From the Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and
the Receptor Systems Unit, Glaxo-Wellcome Research and
Development, Stevenage SG1 2NY, United Kingdom
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ABSTRACT |
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A fusion protein between a pertussis
toxin-resistant (C351G) mutant of the subunit of the G protein
Gi1 and the porcine
2A-adrenoreceptor
was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation
of high affinity GTPase activity, which was partially prevented by
pertussis toxin treatment, demonstrating that the toxin-resistant
component of the GTPase activity was derived from the receptor-fused G
protein and the remainder from endogenous Gi
.
Half-maximal stimulation of the GTPase activity of endogenous
Gi was achieved with lower concentrations of agonist. Although the Km for GTP of the fusion
protein-linked Gi was lower than for the endogenous G
protein, Vmax measurements demonstrated that
adrenaline activated some 5 mol of endogenous Gi/mol of
fusion protein-linked Gi. The isolated
2A-adrenoreceptor could activate Gs;
however, the fusion protein did not. Compared with adrenaline, the
efficacy of a range of partial agonists to stimulate endogenous
Gi
was greater than for the fusion protein-constrained C351G Gi1
.
2A-Adrenoreceptor agonists
could stimulate both p44 mitogen-activated protein kinase and p70 S6
kinase and inhibit forskolin-amplified adenylyl cyclase activity in
untreated
2A-adrenoreceptor-C351G Gi1
fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment.
These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.
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INTRODUCTION |
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G protein-coupled receptors
(GPCRs)1 initiate vectorial
signal transduction cascades via activation of heterotrimeric G
proteins and the subsequent regulation of effector enzymes (1, 2). These function to amplify cellular response to the presence of low
concentrations of extracellular mediators. As part of this process,
agonist-occupied GPCRs have the capacity to catalytically activate G
proteins. Some 20 distinct G protein -subunits are known, and in
many cases they are highly similar in sequence. Many cells co-express a
considerable number of distinct but highly related G proteins. As a
novel means to examine the functional interactions of a GPCR with a
single G protein, we recently generated a fusion protein between the
2A-adrenoreceptor and the
-subunit of Gi1
(3, 4). Since Gi1 is a member of the subfamily of G
proteins that can be modified by ADP-ribosylation catalyzed by
pertussis toxin (5) and a number of these G proteins are routinely
co-expressed by all cells (5), we used a modified version of
Gi1
(C351G Gi1
) that is resistant to the
action of pertussis toxin (6) to generate the fusion protein. Following transient expression of the fusion protein in COS-7 cells, we were able
to treat the cells with pertussis toxin to modify the endogenous
Gi-like G proteins and thus eliminate any potential interactions between the GPCR of the fusion protein and the
endogenously expressed forms of Gi (4). Despite this
precaution, there was no obvious indication that the GPCR constrained
within the
2A-adrenoreceptor-C351G Gi1
(
2AR-C351G Gi1
) fusion protein had
significant capacity to activate endogenous G proteins as well as the
fusion partner G protein in an agonist-dependent manner (3,
4). However, GPCRs that have had other proteins such as green
fluorescent protein linked to their C terminus (7, 8) still appear to
be capable of interacting with and activating cellular G proteins (7, 8). We now examine interactions of the
2AR-C351G
Gi1
fusion protein with endogenous G proteins following
stable expression in Rat 1 fibroblasts. We demonstrate that in this
setting agonist-occupation of
2AR-C351G
Gi1
results in stimulation of the high affinity GTPase
activity of both endogenous Gi
and the fusion protein partner G protein and that the activation of endogenous G protein families is selective. Effective regulation of downstream signaling cascades occurred only via activation of endogenous G proteins rather
than via activation of the GPCR-linked G protein. Despite this, the
relative contribution of the GPCR-linked and endogenous Gi
proteins to agonist-stimulated GTPase activity measured at Vmax provided a highly novel means to
demonstrate that the addition of adrenaline was able to activate some 6 mol of G protein/mol of receptor. Furthermore, studies that have
assumed that agonist-dependent prevention of proliferation
of cancer cell lines following expression of a
2-adrenoreceptor-Gs
fusion protein
reflects activation of the receptor-attached G protein (9) may require
careful reanalysis.
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EXPERIMENTAL PROCEDURES |
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Materials
All materials for tissue culture were supplied by Life
Technologies, Inc. (Paisley, Strathclyde, Scotland).
[3H]RS-79948-197 (90 Ci/mmol) was purchased from
Amersham Pharmacia Biotech. [-32P]GTP (30 Ci/mmol) was
obtained from NEN Life Science Products. Pertussis toxin (240 µg/ml)
was purchased from Speywood. All other chemicals were from Sigma or
Fisons plc and were of the highest purity available. Oligonucleotides
were synthesized on a Millipore Expedite Nucleic Acids Synthesis
System.
Generation of the 2A-Adrenoreceptor-C351G
Gi1
Fusion Construct
A pertussis toxin-resistant C351G form of rat Gi1
was generated (6) and linked to the porcine
2A-adrenoreceptor (10). Briefly, the ORF of the
2A-adrenoreceptor DNA was amplified by PCR using the
following oligonucleotides: sense,
5'-TTGGTACCATGTATCCTTACGACGTTC-3'; antisense,
5'-AAGAATTCCATGGCGATCCGTTTCCTGTCCCCACGGC-3'
(restriction sites for KpnI, EcoRI, and
NcoI are underlined). The PCR-amplified fragment was
digested with KpnI and EcoRI and ligated to
pBluescript (pBS) (Stratagene) through these restriction sites.
Introduction of the NcoI site at the 3'-end of the ORF
resulted in the C-terminal amino acid of the receptor being altered
from Val to Ala and removal of the stop codon. The rat C351G
Gi1
cDNA contains two NcoI sites: one
straddling the ATG start codon and the other 268 bp downstream from
this. This 268-bp fragment was removed from C351G Gi1
in pBS by digestion with NcoI, and the remaining C351G
Gi1
pBS cDNA was religated. The shortened cDNA
was excised from pBS with EcoRI and cloned into the
EcoRI site of the
2A-adrenoreceptor in pBS, adjacent to the 3'-end of the receptor ORF. The 268-bp fragment was
then inserted between the NcoI sites at the 3'-end of the
2A-adrenoreceptor ORF and at the 5'-end of the C351G
Gi1
ORF. This resulted in production of an in-frame
construct whereby the 3'-end of the
2A-adrenoreceptor
ORF was exactly adjacent to the 5'-end of the C351G Gi1
ORF. The full fusion construct was then excised from pBS with
KpnI and EcoRI and ligated into the eukaryotic expression vector pcDNA3.
Cell Culture and Transfection
Generation of Stable Cell Lines Expressing the
2A-Adrenoreceptor-C351G Gi1
Fusion
Protein--
Rat-1 fibroblasts were transfected with
2AR-C351G Gi1
in pcDNA3 using
DOTAPTM (Boehringer Mannheim), according to the protocol
provided by the manufacturer, on cells plated in 10-cm2
dishes at a confluency of 50-60%. Cells were allowed to grow for 3 days before being split 1:10 in medium containing 0.7 mg/ml Geneticin
sulfate (Calbiochem). Individual clones were isolated and expanded in
Geneticin-containing medium.
Preparation of Membranes--
Plasma membrane-containing P2
particulate fractions were prepared from cell pastes that had been
stored at 80 °C following harvest. Cell pellets were resuspended
in 0.5 ml of 10 mM Tris-HCl, 0.1 mM EDTA, pH
7.5 (buffer A), and rupture of the cells was achieved with 50 strokes
of a hand-held Teflon on-glass homogenizer followed by passage (10 times) through a 25-gauge needle. Cell lysates were centrifuged at
1000 × g for 10 min in a Beckman TJ-6 centrifuge to
pellet the nuclei and unbroken cells, and P2 particulate fractions were
then recovered by centrifugation of the supernatant at 200,000 × g for 30 min in a Beckman TL 100 bench-top ultracentrifuge
using a Beckman TLA 100.2 rotor. P2 particulate fractions were
resuspended in buffer A and stored at
80 °C until required.
[3H]RS-79948-197 Binding Studies-- Binding assays were initiated by the addition of 2-4 µg of protein to an assay buffer (75 mM Tris, 1 mM EDTA, 12.5 mM MgCl2, pH 7.4) containing [3H]RS-79948-197 (11) (1 nM). Kd for this ligand at the fusion protein was 0.35 nM (3). For saturation experiments, [3H]RS-79948-197 was used between 0.1 and 10 nM. Nonspecific binding was determined in the presence of 100 µM idazoxan. Reactions were incubated at 30 °C for 45 min, and bound ligand was separated from free by vacuum filtration through GF/C filters. The filters were washed with 3 × 5 ml of assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.
High Affinity GTPase Assays--
These assays were performed
essentially as described in Refs. 3 and 4 using
[-32P]GTP (0.5 µM, 60,000 cpm) and
varying concentrations of agonists (up to 10 µM).
Nonspecific GTPase was assessed by parallel assays containing 100 µM GTP. In a range of experiments, GTPase activity was
measured at Vmax as described previously
(3).
Adenylyl Cyclase Activity-- Intact cell adenylyl cyclase activity was measured as described by Wong (12) and Mercouris et al. (13). Briefly, cells in 12-well plates were loaded for 16 h. with 1 µCi/ml [3H]adenine, in the presence or the absence of 25 ng/ml pertussis toxin. Cyclic AMP formation was stimulated by the addition of 50 µM of forskolin, and regulation of forskolin-induced cAMP formation was assayed in the presence of adrenaline or UK14304 (100 µM).
Reverse Transcriptase-PCR (RT-PCR)--
RT-PCR was performed on
RNA preparations from a range of clones expressing different levels of
the 2AR-C351G Gi1
fusion protein as
determined from binding studies, as well as the parental Rat 1 fibroblast cell line.
Regulation of p44 MAP Kinase Phosphorylation and Mobility-- The phosphorylation of p44 MAP kinase was determined by an electrophoretic mobility shift assay (14). Cells were stimulated with the appropriate ligands for 5 min following maintenance in serum-free medium for 48 h and subsequently lysed at 4 °C in a buffer containing 25 mM Tris-HCl, 40 mM p-nitrophenol, 25 mM NaCl, 10% (v/v) ethylene glycol, 10 µM dithiothreitol, 0.2% (v/v) Nonidet P-40, 1 µg/ml aprotinin, 1 mM sodium orthovanadate, 3.5 µg/ml pepstatin A, and 200 µM phenylmethylsulfonyl fluoride at pH 7.5. Following centrifugation of the lysed samples in a microcentrifuge (13,000 rpm, 5 min), SDS-polyacrylamide gel electrophoresis loading buffer was added to a sample of the supernatant and applied to SDS-polyacrylamide gel (10% (w/v) acrylamide, 0.063% (w/v) bisacrylamide, containing 6 M urea) following heating of the sample in a boiling water bath for 5 min. Samples were transferred from the gel to nitrocellulose and immunoblotted with an antipeptide antiserum raised against amino acids 325-345 of p44 MAP kinase (14). These gel conditions provide excellent "gel shift" of this polypeptide upon ligand regulation, a feature that is synonymous with its phosphorylation and activation (14).
p70 S6 Kinase Activity Measurements-- Cells were grown to confluency in 100-mm cell culture dishes and serum-starved for approximately 16 h before treatment. After treatment with agonist for 10 min, the cells were washed twice with ice-cold phosphate-buffered saline before scraping into 1 ml of lysis buffer (55 mM Tris, pH 8.0, 4 °C, 132 mM NaCl, 22 mM NaF, 1.1 mM EDTA, 5.5 mM EGTA, 11 mM sodium pyrophosphate, 33 mM 4-nitrophenyl phosphate, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml pepstatin A, 2 µg/ml leupeptin). 20 min later, the lysates were centrifuged for 10 min at 14,000 × g. The pellet was discarded, and an aliquot of supernatant was retained for Western blot analysis and protein determination. The remainder of the supernatant was precleared for 1 h with 20 µl (packed volume) of protein A-agarose that had been equilibrated with lysis buffer.
Precleared lysates (250 µg) were immunoprecipitated with 2.5 µl of anti-p70 S6 kinase (15, 16) for 2 h at 4 °C before the addition to 20 µl of protein A for a further 1 h. The immunoprecipitates were then washed twice with lysis buffer and twice with assay buffer (25 mM MOPS, pH 7.2, 30 °C, 1 mM EDTA, 0.05% (v/v) Triton X-100, 1 mM dithiothreitol, 20 mM 4-nitrophenyl phosphate). 40 µl of peptide substrate (32-mer of sequence KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, final concentration 20 µM) and 5 µl of PKI were then added to the beads, and following a preincubation for 2 min at 30 °C, the reaction was initiated by the addition of ATP (250 µM ATP, 100 mM MgCl2 containing 3 µCi of [Data Analysis-- Analysis of data curves was performed using the Kaleidagraph curve fitting package driven by an Apple Macintosh computer.
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RESULTS |
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A fusion protein (2AR-C351G Gi1
) in
which the N terminus of a pertussis toxin-resistant (C351G) mutant of
the
-subunit of the G protein Gi1
was fused to the
C-terminal tail of the porcine
2A-adrenoreceptor was
stably expressed in Rat 1 fibroblasts. 3H-Labeled ligand
binding studies using the high affinity and highly selective
2-adrenoreceptor antagonist
[3H]RS-79948-197 were performed in membranes derived from
a number of individual clones and demonstrated the presence of an
apparently uniform population of sites. In clone RAGI 17, this was
expressed at 1.2 ± 0.2 pmol/mg of membrane protein and bound
[3H]RS-79948-197 with Kd = 4.5 ± 0.7 × 10
10 M and in clone RAGI 77 at
3.3 ± 0.3 pmol/mg membrane protein with Kd = 7.8 ± 0.3 × 10
10 M (means ± S.E., n = 3 in each case). RT-PCR of RNA derived from these cells using a primer pair in which the sense primer was designed
to anneal with sequence contributed from the
2A-adrenoreceptor and the antisense primer from
the sequence of Gi1
allowed amplification of a 465-bp
fragment (data not shown). A fragment of equivalent size was generated
by PCR of the fusion protein cDNA using the same primer pair, but
no equivalent product was obtained from RT-PCR of RNA from parental Rat
1 cells (data not shown).
The addition of the natural ligand adrenaline (100 µM) or
the synthetic agonist UK14304 (100 µM) to membranes of
clone RAGI 77 cells resulted in a substantial stimulation of high
affinity GTPase activity (Table I). When
equivalent experiments were performed on membranes derived from
pertussis toxin-treated (25 ng/ml, 24 h) cells, agonist-stimulated
GTP hydrolysis was still marked although reduced (Table I). This level
of pertussis toxin treatment has been shown to fully attenuate
functional interactions between the 2A-adrenoreceptor
and endogenously expressed Gi proteins (6, 17) in Rat 1 cells. Membranes from a clonal cell line (TAG WT17) derived from Rat 1 cells that express some 4 pmol/mg protein of the isolated porcine
2A-adrenoreceptor produced similar levels of stimulation
of high affinity GTPase upon the addition of adrenaline or UK14304
(data not shown). As such, these results demonstrated both that
adrenaline and UK14304 were capable of causing guanine nucleotide
exchange and subsequent hydrolysis of GTP by the GPCR-linked G protein
and that the GPCR within the fusion protein also had the capacity to
activate endogenous Gi-like G proteins as well as the
linked G protein. Trivial explanations for these data, related to
potential reduction in cellular levels of the
2AR-C351G
Gi1
fusion protein during pertussis toxin treatment, were eliminated by performing [3H]ligand binding studies
(data not shown). Equivalent experiments performed on membranes of
clone RAGI 17 also resulted in agonist stimulation of high affinity
GTPase activity, which was partially pertussis toxin-sensitive (data
not shown). However, since the level of stimulation was lower than in
RAGI 77, clone 77 was used for all subsequent studies.
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Time courses of agonist stimulation of high affinity GTPase activity at
37 °C demonstrated that adrenaline allowed hydrolysis of GTP by the
GPCR-associated C351G Gi1 to proceed in an essentially linear manner for at least 60 min. By contrast, the stimulated GTPase
activity derived from the endogenous Gi
population was noted to decay at time points beyond 30 min (Fig.
1), and no further stimulation was
observed at times beyond 60 min. Using a time period at which the
GTPase activity of both the GPCR-linked and endogenous G proteins was
functioning at an essentially maximal rate in response to adrenaline,
when using 0.5 µM GTP as substrate, 31.7 ± 1.3%
(mean ± S.E., n = 11) of the total stimulated
GTPase activity was derived from the fusion protein-linked G protein. Concentration-response curves demonstrated that half-maximal
activation of the endogenous signal was achieved with 3.6-fold lower
concentrations (EC50 for adrenaline = 6.4 ± 0.6 × 10
7 M) of either adrenaline or
UK14304 than required to cause half-maximal activation of the
receptor-linked G protein (EC50 for adrenaline was 2.3 ± 0.4 × 10
6 M) (Fig.
2A and data not shown). Such
data may appear to indicate that the agonists were less potent in
catalyzing GTPase activity of the fusion protein partner.
However, following transient expression in COS-7 cells of either
2AR-C351G Gi1
or
2AR-C351
Gi1
, adrenaline displayed 4.8 ± 1.0-fold
(mean ± S.E., n = 3) greater potency to stimulate
the high affinity GTPase activity of the fusion protein containing the wild type G protein sequence (Fig. 2B).
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By performing agonist-stimulated high affinity GTPase measurements on
membranes of untreated and pertussis toxin-treated clone RAGI 77 cells,
the efficacy of a range of ligands could be assessed. Adrenaline,
noradrenaline, and -methylnoradrenaline all produced a similar
and maximal stimulation of high affinity GTPase activity. The other
agonists tested produced less stimulation at maximally effective
concentrations and functioned as partial agonists. By comparing the
capacity of each ligand to stimulate high affinity GTPase activity
relative to the stimulation produced by adrenaline, measurements of
agonist efficacy could be obtained. The same rank order of efficacy,
adrenaline = noradrenaline =
-methylnoradrenaline > UK14304 > BHT933 = dexmeditomidine = xylazine > clonidine
guanabenz > oxymetazoline, was obtained for
both the receptor-associated Gi1
and the endogenous
Gi
population (Fig. 3).
However, when compared with adrenaline the efficacy of all of the
partial agonist ligands to stimulate the endogenous Gi
population was greater than for stimulation of the GPCR-associated G
protein (Fig. 3). Indeed, in the cases of guanabenz and oxymetazoline,
there was no significant level of stimulation of the GPCR-linked G
protein. The efficacy assessments for agonist stimulation of the
GPCR-associated C351G Gi1
in membranes of clone RAGI 77 cells were, however, almost identical to those obtained following
transient expression of
2AR-C351G Gi1
in
COS-7 cells (18).
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Maximal GTPase activity of the GPCR-linked Gi1 and the
endogenous Gi
pool was assessed by measuring
adrenaline-stimulated GTPase activity in membranes of untreated and
pertussis toxin-treated clone RAGI 77 cells at varying concentrations
of GTP. The large increase in high affinity GTPase activity caused by
adrenaline in membranes of untreated cells was confirmed to represent
an increase in Vmax without an alteration in
Km for GTP (Fig.
4A). Although pertussis toxin
treatment reduced Vmax of the basal high
affinity GTPase activity (Fig. 4B), this was also produced
without a measurable alteration in the measured Km for GTP (Fig. 4B). However, resolution of agonist-induced
stimulation of the GTPase activity of the fusion protein-coupled and
endogenous Gi populations demonstrated the
Km for GTP of the fusion protein-coupled
Gi1 to be some 2-fold lower (260 ± 7 nM)
than for the endogenous Gi (520 ± 50 nM)
(mean ± S.E. n = 3) (Fig. 4B). Since
the levels of expression of the
2AR-C351G
Gi1
fusion protein could be measured directly from
saturation [3H]antagonist binding studies, this allowed
calculation of the catalytic center activity of the
adrenaline-stimulated fusion protein (3) as 4.2 ± 0.5 min
1 (mean ± S.E., n = 3). Since
amounts of endogenous G protein activated by the fusion protein cannot
be assessed directly, it was not possible to measure catalytic center
activity. However, if this is similar to that of the fusion
protein-linked G protein, then it could be estimated from the
Vmax calculations that the adrenaline-occupied
2AR-C351G Gi1
fusion protein was capable
of causing activation of some 5 mol of endogenous Gi
/mol
of receptor-linked G protein at saturating concentrations of GTP (Fig.
4).
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Following stable expression in Rat 1 fibroblasts, Gi-linked
receptors including the 2A-adrenoreceptor (19, 20) and
the
-opioid receptor (14) have been shown to be able to mediate the
phosphorylation and activation of the extracellularly regulated kinase
(ERK) MAP kinases in response to agonist ligands. The addition of
UK14304 (100 µM, 7. 5 min) to serum-deprived clone RAGI
77 cells resulted in the phosphorylation of virtually the entire cellular complement of p44 MAP kinase as measured by a reduced mobility
of the protein through SDS-polyacrylamide gel electrophoresis (Fig.
5). By contrast, UK14304 was unable to
cause phosphorylation of p44 MAP kinase in pertussis toxin-treated
clone RAGI 77 cells (Fig. 5). This was not due to an alteration in the
time frame of p44 MAP kinase phosphorylation and dephosphorylation
(data not shown). As anticipated from previous studies (14), both epidermal growth factor (10 nM, 7.5 min) (Fig. 5) and
platelet-derived growth factor (10 nM, 7.5 min) (data not
shown) were able to cause effective phosphorylation of p44 MAP kinase
in both untreated and pertussis toxin-treated cells. The
2A-adrenoreceptor is also able to stimulate the activity
of p70 S6 kinase in Rat 1 cells by a pathway that is independent of
activation of the ERK MAP kinases (15, 21). UK14304 (100 µM, 10 min) produced robust activation of p70 S6 kinase
in serum-deprived RAGI 77 cells that could subsequently phosphorylate a
peptide substrate (Fig. 6). The same was
true of PDGF (10 nM, 10 min). As with p44 MAP kinase phosphorylation, this capacity of UK14304, but not that of PDGF, was
completely attenuated following pertussis toxin treatment (Fig. 6).
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Expression of the 2A-adrenoreceptor in Rat 1 cells also
allows agonist-mediated inhibition of forskolin-amplified adenylyl cyclase activity in membrane preparations (17). Following stable expression of
2AR-C351G Gi1
in these
cells, UK14304 and adrenaline were able to mediate inhibition of
forskolin-amplified adenylyl cyclase (Fig.
7). However, following pertussis toxin
treatment, this inhibition was no longer observed (Fig. 7). The
2A-adrenoreceptor has been demonstrated to have the
capacity to interact with the stimulatory G protein Gs as
well as the Gi-like G proteins to regulate adenylyl cyclase
activity (22-24). In most circumstances, the inhibitory effect is
predominant, but following pertussis toxin treatment of cells the
activation of Gs
and thence adenylyl cyclase can often
be unmasked (22-24). In Rat 1 cells stably expressing the isolated
2A-adrenoreceptor, neither adrenaline nor UK14304 produced marked effects on forskolin-amplified adenylyl cyclase activity in intact cells (Fig. 8). This
may reflect the combination of capacity of the receptor to activate
both Gi and Gs. In support of this concept,
following pertussis toxin treatment to prevent interaction of the
receptor with Gi, both agonists produced a strong
activation of forskolin (50 µM)-amplified adenylyl
cyclase activity (Fig. 8). By contrast, the agonist-mediated inhibition of forskolin-amplified adenylyl cyclase activity in intact, untreated clone RAGI 77 cells (Figs. 7 and 8) was not converted into a
stimulation following pertussis toxin treatment (Fig. 8); indeed, under
these conditions no effect of agonist was observed. As anticipated, forskolin-amplified adenylyl cyclase activity was not modified by
adrenaline or UK14304 in either untreated or pertussis toxin-treated parental Rat 1 fibroblasts (Fig. 8). Such data are compatible with an
inability of the agonist-occupied
2AR-C351G
Gi1
fusion protein to interact effectively with
endogenous Gs despite its clear activation of endogenous
Gi.
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DISCUSSION |
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Herein, we demonstrate for the first time the capacity of
a GPCR constrained within a GPCR-G protein -subunit fusion protein to interact with and activate G proteins other than that physically linked to it. Furthermore, this interaction was selective between G
protein families, indeed displaying greater selectivity than the
isolated receptor. Using this system, the ability of the natural agonist adrenaline to stimulate high affinity GTPase activity, and the
capacity of the GPCR-G protein fusion protein to be considered as an
agonist-activated enzyme that contains and retains both GPCR and G
protein function, we show that the porcine
2A-adrenoreceptor has the capacity to activate some 6 mol of Gi protein/mol at saturating concentrations of GTP.
Such measurement could not have been obtained in native cell membranes
without the use of the GPCR-G protein fusion protein. Given that the
measured Km for GTP was some 0.4 µM,
with intracellular concentrations of GTP in the region of 10 µM and the measured stoichiometry of G protein activation being assessed at GTPase Vmax, these estimates
are likely to closely reflect the in vivo situation. We have
also examined the capacity of the GPCR to activate downstream signal
transduction via both the activated fusion protein-linked and
endogenous G proteins.
We produced an 2A-adrenoreceptor-Gi1
fusion protein by simply ligating the 5'-end of the G protein cDNA
to the 3'-end of the receptor cDNA. The open reading frame so
produced is expressed as a single, intact polypeptide (3, 4). Following
transient expression of this construct in COS-7 cells, we have
demonstrated that the addition of an
2-adrenoreceptor
agonist to membranes of these cells results in guanine nucleotide
exchange and its subsequent hydrolysis by the receptor-linked G protein
(3, 4). To prove this latter point conclusively, we generated the fusion protein using a pertussis toxin-resistant form of
Gi1
in which cysteine 351, which is the normal acceptor
site for pertussis toxin-catalyzed ADP-ribosylation, was converted to
glycine. Prior pertussis toxin treatment of the COS-7 cells resulted in
modification of all of the endogenous Gi-like G proteins
(6). Since this modification prevents functional interactions between
GPCRs and Gi family G proteins (5), such treatment
determined that the observed agonist regulation of high affinity GTPase
activity could not reflect activation of the endogenous Gi
proteins. Moreover, since agonist stimulation of high affinity GTPase
activity in COS-7 cell membranes following expression of the isolated
2A-adrenoreceptor was completely blocked by pertussis
toxin treatment (6), any measured activity could not reflect
interactions of the receptor with other endogenously expressed G
proteins, such as Gs
, with which the
2A-adrenoreceptor has been reported to interact (22-24) (also see below). Pertussis toxin treatment of COS-7 cells expressing
2AR-C351G Gi1
resulted in little
diminution of agonist signal (3). These data were consistent with
interpretations centered on ideas that either the GPCR in the fusion
protein could only contact its partner G protein or that the cellular
distribution of the
2AR-C351G Gi1
was
completely distinct from the bulk of the endogenously expressed G
protein. It is certainly worth noting in this regard that a number of
studies on GPCR expression on COS cells have indicated that much of the
expressed protein is not targeted appropriately to the plasma membrane
(e.g. see Ref. 25).
Simple attachment of another protein to the C-terminal tail of a GPCR
is not inherently sufficient to prevent interaction of the GPCR with
endogenous G proteins, as noted in studies in which GPCR-green
fluorescent protein fusion proteins have the capacity to regulate
effector function in an agonist-dependent manner (7, 8).
Therefore, to directly assess possible interactions of the
agonist-occupied GPCR within the 2AR-C351G
Gi1
fusion protein with endogenously expressed G
proteins, as well as the GPCR-associated G protein, it was necessary to
express the fusion construct stably. The cell line selected for these
studies was Rat 1 fibroblasts because we have previously stably
expressed the porcine and human
2A-adrenoreceptors in
these cells and shown them to be capable of allowing
agonist-dependent stimulation of high affinity GTPase
activity (17), inhibition of membrane-delineated forskolin-amplified
adenylyl cyclase activity (17), and phosphorylation and activation of
both the ERK MAP kinases (18-20) and p70 S6 kinase (15, 21).
Following stable transfection of 2AR-C351G
Gi1
, two clones were examined by RT-PCR to confirm
expression. Using a primer pair selected to amplify sequence spanning
the site of GPCR and G protein fusion we demonstrated expression of
mRNA encoding the fusion protein in these clones but not in
parental cells. Furthermore, 3H-antagonist binding studies
demonstrated the fusion protein to be expressed at high levels. The
addition of adrenaline to membranes of cells stably expressing
2AR-C351G Gi1
resulted in a robust stimulation of high affinity GTPase activity. This level of activity was greater per copy of the fusion protein than we had observed in transient transfection studies (3) (Fig. 4A), suggesting either that stable expression improved cellular targeting and function
of the fusion protein or that G proteins other than the fusion partner
were becoming activated. Following pertussis toxin treatment of the
cells, the capacity of adrenaline to stimulate high affinity GTPase
activity was reduced significantly. Since pertussis toxin treatment did
not reduce levels of expression of
2AR-C351G
Gi1
, this confirmed that the GPCR within the fusion protein was capable of interacting with and activating endogenous Gi-like G proteins as well as the C351G
Gi1
linked to the receptor.
This is the first demonstration of such a capacity; thus, to explore it
in detail, we devised a series of approaches to examine similarities
and differences in the regulation of these interactions. In the only
other reported construct of a GPCR-G protein fusion protein, between
the 2-adrenoreceptor and Gs
, its
functionality was demonstrated by examining the ability of agonist to
stimulate adenylyl cyclase activity following expression of the fusion
protein in S49 cyc
cells (26). Since S49
cyc
cells lack endogenous Gs
, this system
was clearly not appropriate to address the types of questions posed
herein.
To examine the relative contribution to the total agonist-stimulated
GTPase activity of endogenous Gi compared with C351G Gi1 of the fusion protein, such assays were performed
for 30 min, a period over which both enzymatic activities were
proceeding in a maximal and linear fashion, in membranes of untreated
and pertussis toxin-treated cells. 68.3 ± 2.6% of the total
signal in response to adrenaline was produced via endogenous
Gi proteins and the remainder from the fusion protein.
These experiments were performed at a single (0.5 µM)
subsaturating concentration of GTP, as is the norm for studies on
agonist regulation of GTPase activity. Since the
2AR-C351G Gi1
fusion protein can be
viewed as an agonist-regulated enzyme, the examination of GTPase
activity with variation in GTP concentration allowed measurements of
Vmax for the GPCR-associated and endogenous
Gi protein and direct assessment for the
Km for GTP as substrate. Interestingly, by resolving the contributions of the fusion protein-associated and endogenous G
proteins to total agonist-induced signal by analyzing results in
membranes of untreated and pertussis toxin-treated cells, we noted that
the fusion protein partner G protein displayed higher affinity for GTP
than the endogenous G proteins. Again, this is a unique set of
observations. Since the levels of expression of the GPCR-linked
C351G Gi1
are known from the 3H-ligand
binding studies this allowed estimates of catalytic center activity
(turnover number) of this protein. The measured
Vmax of the endogenous G protein population
cannot be determined in this way, but assuming a similar catalytic
center activity this would predict that the adrenaline-occupied
2AR-C351G Gi1
fusion protein can activate
some 5 mol of endogenous Gi
protein/mol. As noted above,
this measurement of G protein activation stoichiometry could not have
been obtained in native membranes without use of the GPCR-G protein
fusion strategy.
A range of ligands with agonist activity at the
2A-adrenoreceptor are routinely considered as partial
agonists compared with adrenaline. By examining the rate of GTPase
activity produced by these ligands in membranes from untreated and
pertussis toxin-treated clone RAGI 77 cells, this was observed to be
true for activation of both the endogenous and GPCR-linked G proteins.
The rank order of efficacy was the same for the various compounds at
the two G protein pools, but, interestingly, in comparison with the
effects of adrenaline and the other ligands that acted as full
agonists, the partial agonists all displayed greater efficacy to
activate the endogenous Gi-like G proteins. The basis for
this difference is unclear and may relate to constraints imposed by the
fusion or the C351G mutation that was necessary to distinguish agonist activation of these two G protein pools. Concentration-response curves
for stimulation of high affinity GTPase of the endogenous and
receptor-linked Gi
demonstrated a 3-fold higher affinity for activation of the endogenous G protein pool. This difference, however, is likely to reflect differences in affinity of the
agonist-occupied receptor to activate wild type and the C351G G
protein. We have demonstrated previously that following independent
co-expression of the
2A-adrenoreceptor with either wild
type Gi1
or C351G Gi1
, some 10-fold
higher levels of the agonist UK14304 were required to stimulate the
mutant G protein (6). Furthermore, in the present studies we have
compared the capacity of agonist to stimulate the high affinity GTPase
of
2AR-C351G Gi1
and a fusion protein in
which the receptor is linked to the wild type Gi1
sequence (
2AR-C351 Gi1
) following their
transient expression in COS-7 cells. Some 5-fold higher levels of
agonist were required to produce the same stimulation of the fusion
protein containing the C351G mutation. This should not be considered
inherently surprising, since the C-terminal tail of the G protein
comprises a key contact site for a receptor, and the addition of
ADP-ribose to the cysteine residue by pertussis toxin results in
attenuation of receptor-G protein interactions (5).
2-Adrenoreceptors are classical examples of GPCRs that
mediate inhibition of adenylyl cyclase. Like many other
Gi-linked GPCRs, when expressed in appropriate locations,
they are also able to initiate cascades that result in the
phosphorylation and activation of the ERK family of MAP kinases and of
p70 S6 kinase. We have previously demonstrated that following
expression in Rat 1 fibroblasts the
2A-adrenoreceptor is
able to produce each of these effects in a pertussis toxin-sensitive
manner (21). Following expression of the
2AR-C351G
Gi1
fusion protein in these cells forskolin-amplified
intact cell adenylyl cyclase activity was reduced by
2-adrenoreceptor agonists. However, this was virtually abolished following treatment with pertussis toxin, demonstrating the
effect to be due to activation of the endogenous Gi
population (Fig. 7). As anticipated from studies with expressed
isolated
2A-adrenoreceptors, in clone RAGI 77, agonists
were able to cause the phosphorylation and activation of both the p44
MAP kinase and p70 S6 kinase. However, following pertussis toxin
treatment, there was no ability of
2-adrenoreceptor
agonists to activate either of these kinase cascades, although, as
anticipated, epidermal growth factor and PDGF acted as strong
activators of both kinases in both untreated and pertussis
toxin-treated cells (Figs. 5 and 6). It was further noteworthy that the
stably expressed
2AR-C351G Gi1
fusion
protein did not have the capacity to produce an enhancement of
forskolin-stimulated adenylyl cyclase activity in pertussis toxin-treated intact clone RAGI 77 cells. A number of studies have
noted the capacity of agonists at
2-adrenoreceptors to
stimulate adenylyl cyclase activity in defined circumstances (22-24).
This is often effectively observed following pertussis toxin treatment of cells to eliminate interactions of the receptor with
Gi-like G proteins (23, 24). Comparisons of the capacity in
this regard of the isolated porcine
2A-adrenoreceptor
and the
2AR-C351G Gi1
fusion protein were
also illuminating (Fig. 8). Although
2AR-C351G
Gi1
produced strong agonist-mediated inhibition of adenylyl cyclase in intact untreated clone RAGI 77 cells, the isolated
receptor had little effect in these conditions. By contrast, following
pertussis toxin treatment of the cells, although
2AR-C351G Gi1
now failed to modulate
forskolin-amplified adenylyl cyclase activity, the addition of agonist
to cells expressing the isolated receptor resulted in a substantial
increase in activity. The most obvious interpretation of these results
is that although
2AR-C351G Gi1
has the
capacity to activate endogenous Gi in response to agonist,
it does not interact effectively with endogenous Gs. This
is in contrast to the isolated receptor, which in untreated cells
presumably activates a combination of Gi and Gs
to produce little net effect on forskolin-stimulated adenylyl cyclase
and a strong stimulation in pertussis toxin-treated cells. These are again unique observations and provide rather unexpected differences between the isolated receptor and the fusion protein.
Previous studies with a
2-adrenoreceptor-Gs
fusion protein (26)
demonstrated agonist-mediated stimulation of adenylyl cyclase following
stable expression of the fusion protein in S49 cyc
cells,
which lack expression of endogenous Gs
. It might be
argued that the observed effect was due to activation of the endogenous population of
2-adrenoreceptors present in S49 lymphoma
cells now being able to utilize the provided fusion protein-attached G
protein, as it is able to do when provided with exogenous
Gs
in cyc
reconstitution assays. This
appears unlikely, however, due to the high potency of agonist following
expression of the fusion protein. Furthermore, even if it were an
effect of the endogenous receptor, the stimulation of adenylyl cyclase
would still reflect the capacity of the tethered G protein to access
and activate adenylyl cyclase. The minimal capacity of the
2AR-C351G Gi1
fusion protein to mediate
the inhibition of forskolin-amplified adenylyl cyclase via the
physically linked Gi when the fusion protein-activated
endogenous Gi can do so may then reflect limited spatial
opportunities of the fusion protein-linked G protein or the relative G
protein activation ratios that in the fusion protein must, by
definition, be limited to mol/mol of agonist-occupied receptor.
Certainly the
2A-adrenoreceptor has a short C-terminal tail compared with the
2-adrenoreceptor, and little is
known about the cellular distribution of the polypeptides of G
protein-linked signaling cascades relative to one another (27, 28). It
was perhaps even more surprising that the stably expressed
2AR-C351G Gi1
fusion protein showed no
capacity to mediate activation of the signaling cascades leading to
activation of the ERK MAP kinases and p70 S6 kinase, although
activation of endogenous G proteins by the fusion protein did result in
kinase stimulation. Although the early steps of GPCR-mediated
activation of p70 S6 kinase are still unclear, a vast amount of work
has examined the requirements for GPCR-mediated activation of the ERK
MAP kinases. The current consensus is that a key early component is the
complex released by activation of Gi-like G proteins
(29-31), although the relative importance of other proteins including
the isoforms of Shc (32) and a recently identified 100-kDa protein (33)
is still a matter of debate (34, 35). The
2AR-C351G
Gi1
fusion protein interacts with
complex
provided as a combination of
1 and
2
subunits as measured by an enhancement of agonist-stimulated GTPase
activity following their co-expression (3, 4). Furthermore, interaction of the
2-adrenoreceptor-Gs
fusion protein
with
has also been postulated based on the capacity of that
fusion protein to act as a substrate for cholera toxin-catalyzed
ADP-ribosylation only when provided with
complex (26). In both
cases, this is despite the known role of the tethered G protein N
terminus in
interactions (36, 37). Furthermore, we have
demonstrated that relatively low levels of receptor occupancy, and thus
low levels of G protein activation compared with the maximal levels
that can be obtained, are sufficient to fully activate both kinases in
Rat 1 fibroblasts (14-16). If these kinase cascades simply required
release of
complex for their initiation, they would have been
expected to function in both untreated and pertussis toxin-treated
cells, even if the concentration-response curve for the fusion protein partner G protein might have been anticipated to lie to the right of
that for response via endogenous Gi
. Since they do not,
the reasons for this will require further examination and may provide new insights into the early steps in these pathways.
The generation of fusion proteins between GPCRs and G protein
-subunits is beginning to provide new avenues to examine specificity in both the biochemistry and pharmacology of cell signaling systems and
should provide approaches to study the catalytic activation of G
proteins by a GPCR. As such, we predict that fusions of this form will
become widely used. However, the current studies demonstrate that due
attention must be given to whether an observed activity results from
the activation of the G protein partner of the fusion protein or from
activation of endogenous G proteins.
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FOOTNOTES |
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* This work was supported by the Medical Research Council and the Biotechnology and Biosciences Research Council (UK).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.
§ To whom correspondence should be addressed: Davidson Bldg., University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Fax: 44 141 330 4620; E-mail: g.milligan{at}bio.gla.ac.uk.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; 2AR-C351G Gi1
,
2A-adrenoreceptor-C351G Gi1
fusion
protein;
2AR-C351 Gi1
, wild type
sequence; ERK, extracellularly regulated kinase; PDGF, platelet-derived
growth factor; ORF, open reading frame; PCR, polymerase chain reaction;
RT-PCR, reverse transcription-PCR; bp, base pair(s); MAP,
mitogen-activated protein.
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REFERENCES |
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