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(Received for publication, December
1, 1994; and in revised form, January 9, 1995) From the
Recently, we identified and cloned a human endothelial cell
protein C/activated protein C receptor (EPCR). EPCR was predicted to be
a type 1 transmembrane glycoprotein and a novel member of the CD1/major
histocompatibility complex superfamily with 28% identity with CD1d.
Even greater homology (62% identity) was detected with the murine
protein, CCD41, which was previously characterized as a
centrosome-associated, cell cycle-dependent protein. This raised the
possibility that CCD41 was the murine homologue of EPCR. To address
this possibility, to better understand structure-function
relationships, and to facilitate physiological experiments on EPCR
function, we cloned and sequenced murine and bovine EPCR from
endothelial cell cDNA libraries. The nucleotide sequence of murine EPCR
and CCD41 exhibited five differences corresponding to one base change,
three single-base insertions, and one base deletion in the protein
coding region. As a result, the predicted structures of EPCR and CCD41
differed in their amino and carboxyl termini but were identical in the
central portion of the coding sequence. Based on comparison of the
murine, bovine, and human EPCR sequences and the regions where
discrepancies between murine EPCR and CCD41 were detected, we believe
that CCD41 is probably identical to murine EPCR and that the reported
sequence differences are likely the result of compression on the
sequencing gel. Compared with human EPCR, the murine and bovine
sequences were 69 and 73% identical, respectively, and 57% of the
residues were identical between all three species. Both bovine and
murine EPCR could bind human activated protein C when the cDNA clones
were transfected into 293T cells. Like human EPCR, of the cell lines
tested, the murine EPCR message was restricted to endothelium. Cloning
of the murine and bovine homologue of EPCR will facilitate in vivo and in vitro studies of the role of EPCR in the protein C
pathway.
Protein C is the zymogen of the key anticoagulant enzyme,
activated protein C (APC). ( The predicted protein structure
of EPCR was that of a type 1 transmembrane glycoprotein with 238 amino
acids containing a signal sequence at the amino-terminal, a
transmembrane domain near the carboxyl-terminal, a short cytosolic
domain, and four potential N-glycosylation sites. Protein data
base searches indicated significant homology with CD1/MHC molecules.
The extracellular domain of the CD1/MHC family is composed of three
domains termed The physiological
function of EPCR is unknown, but based on homology with the CD1/MHC
family, it is likely to be involved in regulating inflammation. This
prediction is consistent with in vivo studies that suggest
that protein C/APC may be a negative regulator of inflammatory injury.
Specifically, APC has been shown to block the lethal effects of Escherichia coli infusion in baboons(1) , and blocking
the protein C pathway increases the circulating levels of tumor
necrosis factor In addition to the homology to the CD1/MHC
family, the EPCR cDNA exhibited 75% identity in nucleotide sequence and
62% in protein sequence with a murine protein, CCD41(16) ,
raising the possibility that EPCR is the human homologue of CCD41.
However, the characteristics of CCD41 were quite different from those
of EPCR. CCD41 was characterized as a centrosome-associated protein
that was prevalent at the G2/M phase of the cell cycle. Comparison of
the predicted amino acid sequences of the mature CCD41 and EPCR
proteins revealed that the amino-terminal region and the cytosolic tail
were quite different, while the middle portions of the molecules were
nearly identical. CCD41 was predicted to be involved in cell cycle
regulation because it was originally cloned from libraries enriched in
cell cycle-specific messages, and it contained both a PEST motif (Pro-,
Glu-, Ser-, and Thr-rich region) and potential phosphorylation sites.
The PEST motif and the phosphorylation sites were not conserved in
human EPCR. The high degree of homology between human EPCR and CCD41
raised the question of whether EPCR structure and function were
conserved among different mammalian species. To resolve these issues,
we cloned and expressed the murine and bovine EPCR proteins and
demonstrate that the sequence of murine EPCR, which is shown to bind
human APC, is distinct from that reported for CCD41.
The transformed bacteria (2
Figure 1:
Nucleotide sequence and
predicted protein sequence of murine EPCR. A, the nucleotide
sequence of murine clone MEC4 is shown above the predicted amino acid
sequence. The putative signal sequence is underlined. The
transmembrane region is underlinedtwice. Potential N-glycosylation sites are boxed. Extracellular
cysteine residues are circled. B, hydropathy plots of
murine EPCR were generated by the method of Engelman et al.(27) (solidline) and Kyte and Doolittle (28) (dottedline). The signal sequence and
transmembrane region are indicated with the solidbars. The potential N-glycosylation sites are
indicated by a circledN.
The nucleotide sequence was almost
identical with that of CCD41 (Fig. 2A). Only five
single-base differences were detected. When differences in the
sequences were observed, we used at least two and usually all three of
the sequencing methods described under ``Experimental
Procedures'' to confirm the differences. The first difference was
one base change from T to C at position 75 in the murine EPCR
nucleotide sequence. The substitution was in the second base in the
codon for Pro in EPCR and Leu in CCD41 corresponding to amino acid
residue seven in the signal sequences of both proteins (Fig. 2B). In the next three differences, the C base
was inserted in the murine EPCR sequence at positions 105, 133, and
195. These insertions resulted in different usage of the coding frame,
leading to major differences between the protein sequences of EPCR from
residues 18 to 47 and of CCD41 from residues 18 to 46 (Fig. 2B). The third insertion resulted in the
realignment of the reading frame. As a result, the middle portion of
the two molecules have identical sequences. Relative to the nucleotide
sequence of CCD41, the murine EPCR cDNA also exhibited a deletion of 1
C base between base 775 and 776. The three separate sequencing methods
described under ``Experimental Procedures'' all failed to
detect the C base found in the CCD41 sequence. This 1-base deletion
resulted in the carboxyl-terminal sequence of murine EPCR (Arg-Arg-Cys)
being different from that of CCD41 (Arg-Leu-Leu-Ile-Ile). The sequences
of the cytoplasmic tails of murine and human EPCR were identical.
Figure 2:
Comparisons of the nucleotide and
predicted amino acid sequences of murine EPCR and CCD41. A,
comparison of the nucleotide sequences of murine EPCR (mEPCR)
and CCD41. Sites of differences in the nucleotide sequences are
indicated by the numbers marked with the asterisk above the mEPCR sequence. B, comparison of the amino acid
sequences. The predicted amino acid sequence of murine EPCR (upperline) is compared with that of CCD41 (lowerline).
Protein data base searches revealed that the extracellular domain of
murine EPCR, like its human counterpart, had significant homology with
Figure 3:
Nucleotide sequence and predicted protein
structure of bovine EPCR. A, nucleotide and predicted amino
acid sequence of bovine EPCR clone, BEC3. The putative signal sequence,
transmembrane domain, N-glycosylation sites, and extracellular
cysteine residues are indicated in the same manner as Fig. 1. B, hydropathy plots of bovine EPCR were constructed as in Fig. 1. The signal sequence and transmembrane region are
indicated by solidbars, and the potential N-glycosylation sites are indicated by a circledN.
Figure 4:
Amino acid sequence comparisons of human,
bovine, and murine EPCR. Amino acid sequences of human (firstline), bovine (secondline), and murine (thirdline) are compared. The sequences conserved in
these three species are boxed.
The inserts
of the other two clones, BEC5 and BEC9, were 1.8 kb instead of the
1.1-1.4 kb characteristic of the majority of the inserts. To
identify the basis for these differences, the nucleotide sequence of
BEC5 was determined. The sequence at the 5`- and 3`-end was identical
with EPCR, except that a 259-bp insertion was found between position
745 (G) and 746 (G) (Fig. 5). The sequence at the 5`-end of the
insertion was GT, and the sequence at the 3`-end was AG. Therefore,
this clone probably arose due to an alternative splicing event in which
the intron was not removed. As a result, a stop codon was detected
prior to the membrane-spanning domain. Thus, the predicted protein
structure appears to be that of a soluble form of the receptor. We are
yet to identify a comparable alternatively spliced message in human or
murine EPCR.
Figure 5:
Nucleotide and predicted protein sequence
of the larger bovine EPCR clone. The sequence of the 1.8-kb bovine
clone (BEC5) was determined. The nucleotide and predicted protein
sequence, which differed from the smaller clones, is shown in the
figure. The nucleotide differences are indicated by Intron above the sequence and by lowercaseletters, and the protein coding differences are indicated
by underlining. The probable GT and AG sites that were not
removed by message splicing are indicated by outlining the gt and ag. The stop codon for protein translation is
indicated by a star.
Figure 6:
Flow cytometric analysis of Fl-APC
binding. cDNAs of bovine (upper) and murine (middle)
EPCR were transfected into 293T cells. After 48 h, Fl-APC binding in
the absence (brokenlines) and presence of 1.3 mM calcium (solidlines) was determined by flow
cytometric analysis. The dottedlines indicate the
background (293T cells incubated without Fl-APC). As a control,
transfected cells with pEF-BOS without the insert were used. For
details, see ``Experimental
Procedures.''
Figure 7:
Northern blot analysis of murine EPCR.
Northern blot analysis was performed with RNAs from various murine cell
lines (each 15 µg). As the probe, a 969-bp fragment of murine EPCR
was used (upper). The membrane was rehybridized with a control
probe of
Sequence comparisons among proteins of different species can
make a number of significant contributions, especially when the
structure-function relationships of the protein in question remain to
be elucidated. In the case of EPCR, the analysis of the murine EPCR was
particularly germane since the data base search had revealed that the
sequence of the human EPCR was highly homologous to a murine protein,
CCD41, previously described as a cell cycle-specific
centrosome-associated protein. In human endothelial cell cultures, EPCR
is expressed on the cell surface and is capable of binding protein
C/APC. Therefore, identification of the murine EPCR gained added
importance for determining whether EPCR was conserved structurally and
functionally across species. This was especially relevant since, if
CCD41 were the murine homologue of human EPCR, it would suggest major
differences in apparent function of the proteins between species. By
hybridization screening, four independent murine clones were isolated
and were found to contain identical nucleotide sequences. These clones
appear to code for murine EPCR because of the significant structural
homology with human and bovine EPCR (Fig. 4), the ability of
cells transfected with these cDNAs to bind APC (Fig. 6), and the
endothelium-specific message expression (Fig. 7). We identified
only five nucleotide differences between murine EPCR and CCD41. This
extreme similarity in nucleotide sequence suggests that murine EPCR and
CCD41 are identical proteins and that the differences were likely to
have arisen from sequencing or cloning errors arising at these limited
number of sites. Since murine, bovine, and human EPCR are so homologous
and since we used multiple approaches to sequence murine EPCR, we
presume our sequence to be correct. We cannot, of course, exclude the
possibility that CCD41 is a distinct gene product that we simply failed
to identify. If murine EPCR and CCD41 are identical, the question
arises as to whether EPCR is a centrosome-associated protein as
characterized in the CCD41 report. The nuclear localization and
centrosome association of CCD41 were demonstrated by using antibodies
against bacterially expressed recombinant protein. The fusion protein
contained Among the cell lines tested, message
and expression of murine EPCR, like its human counterpart, seemed to be
relatively specific for the endothelium. On the other hand, CCD41 was
cloned from a cDNA library of Ehrlich ascites tumor cells. In the case
of human EPCR, the message expression was readily demonstrable only
with endothelial cells, but a weak signal could also be detected with
an osteosarcoma cell line, HOS, and lymphoma line, U937. Assuming that
CCD41 and murine EPCR are identical, abnormal expression of EPCR might
be caused due to transformation of the cells. In fact, TM (21) and several proteases (22) that modulate blood
coagulation have been reported to be expressed in tumor cell lines but
to be essentially absent from normal cells from which the tumor lines
originated. CCD41 was originally isolated as a G2 phase prevalent
clone by differential screening with cell cycle phase-specific
probes(16) . Given that EPCR and CCD41 appear to be identical
proteins, it seems quite likely that EPCR message expression in
endothelium is also regulated by the cell cycle. TM and EPCR are both
down-regulated by TNF- The relationship of EPCR to the
anticoagulant functions of the endothelium remains unknown. With
respect to the protein C pathway, the endothelium catalyzes protein C
activation mediated by the thrombin-thrombomodulin complex and
facilitates factor Va inactivation mediated by APC. In the case of
bovine endothelium, protein S was found to be essential for both the
binding of APC and rapid factor Va inactivation(13) . On the
other hand, human APC binding to endothelial cells was protein S
independent(14, 24) . Cultured human endothelium also
exhibits less dependence on protein S to accelerate factor Va
inactivation(25) . Whether EPCR expression levels are related
to these specific differences can now be addressed. Comparison of
the predicted sequences of human, bovine, and murine EPCR reveals
several conserved structural features of this molecule. Based on the
observation that all three species bind human APC, it is likely that
the APC binding site is conserved. Absolute conservation of the primary
sequence of surface regions suggests that these regions are involved in
critical interactions, possibly including protein C binding. The 2 Cys
residues in the second domain were well conserved in the EPCR and
CD1/MHC family of proteins. Both of these domains are predicted to be
globular and rich in
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) L-39017 and
L-39065.
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5571-5577
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
THE STRUCTURAL AND FUNCTIONAL CONSERVATION IN HUMAN, BOVINE, AND
MURINE EPCR (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)Protein C is activated by the
thrombin-thrombomodulin (TM) complex on endothelial cell surfaces
(reviewed in (7) ). APC forms complexes with protein S and
inactivates the coagulation factors Va and VIIIa. Protein C, APC, and
protein S bind specifically to cell surfaces such as platelets (8, 9, 10, 11) and endothelial
cells(12, 13) . In a previous study, we identified a
specific, saturable, and calcium-dependent binding site for protein
C/APC on cultured HUVEC (K
= 30
nM, 7,000 sites/cell). Labeled APC was displaced by both
unlabeled APC and protein C equally but not by factor X or
protein S, two structurally similar, Gla domain-containing proteins.
Therefore, the binding sites were specific for protein C and APC. Since
APC blocked at the active center with covalent inhibitors can bind to
the receptor, it is likely that the APC is proteolytically active.
Expression cloning revealed a 1.3-kb cDNA clone, which was capable of
protein C and APC binding when transfected into mammalian
cells(14) . Of the cell types tested, only endothelium
expressed significant APC binding capacity and message levels.
Therefore, we designated the molecule as an endothelial cell protein
C/APC receptor (EPCR). Like TM, the APC binding function and message
expression of EPCR were down-regulated when endothelium was exposed to
tumor necrosis factor (TNF) 
.1, 2, and 3(15) . In EPCR, the
extracellular region was predicted to contain only two domains
corresponding to the 1 and 2 domains but apparently lacks the
third domain, also known as the immunoglobulin domain. Therefore, EPCR
appeared to be a novel member of this family. that occur in response to low level E. coli infusion(2) . Preliminary clinical studies suggest that
protein C infusion can improve the clinical outcome of patients with at
least certain forms of septic shock (3, 4). In addition, protein C and
APC have been shown to inhibit selectin-mediated adhesion of
neutrophils to endothelium(5) , and APC has been reported to
diminish TNF secretion from monocytes exposed to endotoxin(6) .
EPCR is a candidate to be involved in these functions since it is a
receptor for APC, has homology with the CD1/MHC molecules, and is
down-regulated by TNF.
Cells
HA, a murine hemangioendothelioma
cell line, and NIH3T3 were kind gifts from Dr. Paul Kincade. HA was
maintained in minimal essential media/F12 containing 15% fetal bovine
serum. Bovine aortic endothelial cells were prepared as previously
described (13) and maintained in Earle's minimal
essential media containing 10% fetal bovine serum.Construction and Screening of cDNA
Library
Poly(A) RNAs were prepared from HA and bovine
aortic endothelial cells (each 1 
10
) with a
FastTrack mRNA isolation kit (Invitrogen). cDNAs were prepared from 5
µg each of poly(A) RNA by using a SuperScript plasmid system (Life
Technologies, Inc.) according to the manufacturer's protocol.
Synthesized double-stranded cDNAs were fractionated by gel filtration,
and fractions containing longer than 500 bp were pooled and ligated
into a pSPORT vector. Bacterial transformation was carried out by
electroporation as previously described(14) . The murine
library contained 8 10
independent colonies with an
average insert size of approximately 1.8 kb. The bovine library
contained 4 10 independent colonies with an average
insert size of approximately 2.0 kb. 10
) were cultured on eight dishes (150 mm) of LB
plates containing ampicillin. Screening by colony hybridization was
carried out on a Hybond-N nylon membrane filter (Amersham Corp.) by
standard methods(17) . An EcoRI fragment (560 bp) from
the 5`-end of human EPCR cDNA was labeled with
-[
P]dCTP (Amersham) using a multi-prime
labeling kit (Amersham). High stringency conditions were used for
hybridization. Specifically, hybridization was performed at 42 °C
in 50% formamide containing buffer. Washing was carried out at 45
°C in 5 SSC containing 0.1% SDS.Sequencing Analysis
The isolated clones
were subcloned into the pBlueScript vector (Stratagene). DNA sequencing
was performed from both directions by both dGTP and dITP methods using
a Sequenase version 2.0 kit (U. S. Biochemical Corp.). Sequencing with
7-deaza-dGTP was performed using the Ladderman dideoxy sequencing kit
(Takara). Internal sequencing primers were synthesized with a PCR-MATE
391 DNA synthesizer (Applied Biosystems). Nucleotide and protein
homology searches and sequence comparisons were carried out with the
BLAST (National Center for Biotechnology Information), GCG (Genetics
Computer Group, Inc.), GenBank, EMBL, and SwissProt data bases.Flow Cytometric Analysis of APC Binding to
Transfected Cells
For the amplification of the full protein
coding sequence of murine (M) and bovine (B) EPCR, the following
primers were used: M1, 5`-CGCCTCGAGAGGATGTTGACGAAGTTTC-3`; M2,
5`-CGCGCGGCCGCTTAGATAATTAGCAACGC-3`; B1,
5`-CGCCTCGAGTTGAGAACCTCAGCAAAG-3`; and B2,
CGCGCGGCCGCTGGAGAGAATCAACACCG-3`. M1 and B1 were sense primers that
contained the XhoI site (underlined sequence). The M1 primer
also contained an ATG codon, and the B1 primer was just upstream from
the ATG. M2 and B2 were antisense primers containing the stop codon and
a NotI site (underlined sequence). PCR was performed using the
Vent DNA Polymerase (BioLabs) for 20 cycles with the following times
and temperatures: 95 °C for 1 min, 50 °C for 2 min, and 72
°C for 3 min. A murine clone, MEC4, and a bovine clone, BEC3, were
used as the templates. A XhoI site in the promoter region of a
mammalian expression vector, pEF-BOS(18) , was eliminated by a
conventional PCR mutagenesis method. After double digestion, the
PCR-amplified fragments were ligated into the XhoI and NotI sites. The constructs were transfected into 293T cells by
the calcium-phosphate method, and fluorescein-labeled human APC (human
Fl-APC) binding was detected on a FACScan flow cytometer (Becton
Dickinson) as previously described (14) .Northern Blot Analysis
Total RNAs from HA
and NIH 3T3 were isolated by a method of Chomczynski and
Sacchi(19) . RNAs from murine cell lines, SAKRTLS 12.1 (T
cell)(20) , WEHI-231 (B cell, ATCC CRL 1702), and WEHI-3
(myelomonocyte, ATCC TIB 68) were kind gifts from Dr. Paul Kincade.
Northern blot analysis was performed by using a 969-bp EcoRI-AccI fragment from the 5`-terminal of murine
EPCR cDNA as a probe as previously described(14) .
Cloning and Sequence Analysis of Murine
EPCR
As was observed with human endothelium(14) ,
human Fl-APC bound to a murine HA hemangioendothelioma cell line in a
Ca
-dependent fashion (data not shown). A cDNA library
was constructed from this cell line and screened with human EPCR cDNA
as a probe. Four independent colonies, MEC1-4, were isolated and
found to contain different insert sizes (from 1.2 to 1.4 kb). From the
results of restriction mapping and partial nucleotide sequencing, the
four clones contained identical sequences except for the 5`-ends. The
entire nucleotide sequence of the longest clone, MEC4, was determined (Fig. 1). The 5`-end contained an untranslated sequence of 55
bp. A translation initiation ATG codon (AGGATGT) was found at position
56. The sequence of the murine and human translation initiation sites
were identical. The open reading frame that followed coded a protein of
242 amino acids in length. A typical signal sequence of 17 amino acids
was identified at the amino-terminal, and a transmembrane domain (25
amino acids) was located near the carboxyl-terminal followed by a short
cytosolic domain (3 residues). Therefore, mature murine EPCR appears to
be a type 1 transmembrane protein with 225 amino acids. The
extracellular domain contained 4 Cys residues at positions 19, 115,
119, and 189, all of which were conserved in human. Five potential N-glycosylation sites were detected at positions 46, 63, 140,
166, and 176. Except for the fourth site at 166, the other sites were
conserved in human EPCR.
1 and 2 domains of the CD1/MHC class I family. For instance,
28% identity was observed between murine EPCR and human CD1d.Cloning and Sequence Analysis of Bovine
EPCR
Nine cDNA clones, BEC1-9, were isolated from the
bovine aortic endothelial cell cDNA library. Seven of them contained
inserts of different lengths ranging from 1.1 to 1.4 kb, and two
contained inserts of 1.8 kb. Restriction mapping and partial nucleotide
sequencing revealed that the seven smaller clones contained identical
sequences except for variable sizes at the 5`- ends. The two larger
clones contained apparent insertions in the middle of the sequences
(see below). The entire sequence of the longest clone that did not
contain the insertion, BEC3, was determined (Fig. 3). The clone
contained 1426 bp, and the nucleotide sequence was 79% identical with
that of human EPCR. A potential polyadenylation signal sequence,
AATAAA, at 1380 was identical to that found in human EPCR. The 5`-end
contained 198 bp of untranslated sequence; a translation initiation ATG
codon (AGAATGT) was found at position 199, and a TGA stop codon was
found at position 862. The predicted protein was composed of 241 amino
acids. Like human and murine EPCR, a typical signal sequence was
identified at the amino-terminal (17 amino acids) (Fig. 4), and
the transmembrane domain was near the carboxyl-terminal (25 amino
acids). Relative to human or murine EPCR, the cytoplasmic domain
contained an additional Arg residue (Arg-Arg-Arg-Cys), resulting in a
4-residue cytoplasmic tail. Therefore, the mature protein was predicted
to contain 224 amino acids. The 4 Cys residues in the extracellular
domain at 19, 116, 120, and 188 were conserved among all three species.
All four potential N-glycosylation sites at 49, 66, 138, and
174 were conserved relative to human EPCR. As expected, significant
homology with the CD1/MHC class I family was observed.
Expression of Bovine and Murine EPCR in 293T Cells
Creates a Binding Site for Human APC
The PCR-amplified cDNA
clones of bovine and murine EPCR containing the full protein coding
region were cloned into an expression vector, pEF-BOS. They were
transfected into 293T cells, and the human APC binding was monitored by
flow cytometry (see ``Experimental Procedures''). In both
cases, significant levels of human APC binding was demonstrated, and
binding was calcium dependent. No specific binding was detected with
control-transfected cells (Fig. 6).
Endothelial Cell-specific Message Expression of
Murine EPCR
Message expression of murine EPCR was analyzed
with various types of murine cell lines by Northern blot analysis. EPCR
message was readily detected with HA cells, but little or no message
was detected with the other cell lines (Fig. 7).
-actin (lower).
-galactosidase, part of the signal peptide of CCD41, and
the mature protein sequence. It is possible that the antibodies react
with sequences present in this fusion protein that are absent from the
mature molecule and that these cross-react with other proteins within
the cell. In support of the possibility, the antibodies prepared
against the CCD41 fusion protein recognized a 50-kDa protein in cell
lysate, but the molecular size of the in vitro translation
product was smaller than 45 kDa(16) . When appropriate
antibodies to murine and human EPCR become available, the localization
of EPCR should be reexamined.. In addition to sharing this property, TM
expression has also been reported to be cell cycle
dependent(23) .-sheet structure. Between the 2 domain
and the membrane-spanning domain, there are 24 amino acids that may
form an extended structure. Because this region is serine and threonine
rich, it is likely to be the site of O-glycosylation, which
might help stabilize the extended structure as it appears to do in
thrombomodulin(26) . In addition, the transmembrane region and
the carboxyl-terminal Cys were conserved among the three species,
suggesting that they may play important biological functions. The
availability of the three primary structures provides a useful
framework for further investigation of structure-function relationships
in this receptor.
)
We thank Dr. Tetsuro Fujimoto for making cDNA
libraries and Dr. Naomi Esmon, Dr. Alireza Rezaie, and Tim Mather for
helpful discussion. We also thank Shu Chen, Jeff Box, Clendon Brown,
and Gary Ferrell for technical support.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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J.-M. Gu, K. Fukudome, and C. T. Esmon Characterization and Regulation of the 5'-Flanking Region of the Murine Endothelial Protein C Receptor Gene J. Biol. Chem., April 21, 2000; 275(17): 12481 - 12488. [Abstract] [Full Text] [PDF]
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J.-M. Gu, Y. Katsuura, G. L. Ferrell, P. Grammas, and C. T. Esmon Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo Blood, March 1, 2000; 95(5): 1687 - 1693. [Abstract] [Full Text] [PDF]
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 B. O. Villoutreix, A. M. Blom, and B. Dahlback Structural prediction and analysis of endothelial cell protein C/activated protein C receptor Protein Eng. Des. Sel., October 1, 1999; 12(10): 833 - 840. [Abstract] [Full Text] [PDF]
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R. E. Simmonds and D. A. Lane Structural and Functional Implications of the Intron/Exon Organization of the Human Endothelial Cell Protein C/Activated Protein C Receptor (EPCR) Gene: Comparison With the Structure of CD1/Major Histocompatibility Complex alpha 1 and alpha 2 Domains Blood, July 15, 1999; 94(2): 632 - 641. [Abstract] [Full Text] [PDF]
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J. Xu, N. L. Esmon, and C. T. Esmon Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation J. Biol. Chem., March 5, 1999; 274(10): 6704 - 6710. [Abstract] [Full Text] [PDF]
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 K. Fukudome, X. Ye, N. Tsuneyoshi, O. Tokunaga, K. Sugawara, H. Mizokami, and M. Kimoto Activation Mechanism of Anticoagulant Protein C in Large Blood Vessels Involving the Endothelial Cell Protein C Receptor J. Exp. Med., April 6, 1998; 187(7): 1029 - 1035. [Abstract] [Full Text] [PDF]
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K. Fukudome, S. Kurosawa, D. J. Stearns-Kurosawa, X. He, A. R. Rezaie, and C. T. Esmon The Endothelial Cell Protein C Receptor. CELL SURFACE EXPRESSION AND DIRECT LIGAND BINDING BY THE SOLUBLE RECEPTOR J. Biol. Chem., July 19, 1996; 271(29): 17491 - 17498. [Abstract] [Full Text] [PDF]
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P. C. Y. Liaw, T. Mather, N. Oganesyan, G. L. Ferrell, and C. T. Esmon Identification of the Protein C/Activated Protein C Binding Sites on the Endothelial Cell Protein C Receptor. IMPLICATIONS FOR A NOVEL MODE OF LIGAND RECOGNITION BY A MAJOR HISTOCOMPATIBILITY COMPLEX CLASS 1-TYPE RECEPTOR J. Biol. Chem., March 9, 2001; 276(11): 8364 - 8370. [Abstract] [Full Text] [PDF]
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