Howard Hughes Medical Institute (R.S., U.K., K.W.-S., B.K.K.) and
Division of Cardiovascular Medicine (B.K.K.), Stanford University
Medical School, Stanford, California
The aim of our study was to examine the effects of different purine
nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on
receptor/G protein coupling. As a model system, we used a fusion
protein of the 
2-adrenergic receptor and the 
subunit of the G protein Gs. GTP was more potent and efficient than
ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of
several 2-adrenergic receptor ligands on nucleotide
hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The
efficacy of agonists at promoting GTP hydrolysis correlated well with
the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in
the presence of ITP. The efficacy of ligands at stimulating AC in the
presence of XTP differed considerably from the efficacies of ligands in
the presence of GTP and ITP, and there was no evidence for
receptor-regulated XTP hydrolysis. Our findings support the concept of
multiple ligand-specific receptor conformations and demonstrate the
usefulness of purine nucleotides as tools to study conformational
states of receptors.
 |
Introduction |
The
2-adrenergic receptor
(2-AR) is a prototypical G protein-coupled
receptor (GPCR) that interacts with the G protein
Gs to activate adenylyl cyclase (AC; Gilman,
1987
; Kobilka, 1992). GPCRs activate G proteins by promoting GDP
release from and GTP binding to G protein subunits (Iiri et al.,
1998). GTP-liganded Gs
activates AC, and G
protein deactivation is accomplished by GTP hydrolysis (Cassel and
Selinger, 1976; Gilman, 1987). The extended ternary complex model
assumes that GPCRs exist in an equilibrium between an inactive
state (R) and an active state (R*) (Lefkowitz et al., 1993; Gether and
Kobilka, 1998). According to this model, GPCRs can undergo R to R*
isomerization in the absence of agonist, which gives rise to a
receptor-dependent basal G protein and effector activity. Agonists
stabilize the R* state and increase G protein activity above basal
levels, whereas inverse agonists stabilize the R state and suppress
basal G protein activity (see, e.g., Chidiac et al., 1994; Samama et
al., 1994; Gether et al., 1995; Wenzel-Seifert et al., 1998a). The R*
state is also stabilized by guanine nucleotide-free G protein subunits (De Lean et al., 1980; Seifert et al., 1998a,b). The
agonist-occupied receptor and nucleotide-free G protein subunit
form a ternary complex that is characterized by high agonist affinity.
The ternary complex is disrupted by guanine nucleotide binding to the G
protein (De Lean et al., 1980; Seifert et al., 1998a,b). An increasing number of experimental observations indicate that the extended ternary
complex model cannot sufficiently explain the molecular mechanisms
underlying GPCR activation. First, Chidiac et al. (1994) have shown
that certain 2-AR agonists can either act as
partial agonists or as inverse agonists depending on whether effector system activity is assessed in intact cells or in cell membranes. Second, the extended ternary complex model proposes that inverse agonists stabilize an inactive and G protein-uncoupled state of GPCRs
(Lefkowitz et al., 1993; Gether and Kobilka, 1998). However, the
results from various studies suggest that inverse agonists induce a
specific conformation in the GPCR that actively inhibits G protein
function (Bouaboula et al., 1997; Seifert et al., 1998b). Third, the
extended ternary complex model cannot explain why defined mutations in
the dopamine D2 receptor result in
agonist-dependent changes in signaling (Wiens et al., 1998). Fourth,
the observation that not only agonists but even antagonists can promote
GPCR internalization (Roettger et al., 1997) and that some receptor
ligands behave as antagonists with respect to G protein activation but
as agonists with regard to ternary complex formation (Brown and
Pasternak, 1998) cannot be reconciled with the extended ternary complex
model. Finally, several reports showed that various synthetic and
natural opioids interact differently with the µ-opioid receptor
(Keith et al., 1996; Blake et al., 1997; Yu et al., 1997). Based on
these and several other observations, it has been proposed that there are multiple, ligand-specific GPCR conformations (Kenakin, 1996; Tucek,
1997).
The aim of our study was to explore the usefulness of guanine, inosine,
and xanthine nucleotides as experimental tools to explore
ligand-specific GPCR conformations. Previous studies had shown that
inosine and xanthine nucleotides can bind to various G proteins,
although with lower affinity than guanine nucleotides (Northup et al.,
1982; Kelleher et al., 1986; Florio and Sternweis, 1989; Klinker and
Seifert, 1997). The idea to use nucleotides as tools for analyzing
receptor conformations originated from previous studies showing that
GTP, ITP, and xanthosine 5'-triphosphate (XTP) behave differently with
respect to signaling mediated by different GPCRs that are coupled to
the same G proteins and effector systems (Wolff and Cook, 1973;
Bilezikian and Aurbach, 1974; Klinker and Seifert, 1997). In our study,
we use purine nucleotides to examine ligand-specific differences in
signaling mediated by a single GPCR. We examined the effects of
different classes of ligands on 2-AR-modulated
interactions between the G protein Gs and the purine nucleotides GDP, GTP, IDP, ITP, xanthosine 5'-diphosphate (XDP),
and XTP. As an experimental system, we used a fusion protein of the
2-AR and the long-splice variant of
Gs (GsL) expressed in
Sf9 insect cells. Fusion of the two proteins to each other does
not change the fundamental properties of either the 2-AR or Gs and allows
for sensitive analysis of GPCR/G protein coupling in terms of ternary
complex formation, GTP hydrolysis, and AC regulation (Seifert et al.,
1998a,b). The 2-AR coupled to
GsL, but not the 2-AR
coupled to GsS, possesses the hallmarks of
constitutive activity (high basal GTPase activity and high efficacy of
inverse agonists and partial agonists; Seifert et al., 1998a). The
apparent constitutive activity of the 2-AR coupled to GsL can be explained by the
relatively low GDP affinity of GsL compared
with the short-splice variant of Gs
(GsS). Specifically,
GsL is more often guanine nucleotide-free than
GsS and therefore is more often available to
stabilize the R* state. Here, we report that the potency and efficacy
of a series of 2-AR ligands at the
2-ARGsL fusion protein is dependent on the purine nucleotide that binds to
GsL. Our results provide further evidence for
ligand-specific receptor conformational states.
| |
Experimental Procedures |
Materials.
[
-32P]GTP (6000 Ci/mmol), [-32P]ITP (4000 Ci/mmol), and
[-32P]XTP (4000 Ci/mmol) were custom
synthesized by DuPont-NEN (Boston, MA). ITP, IDP, XTP, and XDP were of
the highest purity available and were purchased from Sigma
Chemical Co. (St. Louis, MO). GTP, guanine
5'-O-(3-thiotriphosphate) (GTPS), guanylyl
imidodiphosphate (GppNHp), GDP, and ATP were of the highest purity
available and were purchased from Boehringer Mannheim (Mannheim,
Germany). Nucleotide stock solutions (10 mM) were stored at 
20°C.
Nucleotide dilutions were prepared fresh daily. Sources of other
materials have been described elsewhere (Seifert et al., 1998a,b). The
construction of the fusion protein of the 2-AR
and GsL is described in Seifert et al.
(1998a,b).
Cell Culture and Membrane Preparation.
The
2-AR or
2-ARGs fusion protein
was expressed in Sf9 cells via recombinant baculovirus, as described
(Seifert et al., 1998a,b). Sf9 membranes expressing
2-ARGs were prepared according to Seifert et al. (1998a,b). The experiments described in
this study were performed in the absence of mammalian complex. The effect of mammalian
12 complex on the
function of 2-ARGs was described previously (Seifert et al., 1998b).
[3H]Dihydroalprenolol (DHA) Binding.
[3H]DHA binding studies were carried out as
described (Seifert et al., 1998a,b). Tubes contained Sf9 membranes
expressing 2-ARGs at
5.0 to 7.5 pmol/mg of protein (15-30 µg of protein/tube), 1 nM
[3H]DHA, 1 µM salbutamol (SAL) and various
nucleotides at increasing concentrations. As reported before, the
Kd value for
[3H]DHA at
2-ARGs is 0.36 ± 0.03 nM (Seifert et al., 1998b). Nonspecific binding with 1 nM
[3H]DHA, as assessed by the binding not
competed for by 10 µM ()-alprenolol, was less than 5% of total binding.
Steady-State Nucleoside 5'-Triphosphatase (NTPase) Activity.
Nucleoside 5'-triphosphate (NTP) hydrolysis was determined according to
Seifert et al. (1998a,b). Unless stated otherwise, assay tubes
contained Sf9 membranes expressing 2-AR at 6.1 pmol/mg of protein or
2-ARGs at 7.0 to 7.5 pmol/mg of protein (10 µg of protein), 1.0 mM
MgCl2, 0.1 mM EDTA, 0.1 mM ATP, 1 mM adenylyl imidodiphosphate, 5 mM creatine phosphate, 40 µg of creatine kinase, and 0.2% (w/v) BSA in 50 mM Tris/HCl, pH 7.4. Tubes additionally contained 2-AR ligands and unlabeled GTP, ITP,
or XTP at various concentrations. Assay tubes (80 µl) were incubated
for 3 min at 25°C before the addition of 20 µl of
[-32P]GTP,
[-32P]ITP, or
[-32P]XTP (0.75-2.0 µCi/tube). Reactions
were conducted for 20 min at 25°C. Reactions were terminated by the
addition of 900 µl of a slurry consisting of 5% (w/v) activated
charcoal and 50 mM NaH2PO4, pH 2.0. Charcoal absorbs nucleotides but not Pi.
Charcoal-quenched reaction mixtures were centrifuged for 15 min at room
temperature at 15,000g. Seven hundred microliters of the
supernatant fluid of reaction mixtures was removed, and
32Pi was determined by
liquid scintillation counting. Enzyme activities were corrected for
spontaneous degradation of [-32P]NTP.
Spontaneous [-32P]NTP degradation was
determined in tubes containing all of the above-described components
plus a very high concentration of unlabeled NTP (1 mM) that, by
competition with the trace concentrations of
[-32P]NTP, prevents
[-32P]NTP hydrolysis by enzymatic activities
present in Sf9 membranes. Spontaneous
[-32P]NTP degradation was <1% of the total
amount of radioactivity added. Note that, for NTPase studies,
2-ARGs was expressed at high levels to increase the sensitivity of the system (Seifert et
al., 1998b).
AC Activity.
Cyclic AMP (cAMP) formation in Sf9 membranes
was carried out as described (Seifert et al., 1998a,b). Tubes contained
Sf9 membranes expressing 2-AR at 6.1 pmol/mg
of protein or 2-ARGs
at 2.3 to 2.7 pmol/mg of protein (15-20 µg of protein/tube), 5 mM MgCl2, 0.4 mM EDTA, and 30 mM Tris/HCl, pH 7.4, and purine nucleotides and 2-AR ligands at
various concentrations. Assay tubes (30 µl) were incubated for 3 min
at 37°C before the addition of 20 µl of reaction mixture containing
(final) 40 µM [-32P]ATP (2.5-3.0
µCi/tube), 2.7 mM mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU of pyruvate kinase, 1 IU of myokinase, and 0.1 mM cAMP. Reactions
were conducted for 20 min. [32P]cAMP was
separated from [-32P]ATP as described
(Seifert et al., 1998a,b). Note that, for AC studies,
2-ARGs was expressed
at considerably lower levels than for NTPase studies. This was done to
avoid AC availability becoming limiting (Seifert et al., 1998b).
Miscellaneous.
Protein was determined with the Bio-Rad DC
protein assay kit (Bio-Rad, Hercules, CA). Data were analyzed by
nonlinear or linear regression with the Prism program (GraphPAD, San
Diego, CA). In this article, we use the term efficacy
to describe the phenomenon that different agonists and nucleotides may
vary in their ability to produce a response, although they may occupy
the same proportion of receptors and G proteins, respectively. The
efficacies of ligands on AC in the presence of GTP versus GTPase and on
AC in the presence of ITP versus ITPase and the effect of
[erythro-DL-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] (ICI) on AC in the presence of XTP were compared with the t test.
| |
Results |
Regulation of High-Affinity Agonist Binding at
2-ARGs by Guanine, Inosine, and Xanthine
Nucleotides and ATP.
One of the earliest steps of the G protein
activation/deactivation cycle is formation of a ternary complex
consisting of agonist, GPCR, and guanine nucleotide-free
G (De Lean et al., 1980; Seifert et al.,
1998a,b). The ternary complex is characterized by high agonist
affinity. On binding of a guanine nucleotide, be it GMP, GDP, GTP, or a
GTPase-resistant GTP analog, the ternary complex is disrupted and
agonist affinity decreases (De Lean et al., 1980; Seifert et al.,
1998a,b). To determine whether inosine and xanthine nucleotides can
also disrupt the ternary complex, we examined binding of a fixed
concentration of the antagonist [3H]DHA in the
presence of a subsaturating concentration of the full
2-AR agonist ()- isoproterenol (ISO; Fig.
1, A and B) and the strong partial
agonist SAL in Sf9 membranes expressing
2-ARGs. Nucleotides
at increasing concentrations were added to the binding assays.
Nucleotide binding to Gs reduces the affinity
of the 2-AR for agonist and thereby increases
[3H]DHA binding.
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Fig. 1.
Effects of GTP, ITP, XTP, and ATP on high-affinity
agonist binding in Sf9 membranes expressing
2-ARGs. Binding experiments were carried
out as described in Experimental Procedures, with
membranes expressing 2-ARGs at 5.0 to 7.5 pmol/mg of protein. Reaction mixtures additionally contained 1 nM
[3H]DHA, 1 µM SAL, and NTPs at the concentrations
indicated on the abscissa. Data are means ± S.D. of three
independent experiments performed in triplicate.
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NTPs inhibited high-affinity binding of both ()-ISO and SAL at
2-ARGs in the order
of potency GTP > ITP > XTP > ATP (ineffective). This
rank order to potency is in agreement with the data obtained for
nonfused Gs (Northup et al., 1982). The lack
of effect of ATP on high-affinity agonist binding indicates that
nucleoside diphosphate kinase-catalyzed transphosphorylation of
endogenous GDP to GTP by NTP cannot account for the effects of ITP and
XTP. In a previous study (Seifert et al., 1998b), we showed that
agonist binding in membranes expressing 2-AR
alone is guanine nucleotide-insensitive, ruling out the possibility that the 2-AR coupling to endogenous insect
Gs-like G proteins is responsible for the
observed NTP effects. In agreement with the concept that the guanine
nucleotide-free G protein subunits support ternary complex
formation (De Lean et al., 1980; Seifert et al., 1998a,b), we found
that nucleoside 5'-diphosphates (NDPs) also inhibited ternary complex
formation (order of potency, GDP > IDP > XDP). Whereas the
observed order of potency of nucleotides to inhibit high-affinity
agonist binding was expected (Northup et al., 1982; Klinker and
Seifert, 1997), differences in potency and efficacy of nucleotides
between ()-ISO and SAL were somewhat unexpected. Specifically, NTPs
and GDP were more potent at disrupting the ternary complex with SAL
than with ()-ISO (compare Fig. 1A with Fig. 1C and Fig. 1B with Fig.
1D). In addition, whereas ITP and GDP were less efficacious at
inhibiting the high-affinity binding of SAL, these nucleotides were
about similarly efficacious at inhibiting the high-affinity binding to
()-ISO.
Regulation of Basal AC Activity by GTP, ITP, and XTP in Sf9
Membranes Expressing 2-AR and
2-ARGs.
Membranes expressing
2-ARGs at 2.3 to 2.7 pmol/mg of protein had ~8-fold higher basal AC activity than
membranes expressing 2-AR alone at a higher
level (6.1 pmol/mg of protein; Fig. 2). In membranes expressing
2-ARGs, GTP increased
AC activity with an EC50 of 0.7 ± 0.1 µM.
Compared with GTP, ITP was considerably less potent
(EC50, 30 ± 5 µM) and effective at
increasing basal AC activity in membranes expressing
2-ARGs. XTP had
virtually no stimulatory effect on basal AC activity in membranes
expressing 2-ARGs.
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Fig. 2.
Effects of GTP, ITP, and XTP on basal AC activity in
Sf9 membranes expressing 2-AR or
2-ARGs. AC activity in membranes
expressing 2-AR (6.1 pmol/mg of protein) or
2-ARGs (2.3-2.7 pmol/mg of protein) was
determined as described in Experimental Procedures. AC
activity was determined in the presence of NTPs at the concentrations
indicated on the abscissa. Data are means ± S.D. of three to six
independent experiments performed in duplicate.
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It has been shown that, in certain systems expressing fusion proteins,
there can be cross talk between the fused GPCR and endogenous G
proteins of the host cell (Burt et al., 1998). Could the stimulatory
effects of GTP and ITP on basal AC activity in membranes expressing the
2-ARGsL fusion
protein be mediated by cross-activation of endogenous
Gs-like G proteins of Sf9 cells by the fused
2-AR? To address this question, we studied the
effects of NTPs on AC activity in membranes expressing nonfused 2-AR. Note that, for these studies, we
expressed the 2-AR at a level more than twice
as high as 2-ARGsL to
increase the effects seen with the nonfused receptor. Despite the high
expression level of 2-AR, the absolute
stimulation of AC by GTP, ITP, and XTP was much less efficient in
membranes expressing 2-AR than in membranes
expressing 2-ARGsL,
and the maximal AC stimulation in membranes expressing nonfused
2-AR did not even approach basal AC activity
in membranes expressing
2-ARGsL in the
absence of added nucleotides. We also had to rule out the possibility that the stimulatory effects of GTP and ITP in membranes expressing 2-ARGsL had been
caused by a nonspecific fusion-dependent perturbation of the
2-AR, resulting in high constitutive activity of the GPCR. If this were the case, we would observe a similar level of
constitutive activity when the 2-AR is fused
to either GsS or GsL.
Therefore, we compared the effects of GTP (1 µM), ITP (10 µM), and
XTP (100 µM) on AC activity in membranes expressing
2-ARGsL (2.3-2.7
pmol/mg of protein) with the corresponding NTP effects in Sf9 membranes
expressing 2-ARGsS at
a similar level (2.6 pmol/mg of protein). In membranes expressing 2-ARGsS, NTPs did not
have substantial effects on basal AC activity; i.e., the AC activities
in the presence of the different nucleotides were in the same range
(17-22 pmol · mg
1
protein · min1; data not shown). In
contrast, with
2-ARGsL, AC
activities varied by 3-fold (4-12
pmol · mg1
protein · min1; Fig. 2). In agreement with
our previous study (Seifert et al., 1998a), the AC activities with
2-ARGsS are
considerably higher than the AC activities achieved with
2-ARGsL.
Collectively, these data indicate that the observed NTP effects on AC
in membranes expressing
2-ARGsL are
attributable to the fused GsL and not to
activation of endogenous Gs-like G proteins.
Regulation of AC Activity by ()-ISO and ICI in Sf9 Membranes
Expressing 2-ARGs in Presence of GTP,
ITP, and XTP.
The full 2-AR agonist
()-ISO further increased AC activity in the presence of GTP, but the
stimulatory effect of ()-ISO did not exceed 50% (Fig.
3A). The inverse agonist ICI suppressed GTP-dependent AC activity by ~50%. In the presence of ITP, ()-ISO increased AC activity by up to 100%, whereas ICI decreased basal AC
activity by not more than 17% (Fig. 3B). The absolute
agonist-stimulated AC activity with ITP was substantially lower than
with GTP. In the presence of XTP (0.1-1 mM), ()-ISO increased AC
activity by up to 110%, but the absolute agonist-stimulated AC
activity with XTP was lower than the corresponding AC activity with ITP (Fig. 3C). Whereas ICI behaved as an inverse agonist by suppressing AC
activity in the presence of GTP and ITP, it behaved as a partial agonist in the presence of XTP. ICI increased AC activity by up to 20%
in the presence of 100 µM XTP. In Fig. 3F, the stimulatory effect of
ICI on AC in the presence of XTP is seen more clearly than in Fig. 3C,
because Fig. 3F shows AC activities normalized to basal values.
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Fig. 3.
Effects of ()-ISO and ICI on AC activity in Sf9
membranes expressing 2-ARGs in the
presence of GTP, ITP, or XTP. AC activity in membranes expressing
2-ARGs (2.3-2.7 pmol/mg of protein) was
determined as described in Experimental Procedures.
A-C, AC activity was determined in the presence of NTPs at the
concentrations indicated on the abscissa without or with ()-ISO (10 µM) or ICI (1 µM). D-F, AC activity was determined in the presence
of purine nucleotides at the indicated concentrations and ()-ISO or
ICI at increasing concentrations. Data are means ± S.D. of four
to six independent experiments performed in duplicate. The dotted lines
in A-C are extrapolations of basal AC activities to illustrate the
relative contributions of ()-ISO and ICI at the ligand-regulated
enzyme activities.
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We also studied the concentration dependence of the effects of ()-ISO
and ICI on AC activity in the presence of GTP, ITP, and XTP at fixed
concentrations. NTPs were used at concentrations that gave the highest
relative agonist stimulation of AC. In the presence of GTP, ()-ISO
increased AC activity, with an EC50 of 18 ± 8 nM (Fig. 3D). Compared with GTP, the concentration-response curves
for ()-ISO were shifted to the right with ITP
(EC50, 233 ± 34 nM; Fig.
4E) and XTP (EC50,
416 ± 44 nM; Fig. 3F). The IC50 values of
ICI to inhibit AC in the presence of GTP and ITP were similar (16 ± 8 and 22 ± 12 nM, respectively). The stimulatory effect of ICI
on AC in the presence of XTP was half-maximal at 7 ± 4 nM and
reached a maximum at 0.1 to 1.0 µM. At 0.1 and 1.0 µM, the
stimulatory effect of ICI on AC in the presence of XTP was significant
(p < .05).
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Fig. 4.
Kinetics of steady-state ITP and XTP hydrolysis in
Sf9 membranes expressing 2-AR or
2-ARGs. ITPase and XTPase activity in
membranes expressing 2-ARGs was
determined as described in Experimental Procedures.
Reaction mixtures contained 0.1 to 2.0 µM [-32P]ITP
(1.0 µCi/tube; A and B) or 0.1 to 100 µM [-32P]XTP
(2.0 µCi/tube; C and D). NTPase activities were determined in
membranes expressing 2-AR (6.1 pmol/mg of protein; A and
C) or 2-ARGs (7.5 pmol/mg of protein).
Reaction mixtures additionally contained solvent (basal) or ()-ISO
(10 µM). Data are means ± S.D. of two experiments performed in
duplicate.
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Analysis of GTPase, ITPase, and XTPase Activity in Membranes
Expressing 2-ARGs.
To obtain insight
into the mechanism by which Gs activation by
ITP and XTP is terminated, we studied ITPase and XTPase activities in
membranes expressing
2-ARGs or
2-AR. The basal ITPase activity in membranes
expressing 2-ARGs was almost twice that of ITPase activity in membranes expressing
2-AR alone (Fig. 4, A and B). Whereas ()-ISO
had no stimulatory effect on ITP hydrolysis in membranes expressing
2-AR, ()-ISO significantly increased ITP
hydrolysis in membranes expressing
2-ARGs. These data
were the first indication that Gs exhibits
substantial ITPase activity.
Membanes expressing 2-AR and
2-ARGs both exhibited
significant basal XTPase activity. However, in contrast to the data obtained for ITPase, the XTPase activity in membranes expressing 2-ARGs was not higher
than the XTPase activity in membranes expressing
2-AR alone (Fig. 4, C and D). In addition, we
did not detect a significant stimulatory effect of ()-ISO on XTP hydrolysis in membranes expressing 2-AR or
2-ARGs. It is unlikely that our failure to detect a stimulatory effect of ()-ISO on
XTP hydrolysis was because of an insensitive method, because we used
high amounts of [-32P]XTP and membranes
expressing 2-ARGs at
high levels (see Experimental Procedures). Varying the
concentration of ()-ISO from 1 nM to 1 mM, with another agonist (SAL
at 10 nM to 1 mM) and changing the concentration of
MgCl2 between 0.1 and 10 mM did not unmask
2-AR-ligand effects on XTP hydrolysis. The
lack of ligand regulation of XTPase activity was also reported for Gi protein-coupled chemoattractant receptors in
HL-60 membranes (Klinker and Seifert, 1997).
The XTPase experiments (Fig. 4, C and D) together with the agonist
competition and AC studies (Figs. 1 and 3C) suggest that XTP binds to
Gs but is not hydrolyzed. To substantiate this hypothesis, we performed competition studies with
[-32P]NTPs and various unlabeled NTPs. In a
previous study, it had already been demonstrated that the
GTPase-resistant GTP analog GTPS efficiently inhibits
-AR-mediated GTP hydrolysis in turkey erythrocyte membranes (Cassel
and Selinger, 1977a).
In a first set of experiments, we studied the effects of the
nucleotidase-resistant GTP analogs GTPS and GppNHp on ITP and XTP
hydrolysis in the presence of ()-ISO in membranes expressing 2-ARGs. GTPS and
GppNHp bind to Gs with high affinity, and
GTPS is ~7-fold more potent in this regard than GppNHp (Northup et
al., 1982). GTPS and GppNHp inhibited ITP hydrolysis according to a
biphasic function (Fig. 5A). About 40%
of the inhibition of ITP hydrolysis by stable GTP analogs was
attributable to a high-affinity interaction, whereas the remaining 60%
was attributable to a low-affinity interaction. GTPS inhibited the
high-affinity component of ITP hydrolysis approximately nine times more
potently than GppNHp, whereas, for the low-affinity component, no such difference in potency between GTPS and GppNHp was observed
(IC50, 402 ± 44 and 250 ± 33 µM,
respectively). These findings show that GTPS and GppNHp potently
compete with ITP for binding to Gs. The ITPase
that is inhibited by GppHNp and GTPS with low affinity presumably
represents the activity of nonspecific nucleotidases of Sf9 cells.
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Fig. 5.
Competition of ITP and XTP hydrolysis by GTPS and
GppNHp in Sf9 membranes expressing
2-ARGs. ITPase and XTPase activity in
membranes expressing 2-ARGs (7.5 pmol/mg
of protein) was determined as described in Experimental
Procedures. Reaction mixtures contained 1 µM
[-32P]ITP (1.0 µCi/tube; A) or 1 µM
[-32P]XTP (1.0 µCi/tube; B). Reaction mixtures
additionally contained ()-ISO (10 µM) and GTPS or GppNHp at the
concentrations indicated on the abscissa. The activities observed in
the presence of ()-ISO were set 100% (control). Data are means ± S.D. of two experiments performed in duplicate.
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In marked contrast to the biphasic competition of ITP hydrolysis by
GTPS and GppNHp, no high-affinity inhibition of XTPase by GTPS
and GppNHp was detected (Fig. 5B). The IC50
values of GTPS and GppNHp for XTP hydrolysis were 294 ± 23 and
351 ± 44 µM, respectively, and were similar to the
IC50 values for the low-affinity inhibition of
ITPase by GTPS and GppNHp.
In a second set of experiments, we compared the effects of GTPS and
XTP on ()-ISO-stimulated GTP hydrolysis (Fig.
6). As expected from previous experiments
(Cassel and Selinger, 1977a), GTPS inhibited GTP hydrolysis
(IC50, 17 ± 4 nM). If XTP binds to but is
not hydrolyzed by Gs, XTP is expected to block GTP hydrolysis, as does GTPS. Indeed, XTP abolished
()-ISO-stimulated GTP hydrolysis, although with a much higher
IC50 value than GTPS (IC50, 139 ± 22 µM). Taken together, the
nucleotide competition data and the similar basal XTPase activities in
membranes expressing 2-AR and
2-ARGs indicate that
basal XTP hydrolysis in Sf9 membranes is caused by endogenous
nucleotidases and that Gs does not hydrolyze
XTP to a measurable extent.
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Fig. 6.
Competition of GTP hydrolysis by GTPS and XTP in
Sf9 membranes expressing 2-ARGs. GTPase
activity in membranes expressing 2-ARGs
(7.5 pmol/mg of protein) was determined as described in
Experimental Procedures. Reaction mixtures contained 100 nM [-32P]GTP (0.75 µCi/tube), 10 µM ()-ISO, and
GTPS or XTP at the concentrations indicated on the abscissa. The
activities observed in the presence of ()-ISO were set 100%
(control). Data are means ± S.D. of two experiments performed in
duplicate.
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Kinetics of Agonist-Stimulated GTP and ITP Hydrolysis in Membranes
Expressing 2-ARGs.
Because of the
fixed 1:1 stoichiometry of GPCR to G protein in fusion proteins, the G
protein concentration can be determined by receptor-antagonist
saturation binding (Wise et al., 1997; Seifert et al., 1998a,b). These
properties of fusion proteins allow calculation of agonist-stimulated
NTP turnover of the fused G protein (Wise et al., 1997; Seifert et al.,
1998b). With GTP at concentrations between 0.01 and 1.00 µM, ()-ISO
stimulated GTP hydrolysis up to 250% (Fig.
7A). For each substrate concentration, the basal GTP hydrolysis rates were subtracted from the GTP hydrolysis rates observed in the presence of ()-ISO and referred to the 2-ARGs expression
level. By doing so, a Vmax of
()-ISO-stimulated GTP turnover of 1.37 ± 0.11 min1 was obtained by nonlinear regression
analysis (Fig. 7C). The Km value of the
()-ISO-stimulated GTPase is 0.18 ± 0.04 µM. These kinetic
properties of 2-ARGs
agree with data obtained for reconstituted purified -AR and
Gs (Brandt and Ross, 1986). Because the affinity
of Gs for ITP is lower than for GTP (Figs. 1 and 2; Northup et al., 1982), ITP hydrolysis was studied with higher
substrate concentrations than GTP hydrolysis. We readily detected
stimulatory effects of ()-ISO on ITP hydrolysis, with substrate
concentrations from 0.1 to 100.0 µM (Fig. 7, B and D). The
Vmax of ()-ISO-stimulated ITP hydrolysis
was 3.06 ± 0.07 min1, and the
Km was 6.3 ± 0.5 µM.
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Fig. 7.
Kinetics of steady-state GTP and ITP hydrolysis in
Sf9 membranes expressing
2-ARGs. GTPase and ITPase
activity in membranes expressing 2-ARGs
was determined as described in Experimental Procedures.
Reaction mixtures contained 0.01 to 1.00 µM
[-32P]GTP (1.0 µCi/tube; A) or 0.1 to 100.0 µM
[-32P]ITP (2.0 µCi/tube; B). Reaction mixtures
additionally contained solvent (basal) or ()-ISO (10 µM). Data are
means ± S.D. of two experiments performed in duplicate. C, basal
GTPase activity for each substrate concentration was subtracted from
GTPase activity in the presence of ()-ISO and normalized for
2-ARGs expression level (7.5 pmol/mg of
protein) to calculate molar GTP turnover of fused Gs. D,
basal ITPase activity for each substrate concentration was subtracted
from ITPase activity in the presence of ()-ISO and normalized for
2-ARGs expression level (7.5 pmol/mg of
protein) to calculate molar ITP turnover of fused Gs.
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Ligand Efficacies at 2-ARGs in
GTPase, ITPase, and AC Studies.
Fig.
8 shows the concentration-response curves
for the effects of ()-ISO and ICI on GTP hydrolysis and ITP
hydrolysis in membranes expressing
2-ARGs (7.5 pmol/mg
of protein). ()-ISO stimulated GTPase, with an
EC50 of 13 ± 3 nM, and ICI reduced GTP
hydrolysis, with an IC50 of 3.0 ± 1.2 nM.
()-ISO increased GTP hydrolysis by up to 230%, whereas ICI reduced
GTP hydrolysis by up to 50%. In agreement with the reduced potency of
()-ISO to stimulate AC in the presence of ITP (Fig. 3, D and E), the potency of ()-ISO to activate ITPase was lower
(EC50, 57 ± 10 nM) than the potency to
stimulate GTPase (Fig. 8, A and B). In the presence of ITP at 3.0 µM,
()-ISO increased ITP hydrolysis by ~30% above basal (Fig. 8B). We
could not detect an inhibitory effect of ICI on ITP hydrolysis, despite
high assay sensitivity and presumably high basal ITPase activity of
Gs (see Fig. 4, A and B).
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Fig. 8.
Concentration-response curves for the effects of
()-ISO and ICI on GTPase and ITPase activity in Sf9 membranes
expressing 2-ARGs. GTPase and ITPase
activity in membranes expressing 2-ARGs
(7.5 pmol/mg of protein) was determined as described in
Experimental Procedures. Reaction mixtures contained 0.1 µM [-32P]GTP (0.75 µCi/tube; A) or 3.0 µM
[-32P]ITP (1.5 µCi/tube; B) and ()-ISO and ICI at
the concentrations indicated on the abscissa. The dotted lines are
extrapolations of basal GTPase and ITPase activities to illustrate the
relative contributions of ()-ISO and ICI at the ligand-regulated
enzyme activities. Data are means ± S.D. of three or four
independent experiments performed in duplicate. The data shown in A are
identical with the data shown in Fig. 2B in Seifert et al. (1998a).
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Table 1 summarizes the efficacies of a
series of 2-AR ligands on GTPase and ITPase
activity and AC activities measured in the presence of GTP, ITP, or
XTP. A highly significant correlation was obtained when the efficacies
of 2-AR agonists at activating GTPase and AC
in the presence of GTP were plotted against each other (Fig. 8A).
However, when the efficacies of 2-AR agonists at activating ITPase and AC in the presence of ITP were plotted against
each other, the correlation was much less stringent than in the
corresponding experiments with GTP (compare Fig.
9, A and B). The efficacies of SAL,
()-ephedrine (EPH), dichloroisoproterenol (DCI), and ()-alprenolol
at activating AC in the presence of ITP differed significantly from the
respective efficacies of the ligands at activating ITP hydrolysis. The
inverse agonists timolol and ICI were significantly more efficacious at
reducing AC activity in the presence of GTP or ITP than at reducing
hydrolysis of the respective NTP (see Table 1). Note that the
efficacies of partial agonists on AC in the presence of XTP, most
prominently the efficacies of dobutamine (DOB) and EPH, were very low.
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TABLE 1
Efficacies of 2-AR ligands at
2-ARGs as assessed by AC Activity in
presence of GTP, ITP, or XTP and by GTPase and ITPase activity
AC activity was determined as described in "Experimental
Procedures" in membranes expressing
2-ARGs (2.3-2.7 pmol/mg of protein) in
the presence of GTP (1 µM), ITP (10 µM), or XTP (100 µM). GTPase
activity was determined in membranes expressing
2-ARGs (7.0-7.5 pmol/mg of protein) in
the presence of 100 nM [-32P]GTP. ITPase activity was
determined in membranes expressing
2-ARGs (7.0-7.5 pmol/mg of protein) in
the presence of 3 µM [-32P]ITP. Reaction mixtures
contained ligands at 0.1 nM to 1 mM as appropriate to obtain saturated
concentration-response curves. The concentration-response curves were
generated by nonlinear regression analysis, and the plateau values for
()-ISO were set 100%. The efficacies of the other agonists are
referred to the efficacy of ()-ISO. The inhibitory effects of TIM and
ICI are referred to the stimulatory effect of ()-ISO as well. Data
are means ± S.D. of three to seven independent experiments
performed in duplicate or triplicate. Comparison of the efficacies of
ligands on AC in the presence of GTP versus GTPase and on AC in the
presence of ITP versus ITPase.
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Fig. 9.
Correlation between the efficacies of agonists at
activating steady-state GTPase or ITPase activity in Sf9 membranes
expressing 2-ARGs with the efficacies of
ligands at activating AC in the presence of GTP and ITP, respectively.
A, the efficacies of partial and full agonists at activating
steady-state GTP hydrolysis in Sf9 membranes expressing
2-ARGs (7.0-7.5 pmol/mg of protein) and
AC in the presence of GTP in Sf9 membranes expressing
2-ARGs (2.0-2.7 pmol/mg of protein) as
shown in Table 1 were plotted against each other. Data were analyzed by
linear regression analysis (r2 = 0.990, p < .0001). The dotted line indicates the 95%
confidence interval of the regression line. B, the efficacies of
partial and full agonists at activating steady-state ITP hydrolysis in
Sf9 membranes expressing 2-ARGs (7.0-7.5
pmol/mg of protein) and AC in the presence of ITP in Sf9 membranes
expressing 2-ARGs (2.0-2.7 pmol/mg of
protein) as shown in Table 1 were plotted against each other. Data were
analyzed by linear regression analysis
(r2 = 0.644; p < .0092). The dotted line indicates the 95% confidence interval of the
regression line. LAB, (±)-labetalol; PRO, ()-propranolol; ALP,
()-alprenolol.
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Table 2 summarizes the
EC50 values of selected
2-AR agonists on GTPase and ITPase activity
and AC activities in the presence of GTP, ITP, or XTP. For comparison
with agonist potencies, Table 2 also contains the high- and
low-affinity Ki values for the agonists
studied. Moreover, we calculated potency ratios for the various
parameters studied. For ()-ISO, (+)-ISO, SAL, and DOB, the
EC50 values for activation of AC in the presence
of GTP were substantially lower than the EC50
values for activation of AC in the presence of ITP. The same was true
for the comparison of agonist potencies for activation of GTPase versus
ITPase activation. With the exception of EPH, the
EC50 values of agonists at activating AC in the
presence of GTP were similar to the EC50 values
of agonists at activating GTPase. Similarly, the
EC50 values of agonists at activating AC in the
presence of ITP were similar to the EC50 values
of agonists at activating ITPase. When AC activation in the presence of
XTP was considered, the EC50 values of ()-ISO, (+)-ISO, and DOB were even higher than the EC50
values for AC activation in the presence of ITP. When EPH was
considered, we noted an unexpectedly low EC50
value of the agonist at activating GTPase. However, the
EC50 values of ()-ISO, (+)-ISO, and EPH in
certain functional assays were even higher than the low-affinity Ki values. In contrast, the
EC50 values for DCI in functional assays were
lower than was expected from the Ki values
in the [3H]DHA competition-binding assay.
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TABLE 2
Potencies of selected 2-AR ligands at
2-ARGs as assessed by AC activity in
presence of GTP, ITP, or XTP and by GTPase and ITPase activity and
agonist affinities
AC activity was determined in membranes expressing
2-ARGs (2.3-2.7 pmol/mg) in the
presence of GTP (1 µM), ITP (10 µM), or XTP (100 µM). GTPase
activity was determined in membranes expressing
2-ARGs (7.0-7.5 pmol/mg) in the
presence of 100 nM [-32P]GTP. ITPase activity was
determined in membranes expressing
2-ARGs (7.0-7.5 pmol/mg) in the
presence of 3 µM [-32P]ITP. Reaction mixtures contained
ligands at 0.1 nM to 1 mM as appropriate to obtain saturated
concentration-response curves. The EC50 values of ligands were
calculated by nonlinear regression analysis. To facilitate the analysis
of the different potencies of ligands for various parameters, we also
calculated potency ratios by dividing EC50 values for two
parameters (dimensionless). For comparison of agonist potencies with
agonist affinities, the Ki values for high-affinity
(Kh) and low-affinity (Kl)
agonist binding are shown as well (expressed in nM). When distinct
high- and low-affinity sites could not be discriminated,
Ki values are listed as Kl
values. Data for ligand affinities were taken from Seifert et al.
(1998a).
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Discussion |
Interaction of Guanine, Inosine, and Xanthine Nucleotides with
Gs.
Guanine, inosine, and xanthine nucleotides
differentially form hydrogen bonds with a highly conserved aspartate
residue in G protein subunits (Noel et al., 1993; Fig.
10). The reduced affinity of
Gs for IDP and ITP compared with GDP and GTP may be because the inosine ring forms only one hydrogen bond with Asp295 in Gs (see Figs. 1, 2, and 10; Northup
et al., 1982). Repulsion of the electronegative groups in the xanthine
ring and Asp295 may explain why XDP and XTP have an even lower affinity for GS than IDP and ITP (see Figs. 1, 2, and
10). Factors that influence nucleotide affinity may also affect the
efficacy of nucleotides. GTP, ITP, and XTP do not have the same maximal effect with respect to disruption of the ternary complex and AC activation (Figs. 1-3). Thus, there may be nucleotide-specific
conformational changes in Gs that influence
interactions with the receptor and/or effector. Studies with
fluorescent guanine nucleotides already provided evidence for the
existence of nucleotide-specific G protein activation states (Remmers
and Neubig, 1996).
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Fig. 10.
Model for the interaction of guanine, inosine, and
xanthine nucleotides with Asp295 in Gs. A, a highly
conserved aspartate residue in G protein subunits (Asp295 in
GsL) forms hydrogen bonds with the substituted nitrogen
at position 1 of the guanine ring and the amino group in position 2. B,
in inosine nucleotides, the amino group at position 2 is missing.
Therefore, hydrogen bonding at this position cannot take place, and the
orientation of IDP/ITP relative to Asp295 is presumably different from
the orientation of GDP/GTP relative to Asp295. C, in xanthine
nucleotides, position 2 of the purine ring is substituted with a keto
group. Therefore, hydrogen bonding with Asp295 at position 2 of the
purine ring cannot take place. The orientation of XDP/XTP relative to
Asp295 may be different from the orientations of IDP/ITP relative to
Asp295, because there is repulsion between the electronegative keto
group of the xanthine ring and the carboxyl group of the aspartate
residue. For the sake of simplicity, the sugar moiety and phosphate
chain of NDPs/NTPs are not shown.
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Differences in the kinetics of interaction of nucleotides with
Gs could also contribute to the different
efficacies of nucleotides. NTP hydrolysis is the principal mechanism by
which Gs is deactivated (Cassel and Selinger,
1976; Gilman, 1987). The faster NTP hydrolysis proceeds, the shorter
the time for which Gs can stay in an active
conformation (Wenzel-Seifert et al., 1998b). Thus, the lower efficacy
of ITP compared with GTP at disrupting the ternary complex and
activating AC could result from the higher ITP hydrolysis rate compared
with GTP hydrolysis rate (see Figs. 1-3 and 7). Based on the
dissociation rates of GDP and IDP from G proteins, it is also likely
that the rate of ITP dissociation from Gs is
greater than the rate of GTP dissociation (Florio and Sternweis, 1989).
Fast dissociation from Gs could be another
factor that contributes to the lower efficacy of IDP and ITP compared
with GDP and GTP. For AC activation by GS in
the presence of XTP, nucleotide dissociation and not rapid hydrolysis
appears to be the major mechanism by which Gs is deactivated (Figs. 4, C and D, and 5B). Because of its low affinity
for Gs, XTP could be thought to dissociate
from Gs even before it can be cleaved. As a
result of the rapid dissociation of XTP, Gs
stays in the active state only for short periods. NTP dissociation as a
major mechanism of G protein deactivation is conceivable in view of the
fact that even highly potent G protein ligands such as GTPS or
GppNHp can dissociate from G protein subunits (Cassel and Selinger,
1977b; Hilf et al., 1992; Breivogel et al., 1998). We could not
directly study dissociation of ITP and XTP because of the low affinity
of these nucleotides for Gs.
Another mechanism that could contribute to the observed differences in
efficacy between GTP, ITP, and XTP is differential 2-AR regulation of NTP binding to
Gs. GTP binding to G protein subunits does
not passively follow GDP release but is actively catalyzed by GPCR
(Iiri et al., 1998). The dual hydrogen bonding of the guanine ring at
Asp295 could be envisaged to stabilize GTP binding to such an extent
that even the agonist-free 2-AR can
effectively increase GTP binding to Gs (see
Fig. 10A). This assumption is supported by the strong stimulatory
effect of GTP on basal AC activity and the high inverse agonist
efficacy of ICI and timolol (see Figs. 2 and 3; Table 1). Because of
the weaker hydrogen bonding of the inosine ring to
Gs (see Fig. 10, A and B), binding of ITP to
Gs is less stable than binding of GTP so that
substantial agonist occupancy of 2-AR is
required to stabilize ITP binding. In accordance with this model are
our findings that ITP is less efficient at increasing basal AC activity than GTP and that the inverse agonist efficacy in the presence of ITP
is lower than in the presence of GTP (see Figs. 2, 3, and 8; Table 1).
Additionally, agonist potency is lower in the ITPase assay and the AC
assay with ITP than in the GTPase assay and the AC assay with GTP (see
Figs. 4, D and E, and 9). Because of the repulsion of the
electronegative groups, the energy barrier for XTP to bind to
Gs may be so high that the agonist-free
2-AR is virtually ineffective at promoting
this XTP binding (see Fig. 10C). In support of this assumption, XTP
only minimally increased basal AC activity, no inverse agonist effects
were observed, and agonist potency was very low (see Figs. 2 and 3, C
and E, and Table 1).
GTP, ITP, and XTP as Tools to Analyze Ligand-Specific GPCR
Conformations.
If receptor-stimulated NTP hydrolysis is assumed to
be a function of receptor-promoted nucleotide binding and the intrinsic nucleotidase activity of the G protein, then the efficacies and potencies of ligands at activating AC and NTP hydrolysis should be
identical. In agreement with previously published data, we found that a
strong correlation exists when AC activation in the presence of GTP and
GTPase activation is considered (see Fig. 9A and Table 1; Pike and
Lefkowitz,
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2-Adrenergic Receptor/Gs Interactions:
Evidence for Multiple Receptor Conformations
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Summary
Introduction
