©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Determinants of Thrombin Receptor Cleavage
RECEPTOR DOMAINS INVOLVED, SPECIFICITY, AND ROLE OF THE P3 ASPARTATE (*)

Kenji Ishii (1), Robert Gerszten (1), Yao Wu Zheng (1) (2), John B. Welsh (1), Christoph W. Turck (1), Shaun R. Coughlin (1) (2)(§)

From the (1)Cardiovascular Research Institute and (2)Daiichi Research Center, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thrombin receptor cleavage at the Arg--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.


INTRODUCTION

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 LDPRSFLLRNPNDKYEPF, 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) .

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--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 LDPRS site.


EXPERIMENTAL PROCEDURES

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-MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSDYKDDDDVD-ATLDPRSFLLR. 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.

The sequence of chimeric and other mutant receptors is shown in . ATE-5HT1cR()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 ().

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).

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.

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 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.




RESULTS AND DISCUSSION

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.


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 LDPRSFLLRN 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.

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--Ser peptide bond (i.e. the LDPRSFLL 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.

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 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.

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 LDPRS 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.

In summary, these studies show that the region of the thrombin receptor's amino-terminal exodomain containing the LDPRSFLL 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 LDPRSFLL 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 LDPRSFLL 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

cDNA construction is described under ``Experimental Procedures.'' , 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.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL44907 and by the Daiichi Research Center, University of California, San Francisco. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Box 0524, HSW-831 University of California San Francisco, 505 Parnassus Ave., San Francisco, CA 94143. Tel.: 415-476-6174; Fax: 415-476-8173.

The abbreviation used is: ATE, amino-terminal exodomain.


ACKNOWLEDGEMENTS

We thank Drs. Henry Bourne and Charles Craik for critical reading of this manuscript.


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