Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow, Scotland, United Kingdom (P.J.W., E.K.,
G.M.); and Molecular Pharmacology and Biochemistry, Department of
Enabling Science and Technology, Astra-Zeneca, Alderley Park,
Macclesfield, Cheshire, England, United Kingdom (G.W.)
 |
Introduction |
Constitutive
activity of G protein-coupled receptors (GPCRs) has been one of the
most highly studied topics in pharmacology in the recent past (Leurs et
al., 1998
; Pauwels and Wurch, 1998
; de Ligt et al., 2000
). Such studies
have provided novel insights into the mechanisms of action of GPCRs and
introduced the term inverse agonist for ligands able to suppress this
activity (Milligan et al., 1995
). So widespread have the studies been
that compounds, generally now described as neutral antagonists, that
bind receptors but fail to alter their activity are considered to be
relatively uncommon.
Direct measures of regulation of the activation of a G protein by a
GPCR can be provided by monitoring either exchange of a poorly
hydrolyzed analog of GTP for GDP on the G protein
-subunit (Wieland
and Jakobs, 1994
) or the subsequent hydrolysis of authentic GTP by the
GTPase activity of this subunit (Gierschik et al., 1994
). This GTPase
activity and its regulation can be analyzed using basic enzyme
kinetics. However, when using both purified G proteins and membrane
preparations, the rate of GTP hydrolysis has routinely been noted to be
much slower than the rate of deactivation of a range of G
protein-mediated events in vivo. Such discrepancies informed searches
for GTPase-activating proteins (GAPs) capable of accelerating the
turn-off reaction. The largest family of such GAPs for heterotrimeric G
proteins in mammals are the regulators of G protein signaling (RGS)
proteins (De Vries et al., 2000
), comprising more than 20 polypeptides
that contain a highly conserved RGS domain within their sequence. These
act as GAPs for many G proteins and can be shown to alter the
effectiveness of downstream signal transduction (Berman et al., 1996
;
Druey et al., 1996
; Doupnik et al., 1997
; Hepler et al., 1997
; Saitoh
et al., 1997
). Wide-ranging experiments (Berman et al., 1996
) and the
crystal structure of the core RGS domain of RGS4 complexed with
Gi1
(Tesmer et al., 1997
) indicated that the
mechanism of these proteins was via stabilization of the transition
state required for GTP hydrolysis.
For many native GPCRs in cell membranes, their degree of constitutive
activity is modest. Thus, when measuring inhibition of GTPase activity
by potential inverse agonists, it may be difficult to obtain precise
information. Because an RGS protein must be expected to function as a
GAP for GTP loaded by the constitutive activity of a GPCR and after
agonist activation, we reasoned that RGS proteins may be used to
enhance the dynamic range of GTPase activity arising from the presence
of constitutively activated GPCRs.
We demonstrate that this is the case, that the extent of this effect
varies dependent upon the identity of the G protein studied, that it
can provide a substantially more robust analysis of compounds with
inverse agonist activity, and that this approach is well suited to the
study of ligands with positive but low intrinsic activity.
 |
Experimental Procedures |
Materials.
All materials for tissue culture were supplied by
Invitrogen (Paisley, Strathclyde, UK). The 5-HT1A
receptor antagonist [3H]MPFF (70.5 Ci/mmol) and
[
-32P]GTP (30 Ci/mmol) were obtained from
PerkinElmer Life Sciences (Boston, MA). The
5-HT1A receptor antagonist
[3H]WAY100635 (83.0 Ci/mmol) was from Amersham
Biosciences (Piscataway, NJ). Pertussis toxin was purchased from Sigma
(St. Louis). Oligonucleotides were purchased from Cruachem (Glasgow,
Strathclyde, UK). All other chemicals were from Sigma and Roche
Molecular Biochemicals (Summerville, NJ).
Construction of Plasmids Encoding
5HT1A-Gi1
and
5HT1A-Go1
Fusion Proteins.
The human
5-HT1A receptor clone in pSP64 (a gift from
Glaxo-Wellcome, Stevenage, UK) was digested with
XbaI/BamHI, and the resulting 1.5-kilobase pair
fragment was ligated to pcDNA3. To obtain the open reading frame of 1.3 kilobase pairs, PCR was carried out using the following primers to
introduce a HindIII restriction site at the 5' end and to
remove the stop codon and introduce a BamHI restriction site
at the 3' end, respectively:
5'-CTGAAGCTTATGGATGTGCTCAGCCCTGGTC-3'; 5'-CTGGGATCCCTGGCGGCAGAAGTTACACTTAATG-3' (restriction
enzyme sites underlined). The PCR fragment was digested with
HindIII and BamHI and ligated into pcDNA3 to make
the plasmid p5HT. To link the Gi1
wild-type
(cys351)cDNA to the 5HT1A
receptor sequence, PCR was carried out on Gi1
to produce compatible restriction sites. The oligonucleotides used to
do this were 5'-CTGGGATCCGGCTGCACACTGAGCGCTGAG-3' at the 5'
end and 5'-GAGAATTCTTAGAAAGAGACCACAGTC-3' for the 3' end.
The plasmid p5HT was digested with BamHI/EcoRI as
was the Gi1
PCR fragment, and the two were
ligated to give the plasmid p5HTGi1. To construct the
5-HT1A-(Gly351) and
(Ile351)Gi1
fusion
proteins, plasmid (Gly or
Ile351)Gi1
in pBS was
digested with SacII/EcoRI, and the 730-base pair fragment was used to replace the corresponding fragment in
p5HTGi1. Equivalent strategies were used to
produce the
5-HT1A-(Ile351)Go1
fusions. The constructs were then sequenced to verify the DNA sequence.
Cell Culture and Stable Expression.
HEK 293 cells were
maintained in Dulbecco's modified Eagle's medium containing 10%
(v/v) newborn calf serum and 2 mM L-glutamine. Cells were
seeded into 100-mm culture dishes and grown to 60 to 80% confluence
(18-24 h) before transfection with 5 µg of appropriate cDNAs using
DOTAP reagent (Roche Molecular Biochemicals). Forty-eight hours after
transfection, the cells were split 1:4 into medium containing 800 µg/ml G418 sulfate (Calbiochem, San Diego, CA). A 100-mm dish of
untransfected HEK 293 cells was also split into the same medium as a
control dish. About 1 week later, after all the cells in the control
dish had died, G418-resistant cells in the transfected dishes were
picked and transferred into 24-well plates using autoclaved pipette
tips. About 20 clones of each cDNA were picked and grown in 1 ml/well
G418 medium (400 µg/ml). Clones were amplified, membrane preparations
were made, and their binding of
[3H]4-(2'- methoxy)-phenyl-1-[2'-(N-2"-pyridinyl)-p-fluorobenzamido]ethyl-piperazine was determined.
Preparation of Membranes.
Plasma membrane-containing P2
particulate fractions were prepared from cell pastes that had been
stored at
80°C after harvesting. Cell pellets were resuspended in
Tris/EDTA buffer [10 mM Tris HCl, pH 7.5, and 0.1 mM EDTA], and
rupture of the cells was achieved with 25 strokes of a hand-held
Teflon-on-glass homogenizer. Unbroken cells and nuclei were removed by
centrifugation at low speed (1600 rpm) in a refrigerated
microcentrifuge. The supernatant fraction was then centrifuged at
50,000 rpm for 30 min in an Optima TLX ultracentrifuge with a TLA100.2
rotor (Beckman Coulter, Inc., Fullerton, CA). The pellets were
resuspended in Tris/EDTA buffer to a final protein concentration of 1 mg/ml and stored at
80°C until required.
[3H]WAY100635 Binding Studies.
Binding assays
were performed by adding 5 µg of membrane protein to an assay buffer
(20 mM HEPES, 10 mM MgCl2, 0.1% ascorbic acid,
and 10 µM pargyline, pH 7.4) containing
[3H]WAY100635 (0.25-12 nM). Nonspecific
binding was determined in parallel in the presence of 100 µM 5-HT.
Samples were incubated at 30°C for 40 min and then terminated by
rapid filtration through GF/C filters. The filters were washed three
times with 5 ml of ice-cold wash buffer (20 mM HEPES, 10 mM
MgCl2, and 0.1% ascorbic acid, pH 7.4) and then
counted. In a number of experiments, recombinant RGS1 was also added to
the binding assays. In competition binding assays,
[3H]WAY100635 was present at 1 nM.
High-Affinity GTPase Assays.
High-affinity GTPase assays
were performed essentially as described previously (Wise et al., 1997a
,
1997b
; Wise and Milligan, 1997
) adapted to a 96-well microtiter plate
assay (Hoffmann et al., 2001
). Nonspecific GTPase activity was assessed
by parallel assays in the presence of 100 µM GTP. GTPase saturation
data were analyzed by nonlinear regression using Prism version 2.01 (GraphPad Software, San Diego, CA).
Purification of GST-Tagged RGS1 and RGS16.
GST-RGS1 (Denecke
et al., 1999
) was kindly donated by Dr. A. Meyerdierks (Department of
Medical Microbiology, Medizinischer Hochschule, Hannover, Germany). The
full coding region of the human RGS1 gene except the initiation codon
was cloned in-frame in the BamHI and SalI sites
of the vector pGEX4T1 (Amersham Biosciences).
GST-RGS16 (Chen et al., 1997
) was kindly donated by Dr. C. W. Fong
(Institute of Molecular and Cell Biology, Singapore). In essence, the
GST-RGS16 was constructed in a similar way as for RGS1. The coding
region of the RGS16 gene was fused to the GST gene of the vector
pGEX-2TK2 (derived from pGEX-2KT, Amersham Biosciences) with
modifications in the multiple cloning region.
GST-RGS fusion proteins were isolated from transformed
Escherichia coli BL21 cells. In brief, E. coli
BL21 cells were transformed with the appropriate plasmids and plated on
LB agar plates containing 100 µg/ml ampicillin. The next day, cells
were washed from the plate and used to inoculate 400 ml of LB medium
(supplemented with 100 µg/ml ampicillin) at an
OD660 of 0.1. Cells were grown for 1 h
before induction of the expression of the GST-RGS fusion proteins by
addition of 1 mM isopropyl
-D-thiogalactoside.
Cells were allowed to express the fusion protein for 4 h, after
which the cells were harvested by centrifugation. Pellets were stored at
80°C or used immediately for GST affinity purification of the
GST-tagged proteins. Pellets were resuspended in BugBuster solution
(Novagen, Madison, WI) with 5 ml/g wet tissue and containing 10 µl of Benzonase (Novagen) to reduce viscosity. Cells were incubated with 1 mg/ml lysozyme for 30 min on ice followed by sonication to
disrupt the cells (4 × 30-s pulses). Dithiothreitol (5 mM) was
added to the lysed cells before the addition of 200 µl of 50% (w/v)
slurry of glutathione-Sepharose 4B (Amersham Biosciences). Samples were
incubated at room temperature on a rotary wheel for 30 min and the
Sepharose harvested by centrifugation at 500g. The beads
were washed three times with 2 ml of ice-cold phosphate-buffered saline
and the GST fusion proteins eluted from the beads with 20 mM reduced
glutathione in a Tris-HCl buffer, pH 7.4. Purity of the isolated
protein was visually inspected after SDS-PAGE. Protein amounts were
determined according to Bradford (1976)
after precipitation of a small
amount of protein with 6% trichloroacetic acid to remove the glutathione.
Miscellaneous.
All experiments were performed on a minimum
of three occasions using cells or membrane preparations derived from
different cell passages. Where appropriate, data are presented as
means ± S.E.M.
 |
Results |
Membranes stably expressing a fusion protein between the human
5-HT1A receptor and a pertussis toxin-resistant
form of Gi1
in which
cysteine351 of the G protein was replaced by
isoleucine have higher levels of basal high-affinity GTPase activity
than those expressing a similar level of a fusion protein between the
5-HT1A receptor and a form of
Gi1
in which this cysteine is replaced by
glycine (Kellett et al., 1999
). This difference reflects a constitutive capacity of the 5-HT1A receptor to activate
(Cys351Ile)Gi1
because
the 5-HT1A receptor inverse agonist spiperone (Barr and Manning, 1997
; Newman-Tancredi et al.,1997a
, 1997b
; Kellett
et al., 1999
; Milligan et al., 2001
) is able to reduce basal
high-affinity GTPase activity in these membranes but not in those
expressing the 5-HT1A
receptor-(Cys351Gly)Gi1
fusion protein (Kellett et al., 1999
).
The 5-HT1A
receptor-(Cys351Ile)Gi1
fusion protein could be immunodetected as an 85-kDa polypeptide (Fig.
1A). Addition of 1 µM recombinant RGS1
(Hoffmann et al., 2001
) to membranes of pertussis toxin-pretreated
cells stably expressing this construct resulted in an increase in basal
high-affinity GTPase activity (Fig. 1A) and a greatly enhanced capacity
to measure stimulation of GTPase activity upon addition of increasing
concentrations of 5-HT (Fig. 1A). The 5-HT1A
receptor selective agonist 8-OH-DPAT was as effective as 5-HT (Fig.
1B), and 1 µM recombinant RGS16 (Hoffmann et al., 2001
) also enhanced
the effects of the agonists (Fig. 1B). This was not observed upon
equivalent additions of the RGS proteins to membranes expressing the
5-HT1A
receptor-(Cys351Gly)Gi1
fusion protein or to membranes of mock-transfected cells (data not
shown). We also constructed and stably expressed fusion proteins
between the human 5-HT1A receptor and both
(Cys351Ile)Go1
and
(Cys351Gly)Go1
. After
pertussis toxin treatment and membrane preparation, addition of
recombinant forms of either RGS1 (Fig. 2)
or RGS16 (data not shown) also elevated basal high-affinity GTPase
activity in the membranes expressing the 5-HT1A
receptor-(Cys351Ile)Go1
fusion protein (Fig. 2). This polypeptide could also be immunodetected
as an 85-kDa polypeptide (Fig. 2). As before, the RGS proteins had
little effect in membranes expressing the Gly351-containing version of this fusion protein
(data not shown). Noticeably, however, compared with the effects (less
than 2-fold) on basal GTPase activity in membranes expressing
5-HT1A
receptor-(Cys351Ile)Gi1
(Fig. 1A), 1 µM RGS1 increased basal activity some 4-fold in
membranes expressing the 5-HT1A
receptor-(Cys351Ile)Go1
fusion protein (Fig. 2). The stimulatory effects of both 5-HT (Fig. 2)
and 8-OH-DPAT (data not shown) on GTPase activity were again greatly
enhanced compared with those observed in the absence of the RGS (Fig.
2). Half-maximal effects of RGS1 required some 50 nM for
5-HT1A
receptor-(Cys351Ile)Go1
and some 90 nM for 5-HT1A
receptor-(Cys351Ile)Gi1
(Fig. 3). Enzyme kinetic analysis of the
high-affinity GTPase activity indicated that increasing concentrations
of RGS16 elevated the Vmax of both
basal (Fig. 4A) and 100 µM
5-HT-stimulated (Fig. 4B) GTPase activity of the
5-HT1A
receptor-(Cys351Ile)Go1
fusion protein. Moreover, although in the absence of an RGS protein,
5-HT increased Vmax of membranes
expressing the fusion protein (Table 1),
it did so without producing an alteration in apparent
Km for GTP (Table 1). By contrast,
both in the absence and the presence of 5-HT, the effects of the RGS
proteins reflected a combination of increased GTPase
Vmax and increases in the observed Km for GTP (Fig. 4; Table 1). To
ensure that the effects of the RGS proteins were not restricted to the
fusion proteins containing the Cys351Ile
mutations similar experiments were performed on membranes expressing
fusion proteins between the 5-HT1A receptor and
wild-type forms of either Gi1
or
Go1
. For both of these constructs RGS1 markedly enhanced basal GTPase activity and synergistically increased the effect of a maximally effective concentration of 5-HT in membranes of cells that had not been pretreated with pertussis toxin (Fig. 5).

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Fig. 1.
Agonist-mediated stimulation of the GTPase activity
of a 5-HT1A receptor-(Cys351Ile)
Gi1 fusion protein; effects of RGS proteins. A, HEK 293 cells stably expressing a 5-HT1A
receptor-(Cys351Ile) Gi1 fusion protein were
pretreated with pertussis toxin (25 ng/ml, 24 h) before harvest
and membrane preparation. The capacity of varying concentrations of
5-HT to regulate high-affinity GTPase activity was measured in the
absence (open symbols) (absence of 5-HT = 28.9 ± 0.6 pmol/min/mg membrane of protein) and presence (filled symbols) (absence
of 5-HT = 47.6 ± 0.1 pmol/min/mg membrane of protein) of 1 µM recombinant RGS1. GTPase activity was measured at 0.5 µM GTP.
Inset, membranes from parental HEK 293 cell (A) and those expressing
either 5-HT1A receptor-(Cys351Ile)
Gi1 (B) or 5-HT1A
receptor-(Cys351Ile) Go1 (C) were resolved
by SDS-PAGE and transferred to nitrocellulose. Immunodetection of
5-HT1A receptor-(Cys351Ile) Gi1
was achieved using an antiserum that identifies the extreme C terminus
of Gi1 . B, the effects of 5-HT and 8-OH-DPAT (both 100 µM) to modulate the GTPase activity of the 5-HT1A
receptor-(Cys351Ile) Gi1 fusion protein in
the absence or presence of RGS1 or RGS16 (both 1 µM) were measured.
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Fig. 2.
5-HT-mediated stimulation of the GTPase activity of a
5-HT1A receptor-(Cys351Ile) Go1
fusion protein: effects of RGS1. HEK 293 cells stably expressing a
5-HT1A receptor-(Cys351Ile) Go1
fusion protein were pretreated with pertussis toxin (25 ng/ml, 24 h) before harvest and membrane preparation. The capacity of varying
concentrations of 5-HT to regulate high-affinity GTPase activity was
measured in the absence (open symbols) (absence of 5-HT = 23.9 ± 0.4 pmol/min/mg membrane of protein) and presence (filled
symbols) (absence of 5-HT = 99.2 ± 2.3 pmol/min/mg membrane
of protein) of recombinant RGS1. GTPase activity was measured at 0.5 µM GTP. Inset, membranes from parental HEK 293 cell (A) and those
expressing either 5-HT1A receptor-(Cys351Ile)
Gi1 (B) or a 5-HT1A
receptor-(Cys351Ile) Go1 (C) were resolved
by SDS-PAGE and transferred to nitrocellulose. Immunodetection of
5-HT1A receptor-(Cys351Ile) Go1
was achieved using an antiserum that identifies the extreme C terminus
of Go1 .
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Fig. 3.
The potency of RGS1 to regulate basal GTPase activity
of 5-HT1A receptor-containing fusion proteins. Varying
concentrations of recombinant RGS1 were added to membranes from
pertussis toxin-pretreated cells expressing either the
5-HT1A receptor-(Cys351Ile) Go1
(squares) or the 5-HT1A receptor-(Cys351Ile)
Gi1 fusion protein (triangles). High-affinity GTPase
activity was then measured at 0.5 µM GTP.
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Fig. 4.
Effects of varying concentrations of RGS16 on basal
and agonist-stimulated GTPase activity of a 5-HT1A
receptor-(Cys351Ile) Go1 fusion protein.
Enzyme kinetic analysis. GTPase activity of membranes expressing the
5-HT1A receptor-(Cys351Ile)Go1
fusion protein was measured at a wide range of concentrations of GTP in
the absence (A) or presence (B) of 100 µM 5-HT. Assays also contained
0 (filled squares), 1 nM (filled triangles), 10 nM (filled diamonds),
50 nM (open triangles), 100 nM (filled circles), 500 nM (open diamonds)
or 1 µM (open squares) RGS16.
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TABLE 1
WAY100635 is a partial agonist at the 5-HT1A receptor
Enzyme kinetic analysis was performed on high-affinity GTPase activity
and its regulation in membranes of HEK293 cells expressing either the
5-HT1A(Cys351Ile)Gi1 or
5-HT1A(Cys351Ile)Go1 fusion proteins. Data
are taken from representative experiments. RGS1 and the ligands were
present at 1 µM.
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Fig. 5.
Agonist and RGS1 regulation of the GTPase activity of
5-HT1A receptor fusion proteins containing wild-type G
proteins. GTPase activity was measured at varying concentrations of GTP
in cell membranes expressing either the 5-HT1A
receptor-Gi1 fusion protein (A) or the
5-HT1A receptor-Go1 fusion protein (B).
Basal activity (squares) and the effects of 100 µM 5-HT (triangles),
1 µM RGS1 (inverted triangles), or both 5-HT and RGS1 (diamonds) were
assessed. Data are shown from a representative experiment.
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This effect of the RGS proteins was extremely useful in demonstrating
weak agonism of WAY100635. In membranes expressing either the
5-HT1A
receptor-(Cys351Ile)Go1
or the 5-HT1A
receptor-(Cys351Ile)Gi1
fusion proteins, this compound acted as either a neutral antagonist or
a very weak partial agonist (Fig. 6) with
little ability to alter basal GTPase activity. However, in the
additional presence of 1 µM RGS1 WAY100635 clearly functioned as a
low-efficacy partial agonist (Fig. 6; Table 1). Furthermore, the
enhanced sensitivity imbued to detection of efficacy in the presence of an RGS protein allowed good estimates to be obtained for
EC50 for WAY100635 (2.8-4.6 × 10
9 M) (Fig. 6). This was not possible without
addition of the RGS. In the absence of RGS1, enzyme kinetic analysis
showed that the effects of 1 µM WAY 100635 were, like 5-HT, achieved
by an increase in Vmax without
alteration in Km for GTP (Table 1). In
the presence of RGS1, WAY100635 further increased enzyme activity over
that achieved by RGS1 alone but had little or no further effect on the
Km for GTP (Table 1). Importantly,
addition of RGS1 to membranes expressing these fusion proteins had
little effect of the affinity or maximal capacity of
[3H]WAY100635 to bind to the receptor (Fig.
7; data not shown).

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Fig. 6.
WAY100635 is a weak partial agonist at the
5-HT1A receptor. The effects of varying concentrations of
WAY100635 to regulate basal high-affinity GTPase activity were measured
in membranes of pertussis toxin-pretreated cells expressing either the
5-HT1A receptor-(Cys351Ile) Go1
fusion protein (circles) or the 5-HT1A
receptor-(Cys351Ile) Gi1 fusion protein
(squares). Experiments were performed in the absence (open symbols) or
presence (filled symbols) of 1 µM RGS1.
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Fig. 7.
RGS1 does not alter the binding characteristics of
[3H]WAY100635. The specific binding of varying
concentrations of [3H]WAY100635 was measured in membranes
of pertussis toxin-pretreated HEK 293 cells expressing the
5-HT1A receptor-(Cys351Ile) Gi1
fusion protein in the absence (squares) or presence (triangles) of 1 µM RGS1.
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Because the majority of wild-type receptors display relatively low
levels of constitutive activity (Lefkowitz et al., 1993
; Rossier et
al., 1999
), it is often difficult to monitor functional differences
between inverse agonists and neutral antagonists. Mutational alteration
of the receptor is often required to boost the level of constitutive
activity for such studies (Lefkowitz et al., 1993
; Samama et al., 1993
;
Scheer and Cotecchia, 1997
). However, as RGS proteins function to
catalyze the GTP hydrolysis rate (De Vries et al., 2000
), the elevated
GTPase activity of the fusion proteins in the absence of ligands but
the presence of RGS reflects a response to constitutive loading of GTP
onto the fusion protein. Dependent on their relative intrinsic
activity, therefore, inverse agonists would be expected to reduce or
eliminate the effect of the RGS proteins. If this is so, then the
capacity to detect inverse agonism should be markedly improved in the
presence of an RGS. To test this hypothesis, we measured the ability of varying concentrations of the previously characterized
5-HT1A receptor inverse agonist spiperone to
reduce basal high-affinity GTPase activity in membranes expressing
either the 5-HT1A
receptor-(Cys351Ile)Gi1
or the 5-HT1A
receptor-(Cys351Ile)Go1
fusion proteins in the absence and presence of RGS1. As anticipated
from previous studies (Kellett et al., 1999
; Milligan et al., 2001
), in
the absence of RGS1, spiperone inhibited basal GTPase activity in
membranes expressing the 5-HT1A
receptor-(Cys351Ile)Gi1
fusion protein by some 50% with an EC50 value of
6.7 × 10
8 M (Fig.
8A). In the presence of 1 µM RGS1,
spiperone displayed a similar EC50 value
(4.6 × 10
8 M). However, with the
elevation in constitutive, receptor-mediated GTPase activity, this was
substantially easier to measure (Fig. 8A). The precision of measurement
was even more pronounced when the experiments were repeated using the
5-HT1A
receptor-(Cys351Ile)Go1
fusion protein. Although spiperone could be shown to be an inverse
agonist at this construct, in the absence of the RGS, this was
difficult to quantitate with precision (Fig. 8B). Because the 4-fold
elevated basal activity in these membranes observed on addition of RGS1
was attenuated almost completely by spiperone, this was easy to measure
and provided an EC50 value of 3.2 × 10
8 M for the ligand (Fig. 8B). Importantly,
the ability and potency of spiperone to compete with
[3H]WAY100635 for binding to the fusion
constructs was also unaltered by the presence of the RGS (Fig. 8C; data
not shown).

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Fig. 8.
RGS1 enhances detection of the inverse agonist
properties of spiperone at the 5-HT1A receptor. Membranes
of pertussis toxin-treated HEK 293 cells expressing either the
5-HT1A receptor-(Cys351Ile) Gi1
fusion protein (A) or the 5-HT1A
receptor-(Cys351Ile) Go1 fusion protein (B)
in the absence (open symbols) or presence (filled symbols) of 1 µM
RGS1 were exposed to varying concentrations of spiperone and
high-affinity GTPase activity measured at 0.5 µM GTP. C, the ability
of spiperone to compete with [3H]WAY100635 for binding to
the 5-HT1A receptor-(Cys351Ile)
Gi1 fusion protein was assessed in the absence (open
symbols) and presence (filled symbols) of 1 µM RGS1.
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To explore the basis of the effect of the RGS proteins, GTPase assays
were again performed at a wide range of concentrations of GTP. Basal
activity in membranes expressing the 5-HT1A
receptor-(Cys351Ile)Go1
fusion protein was adequately described by a single function with
Km for GTP in the region of 100 nM. In
the presence of 1 µM RGS1, the increase in basal activity was shown
to represent both an increase in GTPase
Vmax and an increase in the observed Km for GTP (Fig.
9). Spiperone, at a maximally effective
concentration, reduced Vmax and
returned the observed Km for GTP to a
value close to that of the basal state (Fig. 9).

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Fig. 9.
Mechanism of action of RGS1 on the basal GTPase
activity of the 5-HT1A receptor-(Cys351Ile)
Go1 fusion protein; enzyme kinetic analysis and the
effects of spiperone. The ability of 1 µM RGS1 (squares) to regulate
the basal (circles) GTPase activity of membranes expressing the
5-HT1A receptor-(Cys351Ile) Go1
fusion protein was measured at a wide range of concentrations of GTP in
the absence (open symbols) or presence (filled symbols) of 100 µM
spiperone. In the example displayed, GTPase
Vmax in the basal state was 23.4 ± 0.9 pmol/min/mg membrane of protein, and the measured
Km for GTP was 145 ± 11 nM. RGS1 alone
increased the GTPase Vmax to 146 ± 11.0 pmol/min/mg membrane of protein and increased markedly the
Km for GTP (492 ± 61 nM). Spiperone
reduced basal GTPase (12.7 ± 1.5 pmol/min/mg membrane of protein)
without altering the Km for GTP (100 ± 25 nM). In the presence of both RGS1 and spiperone, the values were
Vmax 23.6 ± 2.7 pmol/min/mg membrane
of protein and Km 143 ± 33 nM.
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A series of other ligands with affinity at the
5-HT1A receptor was also examined. For
methiothepin, (+)-butaclamol, and chlorpromazine, inverse agonist
activity could be easily detected at both the 5-HT1A
receptor-(Cys351Ile)Gi1
and the 5-HT1A
receptor-(Cys351Ile)Go1
fusion proteins when the assays were performed in the presence of RGS1
(Fig. 10). These compounds were without
effect on basal GTPase activity in membranes of parental HEK 293 cells (data not shown). Data on inverse agonism of these compounds was either
impossible to quantitate effectively [5-HT1A
receptor-(Cys351Ile)Go1
]
(Fig. 10A) or was substantially less convincing [the 5-HT1A
receptor-(Cys351Ile)Gi1
]
(Fig. 10B) when the assays were performed in the absence of an
RGS. Only in the case of haloperidol were conclusions that it
functioned as an essentially neutral ligand at the
5-HT1A receptor-fusion proteins confirmed when
the assays were also performed in the presence of RGS1 (Fig.
11A). This was not a reflection of a
lack of binding of haloperidol to the fusion proteins (Fig. 11B). A small inhibition of GTPase activity was noted for the highest concentrations of haloperidol tested (Fig. 11A), but this is probably a
nonspecific effect because these concentrations are not consistent with
the affinity of haloperidol at the 5-HT1A
receptor (Fig. 11B). High concentrations of haloperidol were also able
to reverse the inhibition of GTPase activity produced by spiperone
(Fig. 11C), confirming that spiperone was acting as an inverse agonist at the 5-HT1A receptor-fusion proteins.

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Fig. 10.
A range of ligands act as inverse agonists at the
5-HT1A receptor. Enhanced detection in the presence of
RGS1. Basal GTPase activity in membranes expressing the
5-HT1A receptor-(Cys351Ile) Gi1
(A) or to the 5-HT1A receptor-(Cys351Ile)
Go1 (B) fusion proteins was measured in the absence
(open symbols) or presence (filled symbols) or 1 µM RGS1. The ability
of varying concentrations of methiothepin (squares), (+)-butaclamol
(circles), and chlorpromazine (triangles) to regulate GTPase activity
was then measured at 0.5 µM GTP.
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Fig. 11.
Haloperidol is a neutral ligand at the
5-HT1A receptor. A, The effect of varying concentrations of
haloperidol on GTPase activity in membranes expressing the
5-HT1A receptor-(Cys351Ile)Gi1
(squares) or the 5-HT1A
receptor-(Cys351Ile)Go1 (diamonds) fusion
proteins was assessed in the absence (open symbols) or presence (filled
symbols) of 1 µM RGS1. B, competition by haloperidol for binding of
[3H]WAY100635 to the 5-HT1A
receptor-(Cys351Ile)Gi1 (filled symbols) or
the 5-HT1A
receptor-(Cys351Ile)Go1 (open symbols)
fusion proteins. C, The basal GTPase activity of the 5-HT1A
receptor-(Cys351Ile)Go1 fusion protein was
measured in the presence of 1 µM RGS1 and regulation of this activity
by 0.1 µM spiperone, 10 µM haloperidol, or a combination of the two
ligands assessed. *, significantly different from basal,
p < 0.01.
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Discussion |
5-HT is a key neurotransmitter, and the
5-HT1A receptor is an important target for drug
action. This reflects that presynaptic 5-HT1A
receptors in the raphe nuclei control 5-HT release throughout the brain
and that postsynaptic 5-HT1A receptors are
important in functions that include memory and stress modulation.
Indeed, gene knockout studies have confirmed key roles for the
5-HT1A receptor in anxiety and anti-depressive
actions (Heisler et al., 1998
; Ramboz et al., 1998
).
The capacity of recombinant RGS proteins to elevate basal
high-affinity GTPase activity in membranes expressing fusion proteins between the 5-HT1A receptor and each of wild type
and pertussis toxin resistant (Cys351Ile) forms
of both Gi1
and Go1
,
but not in membranes from mock transfected cells, provides clear
evidence for constitutive information transfer between this receptor
and these G proteins. It is noteworthy that we have previously recorded
constitutive activity of the 5-HT1A receptor-
(Cys351Ile) Gi1
fusion
protein but not for a similar construct containing (Cys351Gly)Gi1
(Kellett
et al., 1999
) because we have also demonstrated that the interactions
between GPCRs and Gi-family G proteins are of
higher affinity and more conducive to productive information transfer
when a hydrophobic amino acid is used to replace the pertussis
toxin-sensitive cysteine (Waldhoer et al., 1999
; Moon et al., 2001a
).
RGS1 was half-maximally effective at some 50 nM, a concentration akin
to that recently noted for its regulation of an agonist activated
2A-adrenoceptor-Go1
fusion protein (Hoffmann et al., 2001
). Interestingly, this effect of
RGS1 was much more pronounced in membranes expressing the
Go1
rather than the
Gi1
-containing fusion protein. Again, this was
previously noted for agonist stimulation of
2A-adrenoceptor-containing fusion proteins
(Cavalli et al., 2000
) and may indicate that in a membrane environment,
it is a more efficient GAP for Go1
than
Gi1
.
Constitutive activity of GPCRs and the capacity of this to be
suppressed by inverse agonists has been very actively studied in recent
years. However, because the level of constitutive activity of many
wild-type receptors is relatively low, many of these studies have used
mutationally modified receptors with enhanced agonist-independent activity to allow amplification of the constitutive signal. There is,
however, clear evidence that at least certain GPCRs do display significant constitutive activity in vivo and that regulation of this
activity may have pathophysiological significance (Morisset et al.,
2001
). An attractive feature of the use of recombinant RGS proteins to
boost the constitutive GTPase activity of the 5-HT1A receptor fusion proteins is that their
accepted mechanism of action is to promote the rate of hydrolysis of
GTP. By contrast, receptor ligands regulate the rate of guanine
nucleotide exchange of the G protein (Gilman, 1987
). Because nucleotide
exchange is the rate-limiting step in the activation/deactivation cycle
(Gilman, 1987
), then the effect of the RGS is simply to boost the
capacity of the fusion protein to load GTP in response to either
constitutive or agonist-induced activity. Proof of this mechanism of
action of RGS1 and RGS16 on the constitutive activity of the
5-HT1A receptor-(Cys351Ile)
Go1
fusion protein was obtained by enzyme
kinetic analysis of the GTPase activity. Addition of these RGS proteins
increased both GTPase Vmax and the
Km for GTP. We have previously shown that acceleration of the GTP hydrolytic activity must be accompanied by
both of these features (Cavalli et al., 2000
; Hoffmann et al., 2001
).
It was, thus, anticipated that an inverse agonist would restore both of
these features and this was observed when a maximally effective
concentration of spiperone was added in concert with RGS1 (Fig. 9). We
selected spiperone for these studies because it has previously been
well characterized as an inverse agonist at the
5-HT1A receptor. These observations and the
related effects of added RGS proteins on 5-HT stimulated GTPase
activity raise an interesting issue. When 5-HT is added to membranes
expressing either the 5-HT1A
receptor-(Cys351Ile) Go1
or the 5-HT1A
receptor-(Cys351Ile) Gi1
fusion proteins the effect is to increase GTPase
Vmax, but it has little effect on the
observed Km for GTP (Table 1; Kellett
et al., 1999
). However, with addition of the RGS proteins, as with the
situation with basal activity, both the GTPase activity and the
apparent Km for GTP are markedly
increased. These observations imply that the membrane preparations
themselves have little functionally relevant RGS activity. It is
currently unclear whether this reflects low levels of cellular
expression of RGS proteins or whether cell homogenization and membrane
preparation results in removal of much of the endogenous RGS. This will
be explored in future studies, but clearly RGS proteins are, at best,
peripheral membrane proteins with anchorage to the membrane being
provided by combinations of post-translational acylation, cysteine
string motifs, N-terminal amphipathic
-helices and possibly other
means (Chen et al., 1999
; Druey et al., 1999
; De Vries et al., 2000
).
It also seems that a number of RGS proteins are not confined to the
plasma membrane but are present in cellular compartments including the
cytosol, Golgi, and nucleus (Wylie et al., 1999
; Chatterjee and Fisher, 2000
).
In the recent past, fusion proteins between receptors and G protein
-subunits (Seifert et al., 1999
; Milligan, 2000
; Guo et al., 2001
;
Wurch and Pauwels, 2001
) have been employed to explore the details of
topics as diverse as the relative intrinsic activity of different
agonist ligands (Jackson et al., 1999
), the selectivity of receptors
for closely related G proteins (Moon et al., 2001b
), and the regulation
of post-translational acylation of both receptors and G proteins
(Loisel et al., 1999
; Stevens et al., 2001
). Herein, the use of such
fusion proteins containing the human 5-HT1A
receptor and pertussis toxin-resistant forms of
Gi-family G proteins allowed prior pertussis
toxin treatment of the cells to define that ligand and RGS regulation
of the high-affinity GTPase activity was a direct monitor only of
effects on the fusion protein-linked G proteins. Because antagonist
binding is generally insensitive to the G protein interaction state of
receptors it is hardly surprising that the affinity for antagonist
ligands was highly similar for each of the fusion constructs employed
in these studies and that this was not altered by addition of the
recombinant RGS proteins (Figs. 7 and 8C).
Furthermore, because ligand occupancy of the
5-HT1A receptor is required to suppress the
effects of the RGS proteins on constitutive GTPase activity of the
fusion proteins, it was also anticipated that the
pEC50 value for the ligands as inverse agonists
should be in good agreement with measures of ligand affinity. Measures of pEC50 were difficult to obtain for such
ligands at the 5-HT1A receptor-(Cys351Ile)Go1
construct in the absence of an RGS because the measurable effects of
the ligands were small. Affinity estimates were, thus, of low
precision. However, for the 5-HT1A
receptor-(Cys351Ile)Gi1
construct, such estimates were not altered significantly by the
presence of the RGS. This is a more complex issue for agonist ligands
because we have previously noted that the presence of an RGS reduces
the potency of high-efficacy agonists at the
2A-adrenoceptor, although it does not alter
their binding affinity (Hoffmann et al., 2001
). When examining a range
of ligands with inverse agonist activity at the
5-HT1A receptor fusion proteins, it was clear that the rank order of potency was the same for their capacity to
inhibit basal GTPase activity and their affinity to bind to the
receptor with methiothepin >+ butaclamol > chlorpromazine. Furthermore, because the only effects of haloperidol on GTPase activity
occurred at substantially higher concentrations than are consistent
with ligand occupancy of the receptor (Fig. 11), this indicates that
these effects are not specific. Thus, over the pharmacologically
relevant concentration range, haloperidol was shown as a true neutral
ligand for the 5-HT1A receptor.
The general strategy adopted in these studies should be suitable for
any Gi/Go-coupled receptor
that possesses a degree of constitutive activity. Furthermore, although
Gs seems to be resistant to the effects of
traditional RGS proteins, it is possible to employ chimeric G proteins
to overcome this limitation. For example, the effects of vasopressin
V2 receptor agonists became sensitive to RGS
regulation when the receptor was constructed into a fusion protein with
a Gi/Gs chimera that is
activated by the receptor and retains high affinity to bind RGS1 (Feng
et al., 2002
).
These studies provide an entirely novel means of enhancing the
sensitivity of studies on regulation of high-affinity GTPase activity
and, thus, of examining the detailed characteristics of weak partial
agonists and inverse agonists.
Financial support for this work was provided by the Medical
Research Council and the Wellcome Trust. P.W. is a recipient of a
"CASE" studentship from the Biotechnology and Biosciences Research Council.
5-HT, 5-hydroxytryptamine;
GPCR, G
protein-coupled receptor;
GAP, GTPase-activating protein;
HEK, human
embryonic kidney;
RGS, regulator of G protein signaling;
8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin;
PAGE, polyacrylamide gel
electrophoresis.