From the Howard Hughes Medical Institute, ** Division
of Cardiovascular Medicine, Stanford University Medical School,
Stanford, California 94305-5428
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ABSTRACT |
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The 2-adrenoreceptor
(
2AR) couples to the G-protein Gs to mediate
adenylyl cyclase activation. The splice variants of Gs
differ by a 15-amino acid insert between the Ras-like domain and the
-helical domain. The long splice variant of Gs
(Gs
L) binds GDP with lower affinity than the
short splice variant (Gs
S), but the impact
of this difference on the interaction of Gs
with the
2AR is not known. We studied the
2AR/Gs
interaction using receptor/G-protein fusion proteins
(
2ARGs
S and
2ARGs
L) expressed in
Sf9 cells. Fusion of the
2AR to Gs
promotes efficient coupling as shown by high-affinity agonist binding
and GTPase and adenylyl cyclase activation and ensures fixed
stoichiometry between receptor and G-protein. Importantly, fusion does
not change the fundamental properties of the
2AR or
Gs
. The
2AR in
2ARGs
L showed hallmarks of
constitutive activity (increased potency and intrinsic activity of
partial agonists, increased efficacy of inverse agonists, and increased
basal GTPase activity) compared with the
2AR in
2ARGs
S. The apparent
constitutive activity of the
2AR in
2ARGs
L may be due to the
lower GDP affinity of Gs
L compared with
Gs
S, i.e. Gs
L is more often nucleotide-free than
Gs
S and, therefore, more frequently
available to stabilize the
2AR in the active (R*) state.
This study demonstrates that subtle structural differences between
closely related G-protein
-subunits can have important consequences
for the functional properties of a G-protein-coupled receptor.
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INTRODUCTION |
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Numerous hormones and neurotransmitters exert their effects
through G-protein-coupled receptors
(GPCRs)1 (1-4). The
2-adrenoreceptor (
2AR), a prototypical
GPCR, interacts with the G-protein Gs, causes GDP/GTP
exchange at its
-subunit (Gs
) and, thereby, leads to
activation of adenylyl cyclase (AC). Recently, the ternary complex
model of GPCR activation has been extended to explain the finding that
GPCRs can activate G-proteins, even in the absence of agonist, and that
certain receptor ligands, namely inverse agonists, can suppress the
G-protein activation mediated by agonist-free GPCRs (5-14). The
agonist-independent activity of a GPCR is referred to as constitutive
activity. The extended ternary complex model (two-state model) assumes
that agonists stabilize GPCRs in the active (R*) state, while inverse agonists stabilize the inactive (R) state. Although constitutive GPCR
activity can be most easily observed when receptors are overexpressed (10-12) or mutated (7, 8, 13), it also occurs at physiological receptor expression levels (5, 6, 9, 14). Hallmarks of constitutive
GPCR activity are increased potency and efficacy of partial agonists,
increased efficacy of inverse agonists, and elevated basal G-protein
activity (5-14). These properties of constitutive activity are
generally associated with GPCR function, and little is known about the
ability of different G-proteins to influence the efficacy and potency
of ligands.
Gs exists as a short (Gs
S)
and a long (Gs
L) splice variant. Compared
with Gs
S, Gs
L
contains additional 15 amino acids inserted at position 72 of the
polypeptide chain, and there is an exchange of glutamate for aspartate
at position 71 (15, 16) (Fig. 1A). Based on the
-carbon
model of the
-subunit of the retinal G-protein tansducin (17), the
sequence within which the 15-amino acid insert is localized in
Gs
L serves as a linker between the Ras-like
domain and the
-helical domain (Fig. 1B). The guanine
nucleotide-binding site is embedded between these two domains. Thus, a
change in this linker sequence might be expected to influence the
binding kinetics of guanine nucleotides. In fact, purified
Gs
L releases GDP more than twice as fast as
Gs
S (18).
The results of a previous study indicate that
GsS may be more effective than
Gs
L in activating AC (19), but with regard to
2AR coupling, studies have not revealed significant
differences between Gs
S and
Gs
L (18, 20, 21). Studying differences in
the interaction of structurally very similar G-proteins with a given
GPCR is technically difficult, because functional interactions between
receptors and G-proteins are strongly influenced by their relative
expression levels (22). Specifically, defined receptor/G-protein stoichiometries have to be achieved to be able to detect subtle differences in GPCR/G-protein coupling.
To facilitate the examination of receptor/G-protein interactions we
constructed fusion protein DNAs in which the C terminus of the
2AR was linked to the N terminus of
Gs
S
(
2ARGs
S) or Gs
L
(
2ARGs
L) (Fig. 1A)
and expressed the fusion proteins in Sf9 cells. Fusion proteins
have a fixed ratio of receptor to
-subunit (23, 24). Thus,
ambiguities in data analysis because of varying stoichiometry of the
signaling partners can be eliminated. Using the fusion protein
approach, we observed that the
2AR coupled to
Gs
L has properties of constitutively active
GPCR.
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EXPERIMENTAL PROCEDURES |
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Materials--
Rat GsL DNA was kindly
provided by Dr. R. R. Reed (Johns Hopkins University, Baltimore,
MD) (25). For generation of recombinant baculoviruses encoding for rat
Gs
L, its DNA sequence was transferred into
the baculovirus transfer vector pVL 1392 (11).
[
-32P]GTP (6000 Ci/mmol) and
[
-32P]ATP (3000 Ci/mmol) were from NEN Life Science
Products. [3H]Dihydroalprenolol ([3H]DHA)
(85-90 Ci/mmol) was from Amersham Corp. Anti-Gs
antibody was from Calbiochem. Guanosine 5'-phosphorothioate (GMPS) was from U. S. Biochemical Corp. All other nucleotides were from
Boehringer Mannheim (Mannheim, Germany). Sources of other materials
have been described elsewhere (11, 13, 26).
Construction of 2ARGs
L
and
2ARGs
S
DNAs--
2ARGs
L DNA was
generated by a two-step PCR protocol using Pfu polymerase. A
set of fusion primers (sense and antisense), encoding 18 base pairs
from the C terminus of the
2AR, 18 base pairs encoding a
hexahistidine tag, and 21 base pairs from the N terminus of
Gs
L, were synthesized. In PCR 1A, the
sequence between a primer 5' of the EcoRV site of the human
2AR and the antisense fusion primer was amplified using
2AR DNA in pGEM-3Z as template. In this vector, referred
to as pGEM-3Z-SF-
2AR-6His, the
2AR is
tagged at the N terminus with the cleavable influenza-hemagglutinin signal sequence followed by the Flag epitope (IBI, New Haven, CT), and
the C terminus of the
2AR is tagged with a hexahistidine tail (Fig. 1A) (26). In PCR 1B, the sequence between the
sense fusion primer and the antisense primer with an extra
SalI site 3' of the stop codon of
Gs
L was amplified using rat
Gs
L DNA in pGEM-3Z as template. In PCR 2, the products of PCRs 1A and 1B were annealed and the sense primer 5' of
the EcoRV site in the
2AR sequence and the
antisense primer 3' of the stop codon of Gs
L
were used. In this way, a fragment encoding the C terminus of the
2AR, a hexahistidine tag, and
Gs
L was obtained. This fragment was digested
with EcoRV and SalI and cloned into
pGEM-3Z-SF-
2AR-6His digested with EcoRV and
SalI to obtain the full-length fusion protein DNA sequence
(pGEM-3Z-SF-
2AR-6His-Gs
L).
For generation of
2ARGs
S DNA,
a set of deletion primers (sense and antisense) and an antisense primer
3' of the EcoRI site of Gs
L were
synthesized. In PCR 3A, the sequence between a primer 5' of the
EcoRV site in the
2AR and the antisense
deletion primer was amplified using pGEM-3Z-SF-
2AR-6His-Gs
L as
template. In PCR 3B, the sequence between the sense deletion primer and
the antisense primer 3' of the EcoRI site of
Gs
L was amplified using the same template as
in PCR 3A. In PCR 4, the products of PCRs 3A and 3B were annealed, and
the sense primer 5' of the EcoRV site in the
2AR and the antisense primer 3' of the EcoRI
site of Gs
L were used. In this way, a DNA
fragment encoding the C terminus of the
2AR, a
hexahistidine tail and the N-terminal portion of
Gs
S, missing the sequence for amino acids
72-86 in Gs
L and encoding the Glu-71
Asp substitution (15), was created (Fig. 1A). This fragment
was digested with EcoRV and EcoRI and cloned into
pGEM-3Z-SF-
2AR-6His-Gs
L
digested with EcoRV and EcoRI. PCR-generated DNA
sequences were confirmed by enzymatic sequencing. Fusion protein DNAs
were cloned into the baculovirus transfer vector pVL 1392 (11).
Recombination of viruses was confirmed by reverse transcriptase
PCR.
Cell Culture--
Recombinant baculoviruses were generated and
amplified as described (11). Sf9 cells were seeded at 3.0 × 106 cells/ml and infected with 1:50 or 1:500 dilutions
of high titer virus stocks. Cells were cultured for 24-48 h to obtain
various expression levels of fusion proteins and 2AR.
For co-expression studies, Sf9 cells were infected with a
1:10,000 dilution of a high titer
2AR baculovirus stock
and a 1:50 dilution of a high titer Gs
L
baculovirus stock to achieve a receptor to G-protein stoichiometry of
~1:100. Cells were cultured for 48 h. Membranes were prepared
according to Gether et al. (11).
[3H]DHA Binding--
For determination of
Kd and Bmax values, Sf9 membranes
(5 µg of protein) were suspended in 500 µl of buffer containing 75 mM Tris/HCl, pH 7.4, 12.5 mM MgCl2,
and 1 mM EDTA, supplemented with 0.1-10 nM
[3H]DHA and 0.2% (w/v) bovine serum albumin. Nonspecific
binding was assessed in the presence of 10 µM
()-alprenolol (ALP). Incubations were performed for 90 min at
25 °C and shaking at 200 rpm. Competition binding experiments were
carried out with 15-30 µg of membrane protein with 1 nM
[3H]DHA in the presence of unlabeled ligands at various
concentrations without or with guanosine
5'-O-(3-thiotriphosphate) (GTP
S) (10 µM).
In some experiments, tubes contained 1 nM
[3H]DHA, 1 µM salbutamol (SAL), and various
nucleotides at increasing concentrations.
GTPase Activity--
Assay tubes (100 µl) contained 10 µg of
membrane protein, 0.1 µM [-32P]GTP
(0.1-0.5 µCi/tube), 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, 0.2% (w/v) bovine serum albumin in 50 mM
Tris/HCl, pH 7.4, and ligands at various concentrations. Reactions were conducted for 20 min at 25 °C and 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. Reaction
mixtures were centrifuged for 15 min at room temperature and
15,000 × g. Seven-hundred µl of the supernatant
fluid of reaction mixtures were removed and [32P]Pi was determined by liquid
scintillation counting.
AC Activity--
Assay tubes (50 µl) contained 15 µg of
membrane protein, 1 µM GTP, 40 µM
[-32P]ATP (2.5 µCi/tube), 2.7 mM
mono(cyclohexyl)ammonium phosphoenolpyruvate, 0.125 IU of pyruvate
kinase, 1 IU of myokinase, 0.1 mM cAMP, 5 mM
MgCl2, 0.4 mM EDTA, 30 mM Tris/HCl,
pH 7.4, and ligands at various concentrations. Reactions were conducted
for 20 min at 37 °C. Separation of [32P]cAMP from
[
-32P]ATP was performed as described (27).
Western Blot Analysis--
Solubilized Sf9 membrane
proteins (5-10 µg of protein/lane) were separated by SDS-PAGE (8%
(w/v) acrylamide). Proteins were visualized using either M1 antibody or
anti-Gs antibody and the ECL Western blotting system
(Amersham). Gs
L expression in Sf9 membranes was quantitated by immunoblotting with anti-Gs
antibody using defined amounts of
2ARGs
fusion protein as standard.
Miscellaneous--
Protein was determined using the Bio-Rad DC
protein assay kit (Bio-Rad). Data were analyzed by nonlinear
regression, using the program Prism (GraphPad, Prism, San Diego, CA).
Statistical comparisons between
2ARGs
S and
2ARGs
L were done with the
Wilcoxon test. Data are given as means ± S.D. of three to seven
independent experiments performed in duplicate or triplicate.
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RESULTS |
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Expression of 2ARGs
S and
2ARGs
L in Sf9
Membranes--
Expression of fusion proteins in Sf9 membranes
was confirmed by SDS-PAGE using the M1 monoclonal antibody to detect
the N-terminal Flag epitope of the
2AR (Fig.
1A). The nonfused
2AR expressed in Sf9 cells runs as a broad
glycosylated 52-kDa protein in SDS-PAGE (11, 26). The apparent
molecular masses of Gs
S and
Gs
L are 45 and 52 kDa, respectively (16).
Accordingly, the apparent molecular masses of
2ARGs
S and
2ARGs
L were expected to be 97 and 104 kDa, respectively. The data obtained are in agreement with this
expectation (Fig. 1C). Immunoblots with an
anti-Gs
antibody confirmed the presence of
Gs
in the fusion proteins and the difference in apparent
molecular mass between
2ARGs
S and
2ARGs
L. In membranes from
uninfected cells, no immunoreactive bands in the 97-104-kDa region
were detected with the M1 and anti-Gs
antibody (data not
shown).
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Ligand Binding Properties of
2ARGs
S and
2ARGs
L, Comparison with the
Nonfused
2AR--
The Kd values of
[3H]DHA for
2ARGs
S and
2ARGs
L were very similar
(Table I). In competition experiments, we
studied the effects of (
)-isoproterenol ((
)-ISO), (+)-isoproterenol ((+)-ISO), SAL, dobutamine (DOB), (
)-ephedrine (EPH),
dichloroisoproterenol (DCI) and ICI 118,551 (ICI) on
[3H]DHA binding. (
)-ISO binds to
ARs with higher
affinity than (+)-ISO, but both stereoisomers are full agonists
(28-30). SAL, DOB, EPH, and DCI are partial
2AR
agonists (7, 10, 11), and ICI is an inverse agonist (8, 11-13). At
both fusion proteins, full and strong partial agonists ((
)-ISO,
(+)-ISO, SAL, and DOB) showed a high- and low-affinity binding
component (Table I). The high-affinity agonist binding was abolished by
GTP
S. For agonists with lower intrinsic activity (EPH and DCI),
high- and low-affinity binding sites were not discriminated by curve
fitting analysis, but GTP
S still reduced the affinity of these
ligands to
2ARGs
fusion proteins. There
were no significant differences in the low- and high-affinity
Ki values of the agonists studied between
2ARGs
S and
2ARGs
L. There was a trend
toward higher fractions of high-affinity agonist-binding sites for full agonists and strong partial agonists at
2ARGs
S compared with
2ARGs
L, but this was
significant only for (+)-ISO.
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Regulation of GTPase Activity in 2ARs
Fusion Proteins, Comparison with a Co-expression System Consisting of
2AR and Gs
--
Activation of the GTPase
of Gs by agonist-occupied
ARs can be studied with great
sensitivity in reconstituted systems (32, 33), but in most plasma
membrane systems, the GTPase stimulation induced by
ARs is small
relative to the high background GTPase activity of other cellular
G-proteins with higher GTP turnover than Gs and the
presence of low-affinity nucleotidases (34, 35). In S49 lymphoma cell
membranes, a prototypical system for studying
2AR/Gs interaction (23, 36), the
2AR and Gs
are expressed at levels of
~0.2 and ~20 pmol/mg, respectively, i.e. there is an
~100-fold molar excess of G-protein compared with receptor (37). We
co-expressed the
2AR at a level of 1.4 pmol/mg with
Gs
L at a level of ~100 pmol/mg in
Sf9 membranes, achieving a similar receptor/G-protein ratio as
in S49 lymphoma cells, and studied the regulation of GTPase activity by
(
)-ISO and ICI. However, despite the relatively high expression of
2AR and Gs
L at a
stoichiometry similar to that in the mammalian cell line, we detected
only marginal activation of GTPase by agonist in Sf9 membranes
and failed to see inhibition by inverse agonist (Fig. 2A). Similar results were
obtained when the expression level of
2AR was increased
to 11.8 pmol/mg (data not shown). In marked contrast, (
)-ISO
increased GTP hydrolysis in membranes expressing
2ARGs
(5.0 pmol/mg) by up to 245% above
basal, and ICI reduced GTP hydrolysis by up to 50% (Fig.
2B). These findings demonstrate that fusion of the
2AR to Gs
greatly facilitates detection
of ligand-regulated GTP hydrolysis in Sf9 membranes.
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The Effects of GsS and
Gs
L on the Efficacy of Agonists and Inverse
Agonists at the
2ARGs
Fusion
Proteins--
The precise determination of the intrinsic activities of
partial agonists constitutes a major problem in the functional
characterization of GPCRs, because the intrinsic activity of a given
ligand may depend on numerous variables, i.e. receptor and
G-protein expression level and the availability of effector molecules
such as AC (11, 22, 38-40). In most studies, the intrinsic activities
of partial
2AR agonists were characterized by measuring
AC activity (7, 10, 11, 41). The AC assay takes advantage of the signal amplification at the Gs level, but it is difficult to
control for the impact of Gs and AC availability on
intrinsic activities of ligands. We reasoned that with the GTPase
activity of
2ARGs
fusion proteins as
parameter, determination of the intrinsic activities of partial
agonists should be less ambiguous because of the fixed stoichiometry of
the signaling components. Moreover, signal amplification by AC is not
required, thereby reducing the number of variables that can influence
the determination of intrinsic activity. To validate this assumption,
we studied the potencies and intrinsic activities of a series of
partial
2AR agonists at the GTPase of
2ARGs
L with expression levels
ranging from 0.6 to 7.6 pmol/mg. Within this broad range of expression,
we did not observe significant differences in the potency and intrinsic
activity of partial
2AR agonists (data not shown).
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Regulation of High-affinity Agonist Binding at
2ARGs
S and
2ARGs
L by Guanine
Nucleotides--
High-affinity agonist binding to GPCRs depends on
their interaction with G-protein
-subunits, presumably in the
nucleotide-free state (1, 42). Occupation of the guanine
nucleotide-binding site of
-subunits disrupts high-affinity agonist
binding (1, 7). To determine the guanine nucleotide binding affinities of Gs
S and Gs
L in
2ARGs
fusion proteins, we examined
binding of a fixed concentration of the antagonist
[3H]DHA in the presence of a subsaturating concentration
of the strong partial agonist SAL in membranes expressing
2ARGs
S and
2ARGs
. Various nucleotides at increasing
concentrations were added to the binding assays. Guanine nucleotide
binding to Gs
reduces the affinity of the
2AR for agonist and, thereby, increases [3H]DHA binding (Fig. 5).
In this way, the affinity of G-proteins for nucleotides can be
measured. It should be noted that our binding experiments were
performed in the absence of a nucleotide-regenerating system, excluding
the possibility that effects caused by nucleoside 5'-monophosphates and
-diphosphates are due to transphosphorylation. GTP was similarly potent
at inhibiting high-affinity agonist binding at
2ARGs
S and
2ARGs
L (EC50,
59 ± 15 and 49 ± 20 nM, respectively). In
contrast, GDP was far more potent at
2ARGs
S (EC50,
83 ± 23 nM) than at
2ARGs
L (EC50,
1.8 ± 0.2 µM). Like GDP, its nucleotidase-resistant phosphorothioate analog, guanosine 5'-O-(2-thiodiphosphate)
(GDP
S), inhibited agonist binding at
2ARGs
S more potently than at
2ARGs
L (EC50,
490 ± 150 nM and 2.2 ± 0.3 µM,
respectively). These data show that in the
2ARGs
fusion proteins,
Gs
S has a higher affinity for guanosine
5'-diphosphates than Gs
L and are in
agreement with data obtained with purified
Gs
L and Gs
S
(18).
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Regulation of AC Activity in Sf9 Membranes Expressing
2ARGs
S and
2ARGs
L by Agonist and Inverse
Agonist--
The analysis of AC activity in Sf9 membranes
expressing
2ARGs
S and
2ARGs
L must take into
consideration the fact that Sf9 cells express endogenous
Gs
-like G-proteins (10, 11, 30, 31). This is of
particular relevance because for AC studies, we expressed fusion
proteins at relatively low levels to avoid AC availability becoming the
limiting factor (38). However, the AC activity in membranes from
uninfected Sf9 cells in the presence of 10 µM
GTP
S was ~5.5-fold lower than in Sf9 membranes expressing
2ARGs
L at 2.6 pmol/mg
(0.089 ± 0.015 nmol/mg/20 min versus 0.491 ± 0.054 nmol/mg/20 min). These data show that even under maximal
stimulation of AC, the contribution of endogenous Gs
-like G-proteins in Sf9 cells to total AC
activity is small.
AC Regulation in Sf9 Membranes Expressing
2ARGs
S and
2ARGs
L in the Absence of
Exogenous Guanine Nucleotides--
In the presence of GTP, agonists at
Gs-coupled GPCRs cause AC activation (1, 2). However, in
the absence of added guanine nucleotides, agonists at
Gs-coupled receptors can reduce AC activity (43,
44). The most likely explanation for these observations is that
agonists induce release of prebound guanine nucleotide from
Gs
, generating guanine nucleotide-free Gs
and, thereby, reducing AC activity. Indeed, (
)-ISO reduced the basal
AC activity in membranes expressing
2ARGs
S by about 30% and with
an IC50 of 40 ± 12 nM (Fig.
6). In contrast, (
)-ISO had no
significant deactivating effect on AC activity in membranes expressing
2ARGs
L. These results suggest
that the nucleotide-binding pocket of Gs
L in
2ARGs
L is already
nucleotide-free.
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DISCUSSION |
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The 2AR Fused to Gs
L Has
Properties of a Constitutively Active Receptor--
Previous studies
have shown that there are subtle differences in the GDP affinities of
purified Gs
S and
Gs
L (18) and that Gs
S may activate AC more efficiently than
Gs
L (19). However, studies aiming to reveal
differences between Gs
S and
Gs
L in their coupling to the
2AR have remained inconclusive because of the
difficulties to ensure exactly defined receptor/G-protein stoichiometry
(18, 20, 21). This is important because functional interactions between
GPCRs and G-proteins are strongly influenced by their relative
expression levels (11, 22, 40). To circumvent this problem, we
constructed fusion proteins in which the C terminus of the
2AR was linked to the N terminus of
Gs
S or Gs
L (Fig. 1A), thereby guaranteeing a defined stoichiometry of
receptor to G-protein and increasing the efficiency of
receptor/G-protein coupling. Using this approach we observed that the
efficacy and potency of partial agonists acting on the
2AR were significantly higher when the receptor was
fused to Gs
L than when the receptor was
fused to Gs
S (Fig. 4 and Table II).
Moreover, the basal GTPase and AC activities in membranes expressing
2ARGs
L were more sensitive to
inverse agonists than the corresponding activities in membranes expressing
2ARGs
S (Fig. 3).
These functional properties of the
2AR fused to
Gs
L are similar to those of the
2ARCAM (7, 8, 13).
Physiological Considerations--
Although constitutive activation
of GPCRs is easily observed with high receptor expression levels
(10-12), this is not a prerequisite. There are several examples in the
literature documenting constitutive GPCR activity at physiological or
near-physiological expression levels (5, 6, 9, 14). These data raise
the possibility that constitutive activity of GPCRs is of relevance
in vivo and that the R* state can be more readily stabilized
or detected by specific G-protein -subunits. In agreement with such
a concept is the finding that increases in expression of specific
G-proteins can increase high-affinity agonist binding and can promote
constitutive receptor activation (40, 46).
Conclusion--
The 15-amino acid insert by which
GsL differs from
Gs
S (Fig. 1B) lowers the GDP
affinity of the G-protein. Using fusion proteins of the
2AR with Gs
splice variants, which ensure
precise receptor/G-protein stoichiometry, we could show that the subtle differences in GDP affinity between Gs
S and
Gs
L have important consequences for the
interaction with the
2AR, i.e.
Gs
L confers to the
2AR some
properties of a constitutively active receptor. Future studies will
have to examine the effects of partial and inverse agonists of the
2AR in tissues and cells expressing
Gs
S and Gs
L at
different levels and to further define the physiological and
pharmacological implications of the differences that we have discovered
for the interaction of the
2AR with the two splice variants of Gs
. Because the overall properties of the
2AR and Gs
and their interaction were not
changed as a result of fusion, this approach may be applied to a broad
variety of receptors and G-proteins to uncover subtle differences in
the interaction of closely related G-protein
-subunits with
GPCRs.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Henry R. Bourne for
providing the three-dimensional model of transducin- and most
helpful discussion. We thank Dr. Hans Schambye for his help with the
preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Research Grant R01-MH34007 (to E. S.-B.).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.
§ Recipients of a research fellowship of the Deutsche Forschungsgemeinschaft.
¶ Present address: Dept. of Cellular Physiology, Institute of Medical Physiology 12.5, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2100 Copenhagen N, Denmark.
Permanent address: Dept. of Pharmacology, Vanderbilt School of
Medicine, Nashville, TN 37232-6600.
To whom correspondence should be addressed: Howard Hughes
Medical Institute, B-157, Beckman Center, Stanford University Medical School, Stanford, CA 94305-5428. Tel.: 650-723-7069; Fax: 650-498-5092; E-mail: kobilka{at}cmgm.stanford.edu.
1
The abbreviations used are: GPCR(s),
G-protein-coupled receptor(s); 2AR,
2-adrenoreceptor;
2ARCAM,
constitutively active mutant of the
2AR;
Gs
,
-subunit of the G-protein Gs;
Gs
L, long splice variant of the
-subunit
of Gs; Gs
S, short splice variant
of the
-subunit of Gs;
2ARGs
L, fusion protein
consisting of the
2-adrenoreceptor and the long splice
variant of Gs
;
2ARGs
S, fusion protein of the
2-adrenoreceptor and the short splice variant of
Gs
; DCI, dichloroisoproterenol; [3H]DHA,
[3H]dihydroalprenolol; EPH, (
)-ephedrine; GDP
S,
guanosine 5'-O-(2-thiodiphosphate); GMPS, guanosine
5'-phosphorothioate; GTP
S, guanosine
5'-O-(3-thiotriphosphate); (
)-ISO; (
)-isoproterenol;
(+)-ISO, (+)-isoproterenol; ICI, ICI 118,551; SAL, salbutamol; DOB,
dobutamine; ALP, (
)-alprenolol; AC, adenylyl cyclase; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain
reaction.
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