(Received for publication, November 27, 1996)
From the Cardiovascular Research Institute, the
§ Daiichi Research Center, and the ¶ Department of
Medicine, University of California,
San Francisco, California 94143-0130
Thrombin activates human platelets and other
cells in part by cleaving an unusual G protein-coupled receptor.
Thrombin cleavage of this receptor's amino-terminal exodomain unmasks
a new amino terminus. This then binds intramolecularly to the body of
the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying
the domains responsible for thrombin receptor-G protein interactions,
we examined the signaling properties of chimeric receptors in which
thrombin receptor cytoplasmic sequences replaced the cognate sequences
in the Gs-coupled 2-adrenergic receptor
(
2AR) or the Gi-coupled dopamine
D2 receptor (D2R). In Xenopus
oocytes, a chimeric
2AR bearing the thrombin receptor
second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic
agonist, whereas a
2AR bearing the cognate C2 loop from
the D2R did not. Similarly, in COS-7 cells, a chimeric
D2R bearing the thrombin receptor C2 loop gained the
ability to trigger phosphoinositide hydrolysis in response to
dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the
thrombin receptor C2 loop was able to confer Gq-like
coupling in several different receptor contexts. These observations
suggest that the thrombin receptor C2 loop specifies Gq
coupling by directly contacting Gq or by contributing to a
structure required for Gq coupling. The ability of the
thrombin receptor C2 loop to function in the context of the
D2R and
2AR strongly suggests that the
transmembrane switching and G protein activation strategies used by the
thrombin receptor must be very similar to those used by the
D2R and
2AR despite the thrombin receptor's
strikingly different liganding mechanism.
Thrombin activates platelets, leukocytes, and endothelial and
mesenchymal cells in part via an unusual proteolytically activated G
protein-coupled receptor (1-3). This receptor is important in
embryonic development (4) and is thought to contribute to thrombotic,
inflammatory, and proliferative responses in the adult. The mechanism
by which thrombin activates its receptor is unusual. Thrombin cleaves
its receptor's amino-terminal exodomain to unmask a new amino
terminus, which then functions as a tethered peptide ligand, binding
intramolecularly to the body of the receptor to effect transmembrane
signaling. The irreversibility of this proteolytic activation mechanism
stands in contrast to the reversible agonist binding that activates
prototypical G protein-coupled receptors such as the
2-adrenergic receptor
(
2AR).1 In addition,
available evidence suggests that the thrombin receptor's tethered
ligand interacts at least in part with the receptor's extracellular
face (5-8), while catecholamines bind in a pocket formed by the
receptor's transmembrane domains (reviewed in Ref. 9). Activation of
the thrombin receptor leads to phosphoinositide hydrolysis and
inhibition of adenylyl cyclase via coupling to Gq and
Gi, respectively (1, 10); coupling to G12 (11,
12) and Go (13) has also been reported. The structural
determinants of the thrombin receptor's G protein specificity are
unknown, and identification of the interactions by which the thrombin
receptor activates G proteins might point out strategies for disrupting thrombin signaling.
Studies of prototypical G protein-coupled receptors such as rhodopsin and adrenergic and muscarinic receptors suggest that multiple sites in the cytoplasmic domains of these receptors contribute directly or indirectly to G protein coupling (reviewed in Ref. 14). Studies in which cytoplasmic domains were deleted (15-17) or exchanged between related receptors (18-27) implicate cytoplasmic loop 2, the juxtamembrane portions of cytoplasmic loop 3, and the carboxyl-teminal tail in receptor-G protein interactions. Studies with synthetic peptides that mimic receptor cytoplasmic structures suggest that these structures directly bind to G proteins. For example, peptides mimicking the second (C2) and third (C3) cytoplasmic loops of rhodopsin and the juxtamembrane portion of its carboxyl-terminal tail bind to the rod cell G protein transducin and block receptor coupling (28), and peptides mimicking the C2 and C3 loops in other G protein-coupled receptors can directly activate G proteins (29-32).
To identify the domains responsible for thrombin receptor-G protein
interactions, we examined the signaling properties of chimeric
receptors in which thrombin receptor cytoplasmic sequences replaced the
cognate sequences in the Gs-coupled 2AR or
the Gi-coupled dopamine D2 receptor
(D2R). In Xenopus oocytes, a chimeric
2AR bearing the thrombin receptor second cytoplasmic
loop gained the ability to trigger intracellular Ca2+
release in response to adrenergic agonist, whereas a
2AR
bearing the cognate C2 loop from the D2R did not.
Similarly, in COS-7 cells, a chimeric D2R bearing the
thrombin receptor C2 loop gained the ability to trigger
phosphoinositide hydrolysis in response to dopaminergic agonist,
apparently by coupling to a Gq-like G protein. No
detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in
several different receptor contexts. Whether this loop specifies
Gq coupling by directly binding Gq or by
contributing to a structure required for Gq coupling
remains to be determined. The ability of the thrombin receptor C2 loop
to function in the context of the D2R and
2AR strongly suggests that the transmembrane switching
and G protein activation strategies used by the thrombin receptor must
be very similar to those used by the D2R and
2AR despite the thrombin receptor's strikingly
different liganding mechanism.
cDNAs were kindly provided by Dr. Olivier
Civelli (University of Oregon, Portland, OR) for the human
D2R (33) and Dr. Brian Kobilka (Howard Hughes Medical
Institute, Stanford University, Stanford, CA) for the human
2AR bearing an N-terminal epitope tag recognized by the
12CA5 monoclonal antibody in the mature protein (34, 35).
cDNA encoding the
wild-type human thrombin receptor (3) bearing an N-terminal epitope tag
recognized by the M1 monoclonal antibody (Kodak Scientific Imaging
Systems) in the mature protein has been described previously (36). The
hemagglutinin epitope sequence YPYDVPDYA (37), recognized by the 12CA5
monoclonal antibody, was engineered between Trp-8 and Asp-9 of the
D2R protein. All engineered cDNAs were produced by
oligonucleotide-directed mutagenesis (38) and were verified by dideoxy
sequencing (39). Specific constructs are described in Fig. 1. Chimeric
receptors were designated by the parent receptor (2 or
D2), followed by the domain replaced with thrombin receptor
sequence. An exception was
2D2C2, in which the
2AR second cytoplasmic loop was replaced with the
cognate D2R domain. Surface expression of mutant receptors ranged from 50 to 85% of that of wild-type
2-adrenergic
and dopamine D2 receptors.
Evaluation of Chimeric Receptor Expression and Signaling in Xenopus Oocytes
cDNAs encoding wild-type and chimeric
receptors were subcloned into pFROGY and transcribed (3). 25-50 ng of
cRNA in 50 nl of 10 mM HEPES (pH 7.4) was injected into
each Xenopus oocyte. After incubation for 24-48 h at
16 °C in modified Barth's solution (3), receptor surface expression
was measured as the specific binding of relevant monoclonal antibody to
receptor N-terminal epitope tag. Agonist-induced Ca2+
mobilization was performed (40) using isoproterenol (Sigma), dopamine
(Sigma), -thrombin (generously provided by Dr. J. Fenton II, Albany
Medical College, Albany, NY), or the human thrombin receptor agonist
peptide SFLLRN at the concentrations indicated in the text. The latter
was synthesized as the carboxylamide and purified by high pressure
liquid chromatography. Oocyte membranes were prepared (41) using a
Beckman TLX-120,000 ultracentrifuge and assayed the same day. Protein
assay was performed with the Bio-Rad Dc protein assay Kit. Adenylyl
cyclase assay (42) was done in a 50-µl volume using 15-25 µg of
membrane protein and 2.5-5 µCi of [
-32P]ATP (DuPont
NEN) per assay, supplemented with 1 mM unlabeled ATP.
Incubation time was 40 min at 30 °C.
For transient expression in COS-7 cells (American
Type Culture Collection), cDNAs were subcloned into pBJ1 (provided
by Prof. Mark Davis, Stanford University). 1 × 106
COS-7 cells were plated in 100-mm dishes in growth medium (Dulbecco's modified Eagle's medium H-16 supplemented with 10% bovine calf serum,
100 µg/ml streptomycin, and 100 units/ml penicillin), transfected the
following day with 9 µg of plasmid DNA using a modified (43) DEAE-dextran transfection procedure (44), and incubated overnight in
growth medium. Cells were then split in 12 × 4-cm2
wells and, 24 h after transfection, were incubated for 18 h
with 2 µCi/ml [3H]myoinositol (Amersham Corp.) in
serum-free Dulbecco's modified Eagle's medium. When indicated, cells
were exposed to 100 ng/ml pertussis toxin (List Biological Laboratories
Inc.) for 5 h at 37 °C before the addition of agonist. Under
these conditions, pertussis toxin inactivated ~95% of endogenous
Gi in COS-7 cells as measured by membrane
ADP-ribosylation. Cells were rinsed with Dulbecco's modified Eagle's
medium supplemented with 20 mM LiCl and incubated at
37 °C in the same medium with or without agonist as follows: 10 nM
-thrombin for 90 min and 10 µM
isoproterenol or 10 µM quinpirole (Research Biochemicals
International) for 40 min. Released inositol phosphates were measured
as described (40). Parallel cultures were assayed for specific binding
of relevant monoclonal antibody to assess surface expression of
wild-type and mutant receptors (36).
Membrane preparation and ADP-ribosylation of COS-7 cells were performed as described (2). Treatment of cells with 100 ng/ml pertussis toxin for 5 h removed >95% of all available pertussis toxin substrate from cell membranes.
To identify the thrombin receptor domains that specify its
interaction with particular G proteins, we initially generated chimeras
between the Gs-coupled 2AR and the human
thrombin receptor, which couples to Gq and Gi
(1, 10) and probably to G12 and Go (11, 13).
2-Adrenergic receptors in which each cytoplasmic domain
was replaced with the cognate thrombin receptor domain (Fig.
1) were expressed in Xenopus oocytes. In this
system, the thrombin receptor did not stimulate adenylyl cyclase, but
caused robust mobilization of cytoplasmic calcium, a reflection of
phospholipase C activation (Fig. 2) (45, 46). By
contrast, the
2-adrenergic receptor strongly activated
adenylyl cyclase (Fig. 3A), but coupled weakly at best to calcium mobilization (Fig. 2A). Thus,
retention of
2-adrenergic receptor-like signaling by the
chimeras was measured as isoproterenol-stimulated adenylyl cyclase
activity, and gain of thrombin receptor-like signaling was assessed as
isoproterenol-stimulated calcium mobilization.
All four chimeric receptors were expressed on the oocyte surface at
levels comparable to the wild-type 2-adrenergic
receptor. However, chimeras bearing the thrombin receptor first or
third cytoplasmic loop or carboxyl-terminal tail failed to either
activate adenylyl cyclase or mobilize calcium in response to
isoproterenol and were thus uninformative. By contrast, the chimeric
2-adrenergic receptor bearing the thrombin receptor
second cytoplasmic loop (designated
2C2)
stimulated adenylyl cyclase like the wild-type
2-adrenergic receptor (Fig. 3A) and gained
the ability to mobilize calcium (Fig. 2A). Both ends of this
loop were required for gain of calcium signaling in that receptors
chimeric for only the N- or C-terminal half of the second cytoplasmic
loop did not mobilize Ca2+ (data not shown). The
2C2 chimera's gain of the ability to
mobilize calcium was consistent with several possibilities. The
thrombin receptor second cytoplasmic loop might provide a new surface
that directly binds a calcium-mobilizing G protein. Alternatively, introduction of this loop might remove structural constraints in the
2AR that normally prevent interaction with such a G
protein, either by contributing a surface specifically shaped to allow docking with a calcium-mobilizing G protein (i.e. the right
"filter") or by simply disrupting the normal structure of the
2-adrenergic receptor's cytoplasmic surface, thereby
allowing promiscuous coupling. The latter has been described in
muscarinic receptor chimeras bearing the
1-adrenergic
receptor third intracellular loop (24); such receptors coupled to all G
proteins tested, and this promiscuity was rectified by the additional
substitution of the
1-adrenergic receptor C2 loop.
To test the specificity of the change in G protein coupling produced in
these chimeras, we generated a chimeric dopamine D2 receptor bearing the thrombin receptor second cytoplasmic loop (designated D2C2) and a
2-adrenergic receptor bearing the D2 receptor second cytoplasmic loop (designated
2D2C2) (Fig. 1). In the oocyte
system, the wild-type Gi-coupled dopamine
D2 receptor was expressed on the cell surface and could
trigger calcium mobilization in response to dopamine when
coexpressed with G
and G
-activated
phospholipase C-
2 (47) (data not shown). As expected,
the wild-type D2R by itself neither mobilized calcium nor
activated adenylyl cyclase in response to dopamine (Fig. 2B and data not shown). By contrast, the D2C2
chimera caused robust calcium mobilization. Activation of adenylyl
cyclase by the D2C2 chimera was not detectable
(Fig. 3B), suggesting that its gain of coupling to calcium
mobilization was not due to a general increase in receptor
promiscuity.
The 2D2C2 chimera was expressed
on the cell surface and retained the ability to activate adenylyl
cyclase (Fig. 3A), but did not gain the ability to cause
calcium mobilization (Fig. 2A). The gain of coupling to
calcium mobilization by the
2C2 receptor thus could not be mimicked by just any cytoplasmic loop substitution. These data suggest that the thrombin receptor second cytoplasmic loop
contains specific information for coupling to a calcium-mobilizing G
protein.
To examine the signaling properties of the
2C2 and D2C2
chimeras in mammalian cells, we expressed the two chimeras and their parent receptors and assessed agonist-induced phosphoinositide hydrolysis in transfected COS-7 cells.
2C2
was expressed at only 20-25% of the level of the wild-type
2 adrenergic receptor on the COS-7 cell surface and did
not cause detectable activation of phospholipase C (data not shown); it
was thus not informative. By contrast, surface expression of the
D2C2 chimera was similar to that of the
wild-type dopamine D2 receptor (Fig.
4B), and this chimera elicited substantial
inositol phosphate release in response to quinpirole, a D2R
agonist (Fig. 4A). The wild-type D2R did not
couple to phosphoinositide hydrolysis under the same conditions. Treatment with pertussis toxin only partially reduced
D2C2-mediated phosphoinositide hydrolysis
(0-50% in three experiments); thus, D2C2
likely mediates this response at least in part via a
pertussis-insensitive Gq-like G protein (48, 49).
At a structural level, the mechanism underlying receptor-G protein
coupling remains to be elucidated. The studies presented above show
that in the context of the 2-adrenergic and
D2 receptors, replacement of the native second cytoplasmic
loop with the cognate thrombin receptor sequence is sufficient to
confer the ability to activate a Gq-like G protein. As
noted above, biochemical and genetic experiments implicate elements in
G protein-coupled receptor second and third cytoplasmic loops and
carboxyl-terminal tail in G protein binding and activation. The ability
of the thrombin receptor C2 loop to provide new coupling
"instructions" to
2-adrenergic and D2
receptors is consistent with the C2 loop itself providing a
Gq-binding site or a specific change in the filter function of the cytoplasmic face of these receptors to permit interaction with
Gq, but not, in the case of the
D2C2 chimera, Gs. More broadly, the
ability of the thrombin receptor C2 loop to function in the context of
the
2-adrenergic and D2 receptors suggests
that the thrombin receptor and aminergic receptors use the same
structural strategies for G protein binding and activation despite
their remarkably different activation mechanisms. This provides
additional support for the notion that while G protein-coupled
receptors use quite varied strategies to sense light, small molecules,
peptides and protein hormones, and proteases (50, 51), these ligands probably all elicit very similar changes in the arrangement of their
receptors' transmembrane helices and cytoplasmic loops to effect G
protein activation (14).
We thank Dr. Henry Bourne (University of California, San Francisco) for helpful discussions and Dr. JoAnn Trejo (University of California, San Francisco) for critical reading of this manuscript.