Proteolytically activated receptors (PARs)
represent an emerging subset of seven transmembrane G protein-coupled
receptors that mediate cell activation events by receptor cleavage at
distinct scissile bonds located within receptor amino termini.
Differential genomic blotting using a yeast artificial chromosome known
to contain the PAR-1 and PAR-2 genes identified the PAR-3 gene within a
PAR gene cluster spanning ~100 kilobases at 5q13. The PAR-3 gene is
relatively small (~12 kilobases); and, like the PAR-1 and PAR-2
genes, it displays a two-exon structure, with the majority of the
coding sequence and the proteolytic cleavage site contained within the
larger second exon. Sequence analysis of the 5'-flanking region
demonstrates that the promoter is TATA-less, similar to that seen with
PAR-1, with the identification of nucleic acid motifs potentially
involved in transcriptional gene regulation, including AP-1, GATA, and
octameric sequences. PAR-3 transcripts were apparent in human vascular
endothelial cells, although at considerably lower levels than those of
PAR-1 and not significantly modulated by the endothelial cell stimulus
tumor necrosis factor-
. Likewise, although PAR-3 mRNA was
evident in human platelets, receptor cell surface expression was modest
(~10%) compared with that of PAR-1. Thus, although PAR-3 is
postulated to represent a second thrombin receptor, its modest
endothelial cell and platelet expression suggest that PAR-3 activation
by
-thrombin is less relevant for physiological responses in these
mature cells. Rather, given its disparately greater expression in
megakaryocytes (and megakaryocyte-like human erythroleukemia cells), a
regulatory role in cellular development (by protease activation) could
be postulated.
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INTRODUCTION |
Proteolytically activated receptors
(PARs)1 are members of a
larger family of seven transmembrane cell surface receptors that mediate cell activation events through heterotrimeric G proteins (1,
2). Until very recently, two PARs had been isolated to date, the
thrombin receptor (PAR-1) (3) and the proteinase-activated receptor
(PAR-2) (4, 5). Unlike the former class of receptors in which cellular
activation events are initiated by standard receptor-ligand binding
interactions, both PAR-1 and PAR-2 are activated by cleavage at
distinct Arg-Ser bonds located within the NH2-terminal
extension of both receptors, and synthetic peptidomimetics representing
the new NH2 terminus after receptor cleavage function as
full receptor agonists irrespective of receptor cleavage. In addition
to displaying similar molecular mechanisms of receptor activation,
functional similarities between PAR-1 and PAR-2 have also been
observed. Thus, both are expressed on vascular endothelial cells (6),
and both mediate proliferative responses when activated by their
protease agonists (7, 8) or synthetic peptide ligands (6, 8). These
structure/function similarities also extend to the gene level; thus,
both PAR-1 and PAR-2 genes are known to co-localize at 5q13, and both
genes are organized similarly, suggesting evolution from a common
ancestral gene (9).
Given evolving evidence for protease activation of multiple cell types
(2, 7) and the corollary that some, if not all, of these cellular
sequelae may be mediated by activation of novel PARs, we adapted a
positional cloning approach as a strategy for the identification of
other putative PARs that may co-cluster within this region of the
genome. Interestingly, we identified the recently characterized PAR-3
gene (10) within this region of the genome and demonstrate that its
structural organization is essentially identical to that of PAR-1 and
PAR-2, reinforcing the concept of a gene family of receptors evolving
from a common ancestral gene and emerging along a distinct evolutionary
pathway. Furthermore, like PAR-1 and PAR-2, PAR-3 is also expressed on vascular endothelial cells, although at considerably lower levels; similar low level but detectable PAR-3 expression is also evident in
human platelets; thus, although PAR-3 appears to be a second "thrombin receptor," its comparatively modest expression in these cell types suggests a functional role distinct from PAR-1 in mediating cell activation events by
-thrombin.
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MATERIALS AND METHODS |
Supplies, Reagents, and Cell Lines--
Restriction enzymes were
purchased from Stratagene (La Jolla, CA) or New England Biolabs
(Beverly, MA). Avian myeloblastosis virus reverse transcriptase was
from Seikagaku America, Inc. (Rockville, MD), and ExpandTM
long template polymerase was from Boehringer Mannheim. Oligonucleotide primers were synthesized on an Applied Biosystems model 381A
single-channel synthesizer (G. D'Angelo, Molecular Biology Core, SUNY
Stony Brook). Human umbilical vein endothelial cells (HUVEC) were
isolated from pooled primary cultures of human umbilical veins and
propagated as described previously (6). Human erythroleukemia (HEL)
cells were propagated in RPMI medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Genomic Analysis and Library Screening--
Total genomic DNA
from normal human volunteers was extracted from peripheral blood
leukocytes as described previously (9) and quantitated by absorption
spectrophotometry at 260 nm. Approximately 5-10 µg of DNA was
digested with various restriction enzymes for Southern blot analysis.
The separation of large DNA fragments on agarose gels was completed
using an inversion field gel electrophoresis apparatus
(SwitchbackTM pulse controller, Hoefer Scientific
Instruments, San Francisco). Yeast artificial chromosomal (YAC) DNA was
isolated as described previously (11), and human genomic fragments were
separated from yeast chromosomes using a 1% agarose gel in 0.5 × TBE (Tris borate-EDTA), run at 6.9 V/cm in 0.5 × TBE buffer at
10 °C with a pulse timer of 1-50 s and a forward/reverse ratio of
3:1 for 24 h. Gels were blotted onto nylon membranes (Schleicher & Schuell) and hybridized to 32P-radiolabeled cDNA
probes. Blots were washed at high (0.1 × SSC, 0.1% SDS, 1 mM EDTA, pH 8.0, and 10 mM sodium phosphate at
68 °C for 1 h) or low (0.1 × SSC, 0.5% SDS, 1 mM EDTA, pH 8.0, 10 mM sodium phosphate at
55 °C for 30 min) stringency and analyzed by autoradiography with
Kodak XAR-5 film with an intensifying screen at
80 °C for 3-10
days. For some experiments, filters were stripped according to the
manufacturer's recommendations and adequacy confirmed by overnight
autoradiography.
The PAR-3 cDNA was isolated from a HUVEC cDNA library cloned
into the bacteriophage vector
gt11, essentially as described previously (6). Positive phage clones were plaque purified, and the DNA
was purified from minilysates by standard methods (9). The PAR-3 gene
was isolated from a cosmid library generated from the ~1.4-megabase
YAC 798D11, shown previously in this laboratory to contain both PAR-1
and PAR-2 genes (11). A size-selected library ranging from ~30 to
~40 kb was generated by partial MboI digestion of 1 µg
of YAC 798D11 DNA followed by ligation to SuperCos-1 vector
(Stratagene) containing flanking BamHI sites. Ligation products were packaged using 25 µl of Gigapack III XL packaging extract for 2 h at 25 °C followed by titering and amplification using Escherichia coli host strain XL1-Blue. The titer of
the unamplified library was ~1 × 105 colony-forming
units/ml, consistent with a ~103-fold representation of
nucleotide sequences. Library screening was completed using the
32P-radiolabeled PAR-3 cDNA as probe, and DNA from
individual cosmids was characterized further by extensive restriction
mapping. Discrete genomic fragments were gel purified and ligated into
pBluescript (Stratagene) for DNA sequence analysis using dideoxy chain
termination. Exon-intron boundaries were defined by comparison of the
genomic DNA sequence with that of the published cDNA (10). Sequence analysis was performed using the Wisconsin Genetics Computer Group Package (12). The identification of PAR-3 promoter nucleotide motifs
potentially involved in transcriptional gene regulation was
characterized using TRANSFAC software (13).
Radiation Hybrid (RH) Mapping Analysis--
Genetic mapping
within the PAR gene cluster was completed using both the Stanford G3
(~500-kb resolution) and TNG3 (~100-kb resolution) RH mapping
panels (11, 14). PCR screening was completed using the following
PAR-3-specific oligonucleotide primers: PAR2064 (5'-3':
TCCATCCTTTCACCTACCGGG, bp 731-751) and PAR2065 (5'-3':
TAGCAGTAGATGATAAGCACA, bp 989-969) (10). PAR-1, PAR-2, and
microsatellite-specific primers encompassing this region of the genome
were as described previously (11). PCR conditions were optimized for
individual primer pairs, but the thermal profile generally included a
1-min 95 °C denaturation step, a 2-min 55 °C annealing step, a
3-min 72 °C primer extension step, with a final extension for 10 min
at 72 °C; after 25 cycles in a thermocycler (Coy Laboratory
Products, Ann Arbor, MI), products were analyzed by Southern blot
analysis in a 3% ethidium-stained agarose gel and scored as present or
absent. Retention patterns were submitted for two-point linkage
analyses to the server maintained by the Stanford Human Genome
Center2 or analyzed using
RHMAP (version 2.02), a software package for multipoint radiation
hybrid mapping (16).
RNA Preparation and Northern and PCR Analysis--
Confluent
HUVEC in second to fifth passage were harvested directly with a rubber
policeman, and total cellular RNA was isolated by immediate
solubilization in guanidine hydrochloride and serial ethanol
precipitation (6). Alternatively, poly(A) mRNA was isolated from
solubilized HUVEC (or HEL cells) by oligo(dT)-cellulose chromatography
(Invitrogen, San Diego). For some experiments, 3 × 107 HUVEC were stimulated with 100 units/ml tumor necrosis
factor-
(17) for 4 or 24 h followed by RNA isolation.
Transcript expression of murine tissues was completed using 1 µg of
poly(A) RNA (Clontech, Palo Alto, CA). Northern blot analysis was
completed by size fractionation in a 1% denaturing agarose gel, and
equivalent RNA loading was confirmed by reprobing with a
-actin
cDNA probe (18). Reverse transcription-PCR was completed by
incubating ~1 µg of total cellular RNA with 1 µg of a 14-mer
oligo(dT) primer at 41 °C for 1 h using 10 units of avian
myeloblastosis virus reverse transcriptase in a solution containing 50 mM Tris/HCl, pH 8.3, 50 mM KCl, 8 mM MgCl2, 10 mM dithiothreitol, and
500 µM individual dNTPs in a final volume of 50 µl. 10 µl was subsequently adjusted to PCR conditions using approximately
0.01 OD unit of each PCR primer and 5 units of Taq
polymerase (Thermus aquaticus DNA polymerase; Perkin-Elmer).
Oligonucleotide primers included the reverse primer PAR2065 (see above)
and the forward primers PAR2066 (5'-3': ATAACGTTTAAGAGACGGGACT, bp
111-132). PCR conditions included a 1-min 15-s denaturation step at
94 °C, a 1-min 55 °C annealing step, and a 3-min primer extension
step at 72 °C. Amplifications consisted of 35 rounds using a DNA
thermocycler, and insert sizes were determined by electrophoresis in a
1% ethidium-stained agarose gel. Platelet PCR was completed in a
similar manner, except for minor modifications, essentially as
described previously (19).
PAR-3 Immunodetection--
A 17-mer peptide encompassing the
PAR-3 cleavage site with an added NH2-terminal cysteine and
COOH-terminal amide (PAR-332-48,
Ac-CKPTLPIKTFRGAPPNS-amide) was custom synthesized by Quality Controlled Biochemicals (Hopkington, MA) and purified by reverse phase
high pressure liquid chromatography. For antibody generation, the
peptide was conjugated to keyhole limpet hemocyanin or to BSA using the
hetero-bifunctional cross-linking reagent mal-sac HNSA, and the keyhole
limpet hemocyanin-conjugate was used to immunize New Zealand White
rabbits. Immunoreactivity of the anti-PAR-3 antibody to the BSA
conjugate was confirmed by solid phase enzyme-linked immunosorbent
assay. Preimmune and immune rabbit IgGs were purified by protein G
affinity chromatography. For immunofluorescent studies, the
immunopurified IgG was labeled directly with FITC (0.2 mg of FITC/mg of
IgG) in 0.1 M sodium carbonate buffer, pH 9.5, for 2 h
at 25 °C, and unbound FITC was removed by gel filtration on a
Bio-Rad 10-DG column developed with degassed PBSA (10 mM
sodium phosphate, 150 mM sodium chloride, 0.05% sodium
azide, pH 7.4).
Immunofluorescent staining of confluent HUVEC was completed in
eight-well chamber slides after fixation in 4% paraformaldehyde for 20 min at 25 °C (for determination of cell surface staining) or
fixation and permeabilization using ice-cold 100% acetone for 60 s. Fixed HUVEC were then washed extensively with PBS followed by
incubation with 100 µg/ml FITC-conjugated anti-PAR-3 IgG or a 1:100
dilution of the FITC-conjugated IgG alone for 60 min at 4 °C. After
the last of five washes in PBS, cells were mounted in Aquamount, and
fluorescent images were obtained using a Nikon Diaphot inverted
microscope equipped with fluorescent optics (Nikon, Garden City, NY).
Images were frame averaged to 256 frames with Image I software
(Universal Imaging, Media, PA) using laser light excited at 529 nm and
a blocking filter of 550 nm. Flow cytometric analysis of HUVEC was
completed by detaching adherent cells using PBS and 10 mM
EDTA and resuspending the cells to a final concentration of 5 × 105/ml PBS and 0.1% BSA. 200-µl aliquots were then
incubated with 100 µg/ml FITC-conjugated anti-PAR-3 IgG for 60 min at
4 °C in PBS and 0.1% BSA, and cells were washed twice and then
fixed in 1% formalin before analysis.
Platelet cell surface PAR-1/PAR-3 expression was completed as described
previously (20). Briefly, platelet-rich plasma was prepared in 10 mM EDTA from healthy human donors by differential centrifugation and adjusted to 3 × 108 platelets/ml
TSE (0.1 M Tris, 0.15 M NaCl, 10 mM
EDTA, pH 7.4). 400-µl aliquots were then incubated with 100 µg/ml
FITC-conjugated anti-PAR-3 IgG, 100 µg/ml anti-PAR-1 IgG (20), or 100 µg/ml nonimmune rabbit IgG for 30 min at 25 °C. For PAR-1
expression, platelets were incubated with a 1:100 dilution of the
FITC-conjugated goat anti-rabbit F(ab')2 secondary antibody
(Tago, Inc., Burlingame, CA) for 20 min at 25 °C; samples were then
washed three times in TSE, fixed in 1% formalin, and analyzed in a
FACStar flow cytometer (Becton Dickinson and Co., Rutherford, NJ).
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RESULTS AND DISCUSSION |
Identification of the Human PAR-3 Gene within the PAR Gene
Cluster--
Previous work from this laboratory demonstrated that
human PAR-1 and PAR-2 genes are contained within YAC 798D11 and
separated maximally by ~100 kb (11). In an attempt to identify
additional PARs that potentially may be clustered in this region of the
genome, differential genomic blotting was adapted using a series of
cDNA probes encompassing distinct regions of the PAR-1 or PAR-2
genes. Using such an approach, PAR-1-specific probes failed to identify any novel cross-hybridizing fragments (not shown). In contrast, a
PAR-2-specific probe encompassing the 5'-region of the cDNA identified a novel band at low stringency which was not evident when
the blot was washed at higher stringency (Fig.
1). By inversion field gel
electrophoresis, this EcoRI fragment was estimated to be
~16 kb and could not be resolved by the known genomic organization of
PAR-2 (5). Furthermore, this band was not present within a P1 genomic
clone (P1142) shown previously by Nystedt and colleagues (5) to contain
the entire PAR-2 gene (not shown), again strongly suggesting that it
represented a previously uncharacterized, homologous PAR or a processed
pseudogene. Concomitant with these initial observations, a routine
BLAST (21) homology search using PAR-2 sequences identified a highly
homologous expressed sequence tag (EST) AA177828, partially
characterized from a murine splenic cDNA library, as part of the
Washington University/HHMI Mouse EST project. This expressed sequence
tag was purchased from Research Genetics, fully sequenced, and found to
contain a 1,238-bp insert encoding a 283-amino acid open reading frame
(representing an incomplete cDNA) demonstrating highest homology to
PAR-1 and PAR-2. This 32P-radiolabeled insert was then used
to screen empirically a HUVEC cDNA library using low stringency
screening to maximize hybridization cross-species, with the isolation
of three cDNA clones, the longest of which (
2B1A, 2,566 bp) was
characterized more fully (see below). As demonstrated in Fig.
1D, this cDNA (now known to be identical to PAR-3) (10),
cross-hybridized to the novel ~16-kb EcoRI fragment recognized by the 5'-PAR-2 probe (Fig. 1C), thereby
establishing its identity as the human PAR-3 gene.

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Fig. 1.
Identification of the human PAR-3 gene within
a PAR gene cluster. 1 µg of DNA from YAC 798D11, YAC 907F6
(known to contain the D5S424 microsatellite marker but not PAR-1 or
PAR-2 genes (9)), P1-4251 (an anonymous P1 genomic clone (9)), or 10 µg of human genomic DNA was digested with EcoRI for
Southern blot analysis using the 32P-radiolabeled PCR
fragments spanning discrete portions (in base pairs, BP) of
the PAR-2 cDNA (panels A-C). Blots were washed to low
stringency to maximize hybridization to homologous sequences. A
distinct ~16-kb fragment is evident only in YAC 798D11 using a
5'-probe (panel C, arrow), which is not evident
using other probes, consistent with a homologous gene residing within
this portion of the genome. Rehybridization of the blot with the hPAR-3
cDNA ( 2B1A, see Fig. 4) identified this band as human PAR-3
(panel D, high stringency wash). The PAR-2-specific ~9-kb
band was evident in genomic DNA after longer exposure (not shown). Size
markers using HindIII-digested DNA are shown.
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Physical and Genetic Mapping within the PAR Gene Cluster--
To
establish more precisely distances among the PAR genes within this
genomic segment, we completed genetic mapping using radiation hybrid
mapping panels (11, 14). Using the lower resolution G3 panel, the
amplification (and retention) patterns for all three genes were
indistinguishable (no breaks were observed in the 83 hybrids typed),
establishing that the closest framework marker to all three PARs was
D5S424 (22), with significant linkage as well to D5S1977, D5S2529, and
D5S2596 (in order of decreasing log of odds scores, see Fig.
2). Thus, all three PARs were linked tightly to the identical microsatellite markers and were at least as
close to each other as the resolution power of this panel (360-500 kb).2 To establish further interorder assignments and
distance, we determined amplification patterns from a second radiation
hybrid panel TNG3 with a higher lengthwise resolution of ~100 kb
(11).2 The amplification patterns obtained using this RH
panel were analyzed using RH2PT (16) and demonstrated that the three
genes could now be resolved, with the likely order
centromere-D5S424-PAR-2-PAR-1-PAR-3-telomere (Fig. 2). Although physical conversions are not available for the TNG3
RH panel, if one assumes an average resolution of 3-6 kb/centiRad
throughout the panel,2 this initial analysis suggested that
the three PARs are separated by ~110 to ~220 kb, which was
confirmed subsequently by inversion field gel electrophoresis and
Southern blot analysis. DNA digests of YAC 798D11 were completed using
various restriction endonucleases (including rare-cutting enzymes) in
an attempt to resolve the three genes within single large genomic
fragments. As revealed in Fig. 3,
Southern blot analysis demonstrated that all three cDNAs were
contained within restriction fragments of ~90 kb (XhoI) or
~120 kb (NotI), consistent with a distance among the genes of less than 100 kb.

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Fig. 2.
Schema of human chromosome 5 with interorder
distances for PAR genes. PAR-1-, PAR-2-, and PAR-3-specific
oligonucleotide primers were used for determination of amplification
(and retention) patterns using both G3 and TNG3 RH panels, followed by
two-point linkage analysis using RH2PT (16). The centromeric/telomeric
representation is a consensus from previous mapping data utilizing SHGC
G3 RH panel; the position of D5S1977 relative to D5S424 and D5S2529 has
not been confirmed in this laboratory and thus is presented in
italics (11). Arbitrary distance measurements are expressed
as cR_8000 (8,000 centiRad, the radiation dosage used for the
generation of the G3 panel) or cR_50,000 (TNG3
panel). Note that distance estimates from the two panels are not
comparable and do not indicate physical distances but simply reflect
more frequent radiation-induced chromosomal breakage events.
Tel, telomere; Cen, centromere; LOD,
log of odds.
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Fig. 3.
Characterization of the PAR gene cluster by
inversion field gel electrophoresis. Panel A, 1 µg of YAC
798D11 DNA was digested with EcoRI (first lane
from left), NotI (second lane),
PvuI (third lane), XhoI (fourth
lane), or XbaI (fifth lane) for Southern
blot analysis using the 32P-radiolabeled PAR-3 cDNA
( 2B1A) as probe followed by high stringency wash and overnight
autoradiography. The table (panel B) delineates the
restriction fragment sizes when the blot was stripped and sequentially
hybridized with the cDNAs for PAR-1 (20) and PAR-2 (6). As
demonstrated, all three genes are contained within the identical
NotI (~120 kb) or XhoI (~90 kb) fragments.
Note the different sized restriction patterns with XbaI or
EcoRI, excluding the possibility that the cDNAs are
cross-hybridizing to other genes within the cluster.
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PAR-3 Genomic Characterization--
To characterize the genomic
structure of PAR-3, a cosmid library was constructed using YAC 798D11
DNA. Approximately 7 × 103 independent clones from
the unamplified library were screened using the PAR-3 cDNA, with
the identification of 10, three of which (COS3-2, COS2-1, and
COS7-1) were characterized further. Only one of the three clones
(COS3-2) contained the entire PAR-3 gene, whereas COS2-1 and COS7-1
lacked the 5'-coding region (Fig. 4).
PAR-3 exons, intron/exon boundaries, and portions of the flanking introns were sequenced bidirectionally for further analysis. Not unexpectedly, PAR-3 displayed a genomic organization similar to that of
the PAR-1/PAR-2 genes (5, 9, 23), containing a small first exon and a
larger second exon encoding the majority of the coding sequence and the
protease cleavage site. The single intronic splice site occurs after
the second nucleotide of the Gly22 codon, indicative of a
type II splice site, although the splice junction sequences are
somewhat atypical (24). Nucleotide analysis of the entire coding
sequence was identical to that described initially (10), with the
identification of an additional 1,451 bp of 3'-untranslated sequence in
the cDNA clone
2B1A. This sequence has been deposited into the
GenBank data base and assigned the accession number AF053124. The size
of the PAR-3 first intron was estimated at ~4 kb, utilizing
restriction mapping and long range PCR from YAC 798D11, COS3-2, and
total human genomic DNA. Thus, although genomic
rearrangements/deletions are not uncommonly found in YACs, the
identical sizes of restriction fragments and PCR fragments confirm
these size estimates and determinations.

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Fig. 4.
Schema outlining the structural organization
of the PAR-3 gene. Panel A, exons are indicated by
solid rectangles, and distances (in kb) with key restriction
enzymes are denoted (X, XbaI; H,
HindIII; RI, EcoRI; S,
SphI). The splice junction sites are delineated (exon
sequences are capitalized) along with the nucleotides and
amino acid residues contained in each exon. The numbering system
empirically starts with the initiator methionine (ATG = +1).
Panel B, the schema of the overlapping cosmid clones used to
characterize the PAR-3 gene and the PAR-3 cDNA clone 2B1A
isolated and characterized from the HUVEC cDNA library.
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A ~5-kb XbaI fragment containing the first exon and
5'-untranslated region (see Fig. 4) was sequenced bidirectionally to
characterize further potential regulatory sequences involved in PAR-3
expression (Fig. 5). Neither a TATA box
nor CCAAT sequences are evident (25), similar to the promoter analysis
of the PAR-1 gene (9). Although GC-rich regions (SP1 binding sites)
potentially involved in transcriptional regulation of housekeeping
genes are not found infrequently in TATA-less promoters (and present in
the PAR-1 promoter, for example (9, 25)), such sequences are absent in
the PAR-3 gene 5'-regulatory sequences, further suggesting
transcriptional regulation by more restricted nuclear binding elements.
Further analysis of the 5'-regulatory region (Fig. 5B)
demonstrated potential cis-acting DNA elements, such as
AP-1-like elements present at
772,
1041, and
1208 (26, 27), and a
single cAMP response element-binding protein (28) sequence present at
638. Eight GATA-like sequences known to represent binding sites for
the erythroid nuclear factor protein NF-E1 are present at
344,
430,
480,
619,
735,
891, and
935 (29). This protein is also known
to be expressed in megakaryocytes (30), an observation of potential
relevance given the observed high level of PAR-3 expression in these
cells (10; see below). Also of relevance for megakaryocytic gene
regulation is the presence of seven distinct octameric sequences,
regulatory elements known to modulate the expression of various
megakaryocyte genes (31, 32).

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Fig. 5.
Nucleotide analysis of the PAR-3 5'-flanking
regions. Panel A, the 5'-regulatory sequence is displayed,
with the start of the previously unpublished sequence depicted by the
arrow and the key HindIII restriction site
delineated. Panel B, the schema summarizes the putative
transcriptional regulatory sequences.
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PAR-3 mRNA Expression in Endothelial Cells--
Initial
evaluation for PAR-3 transcript expression was pursued by Northern blot
analysis using murine tissues. Dual transcripts of ~4.0 and ~2.0 kb
were evident primarily in poly(A) mRNA from spleen, with little to
no expression evident in poly(A) mRNA from brain, kidney, liver,
lung, pancreas, or smooth muscle (not shown), results that were
consistent with those described previously (10). Interestingly, this
initial analysis suggested that PAR-3 may be expressed in tissues of
hematopoietic origin, and cellular sources for PAR-3 expression were
pursued subsequently in megakaryocytes/platelets and endothelial cells.
The latter cell type was studied because of prior evidence for both
PAR-1 and PAR-2 expression in endothelial cells (6) and strong
presumptive evidence for PAR-3 expression obtained during initial HUVEC
cDNA library screening (see above). Accordingly, Northern blot
analysis was completed using total cellular RNA from endothelial cells
and from HEL cells, which display phenotypic features of megakaryocytic
differentiation (33). As shown in Fig. 6,
PAR-3 transcripts were clearly evident in HEL cells, approximately
2-fold higher than those of PAR-1 transcripts. In contrast, although
PAR-3 expression was evident in HUVEC, the relative transcript
expression was considerably less than that of PAR-1, with steady-state
PAR-1 levels nearly 25-fold greater than those of PAR-3. To address the
possibility that PAR-3 expression could be up-regulated during cell
activation, cells were then stimulated with the endothelial cell
stimulant tumor necrosis factor-
, and differential PAR-1/PAR-3
expression was studied further. Minimal change in either transcript
expression was evident, with the PAR-1/PAR-3 ratio remaining skewed
toward enhanced PAR-1 expression at both time points studied.

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Fig. 6.
PAR-3 mRNA expression. 10 µg of
total cellular RNA (HEL) or 1 µg of poly(A) RNA (HUVEC, with or
without 100 units/ml tumor necrosis factor- (TNF), was
sized fractionated in a 1% denaturing agarose gel, transferred to
nylon membranes, and hybridized sequentially with
32P-radiolabeled PAR-3 or PAR-1 cDNA. Blots were washed
at high stringency and analyzed by autoradiography after a 3-day
exposure. Two PAR-3 transcripts of ~4.0 kb and ~2.0 kb are evident
in HEL cells, as demonstrated previously (10), whereas the single PAR-1
transcript is evident (upper panel). In HUVEC, only larger
transcript is identified with this exposure. Densitometric scanning was
used to quantify relative PAR-1/PAR-3 (4.0-kb transcript) expression
(lower panel).
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The evidence for PAR-3 transcript expression in endothelial cells
prompted further studies to elucidate receptor expression in these
cells. For these studies, a PAR-3 peptide spanning the thrombin
cleavage site was synthesized for generation of a polyclonal antibody,
and its immunoreactivity with the synthetic peptide was confirmed by
enzyme-linked immunosorbent assay. To enhance the detection of
apparently low level endothelial cell PAR-3 expression as determined by
Northern analysis (Fig. 6), the purified anti-PAR-3 IgG was coupled to
FITC before subsequent immunofluorescent staining in HUVEC. As shown in
Fig. 7A), this antibody was
optimally immunoreactive with PAR-3 on HEL cells at a concentration of
100 µg/ml, an antibody concentration that was used for evaluation of
PAR-3 expression in HUVEC. Consistent with the results demonstrated by
Northern blot analysis, cell surface PAR-3 expression was readily
evident on vascular endothelial cells as determined by flow cytometry (Fig. 7B). The cellular distribution of PAR-3 was then
pursued directly by immunofluorescent staining (Fig. 7C-E)
and was consistent with both an intracytoplasmic and cell surface pool
of receptors. This similar pattern has been described both for
endothelial cell PAR-1 and PAR-2 and has been postulated to represent a
mechanism of regulating cell surface PAR expression and responsiveness
in these cells (6, 34). Interestingly, the pattern of cell surface immunofluorescence appeared to demonstrate more intense PAR-3 expression in areas of endothelial cell-cell contact. Although the significance of this remains unclear at this time, it may suggest an undefined role for PAR-3 in these cells, possibly distinct from that mediating cell activation events through receptor
proteolysis.

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Fig. 7.
HUVEC PAR-3 immunodetection. Panel
A, the FITC-conjugated anti-PAR-3 IgG (at various concentrations)
or a 1:100 dilution of the FITC-conjugated IgG alone (control) was
incubated with 1 × 106 HEL cells for 60 min at
4 °C before analysis by flow cytometric analysis, with evidence for
optimal antibody reactivity at 100 µg/ml. Panel B,
endothelial cells were detached, and 1 × 105
endothelial cells were incubated with 100 µg/ml FITC-conjugated
anti-PAR-3 IgG (or the secondary antibody alone) for 60 min at 4 °C
followed by flow cytometry for evaluation of cell surface receptor
expression. Panels C-E, endothelial cells were grown until
confluent, serum starved for 1 h, and then fixed and permeabilized
using ice-cold acetone for 60 s (panels C and
D) or fixed without permeabilization using 4%
paraformaldehyde (panel E) before immunofluorescence
staining using 100 µg/ml FITC-conjugated anti-PAR-3 IgG, as outlined
under "Materials and Methods." Distinct intracellular staining is
evident in panel D, whereas cell surface staining is seen in
nonpermeabilized cells (panel E). Note the stippled staining
patterns consistent with cell surface expression and the more intense
staining in areas of cell-cell contact (panel E,
arrows). No staining is seen using the secondary antibody
alone (panel C). Final magnification in all panels is × 1100.
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Platelet PAR-3 Expression--
As described initially, PAR-3
transcripts were expressed prominently in splenic megakaryocytes as
evaluated by in situ hybridization (10). This observation,
combined with our demonstration for considerable PAR-3 expression in
the megakaryocyte-like HEL cells, prompted us to study PAR-3 expression
in platelets, an issue of considerable importance given previous
evidence for dual thrombin receptors or signaling pathway(s) in these
cells (20, 35). As shown by reverse transcription-PCR, a PAR-3 mRNA
transcript was readily detectable in human platelets (Fig.
8). Because this assay as designed was
not quantitative, cell surface receptor expression was then studied by
flow cytometric analysis, designed to elucidate more accurately
relative PAR-1/PAR-3 expression on the platelet cell surface.
Interestingly, these results are comparable to those seen in HUVEC,
demonstrating detectable but low level PAR-3 expression and
considerably less cell surface receptor when compared with PAR-1. Prior
determinations suggest that there are approximately 1,500-2,000 PAR-1
receptors on human platelets (36, 37). Based on the relative mean
fluorescence intensities of PAR-1/PAR-3 as established by flow
cytometry, we would estimate PAR-3 receptor density to be approximately
10% of PAR-1 (~150-200 receptors/platelet), although definitive
comparisons will ultimately require monoclonal antibodies with limited
antigenic epitopes.

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Fig. 8.
PAR-3 expression in human platelets.
Panel A, reverse transcriptase-PCR using total cellular RNA
from endothelial cells (E) or platelets (P) was
completed as outlined under "Materials and Methods" in the presence
(+) or absence ( ) of reverse transcriptase (RT), followed
by PCR using PAR-3 oligonucleotide primers PAR2065 and PAR2066. As
shown, a single band of the anticipated size (878 bp) was evident in
both cells only in the presence of reverse transcriptase, confirming
PAR-3 mRNA expression in these cells. Panel B, flow
cytometric analysis of human platelets was completed using 100 µg/ml
FITC-conjugated anti-PAR-3, anti-PAR-1, or nonimmune rabbit
(NIR) IgG, followed by immunodetection (for PAR-1 and NIR)
using a 1:100 dilution of the FITC-conjugated F(ab')2
secondary antibody. The percent positive cells and the mean
fluorescence intensity (MFI) (inset) were
standardized against the nonimmune IgG/FITC control. The results
demonstrate detectable but modest levels of cell surface PAR-3
expression compared with that of PAR-1. Identical results were obtained
from two volunteers studied on different days.
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Summary and Implications for Future Research--
PARs represent a
unique and rapidly emerging family of G protein-coupled
seven-transmembrane receptors that mediate cell activation events to
serine proteases generated during one of three major protease-generating pathways in humans (inflammatory, fibrinolytic, or
hemostatic). To date, all three PAR genes have been identified as being
structurally conserved and residing within a discrete segment of the
human genome, strongly suggesting evolution from a common ancestral
gene. Given progressive evidence that a larger number of serine
proteases affect cell activation (2), we would speculate that more
structurally similar receptors exist, some of which may be found within
this gene cluster or identified as part of the human genome initiative.
The construction of a cosmid library containing the repertoire of genes
encompassing this region of the genome may facilitate the search for,
and rapid characterization of, other structurally similar receptors
that may co-localize within this region of the genome.
What is the function of PAR-3? Although characterized initially as a
second thrombin receptor (10), the Lys38/Thr39
cleavage site appears somewhat unusual for a thrombin substrate. Nonetheless, this may represent the alternative thrombin-responsive pathway described previously in multiple cells, including platelets (20, 35) and endothelial cells (20). In human platelets, PAR-1 is the
primary regulator of platelet aggregation to thrombin (15); and in both
platelets and endothelial cells, anti-PAR-1 antibodies abrogate the
immediate phase of intracytosolic calcium release, although a moderate
and sustained response remains evident (20). Although PAR-3 is clearly
expressed in both of these cells by mRNA and protein
determinations, the levels appear modest compared with those of PAR-1.
Interestingly, PAR-3 expression appears considerably higher in
megakaryocytes (10) and in the megakaryocyte-like cell line (HEL)
studied as our model system. Thus, we would speculate that activation
of human platelet PAR-3 by
-thrombin (or other unidentified
proteases) may be less important for physiological responses in these
mature cells but potentially more applicable for poorly defined
cellular responses (to proteases) that may regulate megakaryocytic (or
endothelial cell) development.
We thank Shirley Murray for assistance with
the processing of this manuscript, Dr. Barry Coller for assistance and
advice with antibody generation and for critically reviewing the
manuscript, and David Colflesh for assistance with the fluorescent
microscopy and image analysis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF050525.