From the Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
Received for publication, January 22, 2003, and in revised form, February 12, 2003
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ABSTRACT |
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Fusion proteins between heptahelical receptors
(GPCR) and G protein Fusion genes between a G protein-coupled receptor
(GPCR)1 and the corresponding
G protein Often the 1:1 stoichiometry, presumably imposed by fission, was
reported to facilitate the study of GPCR-mediated activation of
The underlying assumption is that the proximal GPCR unit in each fused
assembly can interact with the distal tethered The validity of such assumption was never tested however. Bacterial
toxin inactivation of endogenous G proteins (3, 5, 10) or transfection
in cells lacking functional We prepared a number of fusion proteins consisting of a receptor
sequence ( This indicates that there is no preferential interaction between the
tied units enclosed in one fused assembly and suggests that signaling
at these chimeric proteins is more likely the result of
cross-interactions between receptor and Materials--
Materials came from the following sources: Cell
culture media, G418, and fetal calf serum were from Invitrogen.
Nucleotides (GDP, GTP, GTP Construction of cDNA Coding for Fusion Proteins--
We
gratefully acknowledge the following cDNA gifts: human
Fusion protein mutants carrying modifications in the receptor or in
G Cell Culture and Transfection--
COS-7 and HEK-293 cells were
grown in Dulbecco's modified Eagle's medium supplemented with
10%(v/v) fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml
streptomycin sulfate, in a humidified atmosphere of 5% CO2
at 37 °C. CHO cells were maintained in a 1:1 mix of Dulbecco's
modified Eagle's medium/Ham's F-12,10% fetal calf serum. Transient
transfections of the cells were performed by DEAE dextran/chloroquine,
calcium phosphate precipitation, and FuGENE, for COS-7, HEK-293, and
CHO cells, respectively. In all cases the total amount of transfected
DNA was maintained constant by addition of empty vector. Cells were
allowed to express the transfected gene for 48-72 h before harvesting.
When necessary, pertussis toxin was added to the medium (10-20 ng/ml)
18 h before membrane preparation. To generate stable expressing
HEK-293 and CHO clones, cultures were transfected with FuGENE and
selected under G418 (600 µg/ml active drug) for 3-6 weeks prior to
ring cloning. 20-60 G418-resistant clones were selected and screened by the appropriate radioligand binding assay.
GTP cAMP Determination in Intact Cells--
The determination of
intracellular cAMP levels in transfected cells was made by RIA
following extraction in 0.1 N HCl as described (29).
Receptor Binding Studies--
Radioreceptor binding assays were
made in 1-ml reactions containing 50 mM Hepes-Tris, pH 7.4, 0.2 mM EGTA, 0.2 mM DTT, 5 mM MgCl2, 10 µM leupeptin, 10 µM
bestatin, 0.1 mg/ml bacitracin, 0.1% (w/v) bovine serum albumin, and
suitable amounts of membrane proteins (0.5-20 µg). The following
radiolabeled ligands were used: [125I]pindolol
(50-100,000 cpm) or [3H]dihydroalprenolol (25,000 cpm)
for Toxin-catalyzed ADP-ribosylation--
Bacterial toxin labeling
of Immunoblots--
Proteins separated by SDS-PAGE as above were
electrotranferred onto polyvinylidene difluoride membranes (Millipore
Immobilon). Blots were incubated overnight at 4 °C with primary
antibodies and washed three times with TBST before probing with
alkaline phosphatase or 125I-labeled secondary antibody.
Reactive bands were then visualized by phosphatase staining with
Promega reagents or by phosphorimaging (Packard Cyclone), respectively.
Affinity Pull-down Assays--
Membranes or
[32P]ADP-ribosylated pelleted samples (50-100 µg of
proteins) were solubilized and denatured in 100 µl 0.5% SDS, 10 mM DTT, 5 min at 95 °C. Samples were equilibrated to
room temperature for 1 h and then diluted in 5 volumes of buffer A
(50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 5 mM CaCl2, 0.2% Triton X-100, 0.1% bovine serum albumin, containing protease inhibitor mixture "Complete" from Roche Molecular Diagnostics). Samples were clarified by
centrifugation (40,000 × g, 30 min) and to the
supernatants were added 25-50 µl of a gel slurry (1:1) consisting of
agarose-linked primary antibodies (mouse M1 anti-FLAG, or rat anti-HA)
or wheat-germ agglutinin. Incubation lasted 3 h or overnight under
gentle stirring, at the end of which, agarose beads were pelleted
(14,000 × g, 1 min) and resuspended six times in
Buffer A, prior to discarding the supernatants. Bound proteins were
eluted in 40 µl of Laemmli sample buffer 5 min at 95 °C. For the
batch purification of His6-tagged D1R-G Biochemical Properties of Receptor-G
Fusion proteins appeared as diffuse bands in immunoblots reacted with
either the anti-FLAG or the anti-
The most prominent biochemical feature of membranes transfected with
fusion proteins was enhanced agonist stimulation of GTP
Similar enhancements were observed in the
OP1R-Go system, although in this case
wild-type receptor stimulation of nucleotide exchange is measurable.
Strongly biphasic GDP competition curves in the presence of agonist are
also evident for Go fusion proteins and not observable in
membranes expressing wild-type receptors (Fig. 2, bottom),
even if cotransfected with Interaction of Fusion Proteins with G
To test the competence of tethered Interaction of Fused Receptors with Endogenous
Isoproterenol caused a concentration-dependent increase of
toxin-induced ADP-ribosylation of G
Similarly, in membranes from permanent HEK-293 clones expressing
OP1-G Interaction of Tethered
For the Go system, we used a pertussis toxin-resistant
version (37) of V2TM1-G
For the Gs system, we employed G
In the second approach the cross-interaction was studied more directly
in letting a non-fused wild-type receptor react with the
For the Go system, the toxin-resistant fusion protein
OP1R-G
For the Gs system, we used two experimental schemes, where
in both cases one fusion protein, Cross-interaction between Receptor and G
Next, we investigated cross-interactions between a toxin-resistant
version of G
A strategy that required no functional modifications of either the
receptor or the Effect of Polyanionic Inhibitors of Receptor-G Protein
Coupling--
Molecules carrying rigidly spaced sulfate groups, such
as heparin, suramin, or dextran sulfate, are potent inhibitors of
receptor-G protein interaction (38-40). The mechanism has not been
elucidated yet, but it is conceivably the result of electrostatic
interactions between the charged polymer and polar residues (41)
contributing to the binding of GPCR to
In the case of Gs, the effect of dextran sulfate was
examined using two different systems: in one,
We show in this study that the fusion between GPCR and
Taken collectively, these data provide compelling evidence that when
GPCR and the One interpretation comes from the same intuitive argument that leads us
to assume 1:1 stoichiometry of interaction for the fused construct: the
reaction between receptor and G A trivial consideration is that the peptide bond between the
To question the occurrence of intramolecular coupling in fusion
proteins, Small et al. (12) reported that the signaling properties of A second possible interpretation of the results is that the
supramolecular architecture that organizes the interaction between receptors and G proteins in the membrane (47) plays such a dominant role in driving their reaction, that the effect of a covalent linkage
between interacting partners is irrelevant. A variety of diverse
experimental evidences suggests that the interaction between receptor
and G proteins occurs between preassembled units rather than between
random encounters floating long distances within the plane of the lipid
bilayer (47, 48). It is not known yet what mechanism ensures efficient
"precoupling" of receptor and G proteins in the membrane. However,
data suggesting the formation of stable oligomeric forms for either
G Apart from what inhibits exclusive interactions between the tethered
members of RG fusion proteins, an additional question concerns the
enhancement of signaling efficiency generally observed with such
constructs. If there is no entropic gain in the interaction of
covalently linked receptor and We found that the cotransfection of non-fused receptor with
Restricted membrane mobility, enhanced hydrophobic interactions with
receptor transmembrane domains, improved cytoskeletal interactions
(53), or prevention of G-subunits show enhanced signaling efficiency in
transfected cells. This is believed to be the result of molecular
proximity, because the interaction between linked modules of one
protein chain, if not constrained by structure, should be strongly
favored compared with the same in which partners react as free species. To test this assumption we made a series of fusion proteins (type 1 and
4 opioid receptors with Go and
2
adrenergic and dopamine 1 receptors with GsL) and some
mutated analogs carrying different tags and defective GPCR or G
subunits. Using cotransfection experiments with readout protocols able
to distinguish activation at fused and non-fused
-subunits, we found
that both the GPCR and the G
limb of one fusion protein can freely
interact with non-fused proteins and the tethered partners of a
neighboring fusion complex. Moreover, a bulky polyanionic inhibitor can
suppress with identical potency receptor-G
interaction, either when
occurring between latched domains of a fused system or separate
elements of distinct molecules, indicating that the binding surfaces
are equally accessible in both cases. These data demonstrate
that there is no entropy drive from the linked condition of
fusion proteins and suggest that their signaling may result from the
GPCR of one complex interacting with the
-subunit of another.
Moreover, the enhanced coupling efficiency commonly observed for fusion
proteins is not due to the receptor tether, but to the transmembrane
helix that anchors G
to the membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit are not found in nature, but have been generated
in several laboratories over the past years. First shown by Bertin
et al. (1), if a GPCR with the carboxyl terminus tied to the
amino terminus of an
-subunit are expressed as a single polypeptide,
their functional interactions and the consequent signaling activity are
preserved and even enhanced. Following that observation, similar
constructs were created for a variety of studies, as extensively
reviewed in Refs. 2-4.
-subunits in transfected cells (5-12), or it was exploited to test
the specificity of signals generated by GPCR and multiple
-subunits
(13-20). However, the interpretation of experiments in which fusion
proteins are used as investigational probes requires an understanding
of how receptor and G proteins interact in such chimeric proteins.
-subunit. Thus, what
is normally an intermolecular binding reaction between separate
partners is believed to become, by fusion, an intramolecular interaction between flanked domains of the same protein. This assumption comes from thermodynamic considerations. If the binding surfaces of the receptor and the G
subunit can establish contacts in
the fusion construct, molecular proximity should make this intrinsic
interaction strongly favored over any other.
-subunits (1, 2, 7, 8) were used to show
that receptors and G
subunits of fused proteins are functional and
capable of signaling in host cells. However, this is not proof that
interactions occur intramolecularly in the fusion protein, since it may
simply mean that the receptor of one fusion protein can interact with
the
-subunit of another. In fact, there are data suggesting, to our
view, that intramolecular interactions may not be likely; for example,
the finding that receptors of the fusion proteins interact with
endogenous
-subunits (21) and that agonist binding curves in fusion
proteins show multistate behavior as in non-fused receptors (1, 7, 8, 10, 12). Here we address the question of whether the intrinsic interaction between receptor and the
-subunit of a fusion protein is
entropically favored over the extrinsic interaction with fused or
non-fused partners.
2-adrenergic, D1-dopamine,
-opioid, and nociceptin receptors) chained through a short 15-mer
peptide to the amino-terminal of a G protein
-subunit
(G
sL and G
o). Fusions between mutated analogues of either the receptor or the
-subunit were also
engineered in a similar fashion. Using such constructs and
cotransfection experiments to distinguish the activation of tethered
and non-tethered forms of G
subunits, we find that both components
of one fusion protein freely interact with non-fused units or the fused
elements of another.
-subunits located in
neighboring molecular assemblies.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, ATP), thymidine, phosphocreatine,
creatine phosphokinase, chloroquine diphosphate, DEAE-dextran, dextran
sulfate (500 kDa average mass), l-isoproterenol, anti-FLAG M1, free and
agarose-bound, monoclonal antibody were from Sigma. Pindolol, UK14,304,
N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI 174,864),
and SKF 82958 were from Tocris. Nociceptin,
[D-Ala2,D-Leu5]enkephalin
and vasopressin were from Bachem. Pertussis and cholera (holo and A
protomer) toxins were from List Biologicals. Antibodies to HA epitope
and to G protein
-subunits were from Santa Cruz Biotechnology. Wheat
germ agglutinin and anti-HA monoclonal antibody covalently linked to
agarose, from Vector Labs and Roche Molecular Biochemicals,
respectively. Ni-NTA agarose beads were from Qiagen. Receptor
radioligands, [125I]iodopindolol,
[3H]dihydroalprenolol,
[125I-Tyr14]nociceptin,
[3H]naltrindole, [3H]SCH-23390,
[125I]vasopressin antagonist,
[3H]RS-79948-197, and labeled nucleotides,
[
-32P]GTP (6 Ci/µmol), [35S]GTP
S
(1.2 Ci/µmol), [
-32P]NAD (0.9-1 Ci/µmol) were
from PerkinElmer Life Sciences or Amersham Biosciences. Purified rat
myristoylated Go and a bovine brain purified mix of
Gi/o heterotrimers were from Calbiochem. Purified
-subunits (bovine brain) free of ADP-ribosylating
-subunits were a kind gift from Pat Casey.
2-adrenergic receptor,
2A-adrenergic
receptor, and dopamine D1 receptor (D1R) (Dr. S. Cotecchia), rat OP1R (Dr H. Akil), human OP4R (Dr. G. Calò), rat G
sL (Dr. O. Ugur), rat G
o, and
V2TM1-G
o (Dr. M. Parenti), rat
V2R (Dr. F. Naro). Full-length cDNAs encoding fusion
proteins were all constructed by a similar strategy and subcloned into
the pcDNA3 expression vector (Invitrogen). After removal of the
3'-end stop codon from the receptor cDNA by PCR-based mutagenesis,
the fusion between each receptor (
2AR, OP4R,
OP1R) and
-subunit (G
sL and
G
o1) coding sequence was achieved by insertion of a
linker encoding a 15-mer peptide containing the enterokinase cleavage
site (LDPRSDYKDDDDKGS). The linker, (made by annealing two
synthetic oligonucleotides, sense
5'-CTGGATCCTCTAGACCCCCGGTCAGACTACAAGGATGACGATGACAAGGGCTCCATGGGAATTCGA-3' and the corresponding antisense sequence), was ligated between the carboxyl-terminal of the receptor and the Met initiator codon of
G
via XbaI and NcoI cloning sites,
respectively (underlined in the oligo). The only exception to this
general design is the fusion protein
D1R-G
sL, in which the two coding sequences
were directly linked through two residues Leu and Asp (encoded by a PCR-made XbaI site), which removed the initiator Met of
G
sL.
were constructed by a PCR-based strategy using mismatched primers.
These include: the pertussis toxin-resistant (3)
OP1R-G
o[C350I] and
V2TM1-G
o[C350I] (22), the
receptor-defective (23) OP1R[R146E]-G
o, the G protein-defective (24)
OP1R-G
o[C350R], the GTPase-deficient proteins, OP1R-G
o[Q205L] (25),
OP1R-G
o[D273N] (26) and
OP1R-G
o[Q205L,D273N], the latter of which
contains a xanthine nucleotides binding
o (G
oX) (27). Tagged constructs of
2AR-G
sL and
OP1R-G
o were obtained by placing at the
amino terminus the cleavable prolactin signal peptide tethered to the
FLAG epitope (DYKDDDDK). Upon cleavage, that exposes an amino-terminal
M1 epitope immediately before Gly2 in
2AR,
and upstream Glu2 (with an inserted extra Gly residue) in
rat OP1R. Two diverse tagged versions of the fusion protein
D1R-G
sL were prepared by extending the
receptor amino terminus with either the sequence of the HA epitope
(YPYDVPDYA) or with a hexahistidine tag. Both were placed before
Ala2 of human D1R with an interposed extra Ala
residue. All plasmids used in this study were verified by total
sequencing of the inserted cDNA.
S Binding and GTPase--
Enriched plasma membranes from
transfected cells were prepared by differential centrifugation (28) and
stored (2 mg/ml) at
80 °C. [35S]GTP
S was
determined either in a 100-µl or 1-ml reaction mixture containing 50 mM Hepes-Tris, pH 7.4, 1 mM EGTA, 1 mM DTT, 100 mM NaCl, 5 mM
MgCl2, 1-2 nM [35S]GTP
S, 3 µM GDP (or concentrations varying between 0.1 nM and 100 µM) and 1-2 µg of membrane
proteins, with or without the appropriate full agonist. Samples were
incubated 90 min at 20 °C, filtered onto GF/B (Packard Filterplate)
and washed (6-10 volumes ice-cold buffer) prior to scintillation
counting on a Packard Top Count. Nonspecific binding was determined in
the presence of 10 µM GTP
S. Incubations containing
purified
-subunits were filtered onto polyvinylidene difluoride
membrane plates (Millipore). GTPase assays were done under similar
conditions using 5 µg of membrane protein and a 100-µl reaction
containing 50 mM Hepes-Tris, pH 7.4, 1 mM EGTA,
1 mM DTT, 100 mM NaCl, 5 mM
MgCl2, 1 mM AppNHp, 5 mM
phosphocreatine, 5 units/tube creatine phosphokinase, 0.5 mM ATP, 0.2 mM GTP (spiked with 0.25-0.5 × 106 cpm of [
-32P]GTP). Reactions lasted
10 min at 37 °C, and 32Pi release was
determined by charcoal separation as described (28).
2-adrenergic receptor;
[125I]d(CH2)5-[D-Ile2-Ile4-Tyr-NH29]
Arg-vasopressin (100,000 cpm), for V2R;
[3H]naltrindole (25-50,000 cpm) for OP1R;
[125I-Tyr14]nociceptin (50-100,000 cpm) for
OP4R; [3H]SCH 23390 (50,000 cpm) for
D1R, [3H]RS-79948-197 (30,000 cpm) for
2-adrenergic receptor. Reactions lasted 90 min at room
temperature and were terminated by rapid filtration onto GF/B glass
fiber microplates (Filtermate 196, Packard). Filters were washed three
times with 1 ml of ice-cold buffer and allowed to dry a few hours. The
plates were counted in a Top Count (Packard) following the addition (25 µl) of Microscint 20 (Packard) to each well. Binding isotherms where
fitted by mass-action law models (31) to compute binding affinity and
receptor number (Bmax).
-subunits was performed in 50-µl reactions. The buffer
contained 100 mM Tris-HCl, pH 7.8, 5 mM thymidine, 1 mM ATP, 2.5 mM MgCl2,
and 2-5 µCi of [
-32P]NAD (~1Ci/µmol). For
cholera toxin, we further included 10 mM arginine, 250 ng
of cholera toxin A-protomer, 50-100 µg of membrane proteins, and,
depending on the experiment, the desired concentration of receptor
agonist. For pertussis toxin, we added 25-50 µg of membrane
proteins, 100 µM GTP, 10 mM DTT, 0.5 µg
pertussis holotoxin. Reactions were started by membrane addition,
lasted 30 min at 20 °C, and were arrested by dilution with 0.5-ml
ice-cold Tris-HCl prior to centrifugation (40,000 × g,
10 min at 4 °C). Supernatants were discarded and pellets resuspended
in Laemmli sample buffer at 95 °C. For the experiments carried out
with purified
- and
-subunits the 25-µl reaction volume was
stopped by the addition of sample buffer 4× and heating for 5 min at
95 °C. Proteins were separated by SDS-polyacrylamide gel
electrophoresis (10%) using either regular sized (16 × 18 cm) or
mini slabs (6 × 8 cm). Gels were stained and dried under vacuum
at 60 °C, and the radioactivity incorporated into the separated
protein bands was quantified with a microchannel array detector counter
(Packard Instantimager) with at least 2-sigma counting error accuracy.
s
fusion protein, membranes were extracted and denatured in 0.5% SDS,
containing 70 mM
-mercaptoethanol instead of DTT, diluted in Buffer B (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Titron-X 100, protease inhibitors mixture
EDTA-free) prior to the addition of 25 µl of Ni-NTA agarose and
incubated 1 h at room temperature with gentle agitation. Beads
were washed six times in Buffer B supplemented with 20 mM
imidazole before elution in Laemmli sample buffer. Samples were
separated by SDS-PAGE as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Fusion Proteins--
The
molecular nature of the R-G fusion proteins expressed in host cells was
studied by Western blot analysis, bacterial toxin catalyzed
ADP-ribosylation, and lectin-mediated affinity precipitation, using
amino-terminal FLAG-tagged versions to facilitate immunoblotting and
affinity precipitation.
-subunit antibody, exhibiting
molecular masses consistent with the presence of an added
-subunit
(Fig. 1, a-f). This
microheterogeneity reflects variable glycosylation, because it was also
observed in immunoblots of the proteins after affinity precipitation by
agarose-linked WGA (Fig. 1, c and f). Minor
M1-reacting bands of smaller molecular weights were also detected,
especially in transiently transfected cells with opioid receptor fusion
protein (Fig. 1d) and may represent proteolytic forms,
although neither the inclusion of protease inhibitors during membrane
preparation nor the prolonged incubation of the membranes at 37 °C
did significantly change their abundance. Since the bands were not
retained by WGA-agarose, we suspect that may represent aberrant
deglycosylated and proteasome-degraded forms of the fusion protein,
similar to those previously characterized for wild-type opioid
receptors (32). Broad 90-110 kDa radioactive bands corresponding to
those in Western blotting were labeled, in addition to the endogenous
-subunits, by cholera toxin in membranes expressing
2AR-G
s (Fig. 1g) and pertussis
toxin in those expressing OP1-G
o (Fig.
1h), respectively. Previous treatment of the transfected
cells with the corresponding toxin decreased (30-50%), but did not
abolish, the membrane labeling of the fusion proteins (Fig. 1,
g and h), suggesting that receptor-tethered
-subunits are less susceptible to toxin-mediated ADP-ribosylation. (See more on this below).
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Fig. 1.
Molecular size of fusion proteins in Western
blot analysis and ADP-ribosylation. Upper panel,
Western blot analysis of fusion proteins after SDS-PAGE separation of
total membranes or affinity-precipitated glycoproteins by
agarose-linked WGA. Membranes from control cells (CTR) or a
HEK-293 clone permanently expressing
FLAG 2AR-Gs (RG) were
immunoblotted and visualized with M1 monoclonal (a),
anti-G
s antibody (b), or subjected to a WGA
affinity pull-down procedure (c) (see "Experimental
Procedures") before immunoblotting and staining with
anti-G
s. The blots for
FLAG-OP1R-Go are shown in a corresponding
succession (d-f), and were obtained using membranes
prepared from transiently transfected HEK-293 cells, with empty vector
(CTR) or encoding the tagged fusion protein (RG), except in
e where the control lane (
o) shows membranes
from cells transfected with G
o. Lower panel,
bacterial toxin-catalyzed incorporation of
[32P]ADP-ribose (see "Experimental Procedures") in
membrane from COS-7 transiently transfected with fusion proteins.
g, cells were transfected with pcDNA3 (CTR) or vector
encoding
2AR-Gs (RG) and exposed or not to
cholera toxin (10 ng/ml, 18 h) prior to membrane preparations and
ADP-ribosylation by A-protomer. h, COS-7 transfected with
pcDNA3 (CTR) or vector encoding OP1R-Go
(RG) were similarly exposed and ribosylated with pertussis toxin.
S binding in
the presence of GDP. The effect is striking for
2AR-G
s, since very little adrenergic
stimulation of nucleotide binding can be detected in membranes
expressing even high levels of
2AR, whereas in membranes
containing
2AR-Gs agonist-induced
enhancement of nucleotide binding (Fig.
2, top) and GTPase activity
(not shown) are evident. In addition, a mark of fusion protein-mediated
nucleotide exchange is the biphasic shape of the binding isotherms of
GDP in competition for [35S]GTP
S induced by the
addition of agonist (Fig. 2, top).
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Fig. 2.
Enhancement of agonist-stimulated nucleotide
exchange in receptor-G fusion proteins.
Binding of [35S]GTP
S in the presence of increasing
concentrations of GDP (abscissa) measured in membranes
prepared from COS-7 cells transfected with wild-type receptors or the
corresponding receptor-G protein fusion protein. Upper
panels, membranes (1 µg of protein) expressing
2AR (45 ± 6 pmol/mg), left, or
2AR-G
s (31 ± 4 pmol/mg),
right were tested in the absence (BAS) or presence of 10 µM isoprorerenol (ISO). Lower panels,
membranes (2 µg of protein) expressing OP1R (23 ± 7 pmol/mg) left, or OP1R-G
o
(19 ± 3 pmol/mg) right were tested in the absence
(BAS) or presence of 1 µM DDL. Binding is expressed as
B/T, i.e. ratio of bound versus total (~1
nM) radioligand. Data are representative of at least four
experiments for
2AR-G
s and more than six
for OP1R-G
o.
o (not shown). Thus, they
seem to be a feature of the receptor-tied G protein and indicate that
agonist-induced decrease of GDP affinity is far more noticeable on the
fused than on the non-fused
-subunit.
Subunits--
Reduced
sensitivity to toxin ADP-ribosylation may result from an impaired
ability of the amino-terminal modified
-subunit to interact with
. This point was investigated in more detail using
OP1R-linked Go, since toxin-mediated
ADP-ribosylation of this
-subunit strictly depends on
interaction. As a control to evaluate the role of the amino-terminal
extension in the
o sequence (which blocks amino terminus
myristoylation), we used a fusion protein consisting of the first
transmembrane portion of the vasopressin 2 receptor (amino-terminal
plus TM1) and G
o (V2TM1-G
o). Such TM-anchored
-subunits were previously found localized in the plasma membrane,
despite the lack of fatty acid acylation (22). Thus,
V2TM1-G
o closely mimics the
structural modification of the GPCR-tethered
-subunit, but in the
absence of a functional receptor. Cells transfected with wild-type
G
o, V2TM1-G
o, or
OP1R-G
o, were exposed or not to pertussis
toxin and compared. The level of toxin-catalyzed incorporation of
[32P]ADP-ribose detected in membranes indicates that when
G
o is N-tethered either to a full receptor or
to a single transmembrane domain, it is less ribosylated in
vivo than native resident Gi or exogenously
transfected Go (Fig.
3A). This implies that the presence of the amino-terminal transmembrane anchor reduces the accessibility of
o to toxin-mediated modification.
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Fig. 3.
Pertussis toxin resistance of
OP1R-G o and
interaction with
-subunits.
A, duplicate T75 flasks of COS-7 cells transfected with
empty plasmid (CTR) or vectors expressing wild-type
G
o (Go), or the fusion proteins
V2TM1-G
o
(TM-Go) and OP1R-G
o
(R-Go), were exposed or not to pertussis toxin
(20 ng/ml, 18 h) before harvesting. Membranes were
[32P]ADP-ribosylated with pertussis toxin and separated
by SDS-PAGE. Histograms indicate the radioactivity determined
(Instantimager) in the 40-kDa bands for CTR and Go, in the
50-55-kDa range for TM-Go, and in the 80-110-kDa range for R-Go
(inset). The percent of ribosylable protein that survives
toxin exposure of intact cells is indicated on top of each
bar. The experiment was repeated twice with similar results.
B, membranes (25 µg) from COS-7 cells expressing the
fusion protein OP1R-G
o, and membranes from
pertussis toxin-treated control cells supplemented with 5 ng of
purified G
o (
o) were ADP-ribosylated in
the presence of increasing concentrations of purified bovine
(x-axis), separated by SDS-PAGE, and counted in a Packard
Instantimager. The radioactivity incorporated into purified
o-subunit (
) or fusion protein (
) is plotted as a
function of
concentrations. The experiment was repeated two
additional times, (using
V2TM1-G
o in one case), with
similar results.
o to interact with
, we measured the ability of increasing concentrations of
purified
-subunits to support pertussis toxin-catalyzed
incorporation of [32P]ADP-ribose into
OP1R-G
o (Fig. 3B). Purified
myristoylated G
o added to membranes of pertussis
toxin-treated COS-7 cells (to suppress background labeling of
endogenous Gi) served as a reference. G
was equally
effective in enhancing ADP-ribosylation of the two proteins (Fig.
3B), and similar results were obtained when
was added
to V2TM1-G
o (not shown). Thus, the presence
of the amino-terminal tethered sequence does not disrupt the
interaction of the
-subunit with
in isolated membranes.
-Subunits--
Fusion proteins between
2-adrenergic
receptors and Gi were found to interact with endogenous G
proteins in the membrane of host cells (21). We wondered whether there
was any preference in the interaction of fused receptors for tethered
and non-tethered
-subunits. To distinguish the two forms we used
agonist-enhancement of cholera toxin-catalyzed ADP-ribosylation, which
reflects receptor-mediated activation at several G proteins, such as
Gi/o, Gs, and Gt (33-36), and
gives the molecular mass of the activated
-subunit.
s (Fig.
4A, left) in
membranes of CHO cells expressing wild-type
2-adrenergic
receptor. At the same concentrations, it enhanced the labeling of both
the endogenous Gs and of the 110-kDa bands corresponding to
the fusion protein in membranes expressing
2AR-Gs (Fig. 4A,
right). This means that the tethered
2-adrenergic receptor can freely interact with both fused and non-fused G
s subunits and implies little
preference for the two types of interactions. Analogous results were
observed in experiments performed by transient expression in COS cells (not shown).
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Fig. 4.
Interaction of receptor-G
fusion proteins with endogenous
-subunits. A, membranes prepared
from CHO clones permanently expressing wild-type
2-adrenergic receptor (left) or
2AR-Gs fusion protein (right)
were ADP-ribosylated with [32P]NAD and cholera toxin
A-protomer in the presence of increasing concentrations of
isoproterenol (graph x-axis). The radioactivity (120 min
counting time) incorporated into endogenous Gs (46 and
52-kDa bands,
) and fusion protein (97-115 kDa,
) is plotted as a
function of the log of isoproterenol (ISO) concentration
beneath. The curves (solid lines) were fitted with a
4-parameter logistic function (30) to compute the EC50 for
agonist-mediated enhancement of ADP-ribosylation: (in
2AR CHO, 7 ± 2 nM; in
2AR-G
s CHO, 16 ± 6 nM
for
s stimulation and 8 ± 3.2 nM for
2AR-
s stimulation, respectively). The
experiment is representative of two independently made membrane
preparations of the same CHO clone. An additional experiment made on
transiently transfected COS-7 cells also gave comparable results.
B, membranes from HEK-293 clones permanently expressing
Go fused to two different opioid receptors,
OP1R-G
o or
OP4R-G
o, were
[32P]ADP-ribosylated as above under basal conditions
(B) or in the presence of the respective agonists,
[D-Ala2,D-Leu5]enkephalin
(D) 1 µM and nociceptin (N) 1 µM for OP4R-G
o.
Left, SDS-PAGE separation of total membranes
(tot); right, samples were first solubilized and
affinity-precipitated with WGA before electrophoresis. Note that after
affinity purification of the fusion protein agonist enhancement of
ADP-ribose incorporation in tethered
o is evident.
o or OP4-G
o,
the addition of agonist strongly enhanced cholera toxin-mediated
ADP-ribosylation of endogenous 40/41-kDa bands, demonstrating
interaction of the fused receptor with endogenous Gi (Fig.
4B). The extent of stimulation was comparable to that observed in cells transfected with non-fused receptors. The concurrent incorporation of ADP-ribose into the fusion protein was not detectable in this experimental protocol, because the lower level of labeling of
the tethered
o-subunit by cholera toxin was obscured
by an endogenous ADP-ribosylated band of similar molecular mass. This
band, particularly abundant in HEK-293 cells, does not correspond to a
bacterial toxin substrate, since it was also ribosylated in its absence
(not shown). However, agonist enhancement of cholera toxin
ADP-ribosylation of the fused G
o becomes clearly
detectable if the fusion protein is separated from the bulk of membrane
proteins by WGA-agarose (Fig. 4B). Thus, both
Gs- and Go-linked receptors can liberally and
efficiently interact with endogenous free
-subunits of the membrane.
-Subunits with Non-fused
Receptors--
To determine the accessibility of the
-subunit of a
fusion protein to an external receptor we used two approaches. In the first approach, we evaluated how efficiently a wild-type GPCR can
activate a cotransfected
-subunit that is linked to a transmembrane domain peptide.
o
(TM-G
o[C350I]) (22). This was cotransfected with
wild-type OP1R, and cells were exposed to pertussis toxin to eliminate the contribution of endogenous Gi.
Agonist-induced nucleotide exchange was abolished by pertussis toxin
treatment in membranes expressing only OP1 receptors, but
not in those co-expressing OP1R and
TM1-G
o[C350I] (Fig.
5). Moreover, the curve for GDP inhibition of [35S]GTP
S binding induced by agonist in
OP1R/V2TM-G
o[C350I]
co-expressing membranes was biphasic as that typically observed for
transfected OP1R-Go fusion protein (compare
with Fig. 2). Thus, transmembrane-anchored Go interacting
with a non-fused receptor can emulate the enhancement of
receptor-mediated nucleotide exchange observed for fusion proteins. Cotransfection of OP1R with wild-type Go (which
was expressed at levels 2-3-fold greater than
V2TM-G
o as judged by Western blots) could
not reproduce the same type of agonist-induced GDP shift (data not
shown), indicating that a transmembrane tether is important for such an
effect.
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Fig. 5.
Single transmembrane domain
(V2TM1)-tethered
o cotransfected with opioid receptor
emulates the enhanced nucleotide exchange observed with the
corresponding fusion protein. Binding of
[35S]GTP
S in the presence of increasing concentrations
of GDP measured in membranes prepared from transiently expressing
HEK-293 cells, individually transfected with wild-type OP1R
(top) or cotransfected (1:1) with OP1R and the
pertussis toxin-resistant fusion protein
V2-TM1-G
o[C350I]. Cells were
treated with pertussis toxin (20 ng/ml, 18 h) before membrane
preparation. Binding was measured in the absence of ligand
(Bas) or in the presence of either DDL 1 µM,
or the negative antagonist ICI 174,864 1 µM. Data are
representative of three independent transfections.
s fused to
OP1R (OP1R-G
s), acting as
transmembrane carrier of the appended
-subunit. In fact, in
membranes expressing OP1R-Gs, pertussis toxin
suppressed opioid agonist-stimulated GTPase activity (Fig.
6A, left),
indicating that it entirely result from the interaction of the
Gs-fused receptor with endogenous Gi proteins.
Moreover, transfection of OP1R-Gs in intact
cells caused enhancement of basal cAMP levels, which were inhibited by
opioid agonist, just like in cells cotransfected with OP1R
and Gs as separate proteins (Fig. 6A,
right). Therefore Gs attached to the opioid
receptor neither interacts with it nor disturbs its interaction with
endogenous Gi, and this fusion protein can thus be used as
a form of Gs linked to an inert transmembrane sequence,
exactly like V2TM-Go described above. In
membranes co-expressing OP1R-G
s and
wild-type
2-adrenergic receptor, isoproterenol promoted at similar concentrations cholera toxin-catalyzed incorporation of
ADP-ribose in both endogenous Gs and
OP1R-linked
s (Fig. 6B, right), as observed for the
2AR-G
s fusion protein. In addition, in
the same membranes isoproterenol induced GDP inhibition curves very
similar to those measured for
2AR-G
s
(data not shown). This again indicates that enhanced nucleotide
exchange can be generated when a wild-type receptor interacts with the
transmembrane-anchored
-subunit.
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Fig. 6.
Interaction of opioid receptor-tethered
Gs
(OP1R-G s) with
non-fused
2AR.
A, left, GTPase activity in membranes
prepared from COS-7 cells transfected with OP1R
(R) or the fusion protein OP1R-G
s
(RG) that were treated or not with Cholera (CT)
or pertussis toxins (PT) (both, 10 ng/ml 18 h).
Activity was measured in the absence (bas) or presence of
opioid agonist (DDL) and negative antagonist
(ICI) (both 1 µM). Data are means (± S.E.) of
three experiments. Right, HEK-293 plated in 24-well dishes,
were individually transfected with control vector (CTR),
OP1R (R), OP1R-G
s (RG),
G
sL (Gs), or cotransfected with both
OP1R and Gs (R+Gs). Cells were
incubated (30 min) 48 h later in the absence (bas) or presence of
1 µM DDL, and the intracellular levels of cAMP were
measured by RIA. Data are means (± S.E.) of four experiments.
B, COS-7 cells membranes cotransfected with
2AR and OP1R-G
s were
ADP-rybosylated with [32P]NAD and A-protomer of cholera
toxin in the presence of increasing concentrations of isoproterenol as
in Fig. 4. The gel shown is representative of two independent
experiments, both of which were quantified (microarray detector), and
the incorporation of ADP-ribose into endogenous Gs (
)
and OP1R-Gs (
) is plotted as a function of
the log of isoproterenol (ISO) concentration. Points are
means (± range) of c.p.m. values normalized as ratios over the values
in the absence of agonist.
-subunit
linked to another functional GPCR. To do so, we co-expressed pairs of
pharmacologically distinct receptors, both able to couple, but only one
of which covalently docked to the same G protein.
o[C350I] was co-expressed with
wild-type
2-adrenergic receptors. In pertussis
toxin-treated membranes, the OP1R-tethered
G
o displayed enhanced nucleotide exchange in response to
either
2-adrenergic and opioid agonists (Fig.
7A). The effects of the two
agonists were not additive, indicating that there was no apparent
co-partitioning of G
between the two types of receptors (Fig.
7A).
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Fig. 7.
Interaction of a non-fused receptor with
the -subunit tethered to a different
GPCR. A, binding of [35S]GTP
S in
the presence of increasing concentrations of GDP in membranes prepared
from transiently expressing HEK-293, either transfected individually
with wild-type
2BAR (left) or cotransfected
(1:1) with
2BAR and the fusion protein
OP1R-G
o[C350I]. Cells were treated with
pertussis toxin (10 ng/ml, 18 h) before membrane preparation. The
binding was measured in the absence of ligand (Bas), and in
the presence of 1 µM DDL, 1 µM
2-adrenergic agonist UK14,304 (UK), or both. The
Bmax of expressed receptors was 13 pmol/mg for
2AR and 15.8 pmol/mg for
OP1R-G
o[C350I]. Data are representative of
three independent experiments. B, agonists effect on
cholera toxin-catalyzed ADP-ribosylation of COS-7 membranes
cotransfected with vasopressin receptors (V2R)
(Bmax, 1.7 pmol/mg) and the fusion protein
2AR-Gs (Bmax, 9.8 pmol/mg).
Experiments were performed and quantified as in Figs. 4A and
6B. The inset shows a representative
autoradiogram displaying the effect of varying concentration of
arginine vasopressin (AVP). The graph shows concentration-response
curves for AVP-induced enhancement of the ribosylation of the
2AR-Gs band in the absence (
) or presence
(
) of isoproterenol (1 µM). Data are averaged (± range) from two independent experiments normalized as ratios of cpm in
the presence of ligand versus cpm in the absence.
C, a, Stimulation of cholera toxin
ADP-ribosylation in membranes from COS-7 cells cotransfected with
D1R (Bmax, 6.4 pmol/mg) and
2AR-Gs (10.2 pmol/mg). Histograms are the
radioactivity incorporated into endogenous Gs
(Gs band) or the fusion protein
(
2AR-Gs band). bas, no ligand;
skf, 100 nM SKF 82958; iso, 1 µM isoproterenol; skf+iso, both ligands. Data
are representative of two independent experiments. b,
stimulation of [35S]GTP
S binding in the presence of 3 µM GDP determined in COS-7 membranes individually
transfected with D1R or cotransfected with either
D1R and G
s (D1R + Gs) or
D1R and
2AR-Gs. Agonists are
indicated as in a. Data are means (± S.E.) of
three experiments.
2AR-Gs,
was co-expressed with a second Gs-competent receptor.
Vasopressin V2 receptor (VP2R) was used in one
protocol and D1R in the other. As shown in Fig. 7B vasopressin enhanced in a
concentration-dependent manner cholera toxin-catalyzed
labeling of the fusion protein band in membranes expressing
V2R and
2AR-Gs, with an effect
non-additive to that of isoproterenol. Similarly, in membranes
co-expressing D1R and
2AR-Gs,
both isoproterenol and the dopamine selective agonist SKF-82958
increased ADP-ribosylation of endogenous and
2-adrenergic receptor-tethered Gs (Fig.
7C, a), and produced non-additive enhancements in
GTP
S/GDP binding exchange (Fig. 7C, b).
Linked to Distinct
Fusion Proteins--
Finally, we evaluated the existence of
interactions between the receptor unit of one fusion protein and the
G
unit of the other. First, we examined a couple of fusion proteins
in which the receptor of one and the
-subunit of the other carried
inactivating mutations. An inactive OP1R mutant
(Arg146 in the highly conserved DRY motif replaced with
Glu), was fused to wild-type G
o
(OP1R[R146E]-G
o). The matching G
protein-defective counterpart was made by tethering intact
OP1R to a G
o subunit that was inactive
because of the substitution of Cys350 by Arg
(OP1R-G
o[C350R]) (24). Since, as shown
before, a large portion of receptor-tethered G
o can
survive ADP-ribosylation in vivo, cells cotransfected with
these two constructs were exposed to pertussis toxin to eliminate
endogenous Gi response. As shown in Fig.
8A, neither the
receptor-defective nor the G
-defective fusion proteins displayed
agonist-mediated enhancement of GTP
S binding when they were
individually expressed in membranes of pertussis toxin-treated cells.
However, co-expression of the two proteins restored efficient agonist
effect, indicating that the intact receptor of one protein can easily
interact with the functional G
of the other.
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Fig. 8.
Interaction of non-fused receptors with
-subunits fused to a different GPCR.
A, HEK-293 were individually transfected or cotransfected
(as indicated) with two mutants of the opioid receptor-Go
fusion protein: OP1R[R146E]-G
o (carrying
an inactive opioid receptor) and
OP1R-Go[C350I] (carrying an inactive
G
o). Control cells were transfected with empty plasmid
(CTR) or wild-type opioid receptor
(OP1R). Cells were treated with
pertussis toxin, and the stimulation of [35S]GTP
S
binding in the presence of agonist (DDL, 1 µM)or negative
antagonist (ICI, 1 µM) was studied in the presence of GDP
3 µM. Data are means (± S.E.) of three experiments.
B, binding of [35S]GTP
S in the
presence of increasing concentrations of GDP in membranes prepared from
HEK-293 cells individually transfected with a fusion protein between
delta opioid receptor and a constitutive active mutant of
o (OP1R-G
o[Q205L])
(a), a fusion protein between nociceptin receptor and the
pertussis-resistant mutant of
o
(OP4R-G
o[C350I]) (b), or
cotransfected with both fusion proteins (c). Cells were
exposed to pertussis toxin (10 ng/ml, 18 h) before membrane
preparation. Binding is shown in basal conditions (Bas), and
in the presence of agonists, DDL or nociceptin (NOC), 1 µM both. Bmax (pmol/mg) of expressed
receptors were 31 for OP1R-G
o[Q205L], and 8.1 for
OP4R-G
o[C350I], when individually
expressed, and 22 and 3.2, respectively, when co-expressed. Data are
from one experiment, which was replicated two additional times by
measuring GTP
S binding at 3 µM GDP. C,
control HEK-293 cells (left) and a stable HEK-293 clone
(right) expressing a fusion protein between
-opioid
receptor and the XTP-shifted
o mutant
(OP1R-G
oX) were transfected with increasing
(1.25-40 µg/flask) concentrations of cDNA encoding
OP4R-G
o[C350I]. cDNA was mixed with
non-coding plasmid to maintain the total amount constant. Cells were
exposed to pertussis toxin before membrane preparation. Agonist
stimulation of [35S]GTP
S binding (DDL or Noc, both 1 µM) was determined in the presence of 3 µM
GDP using 2 µg of membrane proteins. The levels of the resident
OP1R-G
oX and the transfected
OP4R-G
o were measured by radioligand binding
assays ("Experimental Procedures"). The expression of
OP1R-G
oX (47.3 ± 6.2 pmol/mg) did not
change significantly with OP4R-G
o[C350I]
cDNA transfection, but the latter produced less expression in the
permanent clone than in control cells (note the difference in axis
scale between the graphs). The binding is plotted as a function of
OP4R binding activity. Data are means of quadruplicate
determinations of a single experiment in which control and clone cells
were transfected and assayed simultaneously. The experiment was
repeated two additional times using different protocols. In one, both
fusion proteins were transiently transfected in COS-7 cells, whereas in
the other increasing amounts of OP1R-G
oX
cDNA were transiently transfected in a HEK293 clone permanently
expressing OP4R-G
o[C350I]. Similar results
were obtained in all cases.
o-fused nociceptin receptor
(OP4R-G
o[C350I]) and a series of
constructs consisting of OP1R linked to GTPase-defective mutants of
o. In some experiments
OP4R-
o was co-expressed with OP1R-fused to the constitutively active
G
o[Q205L] mutant (25). In pertussis toxin-treated
membranes expressing only OP1R-G
o[Q205L] there was no DADLE-mediated enhancement of GTP
S (Fig.
8B), presumably because the mutation abolishes any change of
GDP affinity in response to receptor activation. However, in membranes
cotransfected with the two fusion proteins, both DADLE and nociceptin
enhanced GTP
S binding, indicating that receptors from both fusion
proteins can interact with the OP4R-tethered G
. There
was no nonspecific stimulation of OP4 receptors by DADLE,
as verified using cells expressing only
OP4R-G
o[C350I] (Fig. 8B).
Identical results (data not shown) were observed when the nociceptin
receptor-Go construct was cotransfected with
OP1R tethered to G
o[D273N], a mutant with
reduced guanine nucleotide affinity (26). In still other experiments,
the cross-interaction was monitored using OP1R joined to
G
oX (the xanthine nucleotide-shifted mutant of
o resulting from the association the two mutations above) (27). When increasing concentrations of cDNA coding for OP4R-G
o[C350I] were transfected in a
HEK-293 clone permanently expressing
OP1R-G
oX, both DADLE and nociceptin-mediated
enhancements of nucleotide exchange in toxin-treated membranes were
increased in parallel (Fig. 8C), demonstrating that the
o-subunit of the co-expressed fusion protein can be
activated by both tethered receptors.
-subunit of the fusion construct was also used.
Differentially amino-terminal-tagged fusion proteins consisting of
FLAG-
2AR-Gs and dopamine
D1R-Gs (carrying either a HA or a poly(His)6 sequence) were co-expressed. The activation of
Gs (studied as agonist-enhanced ADP-ribosylation catalyzed
by cholera toxin) was individually estimated for each fusion protein
band by gel electrophoresis after solubilization and a selective
pull-down procedure with affinity gels. To suppress cross-precipitation possibly due to receptor oligomerization or other protein-protein interactions, membranes were solubilized under strong denaturating conditions (0.5% SDS, 10 mM DTT at 95 °C) prior to
affinity precipitation. As shown in Fig.
9A for co-expressed
FLAG-
2AR-Gs and
HA-D1R-Gs, the enhancement of ADP-ribose
incorporation into the
2AR-Gs band immunoprecipitated by M1 antibody was stimulated by both isoproterenol and SKF-82958. Likewise, both agonists were capable of enhancing the
ADP-ribosylation of D1R-Gs, which was
selectively recovered by the anti-HA agarose. Similar results were
obtained using membranes co-expressing
FLAG-
2AR-Gs with
(His6)-D1R-Gs, which was
affinity-precipitated by nickel-agarose (Fig. 9A). Western
blots of the M1-immunoprecipitate with anti-HA antibody or of the
HA-immunoprecipitate with M1 antibody did not show any detectable
cross-contamination (not shown). Although quantification of the labeled
bands suggests some degree of preference (Fig. 9B), the data
clearly indicate that each receptor can interact and activate both the
homologous and the heterologous tethered Gs.
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Fig. 9.
Cross-activation of
-subunits from receptor tethered to distinct fusion
proteins. A, ligand-induced enhancement of cholera
toxin ADP-ribosylation by agonists of two different GPCR-Gs
fusion proteins co-expressed in the same cells. Membranes were prepared
from COS-7 cells transfected with pairs of cDNA (1:1 mix) encoding
Gs-fused receptors tagged with different epitopes:
FLAG-
2AR-Gs and
HA-D1R-Gs (left), or
FLAG-
2AR-Gs and
His-D1R-Gs (right). Membranes were
[32P]ADP-ribosylated by cholera toxin A-protomer in the
absence (Bas) and presence of the indicated agonists, Iso, 1 µM; Skf, 100 nM; and equal aliquots (100 µg) were affinity-precipitated under denaturating conditions with the
appropriate agarose-linked probe (anti-FLAG mouse monoclonal M1,
anti-HA rat monoclonal, and nickel-NTA) to selectively recover each
labeled fusion protein (see "Experimental Procedures").
B, radioactivity of the bands of immunoprecipitated
fusion proteins (Instantimager) is averaged after normalization (ratios
of incorporated cpm in the presence versus absence of
agonist). Shown are means (± range) of four experiments for M1
precipitates and two experiments for nickel.
-subunit. We reasoned that if
the fusion process converts the reaction between receptor and
-subunit from intermolecular to intramolecular, steric hindrance
should reduce the efficacy of a very high molecular weight inhibitor.
Thus, the potency of 500 kDa dextran sulfate to block G protein
activation may significantly differ when receptor and G
interact as
separated proteins or as partners of a fused construct.
2AR-Gs was confronted with co-expressed
2AR and OP1R-Gs, (Fig.
10A); in the other,
D1R-Gs was compared with cotransfected
D1R and
2AR-Gs (Fig.
10B). In the case of Go, membranes expressing
either OP1R-G
o or a mixture of
OP1R and V2TM1-G
o
were examined (Fig. 10C). In all the experiments dextran
sulfate inhibited agonist-induced nucleotide exchange at fused and
non-fused interactions with virtually identical IC50 (Fig.
10, A-C). At the range of concentrations used the inhibitor did not affect the binding of GTP
S to recombinant G
o
or to a Gi/o mixture purified from bovine brain (Fig.
10D), nor did it change GDP affinity (Fig. 10E),
indicating that the block of agonist-enhanced nucleotide binding
observed in transfected membranes does indeed reflect disruption of
receptor-G
interactions. These data indicate that the surface of
interaction between a GPCR and an
-subunit is equally accessible to
bulky inhibitors either when they react as part of the same protein or
as distinct molecular entities.
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Fig. 10.
Inhibitory effect of dextran sulfate at
receptor and G reacting as fused partners or
as separated proteins. Concentration-response curves for the
inhibition by 500-kDa dextran sulfate (DS) of
agonist-mediated enhancement of GTP
S binding in the presence of GDP
(3 µM). Each panel (A-C) shows a comparison
between fused and non-fused interactions, using membranes prepared from
cells, either individually transfected with a GPCR-G
fusion proteins
or cotransfected with the same proteins in non-fused condition. Data
were fitted (lines) to a 4-parameter logistic function (30)
to compute the IC50 (± S.E.). A, HEK-293 cells
expressing
2AR-Gs (IC50,
233 ± 35 pM) or
2AR together with
OP1R-Gs (IC50, 328 ± 48 pM).
B, COS-7 cells expressing
D1R-Gs (IC50, 214 ± 13 pM) or a combination of D1R and
2AR-Gs (IC50, 113 ± 19 pM). C, HEK-293 cells (treated with
pertussis toxin before membrane preparation) expressing
OP1R-Go[C350I] (IC50, 204 ± 5 pM) or the combination of OP1R and
V2TM-Go[C350I] (IC50, 305 ± 18 pM). Data points (means of triplicate determinations)
are net agonist-stimulated binding (after subtraction of the binding
measured in the absence of ligand at each dextran sulfate
concentration) and are representative of experiments that were
replicated at least twice with identical results. Panels D
and E, lack of effect of DS on the nucleotide-binding
site of purified G proteins. D, binding of
[35S]GTP
S (1 nM) to myristoylated
o (recombinant, 1 nM) or Gi/o
heterotrimers (from bovine brain, 100 ng/tube) at the indicated
concentrations of DS. E, binding of
[35S]GTP
S to Gi/o at the indicated
concentrations of GDP in the absence or presence of 100 nM
DS.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit, although ensuring their expression as tethered units in the plasma membrane, does not force their interaction to the 1:1 stoichiometry that molecular proximity would imply. This conclusion is
based on a variety of diverse experimental evidences. (a) We found that agonists acting through a fused GPCR can activate with identical EC50 both fused and endogenous
-subunits,
which suggests that the receptor of the fusion protein can bind to
either
-subunit with equal efficiency. (b) The
characteristic enhancement of GTP
S stimulation observed in fusion
proteins could be mimicked when a non-fused GPCR and a
-subunit
tethered to a single transmembrane polypeptide are co-expressed in the
membrane, which indicates that this enhanced stimulation does not
require molecular proximity but simply that the
-subunit be inserted
in the membrane via a transmembrane handle. (c) Similarly,
the GPCR-tethered
-subunit of a fusion protein can be activated by a
second non-tethered GPCR receptor cotransfected in the same membrane,
and the effects are not additive when both agonists at saturating
concentrations are present. This means that tethered and not GPCR can
compete for the same receptor-linked
-subunit. (d)
Moreover, we found extensive cross-reactions between the GPCR of one
fusion protein and the
-subunit of another, when cotransfected in
the same membrane. This cross-reaction was observed regardless of
whether in one of the cotransfected pair of fusion proteins the
-subunit was made inactive or constitutively active. It was also
verified by individually measuring G
activation in two
Gs-attached co-expressed receptors. (e) Finally,
the fact that a bulky polyanionic inhibitor blocks with identical
potency receptor and G protein reacting as fused or as separate
partners is additional evidence that the interacting surfaces are
equally accessible in both cases.
-subunit are linked into one protein, interactions can
occur with equal chance within the same or between separate macromolecules. Therefore, the improved "coupling efficiency" generally observed for fusion proteins (1-4) does not result from the
tethered status of the reactants. These results are surprising and
suggest two equally possible but radically different interpretations.
linked by a covalent bond should be
thermodynamically favored over that between unattached molecules. Since
it is not so, it means that when GPCR and the
-subunit are part of
the same polypeptide backbone their binding surfaces cannot easily make
contact. Hence, binding to partners belonging to adjacent molecules is
just as possible or even predominant, and the interactions between
fused and non-fused species appear equally probable simply because they
are intermolecular in both cases. While this a rational explanation,
the reason why the fused status would exert hindrance to intramolecular
but not to intermolecular contacts is, however, not obvious.
-subunit and the receptor carboxyl terminus may significantly reduce
the degree of freedom of G
, compared with its native lipid-tethered status. This may impose enough rotational constraints to make the
proper alignment of the electronegative binding surfaces of G
with
the footprint of its tethered GPCR difficult, without however limiting
its orientation with respect to a neighboring GPCR molecule. As long as
orientation is a crucial factor for receptor-G
interaction, this is
a sound explanation. Alternatively, it may be the binding to
that makes the interactions within the fused construct unfavorable. We
have verified, in agreement with others (1, 5, 7, 12), that
GPCR-tethered
-subunits can interact with
, and do so just as
effectively as purified
-subunits, when judged by ADP-ribosylation.
There is ample evidence that the binding surface of the G protein for
the receptor comprises residues from both
- and
-subunits
(42-46). It is possible that the formation of the heterotrimer with
membrane-attached
attracts the tethered
-subunit away from
its intramolecular GPCR and facilitates the interaction with that of a
nearby molecules, thus largely offsetting the entropic advantage of
their tied condition. Unfortunately, we have not been able to design an
experiment capable of distinguishing between these two possibilities,
although a fusion protein that includes a GPCR and both
- and
-subunits, if still signaling-competent, might help.
2AR-Gs expressed at
physiological levels in cells were not consistent with a significant
shift of the intrinsic equilibrium of the receptor toward the active
form. Yet, if the covalently linked
-subunit could interact with the
tethered GPCR, that propensity should induce a high degree of
constitutive activation (12).
subunits (48) and GPCR (49, 50), including opioid (51) and
2-adrenergic (52) receptors, have been reported. This
process may represent a primary mechanism of concentrating interactions
for such signaling macromolecules. If receptors and
-subunits
interact normally as dimeric or higher order oligomeric forms, and if
the assembly of oligomers is not disturbed by the covalent linkage
between GPCR and
-subunit, the spatial proximity that such an
arrangement can generate may render the effect of fusion virtually unnoticeable.
-subunit why do they appear to react
more efficaciously than the cotransfected native molecules?
o tethered to a transmembrane polypeptide (but not
with native
o) can reproduce the enhancement of
agonist-induced nucleotide exchange observed when the same GPCR and
-subunit are expressed as a fusion protein. Similar observations
were made following the cotransfection of
2AR or
D1R with a single transmembrane domain-tethered form of
Gs (53). Therefore, it is the
-helical anchored status
of the
-subunit, not fusion to the receptor, which causes the
increase in receptor coupling. Yet, the reason for this enhancement
remains unclear.
release from the membrane (2, 7, 53) are
all possible mechanisms that have been proposed to explain the enhanced
coupling efficiency of transmembrane-tethered
-subunits. Here we
offer an additional suggestion. Unlike native G proteins, either
V2TM-
o (22) and the OP1R and
2AR-tethered
-subunits used in this
study2 were found excluded
from low density Triton-resistant vesicles, representing
cholesterol-rich microdomains of the plasma membrane (54). Moreover, it
was shown that a docked GPCR provides the sole means of membrane
insertion for the otherwise cytosolic acylation-defective mutants of
-subunits (55, 17). Thus, an interesting possibility is that the
transmembrane anchor of the
-subunits, by targeting such molecules
to the plasma membrane outside of the caveolin-rich rafts where they
are naturally located, can shield them from a constitutive inhibitory
influence and favor receptor interactions.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Marco Parenti (University of
Milan, Bicocca) for kindly supplying the transmembrane tethered
Go and to Dr. Patrick J. Casey (Duke University) for the
generous gift of purified -subunits.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Telethon Grant D129 and the EU BIOMED 2 Programme.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. Tel.: 39-06-49902386;
Fax: 39-06-49387104; E-mail: tomcosta@iss.it.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M300731200
2 M. C. Gro and T. Costa, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
AR, adrenergic receptor;
OP1R and
OP4R, opioid receptors type 1 and 4, also known as delta
and nociceptin receptors, respectively;
V2R, vasopressin 2 receptor;
D1R, dopamine 1 receptor;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
NTA, nitrilotriacetic
acid;
HEK, human embryonic kidney cell;
CHO, chinese hamster
ovary;
DTT, dithiothreitol;
RIA, radioimmunoassay;
WGA, wheat germ
agglutinin;
DDL, [D-Ala2,D-Leu5]enkephalin;
HA, hemagglutinin.
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