From the
Thrombin receptor cleavage at the
Arg
Studies of the thrombin receptor have provided a framework for
understanding how thrombin and perhaps other proteases communicate with
cells(1, 2) . The thrombin receptor is a member of the
seven-transmembrane domain family of receptors but is activated by a
novel mechanism. The receptor's amino-terminal exodomain contains
the amino acid sequence LDPR
The rate of
thrombin receptor cleavage appears to be a major determinant of the
magnitude of a cell's response to thrombin(8) ; thus, an
understanding of thrombin binding and receptor cleavage is key to
understanding receptor function. Studies with mutant receptors and
receptor peptides (3, 4, 9, 10) revealed
two domains within the receptor's amino-terminal exodomain, the
cleavage recognition sequence LDPR and the ``hirudin-like
domain'' DKYEPF, to be important for thrombin-receptor
interaction. In the context of previous studies of thrombin interaction
with substrates and inhibitors(11, 12) , these studies
suggested a bidentate interaction between receptor and thrombin.
Specifically, the receptor's LDPR and DKYEPF sequences were
postulated to dock with thrombin's active center and anion
binding exosite, respectively, with the DKYEPF sequence docking in a
manner analogous to hirudin's carboxyl
tail(1, 3, 9) . Recent x-ray crystallographic
studies of thrombin co-crystallized with receptor peptides showed that
the LDPR sequence can indeed bind productively in thrombin's
active center and that the DKYEPF sequence can bind with
thrombin's anion binding exosite in the anticipated
manner(13) .
Several questions regarding thrombin-receptor
interaction remain to be addressed. First, do parts of the receptor
other than its cleavage recognition sequence and hirudin-like domain
participate in thrombin-receptor interaction and influence receptor
cleavage? Second, cleavage of the thrombin receptor's
amino-terminal exodomain has not been examined biochemically. Is it
really cleaved enzymatically, with one thrombin molecule activating
many receptors? How specific is the cleavage? Do secondary cleavage
events remove the tethered ligand domain, a possible mechanism for
terminating receptor signaling? Last, the LDPR cleavage site itself
presents a paradox. This sequence is also utilized as the thrombin
cleavage site in protein C, a natural thrombin substrate that is
efficiently cleaved by thrombin only when thrombin is complexed with
thrombomodulin(14) . The inability of free thrombin to cleave
protein C is in part due to an inhibitory effect of the P3 aspartate
residue in DPR(15, 16) . Docking with thrombomodulin is
thought to cause a conformational change in thrombin's active
center that accommodates the DPR sequence. Unlike protein C and despite
the common DPR cleavage site, thrombin receptor cleavage is not
dependent upon thrombomodulin. Previous biochemical (9) and
structural (13) studies suggest that occupancy of
thrombin's anion binding exosite by the receptor's
hirudin-like domain can cause a conformational change in
thrombin's active center. This raises the possibility that the
thrombin receptor may have an endogenous thrombomodulin-like function;
specifically, the hirudin-like domain's binding to thrombin may
alter thrombin's active center such that it can bind and
efficiently cleave the LDPR sequence.
We addressed these questions
by examining cleavage of chimeric and other mutant thrombin receptors
on intact cells. Most if not all thrombin-receptor interaction appears
to be specified by the cleavage recognition sequence and hirudin-like
domains located in the receptor's amino-terminal exodomain. A
soluble protein representing this domain was cleaved enzymatically by
thrombin exclusively at the LDPR cleavage site at
Arg
The sequence of chimeric and other mutant receptors
is shown in . ATE-5HT1cR
Receptor cDNAs were subcloned into the mammalian
cell expression vector pBJ1 (provided by Mark Davis, Stanford). For
stable expression, Rat1 fibroblasts were co-transfected with mutant
receptor cDNA in pBJ1 and pSVneo; clones were selected for G418 (800
mg/liter) resistance and screened for receptor expression by immunoblot
and by surface antibody binding(8) . For transient expression,
Cos 7 fibroblasts were transfected by DEAE-dextran/chloroquine or
DEAE-dextran/adenovirus (22).
To convert M1 antibody binding data into an estimate of
the number of receptors expressed on the surface of transfected cells,
we utilized the M1 epitope-tagged 5HT1c receptor and the tagged
ATE-5HT1cR chimera constructs. These constructs encode proteins that
both display the M1 epitope on their amino termini and act as
functional serotonin receptors. We determined the number of high
affinity binding sites for the 5HT1c receptor ligand
To identify the receptor domains important for thrombin
receptor cleavage, we examined the rate of cleavage of wild-type and
mutant thrombin receptors on the surface of transfected mammalian
cells. Receptor cleavage was followed as loss of binding sites for
antibody to the receptor's activation peptide, the fragment
cleaved from the receptor by thrombin. We first compared the rate of
wild-type receptor cleavage with that of WT5, a modified thrombin
receptor in which native residues 1-35 were replaced by the
prolactin signal sequence followed by an epitope for the M1 monoclonal
antibody (see ``Experimental Procedures'' and ).
Loss of binding of antiserum 1638, which recognized both receptors in
the uncleaved state and neither in the cleaved state, was used to
assess receptor cleavage. The rates of cleavage of these two receptors
by various concentrations of thrombin were indistinguishable (data not
shown), demonstrating that receptor residues 1-35 do not
contribute significantly to thrombin-receptor interactions. For this
reason and because of antibody availability and an excellent
signal-to-noise ratio for M1 binding, we utilized WT5 as the control
receptor in subsequent experiments.
To determine if domains outside
of the thrombin receptor's amino-terminal exodomain are important
for thrombin-receptor interaction, we examined the rate of cleavage of
the amino-terminal exodomain expressed on the cell surface independent
of the rest of the thrombin receptor. Two chimeric molecules were made (Fig. 1A and ). In one, the thrombin
receptor's amino-terminal exodomain was joined to the
amino-terminal exodomain of a different seven-transmembrane domain
receptor, the serotonin 1c receptor (ATE-5HT1cR). In the other, the
thrombin receptor's amino-terminal exodomain replaced the
extracellular domain of the single transmembrane domain molecule CD8
(ATE-CD8). In either context, the rate of cleavage of the
amino-terminal exodomain by varying concentrations of thrombin was not
significantly different from that of the native receptor (Fig. 1B). These data show that when displayed on the
cell surface, the thrombin receptor's amino-terminal exodomain
between residues 36 and 82 is sufficient to confer efficient cleavage
by thrombin. No contribution from the remainder of the receptor
molecule appears to be necessary.
Cleavage of the thrombin receptor has not been examined
biochemically largely because of the difficulty of expressing and
purifying a sufficient amount of this hydrophobic membrane protein. The
studies described above demonstrated that most if not all
thrombin-receptor interaction occurs via the receptor's
amino-terminal exodomain, independent of the hydrophobic
seven-transmembrane domain segment. This suggested that a soluble
amino-terminal exodomain might serve as a surrogate for the intact
receptor for biochemical and kinetic studies of receptor cleavage.
Accordingly, we expressed the amino-terminal exodomain as a soluble
protein dubbed ATE-H6 and examined proteolytic cleavage by thrombin
(see ``Experimental Procedures'' and Fig. 3). The
uncleaved protein migrated with an apparent molecular mass of 11 kDa on
SDS-polyacrylamide gel electrophoresis. When 600 pmol of protein (60
µM) was incubated with 0.5 pmol of thrombin (50
nM) for 60 min at 25 °C, the 11-kDa band disappeared, and
two new bands migrating at approximately 9 and 2 kDa appeared ( Fig. 3and data not shown). Immunoblot with antiserum 1638 to the
activation peptide domain (AP) or 1809 to the hirudin-like
domain (HIR; Ref. 8) revealed that the 9-kDa band lacked the
activation peptide but retained the hirudin-like domain (Fig. 3A). Immunoblot with the 12CA5 monoclonal antibody
showed that the 9-kDa band retained the ATE-H6 carboxyl tail
(``Experimental Procedures'' and data not shown).
Amino-terminal sequencing confirmed that the 9-kDa band starts with the
sequence SFLLRN (see ``Experimental Procedures''). To detect
possible cleavage at other sites, 600 pmol of ATE-H6 (60
µM) was incubated with 0.5 pmol of thrombin (50
nM) for 1 h at 37 °C, and the unfractionated mixture was
subjected directly to sequencing. Only two sequences were obtained: the
original amino terminus and SFLLRN. Moreover, when a solution of
thrombin-cleaved ATE-H6 was repurified on a nickel column and subjected
to N-terminal sequencing, only a single amino terminus beginning with
the sequence SFLLRN was obtained. The identity of this new
amino-terminal sequence, the lack of other amino-terminal sequences in
this preparation, and the persistence of just two discrete cleavage
products in these digests shows that thrombin cleaves ATE-H6
efficiently only at the Arg
The observation that 0.5 pmol of thrombin completely
cleaved 600 pmol of ATE-H6 proves that thrombin is acting
enzymatically, with one thrombin molecule cleaving many substrate
molecules. Indeed, the kinetics of ATE-H6 cleavage by thrombin revealed
a k
As discussed in the Introduction, the identity of the
thrombin receptor's LDPR cleavage recognition sequence with that
of protein C raised the question of whether the receptor plays a role
analogous to thrombomodulin, causing a conformational change in
thrombin to allow efficient cleavage of the LDPR
In summary, these
studies show that the region of the thrombin receptor's
amino-terminal exodomain containing the LDPR
cDNA
construction is described under ``Experimental Procedures.''
We thank Drs. Henry Bourne and Charles Craik for
critical reading of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-
-Ser
peptide bond in the
receptor's amino-terminal exodomain is necessary and sufficient
for receptor activation. The rate of receptor cleavage at this site is
a critical determinant of the magnitude of the cellular response to
thrombin. These observations underscore the importance of defining the
molecular basis for thrombin-receptor interaction and cleavage. We
report that chimeric proteins bearing only thrombin receptor
amino-terminal exodomain residues 36-60 are cleaved at rates
similar to the wild-type thrombin receptor when expressed on the cell
surface. A soluble amino-terminal exodomain protein was also cleaved
efficiently by thrombin with a K
of
15-30 µM and k
of approximately 50
s
, with cleavage occurring only at the
Arg
-
-Ser
peptide bond. In the context
of previous studies, these data suggest that the receptor's LDPR
cleavage recognition sequence and DKYEPF hirudin-like domain account
for thrombin-receptor interaction. Because a P3 aspartate in protein
C's cleavage site inhibits cleavage by free thrombin, we
investigated the role of the P3 aspartate in the receptor's LDPR
sequence. Studies with mutant receptors revealed an inhibitory role for
this residue only in the absence of the receptor's hirudin-like
domain. These and other data suggest that the receptor's
hirudin-like domain causes a conformational change in thrombin's
active center to accommodate the LDPR sequence and promote efficient
receptor cleavage. Taken together, these studies imply that the
thrombin receptor's amino-terminal exodomain contains all the
machinery needed for efficient recognition and cleavage by thrombin.
Thrombin appears to bind and cleave this domain independently of the
rest of the receptor, with one thrombin molecule probably activating
multiple receptors.
SFLLRNPNDKYEPF, residues 38-55
in this 425-amino acid protein. Thrombin triggers receptor activation
by cleaving the Arg
-
-Ser
peptide bond.
This event unmasks a new amino terminus that then functions as a
tethered peptide ligand, binding intramolecularly to the body of the
receptor to effect receptor
activation(1, 3, 4) . The synthetic peptide
NH
-SFLLRN-COO
, which
mimics this new amino terminus, is a full agonist for receptor
activation(1, 5, 6, 7) .
-
-Ser
peptide bond. The
receptor's P3 aspartate was inhibitory for receptor cleavage by
thrombin only when the receptor's hirudin-like domain was
deleted, suggesting that docking of this receptor domain with
thrombin's anion binding exosite may cause a conformational
change in thrombin's active center to promote efficient cleavage
of the LDPR
S site.
Receptor Constructs and Expression
Mutagenesis
of the human thrombin receptor cDNA () was performed by the
method of Kunkel (17) or by insertion of double-stranded linkers or
polymerase chain reaction products(18) . Mutations were
confirmed by dideoxy sequencing (19). All constructs except the
wild-type thrombin receptor included the prolactin signal sequence
followed by an epitope for the M1 monoclonal antibody joined to
thrombin receptor residue 36(8) . The latter is located in the
thrombin receptor's amino-terminal exodomain amino-terminal to
the thrombin cleavage site. The resulting amino terminus reads as
follows:
NH-MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS
DYKDDDDVD-ATLDPR
SFLLR.
The prolactin signal is underlined; the M1 epitope is in boldface;
marks the signal peptidase site, - marks the junction with
native receptor sequence, and
marks the endogenous thrombin
cleavage site.
(
)encoded a
protein consisting of the epitope-tagged thrombin receptor's
amino-terminal exodomain up to and including thrombin receptor residue
Lys
fused via a two-amino acid spacer to Val
at the extracellular aspect of the 5HT1c receptor's first
transmembrane domain (20) (). ATE-CD8 encoded a
protein consisting of the epitope-tagged thrombin receptor's
amino-terminal exodomain up to and including receptor residue
Asp
fused to Ile
at the extracellular aspect
of CD8's single transmembrane domain(4, 21) .
S-CD8 and L-CD8 encoded altered versions of ATE-CD8 in which the
``tether sequence'' was deleted or replaced by SGA spacer
sequence (). ATD4 encoded a thrombin receptor in which the
tether sequence was deleted from the amino-terminal exodomain ().
Assessment of Receptor Cleavage on Intact
Cells
Rabbit antiserum 1638 was raised against a peptide
representing the native receptor's activation peptide and
cleavage site (PESKATNATLDPRSFLLC) conjugated to keyhole limpet
hemocyanin(23) . Preliminary cell surface binding studies showed
that this antiserum recognized both wild type and the epitope-tagged
WT5 receptors in their uncleaved state but did not recognize the
cleaved versions of either receptor. The DYKDDDD epitope within the
tagged receptor's activation peptide was recognized by the
commercially available monoclonal antibody M1 (IBI-A Kodak Co.) (8).
Thrombin receptor cleavage was assessed as loss of binding sites for
the 1638 or M1 antibodies as described previously(8) . In brief,
cells were split into 24-well plates (Falcon) at 5 10
cells/well. One day later cells were washed with Dulbecco's
modified Eagle's medium with 10 mM Hepes (pH 7.4) and 1
mg/ml bovine serum albumin. Cells were then exposed to various
concentrations of
-thrombin (a gift from J. Fenton II, Albany
Medical College, Albany, NY) for 15 min. at 37 °C and then fixed
with 4% paraformaldehyde in phosphate-buffered saline for 5 min at 4
°C, a fixation protocol that itself had little effect on number of
binding sites for M1 and 1638(8) . Plates were washed twice with
phosphate-buffered saline and then incubated with primary antibodies
(M1 IgG at 0.5 µg/ml or 1638 IgG at 1 µg/ml) in
Dulbecco's modified Eagle's medium/Hepes/bovine serum
albumin for 1 h at room temperature. After washing with
phosphate-buffered saline, plates were incubated with horseradish
peroxidase-conjugated second antibodies (Bio-Rad; 1:1000 dilution in
Dulbecco's modified Eagle's medium/Hepes/bovine serum
albumin) for 30 min at room temperature. After additional washing with
phosphate-buffered saline, plates were developed with the horseradish
peroxidase chromogenic substrate
2,2`-amino-bis(3-ethylbenzthiazinoline-6-sulfonic acid (1 mg/ml) in
citrate/phosphate buffer (pH 4.0) with 0.03% hydrogen peroxide. A
was read after 5-30 min. Antibody
binding data were expressed as specific binding (total minus
nonspecific, with nonspecific being defined as the level of binding
seen with untransfected control cells; nonspecific binding was usually
<5% of total binding seen with transfected cells expressing the
various receptor constructs prior to thrombin treatment). Receptor
cleavage was calculated as percentage loss of specific antibody binding
with thrombin treatment. Results shown represent the mean of five
replicate experiments. In individual experiments each point was done in
duplicate, and standard deviations were usually <5% of the means.
Comparison of Surface Expression Levels of Mutant versus
Wild-type Receptors in Transfected Cells
For the fractional
cleavage rates of the various receptor constructs to be meaningfully
compared, their surface expression levels should be comparable.
Relative levels of surface expression of wild-type and mutant receptors
were compared in each experiment using the cell surface enzyme-linked
immunosorbent assay described above. The stable Rat1 cell lines
expressing various mutant receptors employed were selected for surface
expression levels comparable to that of the WT5 wild-type
receptor-expressing Rat1 line; in individual experiments the level of
mutant receptor expression ranged from 50 to 200% of wild type. In
transient expression experiments, mutant receptor expression levels
ranged from 85 to 300% of wild type. Even when the mutant constructs
such as ATE-CD8 chimeras were expressed at 3 times the wild-type
receptor levels, the fractional cleavage rate as a function of thrombin
concentration remained comparable to that seen for the wild-type
receptor.
H-lysergic acid diethylamide (DuPont NEN) on the surface of
Cos cells expressing these constructs by whole cell binding and
Scatchard analysis(20) . In parallel cultures, we determined M1
antibody binding by cell surface enzyme-linked immunosorbent assay. In
Cos expressing the M1 epitope-tagged 5HT1c receptor or the ATE-5HT1cR
chimera, M1 binding correlated well with
H-lysergic acid
diethylamide binding over a range of receptor expression levels. M1
binding to the WT5 Rat1 line performed in parallel matched the level of
binding seen in cultures expressing 5
10
M1
epitope-tagged 5HT1c receptors/cell. A comparison of thrombin receptor
antibody 1809 binding to WT5 versus HEL cells (8, 24) also yielded an estimate of 5
10
receptors/cell for WT5. This translates to receptor and thrombin
being present at equimolar concentrations at 0.5 nM thrombin
under the conditions used to follow receptor cleavage in this study.
Thus we were not able to operate at a convincing receptor to thrombin
molar excess, precluding a rigorous demonstration that one thrombin
molecule cleaves many receptors by following receptor cleavage on
intact cells under ``physiological'' conditions (see
``Results'' for additional discussion). This does not impact
the structure-activity comparisons reported here and prompted our
determining the kinetics of cleavage of a receptor surrogate (ATE-H6;
see below) that could be employed at a clear cut molar excess over
thrombin.
Bacterial Expression and Cleavage of the Amino-terminal
Exodomain of the Thrombin Receptor
ATE-H6 cDNA encoding a start
methionine followed by human thrombin receptor amino-terminal exodomain
sequence Ala-Gln
, the epitope for the
monoclonal antibody 12CA5 (25) (GDVPDYASGG), and a hexahistidine
tag at the carboxyl end was prepared by polymerase chain reaction
mutagenesis and subcloned into the T7 promoter-based Escherichia
coli expression vector pET-3a(26) . Nucleotide sequence was
confirmed by the dideoxy method. The ATE-H6 protein was expressed in
JM109(DE3) by induction of T7 RNA polymerase with 0.4 mM
isopropyl
-D-thiogalactopyranoside for 3 h at 37 °C.
The bacteria were lysed in 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.27 mg/ml lysozyme, 1 mg/ml deoxycholic
acid. After treatment with DNase I (7 mg/ml) for 10 min at 25 °C,
the lysate was centrifuged at 14,000
g for 10 min at 4
°C. The expressed protein was purified from the supernatant using
nickel-bound Sepharose resin (Pharmacia Biotech Inc.) and then dialyzed
extensively against 20 mM HEPES, pH 7.4, 150 mM NaCl
at 4 °C. Protein concentration was estimated by Amido Black
staining using bovine serum albumin as a standard.
Peptide Sequencing
ATE-H6 was incubated with
-thrombin as in Fig. 3at 25 or 37 °C. Solutions were
then subjected to SDS-polyacrylamide gel electrophoresis and subsequent
electroblotting onto a polyvinylidene difluoride membrane (Millipore
Corp.). Proteins were visualized by staining the membrane with Ponceau
S; bands were excised and subjected to Edman degradation using an ABI
Procise sequencer, model 492 (Applied Biosystems, Foster City, CA).
This served to identify the cleavage fragment as the product of
cleavage at the Arg
-
-Ser
peptide bond.
In other experiments, the unfractionated digestion mix was immobilized
directly on polyvinylidene difluoride membranes and sequenced, or the
digestion mix was subjected to purification and concentration using
nickel-bound Sepharose and then directly immobilized on polyvinylidene
difluoride and sequenced.
Figure 3:
Cleavage of soluble amino-terminal
exodomain (ATE-H6) by thrombin. A, prolonged incubation with
thrombin suggests a single cleavage site. 600 pmol (60 µM)
of ATE-H6 protein was incubated with 0.5 pmol (50 nM) of
thrombin at 25 °C for 60 min in 10 µl of phosphate-buffered
saline containing 0.5% polyethylene glycol 8000. Samples were subjected
to 20% SDS-polyacrylamide gel electrophoresis and analyzed by staining
with Coomassie Blue R-250 or immunoblot with antibodies recognizing the
receptor's activation peptide (AP; antiserum 1638), its
hirudin like domain (HIR; antiserum 1809), or an epitope added
to its carboxyl tail (12CA5; not shown). Similar results were obtained
at 37 °C. B, rapidity of ATE-H6 cleavage by thrombin. 600
pmol of ATE-H6 was incubated as above at 37 °C with the indicated
concentration of thrombin for the indicated times. After
SDS-polyacrylamide gel electrophoresis, substrate and cleavage product
were detected by immunoblot with 1809. Note that 2 nM thrombin
(0.02 pmol) caused half-maximal cleavage of 600 pmol of ATE-H6 in 5
min.
Figure 1:
Cleavage of the thrombin
receptor's ATE expressed on the surface of intact cells in
various protein contexts. A, diagram of fusion proteins. Two
strategies were used to express the thrombin receptor's ATE on
the cell surface but independent of the rest of the thrombin receptor.
The ATE was fused to the amino terminus of the serotonin 5HT1c receptor
(ATE-5HT1cR) or to the extracellular aspect of CD8's
transmembrane domain (ATE-CD8) as described under ``Experimental
Procedures'' and in Table I. B, cleavage of the
amino-terminal exodomain. Stable Rat1 cell lines expressing ATE in its
native receptor context (WT5) or other contexts (ATE-5HT1cR or ATE-CD8)
were exposed to the indicated concentrations of thrombin for 15 min. at
37 °C. Cleavage was determined as described under
``Experimental Procedures.''
To further define the domains
within the amino-terminal exodomain required for efficient receptor
cleavage by thrombin, we determined cleavage rates for altered ATE-CD8
molecules lacking portions of the amino-terminal exodomain. Deletion of
amino-terminal exodomain residues Lys-Asp
(S-CD8) or replacing these residues with spacer sequence (L-CD8)
had no appreciable effect on cleavage rate (Fig. 2). A similar
deletion mutant in the thrombin receptor itself
(
Lys
-Leu
) (ATD4) was also cleaved
with kinetics similar to those of wild type. In this mutant, amino
acids Pro
-Asp
were retained because of
the importance of this region for agonist function. When expressed in Xenopus oocytes, this mutant exhibited a dose response curve
to thrombin indistinguishable from that of the wild-type receptor (not
shown).
Figure 2:
Leu-Glu
of
the thrombin receptor amino terminus is sufficient for effective
receptor cleavage. Cos 7 fibroblasts transiently expressing the
indicated constructs were incubated with thrombin for 15 min at 37
°C. A, cleavage of ATE-CD8 versus ATE-CD8
deletion (S-CD8) and substitution (L-CD8) mutants (see Table I and
``Experimental Procedures''). B, cleavage of
wild-type and amino terminus deletion mutant thrombin
receptors.
Taken together, these studies show that the thrombin
receptor sequence between Ala and Glu
is
sufficient to specify efficient receptor cleavage by thrombin. This
receptor region contains the L
DPR
SFLLRN cleavage
site and tethered ligand as well as the DKYEPF
hirudin-like domain. In the context of previous functional
studies of mutant receptors(3) , biochemical studies with
receptor peptides(9) , and x-ray crystallographic studies of
these peptides docked with thrombin(13) , these data suggest
that virtually all thrombin-receptor interaction is accounted for by
the LDPR (residues 38-41) and DKYEPF (residues 50-55)
sequences.
-
-Ser
peptide bond (i.e. the LDPR
SFLL site) despite the
presence of other basic amino acids representing potential thrombin
cleavage sites in this sequence. Thus in this defined system, there is
no evidence for a thrombin-mediated second cleavage event that would
relieve the receptor of its agonist peptide as a possible shut off
mechanism.
of approximately 50 s
and
a K
of 15-30 µM (Fig. 3B and data not shown). These data are
comparable with those obtained in a previous study in which thrombin
cleaved a smaller synthetic receptor peptide containing both the LDPR
and DKYEPF sequences with a k
of 130
s
and a K
of 10
µM. While extrapolating from cleavage of a soluble
receptor fragment in solution to cleavage of the intact receptor on the
cell membrane must be done with caution, these data suggest that the
relationship between thrombin and its receptor is probably that of
enzyme and substrate, with one thrombin molecule cleaving multiple
receptors. A rigorous demonstration that thrombin cleaves its receptor
enzymatically on intact cells under physiological conditions has not
been possible. As noted under ``Experimental Procedures,''
even the relatively high thrombin receptor expression levels on the
cells used in these studies (5
10
/cell) does not
predict a convincing molar excess of receptor to thrombin at thrombin
concentrations that are effective in eliciting biological responses
(0.1-10 nM), and the existence of abundant endogenous
thrombin inhibitors such as protease nexins (27) makes
interpretation of studies performed at very low thrombin levels
problematic.
S sequence. Given
that the receptor appears to interact with thrombin via only the LDPR
and KYEPF domains, the latter domain was a candidate for mediating this
putative conformational change. To test whether the receptor's P3
aspartate residue plays an inhibitory role analogous to that in protein
C, we replaced the receptor's P3 aspartate with glycine (D39G)
with or without simultaneous deletion of the KYEPF sequence. Deletion
of the KYEPF sequence in the setting of the wild-type receptor caused a
marked decrease in the efficiency of cleavage by thrombin (EC
of 0.1 nM for wild type versus 20 nM for
KYEPF), consistent with the hypothesis that the KYEPF
sequence is an important site for thrombin binding and receptor
cleavage (Fig. 4). Substitution of glycine for aspartate 39 in
the LDPR sequence caused no significant change in the rate of wild-type
receptor cleavage by thrombin, but, strikingly, it did cause a
remarkable improvement in cleavage of the
KYEPF receptor
(EC
of 1 nM for D39G+
KYEPF versus 20 nM for
KYEPF; 20-fold difference). The receptor
cleavage site's P3 aspartate thus appears to inhibit receptor
cleavage in the absence of the receptor's KYEPF sequence but not
in its presence.
Figure 4:
Role of
aspartate 39 in the receptor's LDPR sequence: inhibition of
thrombin receptor cleavage in the absence of receptor's
hirudin-like domain. Rat1 fibroblasts stably expressing wild-type
(), D39G (
),
KYEPF (
), and D39G+
KYEPF
(
) mutant thrombin receptors were incubated with the indicated
concentrations of thrombin, and receptor cleavage was followed as in
Fig. 1.
Previous studies revealed that a peptide
representing the thrombin receptor's hirudin-like domain changed
the V for thrombin cleavage of various small
substrates. This same peptide also changed the fluorescence emission of
active-site dansylated thrombin(9) . Deletion of the
receptor's hirudin-like domain or alanine substitutions within
this domain at Tyr
, Glu
, and Phe
markedly decreased signaling to thrombin but not to agonist
peptide(3) , presumably due to decreased cleavage of these
mutant receptors by thrombin (Fig. 4). Cleavage studies with
receptor peptides showed both increased K
and decreased k
upon deletion of the
hirudin-like domain(3) . These observations suggested that the
receptor's hirudin-like domain might effect a conformational
change in thrombin's active center. Recent x-ray crystallographic
studies showed that occupancy of thrombin's anion binding exosite
by its receptor's hirudin-like domain was indeed associated with
a conformational change in thrombin's active center in the
Ala
-Gly
region with a disordering of the
Glu
side chain(13) . The latter is thought to
participate in inhibitory interactions between thrombin and protein
C's P3 aspartate(16) . Taken in the context of these
studies, the present study suggests that, in addition to binding
thrombin, the KYEPF sequence causes a conformational change in
thrombin's active center that is functionally important for
accommodating the LDPR sequence and promoting efficient receptor
cleavage. This complex relationship between thrombin and its
receptor/substrate may have evolved to enhance specificity, promoting
cleavage by thrombin but not by other proteases.
SFLL cleavage site
through the DKYEPF hirudin-like domain accounts for efficient receptor
cleavage by thrombin. In the context of the previous studies discussed
above, these results suggest that the LDPR and DKYEPF sequences in the
thrombin receptor's amino-terminal exodomain mediate most if not
all of the receptor interactions with thrombin. A soluble polypeptide
representing the receptor's amino-terminal exodomain was thus
constructed as a receptor surrogate for biochemical studies. This was
cleaved enzymatically by thrombin and only at the LDPR
SFLL site.
At face value these observations suggest that a single thrombin
molecule can cleave many receptor molecules, with the caveat that
extrapolating the kinetics of cleavage in solution to cleavage of the
amino-terminal exodomain domain displayed on the intact receptor on a
cell surface must be done with caution. The ability of thrombin to
efficiently cleave its receptor at the LDPR
SFLL site, at first
paradoxical given the known inhibitory influence of a P3 aspartate,
appears to be due in part to a thrombomodulin-like function contributed
by the receptor's hirudin-like domain.
Table: Amino
acid sequences of thrombin receptor mutants and chimeras
, the thrombin cleavage site; - - -, deleted
sequence; introduced spacer sequences are underlined; amino acids
derived from 5HT1c receptors or CD8 are in boldface, indicating the
junctions of chimeric molecules. Numbers at the top indicate the amino
acid number of the wild type receptor with the start methionine being
1.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.