toward the active conformation has already occurred. A
mutation-induced shift of a receptor toward R* will, by definition,
facilitate agonist-independent coupling to G proteins, thus causing
constitutive activation of effector pathways. Based on these tenets of
the model, the higher than WT binding affinity of common
2-agonists seen for the C116F
2AR (Table
I) is strongly suggestive of constitutive receptor activity. Consistent
with receptor theory (6), the degree of high affinity shift was
proportional to the intrinsic efficacy of the agonist tested. Also
supporting this hypothesis, the
2-inverse agonist ICI
118551 exhibited a higher affinity for the WT receptor than for the
C116F mutant (Table I). Again, it appears that the degree of shift to
lower affinity was proportional to the inverse efficacy of the
antagonist tested. ICI 118551, which is known to be a highly efficacious inverse agonist (24), showed the largest rightward shift in
affinity for the mutant receptor (Table I). As expected, alprenolol and
propanolol, which are classified either as weak inverse agonists (23)
or as neutral antagonists (24), exhibited smaller rightward affinity
shifts. Overall, these changes in ligand binding affinity are intrinsic
to the mutant receptor and are not due to altered G protein precoupling
as evidenced by the inability of Gpp(NH)p to affect
Ki values or Hill coefficients (data not shown).
The C116F mutation could evoke these shifts in ligand binding affinity
in one of two ways. First, since the 116th residue is located in the
third transmembrane domain only one helical turn below Asp-113, the
counterion for the protonated amine of catecholamines (11, 12), it
seems possible that it could indirectly affect interaction of the
receptor with ligand per se. However, since the function of
the receptor has also been altered and correlates to an activational
paradigm, this scenario seems unlikely. Rather, we suggest that the
mutation has affected receptor conformation to mimic the activated
state. Since we have postulated that the equivalent mutation in the
1bAR (C128F) is involved in modulation of an important
salt bridge constraint that stabilizes the inactive conformation (25),
it is possible to envision that C116F is influencing a similar
constraining factor.
Even though the binding data allude to a constitutive activity induced
by the C116F mutation, agonist-independent generation of a second
messenger signal must be identified to firmly establish this
hypothesis. Classically, it is held that the 2AR evokes cellular effects through a coupling to cAMP production via
G
s (2, 3). Based on this, agonist-dependent
and -independent effects on cAMP production were compared in WT and
C116F receptor-transfected COS-1 cells. Not only did the C116F mutation
fail to potentiate the isoproterenol dose response (EC50)
relative to the WT receptor, it also failed to evoke a response in the
absence of the agonist (Fig. 2). Similar results were seen with
epinephrine (data not shown). These findings indicate that the C116F
mutation does not facilitate constitutive coupling to
G
s. Rather, the mutant and WT receptors show equal
competency in the generation of agonist-evoked cAMP responses. If the
2AR indeed only signals through a coupling to
G
s, these data would suggest that the C116F mutation
affects ligand binding without inducing the receptor to assume an
activated conformation that is competent to couple to a second
messenger signal.
As was mentioned, a coupling of 2ARs to
G
s has been convincingly established in the literature.
Because of this, when studying
2-receptor pharmacology,
the inclination has been to measure cAMP as a marker of G protein
coupling. Over the past decade, however, a strong case for an alternate
pathway has emerged. Early work by Strader et al. (7) showed
that
-receptor mutants, uncoupled from G
s due to
deletion of G protein-interacting domains (residues 222-229 and
258-270), exhibited marked increases in agonist affinity. Similarly,
mutagenesis of Asp-130 (in the third transmembrane domain) also led to
increases in agonist binding affinity despite receptor uncoupling from
G
s (26). The G
s-uncoupled 222-229
deletion mutant was later shown by Barber and Ganz (8) to activate
Na+/H+ exchange via an unknown G protein. This
study established the novel hypothesis that
G
s-independent cellular responses were evoked by
2ARs. Since then, Barber and co-workers (9) have identified G
13 as responsible for activating a pathway
that stimulates Na+/H+ exchange through the
ubiquitous NHE1. At least one other member of the G
subfamily, G
12, has since been implicated in the
regulation of various isoforms of NHE1 (27), although the G
12 effects are inhibitory. These studies collectively
support a hypothesis implicating G
13 in coupling of the
2AR to Na+/H+ exchange.
With Gs-independent coupling of
2ARs to
NHE1 having been established, questions are raised about possible
effects of the C116F
2AR mutant on
Na+/H+ exchange. NHE1, which is present on the
plasma membrane of all mammalian cells, fulfills several distinct
physiological functions including control of pHi and
maintenance of cellular volume (28). NHE1 function, which is commonly
measured by monitoring pHi with the fluorescent dye BCECF, was
examined via two approaches. Basal pHi was determined in WT and
C116F receptor-transfected COS-1 cells. Due to continuous
H+ extrusion during the post-transfection period, possible
constitutive activation of NHE1 induced by the C116F mutation would be
detected via identification of a cytosolic alkalosis relative to
pHi values seen in the WT case. However, since pHi is
not a direct measure of NHE1 activity, the actual rate of NHE1 function was also determined in WT and C116F transfectants by measuring the rate
of pHi recovery following NH4Cl-induced cellular acid load.
In the first experiment, intracellular alkalosis was evident in C116F
receptor-transfected COS-1 cells relative to pHi levels seen in
WT transfectants (Fig. 3). This was despite a 6.8-fold lower number of
receptors in the C116F case. Locking the mutant receptor in the
inactive state with the inverse agonist ICI 118551 blocked the effect,
with treated mutants exhibiting a basal pHi similar to the WT
case (Fig. 3). These findings not only provide strong evidence of a
constitutive activation of Na+/H+ exchange in
C116F receptor-transfected cells, but the effect is shown to be
2-receptor-specific based on inhibition by the
2-selective inverse agonist.
In the second experiment, cells transfected with the C116F
2AR showed a significantly faster rate of pHi
recovery from acid load than WT transfectants. Confirming facilitation by NHE1, H+ extrusion during pHi recovery was
blocked by dimethylamiloride, a specific NHE1 antagonist (22) (Fig.
4). Recovery rate, quantitated by calculating
dpHi/dt, was >2-fold greater in mutant transfectants than in WT transfectants (Fig. 5). As before, this significant difference was seen despite a 6.8-fold lower receptor number in the C116F receptor-transfected cells. Further implicating the
2AR as a regulator of NHE1, activation of the WT
receptor with epinephrine caused an increase in
dpHi/dt to values seen in the C116F case, whereas
inactivation of the C116F receptor with ICI 118551 reduced
dpHi/dt to values seen in the WT case. These results
(a) corroborate earlier findings demonstrating coupling of
2ARs to NHE1, (b) indicate selective
constitutive coupling of the C116F
2AR to NHE1, and
(c) establish a role for Cys-116 in regulating selective
coupling of the
2AR to either the cAMP pathway or NHE1.
It should be noted that coupling of the
2AR to NHE1 has
been demonstrated in cell lines that endogenously express the receptor
(29). Therefore, it is unlikely that the results presented in this
report are due to a promiscuous coupling of the receptor to
G
13 in COS-1 cells.
Interestingly, epinephrine did not affect the rate of H+ extrusion seen in C116F receptor-transfected cells (Fig. 5). It is possible that the C116F mutation had itself induced a maximal shift of receptor equilibrium toward R*. Treatment with agonist under these conditions would not evoke additional stimulation. Alternatively, it is also possible that NHE1 was functioning at its maximal rate either because of strong constitutive activation induced by the mutant or due to the action of other mechanisms compensatory to the large H+ efflux. These factors would render additional stimulation of the signal by agonist undetectable. Overall, the present findings cannot distinguish between these two possibilities. One possible compensating factor, extracellular Na+ concentration, is not likely rate-limiting in this case because Na+/H+ exchangers normally operate at saturation with respect to Na+ at physiological Na+ levels (30).
Based on the data presented in this report, a model is established that
shows selective constitutive activation of NHE1 over cAMP in
2ARs possessing a C116F mutation (Fig.
6). The model predicts that under basal
conditions, the C116F mutant will preferentially isomerize to a
conformation that couples putatively to G
13 (R*·G
13). Fewer receptors will isomerize to the
uncoupled state (R), while fewer still will reside in the
G
s-coupled conformation (R*·G
s). In the
presence of agonist, competent coupling to G
s is finally
achieved due to a shift in equilibrium that leads to a significant
number of receptors in the G
s-coupled conformation.
Therefore, according to our model, the mutation selectively induces a
putative R*·G
13 conformation that does not restrict
agonist induction of the WT receptor activation scenario (i.e. coupling to both second messenger pathways).
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The importance of Cys-116 in controlling the transition of the receptor
to a selectively coupled conformation suggests a role for this amino
acid position as a "switch" that determines the trafficking of the
receptor between multiple active states. The hypothesis that Cys-116
can function as a switch is supported by earlier studies of
1b-adrenergic and AT1 receptors. Cys-116 in
the
2AR is 14 residues N-terminal to the highly
conserved DRY sequence found on the cytoplasmic border of the third
transmembrane domain. DRY, an important functional domain for the
binding of G proteins and the catalysis of GDP release (31), provides a convenient reference for comparing the C116F
2 mutation
with similar point mutations in
1bARs, AT1
receptors (Fig. 7), and possibly other G
protein-coupled receptor systems.
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In the 1bAR system, the 14th position N-terminal to the
DRY domain is Cys-128 (Fig. 7). Analogous to the C116F
2AR, a C128F mutation of the
1bAR
exhibited a selective constitutive coupling to one second messenger
over another. In the case of the
1bAR mutant,
constitutive coupling to the inositol pathway via a pertussis toxin-insensitive G protein was achieved, whereas only competent coupling to the pertussis toxin-sensitive arachidonic acid pathway occurred (13). Comparatively, an A293E mutation of this receptor exhibited constitutive activity (32) that was distinct from the C128F
mutant because of constitutive coupling to both pathways (13). The
ability of the C128F mutant to achieve a presumed Gq-coupled/Gi/o-uncoupled state in the absence
of agonist could simply be a manifestation of more efficient coupling
to Gq, the supposed primary second messenger pathway in the
1bAR system. It was argued, however, that the R state of
the C128F mutant receptor did not isomerize to a single active
conformation. Rather, it was postulated that R can isomerize to at
least two distinct active states: one coupled to Gq and one
coupled to Gi/o. Given the results of this earlier study,
either the G
13/NHE1 pathway is a more efficient coupler
to
2ARs than the Gs/cAMP pathway, or the
conclusions of the C128F
1bAR study are valid.
Analogous to both Cys-128 in the 1bAR and Cys-116 in the
2AR, Asn-111 resides at the 14th position N-terminal to
the DRY domain of the AT1 receptor (Fig. 7). In the
AT1 system, the constitutively active mutant N111G
exhibited higher than WT activation of inositol metabolism under basal
conditions (33). However, this constitutive activation was only ~50%
of the agonist-evoked response seen in the WT receptor. Noda et
al. (33) suggested that the N111G mutation induced a transition of
the receptor from R to a partially activated intermediate (R
).
Supporting their model, agonistic ligands induced full activation of
inositol metabolism by the N111G mutant, suggesting that transition of
the mutant receptor from R
to the fully activated R* conformation
could be achieved. In agreement with conclusions drawn in the present
study, the authors postulated that Asn-111 is a switch that controls
isomerization between multiple and distinct activation states of the
receptor. Given the diverse nature and lack of amino acid conservation
between G protein-coupled receptors, the behavior of Asn-111 in the
AT1 receptor and of Cys-128 and Cys-116 in the adrenergic
receptors suggests that, in general, this particular amino acid
position may function as a switch that regulates trafficking between
distinct receptor conformations.
The findings presented in this report corroborate previously published
work (7-9) describing a coupling of 2ARs to NHE1, putatively through G
13. Additionally, we identify novel
selective constitutive activation of NHE1 over the cAMP pathway induced by a Cys-to-Phe mutation at residue 116 of the
2AR.
Based on this, we suggest an important role for Cys-116 as a switch
residue that controls isomerization between at least two G
protein-specific activation states of the
2AR. When
considering the present results along with the data of earlier studies,
we hypothesize that, in general, the amino acid located at the 14th
position N-terminal to the conserved DRY domain of G protein-coupled
receptors is a critical entity in the control of receptor isomerization
between R, R*, and other distinct activation states that may exist.
This work supports Kenakin's "cubic" ternary complex model (34) by predicting the formation of two different ternary complexes when a single receptor interacts with two different G proteins (Fig. 8A). In contrast to this model, however, we argue that such a receptor can adopt two distinct activated conformations (R*1 and R*2), each selectively interacting with a different G protein. Based on this, we offer the bicubic model (Fig. 8B) as a revision of Kenakin's current model, which predicts only a single active conformation (R*) that partitions among the G proteins. In either scheme, agonists may be developed that selectively traffic the receptor to a particular activated conformation. Based on this, such signaling-specific agonists would offer vast advantages over promiscuous drugs. These compounds would not only extend our understanding of receptor-pathway-response coupling, but they might also belong to an important new class of therapeutic agents.
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ACKNOWLEDGEMENTS |
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We thank Dr. Derek Damron (Anesthesiology Department, Cleveland Clinic Foundation) for use of the fluorescence microscopy facility and Dr. Robert Graham (Victor Chang Institute, Sydney, Australia) for critique of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant RO152544 and an unrestricted grant from Glaxo Wellcome (to D. M. P.) and by a fellowship from the Northeast Ohio Affiliate of the American Heart Association (to J. E. P).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Performed this work during the tenure of an Established
Investigator award from the American Heart Association. To whom
correspondence should be addressed: Dept. of Molecular Cardiology,
FF3-01, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH
44195. Tel.: 216-444-2058; Fax: 216-444-9263; E-mail:
perexd{at}cesmtp.ccf.org.
1
The abbreviations used are: 2AR,
2-adrenergic receptor;
1bAR,
1b-adrenergic receptor; NHE1,
Na+/H+ exchanger; [125I]CYP,
(
)3-[125I]iodocyanopindolol; WT, wild-type; BCECF,
2
,7
-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; pHi,
intracellular pH; Gpp(NH)p, guanosine 5
-(
,
-imido)triphosphate; AT1 receptor, angiotensin 1 receptor.
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REFERENCES |
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