From the
Activated thrombin receptors on human umbilical vein endothelial
cells rapidly undergo homologous desensitization, leaving the cells
unable to respond to thrombin. The present studies examine the fate of
activated thrombin receptors on endothelial cells and the mechanisms
that restore intact receptors to the cell surface. The results show
that: 1) at biologically relevant concentrations, thrombin rapidly
cleaves all of its receptors on the cell surface. 2) The cleaved
receptors are cleared from the cell surface in a two-phase process,
with 60% being internalized within 10 min, the remainder requiring
several hours. 3) The restoration of intact, thrombin-responsive
receptors on the cell surface initially occurs from an intracellular
pool of receptors in a process that is independent of protein
synthesis. 4) Recycling of cleaved receptors either does not occur on
endothelial cells or is masked by receptor clearance. 5) Subconfluent
endothelial cells re-express intact receptors on the cell surface at a
slower rate than confluent cells. 6) The agonist peptide, SFLLRN, also
causes receptor internalization, although at concentrations greater
than those required for receptor activation and desensitization. These
results are distinctly different from those observed with
megakaryoblastic cell lines, where >90% of the activated thrombin
receptors are internalized rapidly, up to 40% of the cleaved receptors
are recycled, and no intracellular pool of intact receptors has been
detected. Since the primary structure of the thrombin receptor is the
same in all the cell types studied, these results demonstrate that
there can be substantial differences between cell types in the
mechanisms involved in the clearance of activated receptors and the
re-expression on the cell surface of intact receptors capable of
responding to thrombin.
Thrombin is a potent agonist for a number of cells including
endothelial cells, platelets, vascular smooth muscle cells, and
fibroblasts. In all of these cells, thrombin elicits responses by
cleaving the N terminus of a high affinity G protein-coupled receptor
(1, 2) . To date, only a single subtype of the thrombin
receptor has been detected and cloning studies suggest that its primary
structure is identical in different cells within a given animal
species. Thrombin is thought to bind to the human form of the receptor
via its anion-binding exosite, cleaving the receptor between residues
Arg
Even before the sequence and
mechanism of activation of the thrombin receptor had been determined,
it had been noted in a variety of cell types that thrombin responses
are subject to homologous desensitization
(4, 6, 7, 8, 9) . In the
megakaryoblastic HEL and CHRF-288 cell lines, desensitization occurs
whether the receptors are activated proteolytically with thrombin or
non-proteolytically with an agonist peptide, such as SFLLRN, and
activation by either agonist prevents a subsequent response to the
other
(9, 10) . As with other G protein-coupled
receptors, desensitization of signaling through thrombin receptors is
thought to be due in part to receptor phosphorylation by a member of
the receptor kinase family, such as
This model of receptor
desensitization, internalization, and recycling is based upon studies
in megakaryoblastic cell lines. That it may not be universal is
suggested by studies with human thrombin receptors over-expressed in
Rat-1 fibroblasts, in which the loss of receptors from the cell surface
was incomplete
(14) . In the present studies, we have examined
the fate of activated thrombin receptors on early passage human
umbilical vein endothelial cells (HUVEC),
As has also been noted by other investigators
(4, 17) , the addition of thrombin to human umbilical
vein endothelial cells caused a rapid, but transient increase in the
cytosolic free Ca
Finally, to determine whether the newly restored
receptors were functional, endothelial cells that had been transiently
exposed to thrombin were re-stimulated with thrombin and changes in the
cytosolic free Ca
Unlike other G protein-coupled receptors, the activation of
thrombin receptors by thrombin is essentially a ``one time''
event since cleaved thrombin receptors are normally unable to respond
to thrombin a second time
(1, 9) . For a cell to recover
responsiveness to thrombin, new receptors must be expressed on the cell
surface. This process is presumably initiated by receptor activation.
In the present studies, we have examined the clearance and replacement
of activated thrombin receptors on human umbilical vein endothelial
cells. Endothelial cells were of particular interest for several
reasons. As with platelets, thrombin is a biologically important
agonist for endothelial cells, stimulating the synthesis and release of
vasoactive compounds such as prostaglandin I
The results that were obtained demonstrate
some notable differences between endothelial cells, the
megakaryoblastic cell lines, and platelets. In HEL and CHRF-288 cells,
receptor cleavage is followed by rapid internalization of essentially
all of the receptors and recycling of up to 40%, none of which can be
re-activated by thrombin. Recovery of thrombin-responsive receptors is
dependent upon protein synthesis and requires up to 16 h for
completion. On platelets, thrombin receptors are cleaved and
internalized, but not replaced.
In endothelial cells, two mechanisms of internalization appear to
coexist, one of which is rapid and becomes saturated when only 60% of
the receptors have been internalized, while the second is responsible
for the much slower clearance of the remaining receptors. Rapid partial
receptor clearance has also been observed in Rat-1 cells transfected
with epitope-tagged thrombin receptors
(11, 14, 18) . In HEL and CHRF-288 cells, a
substantial population of cleaved receptors recycles back to the cell
surface. The extremely rapid and essentially complete internalization
of activated thrombin receptors in these cells may be responsible in
part for the recycling of cleaved receptors because the very large
receptor load may overwhelm the sorting mechanisms in the endosomes, a
hypothesis that remains to be tested. A biological role for the
recycled cleaved receptors has not been identified. Since we never
observed an increase in the number of cleaved receptors on the
endothelial cell surface, we could not establish that thrombin receptor
recycling occurs in endothelial cells. However, it is theoretically
possible that the slower phase of receptor removal reflects the
difference in rate between continued removal and receptor recycling,
which would not be detected by the methods that were used.
One
factor that appears to affect the rate of thrombin receptor clearance
and recovery on HUVEC was cell density. The rate of recovery of intact
receptors on the cell surface was faster in confluent cultures when
compared to subconfluent cultures, as was the rate of clearance of the
cleaved receptors during the second, slower phase of receptor
internalization (Fig. 5). Endothelial cells normally exist in
vivo as a confluent, quiescent monolayer unless there is damage to
the vascular wall, caused either by local pathology or a therapeutic
intervention such as angioplasty. Based on our data obtained in
vitro, there may be a slower recovery of thrombin responsiveness
under conditions in which the endothelial lining of the vascular wall
is disrupted. Whether these differences actually occur in vivo and whether they apply to arterial, as well as venous, endothelial
cells will have to be determined.
A third issue is the extent to
which activated thrombin receptors on endothelial cells become
refractory to further stimulation. The refractoriness of activated
thrombin receptors to reactivation by thrombin is thought to be due to
two events: phosphorylation of sites in the cytoplasmic domains of the
receptor
(1, 11) and cleavage of the receptor N
terminus. Cleavage prevents thrombin from activating the receptor a
second time, but does not inhibit responses to SFLLRN. Phosphorylation
would be expected to prevent responses to both. When endothelial cells
were incubated with thrombin at concentrations sufficient to cleave all
of the cell surface receptors, they were unable to respond a second
time to thrombin, but showed a continued, although diminished,
responsiveness to SFLLRN. Conversely, when the cells were initially
activated by SFLLRN they showed a diminished response to both thrombin
and SFLLRN, but in order to see this response it was necessary to wash
out the peptide. Since SFLLRN does not inhibit receptor cleavage by
thrombin, several possible mechanisms might account for these
observations. The first possibility is that although thrombin may
cleave and activate all of its receptors, it may not desensitize all of
them, in which case those remaining on the cell surface can respond to
SFLLRN even when a cleaved N terminus precludes a second response to
thrombin. The second possibility is that all of the receptors have, in
fact, initially been desensitized, but some of the receptors remaining
on the cell surface have become resensitized, perhaps after
dephosphorylation. A third possibility, that the second response is due
to expression of new receptors from the internal pool, seems less
likely in view of the short time between stimuli and the failure of the
cells to respond a second time to thrombin after initially being
exposed to thrombin. What is less clear is the biological relevance of
the continued response since it isn't seen with further additions
of thrombin. Theoretically, either the tethered ligand itself or a
locally generated homologous peptide could cause receptor reactivation,
but whether this occurs in vivo remains to be demonstrated.
Finally, the endothelial cells were able to restore intact thrombin
receptors to the cell surface at a rate far faster than HEL or CHRF-288
cells. Following a transient exposure to thrombin, new receptors
appeared almost immediately and a nearly full complement of receptors
was restored within a few hours. Since this process occurred
independently of protein synthesis, it implies that endothelial cells
possess an intracellular pool of intact receptors that can rapidly
replace those cleaved by thrombin (Fig. 8). This pool appears to
contain at least as many receptors as are initially present on the cell
surface, allowing endothelial cells to quickly recover following
exposure to thrombin. In addition to our data, two sets of observations
from other investigators appear to support this conclusion. In the
study by Hein and co-workers
(18) , fibroblasts expressing
epitope-tagged human thrombin receptors were found to contain an
intracellular receptor pool, visible as immunoreactivity in the
perinuclear space that appears similar to observations we have made.
Corroborative studies with an endothelial cell line suggested that a
similar pool was also present in those cells as well. Functional
studies with the endothelial cell line were also presented in that
study and are consistent with our findings with human umbilical vein
endothelial cells. In the second set of studies, Horvat and Palade
(25) stained permeabilized endothelial cells with
receptor-directed antibodies and noted a large number of intracellular
thrombin receptors associated with a branching tubulovesicular network.
They concluded that most of the thrombin receptors in resting
endothelial cells are intracellular, rather than on the cell surface.
Of note, their localization of thrombin receptors on the branching
tubulovesicular network appears different from the perinuclear staining
observed by either Hein et al. (18) or by us. The
reason for this difference is currently unknown.
In conclusion,
these results demonstrate that there are substantial differences in the
regulation of thrombin receptors in different cells. Biologically, this
may make a great deal of sense. Platelets are activated by thrombin,
participate in the formation of the hemostatic plug, and then have no
further need (that has been identified) to be activated by thrombin.
Endothelial cells, on the other hand, do not typically undergo
irreversible changes in response to local thrombin generation. Given
their need to be able to synthesize and release vasoactive products, it
is reasonable that they would have a mechanism for rapidly restoring
thrombin responsiveness once the initial thrombin-generating event has
passed. Replacement of cleaved receptors with intact receptors from an
existing pool provides one mechanism for accomplishing this.
We thank Dr. A. Sue Menko for the use of the Nikon
microscope for the indirect immunofluorescence studies.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and Ser
and exposing a new N terminus
that serves as a tethered ligand
(1, 2) . Peptides
corresponding to at least the first five residues of the tethered
ligand sequence (SFLLR) can, when added to thrombin-responsive cells,
elicit many of the effects of thrombin. In the case of endothelial
cells, such effects include activation of phospholipases A
and C
(3, 4, 5) , and regulation of cAMP
formation.
(
)
-adrenergic receptor kinase 2,
a kinase that has been shown to phosphorylate thrombin receptors when
co-expressed in Xenopus oocytes
(11) . In addition,
thrombin receptors that have been activated by thrombin are left with a
cleaved N terminus which, under most circumstances, precludes a second
response to thrombin
(10, 12) . Activated thrombin
receptors are also subject to sequestration into intracellular
compartments, irrespective of whether they were activated by thrombin
or an agonist peptide. In the case of HEL and CHRF-288 cells, >90%
of the thrombin receptors are cleared from the cell surface via coated
pits within 5 min of activation
(13) . Once internalized, most
of the receptors are routed through endosomes to lysosomes, ultimately
to be degraded. However, a substantial population (25-40%)
returns to the cell surface. These ``recycled'' receptors are
no longer desensitized, suggesting that they have been dephosphorylated
by endosomal phosphatases, although this has not been demonstrated.
Lacking an intact N terminus, they can be activated by SFLLRN, but not
by thrombin
(13) . Full recovery of the thrombin response
requires the synthesis and expression on the cell surface of new
receptors over a period of many hours.
(
)
non-transformed cells that are exposed to thrombin under
normal physiological conditions. The results demonstrate that all of
the thrombin receptors on endothelial cells are cleaved upon thrombin
addition. However, although many of the cleaved receptors are
internalized rapidly, the remainder are internalized slowly, in sharp
contrast to the megakaryoblastic cell lines. Furthermore, the
reappearance of thrombin-responsive receptors on the surface of
endothelial cells is detectable within 30 min and is due to the
presence within these cells of an intracellular reserve of intact
receptors that is not present in the megakaryoblastic cell lines. The
rate of receptor mobilization from this reserve is affected by the
proliferative state of the endothelial cells, occurring more rapidly
when the cells are confluent than when they are subconfluent.
Materials
The peptide-directed IgGmonoclonal antibodies SPAN12, WEDE15, and ATAP2 are directed
against sequences within the N terminus of the human thrombin receptor
(10) . Antibody SPAN12 recognizes an epitope that is lost when
thrombin cleaves the receptor. Antibodies WEDE15 and ATAP2 recognize
epitopes that are retained following receptor cleavage, allowing these
antibodies to bind to both intact and cleaved receptors
(13) .
Monoclonal antibody SSA6 is directed against human glycoprotein IIIa
(integrin
; Ref. 15). Antibody EH1, used as a
isotype-matched negative control, is reactive with the HIV-1 Nef
protein
(13) . Highly purified human
-thrombin (
3000
units/mg) was provided by Dr. J. Fenton (New York State Department of
Health, Albany, NY). Hirudin was obtained from Sigma or Calbiochem.
Culture of Endothelial Cells
Endothelial cells
were isolated from human umbilical cord veins by the procedure of Jaffe
et al. (16) and were cultured on fibronectin-coated
dishes in complete medium, Medium 199 containing 10% fetal calf serum
(Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin, 0.6
mM glutamine, 12 units/ml heparin, and 200 µg/ml crude
endothelial cell growth factor at 37 °C under 5% CO.
The cells were routinely passaged with trypsin-EDTA and used for
experiments at passages 1-6. Since trypsin can cleave thrombin
receptors
(1) , exposure of the cells to trypsin was avoided for
at least 2 days prior to the experiments.
Cytosolic Calcium Measurements
Cells were washed
twice with phosphate-buffered saline, then loaded with 5
µM Fura-2/AM (Molecular Probes) in phenol red-free RPMI
1640 medium for 60 min at 37 °C. The endothelial cells were
released from culture dishes by incubation at 37 °C for 5-15
min in phosphate-buffered saline containing 5 mM EGTA and 1
mM EDTA. The cells were resuspended at 1 10
cells/ml in phenol red-free RPMI 1640 medium and allowed to
equilibrate 30 min, washed, and resuspended in fresh medium at 2
10
cells/ml. Fluorescence was detected in a
SLM/Aminco AB2 spectrophotometer and approximate values for
[Ca
]
were calculated
using an assumed k
of 224 nM.
Flow Cytometry
Cell suspensions (2
10
cells/ml in RPMI 1640 medium) were prepared as described
above. Samples were treated with agonists at 37 °C. Cells (2
10
) were incubated with undiluted hybridoma
supernatant or affinity purified monoclonal antibodies (10-20
µg/ml) for 30 min at 4 °C, washed with staining buffer
(phosphate-buffered saline, 0.02% sodium azide, 0.1% bovine serum
albumin, pH 7.4), then incubated with a 1:40 dilution of fluorescein
isothiocyanate-labeled goat anti-mouse IgG (TAGO) for an additional 30
min at 4 °C. Cells were washed and resuspended in staining buffer.
Antibody binding was analyzed on a FACScan flow cytometer (Becton
Dickinson). When the monoclonal antibodies directed against the
thrombin receptor were preincubated with the appropriate immunizing
peptide, binding to HUVEC decreased to the level of the negative
control as measured by flow cytometry. Preincubation of the antibodies
with an irrelevant peptide had no effect upon their binding. For
thrombin receptor recovery experiments, cell monolayers were treated
with thrombin in RPMI, then allowed to recover for different periods of
time in complete medium containing hirudin. Cell suspensions were then
prepared, incubated with antibodies, and processed for flow cytometry
as described above.
Immunofluorescence
The endothelial cells were
cultured on fibronectin-coated 35-mm dishes. For surface staining of
intact cells, cell monolayers were washed in Medium 199, incubated in
the presence or absence of agonist 10 min at 37 °C, placed on ice,
and washed with ice-cold staining buffer. Cells were incubated with
undiluted hybridoma supernatant or affinity purified monoclonal
antibodies (10-20 µg/ml) for 30 min at 4 °C, washed with
staining buffer, then incubated with 1:100 dilution rhodamine-labeled
donkey anti-mouse IgG (Jackson Immunoresearch Laboratories) for 30 min
at 4 °C. Cell monolayers were washed with staining buffer, fixed in
1% formalin (Polysciences, Inc.) for 10 min, washed with staining
buffer, then distilled water, and coverslips mounted with Elvanol.
concentration
(Fig. 1 A). When maximally-effective thrombin
concentrations were used, the initial increase in the intracellular
calcium concentration subsided after 3-4 min, approaching a
plateau somewhat greater than baseline. A second addition of thrombin
after the initial spike produced no response. However, the addition of
SFLLRN after the initial thrombin spike resulted in a second transient
increase in Ca
whose magnitude was
50% of the
initial response, even when the initial thrombin concentration was more
than sufficient to cleave all of the receptors on the cell surface (see
below). Fig. 1 B shows the response of endothelial cells
to SFLLRN. Like thrombin, SFLLRN caused a transient increase in the
cytosolic Ca
concentration and a second addition of
SFLLRN had little effect. Addition of thrombin after SFLLRN also had no
effect unless the cells were washed to removed the agonist peptide, in
which case a diminished second response to both thrombin
(Fig. 1 C) and SFLLRN (not shown) was observed. The
magnitude of this second response was similar to that seen when the
cells were initially exposed to thrombin and then restimulated with
SFLLRN (Fig. 1 A).
Figure 1:
Desensitization of the Caresponse in endothelial cells. Early passage human umbilical vein
endothelial cells were loaded with Fura-2/AM, incubated in the absence
( A and B) or presence of 100 µM SFLLRN
( C), washed briefly, and then stimulated with thrombin (5
units/ml) or SFLLRN (100 µM), as indicated. These results
are representative of two studies.
These results support observations
made in other cell systems that thrombin receptor desensitization
follows receptor activation by either thrombin or SFLLRN and,
therefore, does not require receptor cleavage. However, the capability,
although attenuated, of thrombin-activated cells to respond to SFLLRN
and SFLLRN-activated cells to respond to both SFLLRN and thrombin after
washing suggests that some of the receptors were either never
desensitized or were rapidly resensitized after an initial
desensitization. This result is consistent with studies of transfected
Rat-1 fibroblasts, but is distinctly different from the results
obtained with the megakaryoblastic cell lines where the
cross-desensitization between thrombin and SFLLRN is essentially
complete
(10, 13, 14) .
Receptor Cleavage and Internalization
Three monoclonal
antibodies directed at defined epitopes within the thrombin receptor N
terminus (Fig. 2) were used to discriminate intact from cleaved
receptors and to examine the fate of activated receptors on endothelial
cells in greater detail. Antibody SPAN12, which was prepared against a
peptide whose sequence spans the receptor cleavage site, binds only to
intact receptors. Antibody ATAP2, which was prepared against a peptide
analogous to the tethered ligand domain, binds to cleaved as well as
intact receptors, as does antibody WEDE15, whose epitope includes the
portion of the receptor that interacts with thrombin's
anion-binding exosite. Flow cytometry was used to detect antibody
binding. In the studies shown in Fig. 3 A, endothelial
cells were incubated with different concentrations of thrombin for 10
min, after which the thrombin was inactivated with an excess of
hirudin. Binding of the cleavage-sensitive antibody SPAN12 decreased as
the thrombin concentration increased, with little binding detected at
thrombin concentrations 0.3 units/ml. Exposure of the endothelial
cells to increasing concentrations of thrombin also caused diminished
binding of the cleavage-insensitive antibodies, ATAP2 and WEDE15.
However, in contrast to the results with antibody SPAN12, the binding
of antibodies WEDE15 (Fig. 3 A) and ATAP2 (not shown)
plateaued at 40-50% of control values at thrombin concentrations
1 unit/ml. The loss of SPAN12 binding sites was complete within 3
min, while the decrease in WEDE15 binding sites was complete after 10
min (Fig. 3 B). These results suggest that thrombin
rapidly cleaves essentially all of its receptors on the surface of
endothelial cells, after which approximately half of the cleaved
receptors are internalized.
Figure 2:
N terminus of the human thrombin receptor.
The approximate location of the epitope for each of the monoclonal
antibodies used in the present studies is indicated by the antibody
designations. The solid bars indicate the peptides used for
the production of the corresponding antibody. Antibody SPAN12 binds
only to intact receptors. Antibodies ATAP2 and WEDE15 bind to both
intact and cleaved receptors.
Figure 3:
Loss of binding sites for thrombin
receptor antibodies following incubation with thrombin. A, the
binding of antibodies WEDE15 and SPAN12 to HUVEC was measured by flow
cytometry after incubating the cells for 10 min with thrombin and then
adding 5-fold excess hirudin. The results shown are the mean ±
S.E. of four studies and are expressed as percentage of the mean
fluorescence obtained in the absence of thrombin. B, the
endothelial cells were incubated with thrombin (1 unit/ml) for up to 30
min, after which hirudin (5 units/ml) was added. The results shown are
the mean ± S.E. of three studies.
Similar studies were performed with
SFLLRN to see whether the loss of receptors requires only activation,
as opposed to proteolysis. The results are shown in Fig. 4. Like
thrombin, SFLLRN caused a time- and concentration-dependent loss of
antibody binding sites. As would be expected given the peptide's
ability to activate the receptor without cleaving it, there was a
comparable loss of binding sites for antibodies WEDE15 (Fig. 4)
and SPAN12 (not shown). Both SFLLRN and thrombin caused a decrease in
WEDE15 binding over a similar time course, but only at concentrations
of SFLLRN 2 mM were the majority of the thrombin receptors
lost from the cell surface. In contrast, the Ca
response to SFLLRN was maximal at
100 µM SFLLRN
(not shown). This suggests that SFLLRN, in contrast to thrombin, is
less effective at causing receptor internalization than it is at
causing receptor activation.
Figure 4:
Loss of binding sites for thrombin
receptor antibodies following incubation with SFLLRN. A, the
binding of antibody WEDE15 was measured after incubating endothelial
cells with SFLLRN for 15 min. The results shown are the mean ±
S.E. of three studies. B, the time course of the loss of
WEDE15 binding sites following the addition of SFLLRN. The results
shown are the mean ± S.E. of five
studies.
Restoration of Thrombin Receptors on the Cell
Surface
In order to study the recovery of thrombin receptors on
the cell surface following activation, endothelial cells were incubated
with 1 unit/ml thrombin for 10 min, sufficient to cleave all of the
receptors, after which the medium was removed and replaced with fresh
medium containing excess hirudin. At intervals up to 5 h, the total
number of receptors on the cell surface was determined with antibody
WEDE15 and the number of intact receptors was determined with antibody
SPAN12. The difference between the total and intact receptor numbers
reflects the number of cleaved receptors remaining on the cell surface
at any given time. The studies shown in Fig. 5 A were
performed with confluent monolayers of endothelial cells. At the end of
the 10-min incubation with thrombin, SPAN12 binding was 10% of control
and WEDE15 binding was 40% of control. By 30 min there was a detectable
increase in the number of SPAN12 binding sites, which rose to 60% of
control by 2 h and 90% of control by 5 h. This process was unaffected
by the protein synthesis inhibitor, cycloheximide (not shown). Binding
sites for antibody WEDE15 also increased from 40% relative to control
immediately after thrombin exposure to 90% of control by 5 h. As new
receptors appeared on the cell surface, the number of cleaved receptors
gradually decreased and was undetectable by 5 h
(Fig. 5 C).
Figure 5:
Recovery of antibody binding sites after
exposure to thrombin. Confluent ( A) or subconfluent
( B) endothelial cells were incubated with thrombin for 10 min,
after which the protease was removed. The data show the recovery of
binding sites for antibodies WEDE15 and SPAN12 over the next 5 h
expressed as a percentage of antibody binding to cells not exposed to
thrombin. The results are the mean ± S.E. of four ( A)
and three ( B) studies. Part C shows the number of
cleaved receptors remaining on the cell surface calculated as the
difference between the results obtained with antibodies WEDE15 (total
receptors) and SPAN12 (intact receptors).
Interestingly, when these studies were
repeated with subconfluent endothelial cells (70-80% coverage),
two notable differences were observed. First, although the kinetics of
cleavage and initial clearance of receptors from the cell surface
appeared unaffected by cell density, the reappearance of intact
receptors on the cell surface, as determined by antibody SPAN12
binding, occurred more slowly on the subconfluent cells
(Fig. 5 B). Also, in contrast to the confluent cells, the
number of cleaved receptors detected on the cell surface after the
10-min thrombin incubation was constant over the 5-h period of
observation (Fig. 5 C). This suggests that subconfluent
cells are either less capable of clearing the residual cleaved
receptors remaining on the cell surface or can recycle cleaved
receptors at a rate equivalent to receptor internalization during the
second phase of receptor clearance. This also suggests that either the
proliferative status of the endothelial cells or the presence of
uniform cell contacts within an intact monolayer can affect the
movement of cleaved receptors to and from the cell surface after
thrombin treatment.
concentration were measured. The
results are shown in Fig. 6, expressed as a percentage of the
initial response to thrombin. The recovery of a thrombin response could
be detected as early as 30 min after exposure to thrombin. By 2 h the
magnitude of the Ca
response reached approximately
60% of control.
Figure 6:
Recovery of Caresponse
after exposure to thrombin. Endothelial cells loaded with Fura-2/AM
were exposed to thrombin (1 unit/ml) for 10 min, hirudin (5 units/ml)
was added, and the incubation was continued. The cells were
re-stimulated with thrombin (6 units/ml) at the time points indicated.
The results shown are the mean ± S.E. from three studies
expressed as a fraction of the Ca
response obtained
in cells not exposed to thrombin.
Receptor Localization
Fluorescence microscopy was
used to complement the results obtained by flow cytometry and examine
the distribution of thrombin receptors in resting and activated
endothelial cells. Intact unstimulated cells exhibited a uniform
punctate staining with antibodies SPAN12 and ATAP2 (Fig. 7, A and D). Exposure to thrombin for 10 min abolished the
binding of SPAN12 (Fig. 7 E). Exposure to thrombin also
caused a decrease in the binding of antibody ATAP2
(Fig. 7 B). Note that this figure tends to underrepresent
the amount of cleaved receptor remaining on the cell surface following
exposure to thrombin. A more quantitative measure is provided by the
flow cytometry data presented in Fig. 3. Two h after the thrombin
was removed there was substantial recovery in the binding of both
antibodies (Fig. 7, C and F). Binding of a
control antibody SSA6, directed against the integrin,
glycoprotein IIIa, was unchanged following exposure of the endothelial
cells to thrombin (Fig. 7, G and H) and there
was negligible staining by secondary antibody alone
(Fig. 7 I). In preliminary studies with permeabilized
cells, staining of small intracellular vesicles with antibody ATAP2,
but not with antibody SPAN12, was seen 10 min after the addition of
thrombin (not shown). The presence of cleaved thrombin receptors in
vesicles has been demonstrated in thrombin-treated HEL and CHRF-288
cells as well as in transfected Rat-1 cells and an endothelial cell
line
(10, 13, 18) . Thrombin and transferrin
receptor colocalization studies performed with the megakaryoblastic
cell lines have shown the intracellular structures to be endosomes, and
this is presumably also the case in endothelial cells
(13) .
Figure 7:
Immunofluorescent detection of the
thrombin receptor on intact, naive, or thrombin-treated HUVEC.
Panels A-C show HUVEC that were stained with antibody ATAP2:
control ( A), after 10 min incubation with thrombin
( B) or 2 h after a 10-min exposure to thrombin ( C).
Panels D-F show cells prepared the same way, but stained with
antibody SPAN12. Panels G and H show control and
thrombin-treated endothelial cells, respectively, that were stained
with antibody SSA6 directed against the chain of the endothelial
cell vitronectin receptor (
).
Panel I shows the result obtained in the absence of the
primary antibody. The results shown are representative of seven such
studies.
and NO
(4, 19, 20) . However, in contrast to platelets,
endothelial cells may be exposed to thrombin repeatedly during their
life cycle. Also, until recently most of the information available
about the trafficking of thrombin receptors has been obtained with
megakaryoblastic cell lines or with epitope-tagged thrombin receptors
expressed in cell lines. HUVEC provided an opportunity to examine the
behavior of endogenous thrombin receptors in early passage,
non-transformed cells.
(
)
In contrast, in
this study we observed that cleaved thrombin receptors on endothelial
cells are cleared in a biphasic manner, the first phase involving the
rapid clearance of 50-60% of the receptors within 10 min, the
second resulting in the removal of the remaining cleaved receptors over
a 5-h period. The reappearance of intact receptors on the cell surface
was detectable within 30 min, reached 60% by 2 h and 90% by 5 h, and
was accompanied by a recovery of thrombin responsiveness. This process
was independent of protein synthesis and appears to be due to an
intracellular pool of intact receptors that can be mobilized to the
cell surface (Fig. 8).
Figure 8:
A
model for thrombin receptor clearance and replacement on human
umbilical vein endothelial cells. In endothelial cells, cleaved
thrombin receptors are cleared from the cell surface in a two-phase
process that is complete within a few hours. Replacement of the cleaved
receptors with intact receptors initially occurs from an intracellular
reserve of receptors. Recycling of cleaved receptors has not been
resolved, but some of the cleaved receptors that are not initially
internalized appear to retain or recover the ability to be activated a
second time by SFLLRN.
These observations raise several
issues. First, despite the marked differences in events following
thrombin receptor activation in endothelial cells, platelets, and HEL
cells, the primary structure and activation mechanism of the thrombin
receptor is apparently the same in all the cells
(1, 13, 21) . This implies that cell-specific
factors unrelated to the thrombin receptor sequence dictate the manner
in which cleaved thrombin receptors are cleared and replaced, although
what these factors are remains to be determined. Phosphorylation by
members of the G protein-coupled receptor kinase family, which plays an
important role in receptor desensitization, does not appear to be
required for G protein-coupled receptor internalization
(22) .
Recent studies have implicated a conserved P XXY sequence near
the beginning of the cytoplasmic tail of many G protein-coupled
receptors in receptor internalization and have shown that swapping a
proline-rich domain in the third cytoplasmic loop of the
- and
-adrenergic receptors results
in a switch in their patterns of internalization
(23, 24) . At present, little is known about the
structural features involved in thrombin receptor internalization
beyond the presence of a PLIY sequence at the end of the last
transmembrane domain, and nothing is known about proteins that might
directly interact with the receptor causing it to be internalized.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.