From the Cardiovascular Research Institute and the
Departments of ¶ Medicine and
Cellular and Molecular
Pharmacology, University of California,
San Francisco, California 94143
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ABSTRACT |
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The G protein-coupled receptor (GPCR) for
thrombin, protease-activated receptor-1 (PAR1), is activated when
thrombin cleaves its amino-terminal exodomain. The irreversibility of
this proteolytic mechanism raises the question of how desensitization
and resensitization are accomplished for thrombin signaling. PAR1 is
phosphorylated, uncoupled from signaling, and internalized after
activation like classic GPCRs. However, unlike classic GPCRs, which
internalize and recycle, activated PAR1 is sorted to lysosomes. To
identify the signals that specify the distinct sorting of PAR1, we
constructed chimeras between PAR1 and the substance P receptor.
Wild-type substance P receptor internalized and recycled after
activation; PAR1 bearing the cytoplasmic tail of the substance P
receptor (P/S) behaved similarly. By contrast, wild-type PAR1 and a
substance P receptor bearing the cytoplasmic tail of PAR1 (S/P) sorted
to lysosomes after activation. Consistent with these observations, PAR1
and the S/P chimera were effectively down-regulated by their respective
agonists as assessed by both receptor protein levels and signaling.
Substance P receptor and the P/S chimera showed little down-regulation.
These data suggest that the cytoplasmic tails of PAR1 and substance P
receptor specify their distinct intracellular sorting patterns after
activation and internalization. Moreover, by altering the trafficking
fates of PAR1 and substance P receptor, one can dictate the efficiency
with which a cell maintains responsiveness to PAR1 or substance P
receptor agonists over time.
Thrombin, a multifunctional serine protease generated at sites of
vascular injury, regulates a variety of cellular processes important in
cardiovascular biology and disease (1). The actions of thrombin on
human platelets, endothelial cells and fibroblasts are mediated at
least in part by a family of G protein-coupled protease-activated
receptors (2-5). Protease-activated receptor-1 (PAR1)1 is prototypical of
this family (6-8). PAR1 is activated by an irreversible proteolytic
mechanism in which thrombin binds to and cleaves the amino-terminal
exodomain of the receptor. Receptor cleavage results in the generation
of a new amino terminus that functions as a tethered ligand by binding
to the body of the receptor to cause transmembrane signaling (2, 9,
10). A soluble peptide with the sequence SFLLRN representing the first
six amino acids carboxyl-terminal to the cleavage site (P1'-P6')
mimics the tethered ligand of PAR1 and acts as a PAR1 agonist. The
irreversible proteolytic mechanism of PAR1 activation raises the
questions of how this receptor is shut off and how cells maintain the
ability to respond to thrombin over time.
The Like the Antibodies and Reagents--
Monoclonal anti-FLAG antibodies, M1
and M2, were purchased from Eastman Kodak. Rabbit polyclonal anti-FLAG
antibody was obtained from Santa Cruz Biotechnology. Polyclonal 1809 antibody was raised to a peptide representing the hirudin-like sequence
in the amino-terminal exodomain of PAR1 (6). The GM10 monoclonal
antibody against the lysosomal membrane glycoprotein (lgp120) was
generously provided by John Hutton (University of Colorado, Boulder,
CO) and Samuel A. Green (University of Virginia, Charlottesville, VA)
(25). Texas Red-conjugated goat anti-mouse antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody were obtained from
Molecular Probes. The fluorescein isothiocyanate-conjugated goat
anti-mouse antibody was from Life Technologies, Inc. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were
purchased from Bio-Rad and 125I sheep anti-mouse antibody
from Amersham International, UK.
The PAR1 agonist peptide SFLLRN was synthesized as the carboxyl
amide and purified by reverse phase high pressure liquid
chromatography. Substance P peptide was purchased from Phoenix Pharmaceuticals.
cDNAs and Cell Lines--
A PAR1 cDNA containing a
prolactin signal sequence followed by a FLAG epitope (DYKDDDD) was used
for generating mutants (17). A SPR cDNA with a prolactin signal
sequence and FLAG epitope identical to that of PAR1 was generated as
follows. NcoI and SalI sites were introduced into
a wild-type rat SPR cDNA (gift of Nigel Bunnett, University of
California, San Francisco). The NcoI site was positioned such that its ATG sequence coincided with the native SPR start codon
and the SalI site was positioned coincident with SPR codons ATGGAT (nucleotide numbers 556-661 in Ref. 26). A 114-base pair NcoI/SalI fragment encoding the amino acid
sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSDYKDDDDVD was then subcloned
into these sites. To generate chimeric receptors, a SacI
site was inserted in the epitope-tagged PAR1 and SPR cDNAs just 3'
to the sequence encoding the putative palmitoylation sites in these
proteins (ILCCKESS for PAR1 and FRCCPFISA for
SPR, where the location corresponding to the SacI sites are
underlined). These SacI sites were then used to exchange
cDNA fragments encoding the carboxyl tails of PAR1 and SPR (Fig.
1B). Mutations in all constructs were confirmed by dideoxy
sequencing. cDNAs encoding wild-type and chimeric receptors were
subcloned into the mammalian expression vector pBJ1 (provided by Mark
Davis, Stanford University, Stanford, CA) for transfection into cells.
Rat1 fibroblasts were cotransfected with a plasmid encoding a neomycin
resistance gene and stable transfectants were selected in 800 µg/ml
geneticin and screened by surface antibody binding (27). Cell lines
were maintained in Dulbecco's modified Eagle's media (DMEM)
supplemented with 5% bovine calf serum, 100 µg/ml streptomycin, 100 units/ml penicillin, and 800 µg/ml geneticin (Life Technologies,
Inc.).
Immunoblotting--
Following various treatments, cells were
rinsed once with ice-cold phosphate-buffered saline (PBS) and lysed in
2× SDS-gel loading buffer (100 mM Tris-HCl, pH 6.8, 4%
SDS, 0.2% bromphenol blue, and 20% glycerol). Lysates were sheared by
passage through a 28.5-gauge syringe needle, resolved by
electrophoresis on a SDS-9% polyacrylamide gel, and transferred to
polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked
with 5% nonfat dry milk diluted in 50 mM Tris-HCl, pH 7.4, 15 mM NaCl, 0.1% Tween 20 (wash buffer) and then incubated
overnight at 4 °C with 3 µg/ml M1 anti-FLAG antibody diluted in
the same blocking solution. Membranes were washed, incubated with a
horseradish peroxidase-conjugated goat anti-mouse antibody (1:5000) for
1 h at 25 °C, washed again, and then developed using Enhanced
Chemiluminescense-ECL (Amersham Pharmacia Biotech) according to the
manufacturer's instructions.
To determine receptor half-lives, lysates of cells incubated for
various times in the presence or absence of agonist were analyzed by
immunoblot in parallel with various equivalents of time 0 samples (100, 80, 40, 20, and 10%). Membranes were analyzed as above but 2 µCi/ml
125I sheep anti-mouse antibody was used in place of
horseradish peroxidase-conjugated secondary antibody. Radioactivity
associated with receptor bands was quantitated using the Molecular
Dynamics Storm imaging system. Standard curves of the time 0 samples
were fitted using Microsoft Excel 5.0 regression function, and
R values ranged from 0.98 to 1.00.
Receptor Phosphorylation--
Receptor phosphorylation was
examined using a modification of a previously described procedure (18).
Rat1 fibroblasts plated in 6-well dishes (Falcon) were labeled with 250 µCi/ml [32P]orthophosphate (NEN Life Science Products)
in phosphate-free DMEM containing 1 mg/ml bovine serum albumin for
3 h at 37 °C. Cells were then washed, incubated with agonists,
and lysed in 1% Triton X-100 in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 50 mM NaF,
10 mM sodium pyrophosphate, 200 µM sodium
orthovanadate, and protease inhibitors (18). 1809 antibody was used to
immunoprecipitate PAR1 and P/S chimera, and the M2 anti-FLAG antibody
was used to immunoprecipitate SPR and the S/P chimera.
Immunoprecipitates were resuspended in 2× SDS gel loading buffer
containing 6 M urea, resolved on a SDS-9% polyacrylamide
gel, and analyzed by autoradiography.
Microscopy--
Stably transfected Rat1 cells were plated on
glass coverslips (22 × 22 mm) and cultured overnight in normal
DMEM. To follow lysosomal sorting of cell surface receptors, cells were
first incubated with primary antibodies diluted in DMEM containing 1 mg/ml bovine serum albumin and 10 mM HEPES, pH 7.4, for
1 h at 4 °C. Rabbit polyclonal antibody 1809 (1:200) was used
for immunostaining of PAR1 and P/S chimera, and the rabbit polyclonal
anti-FLAG antibody (1:100) was used for staining SPR and S/P chimera.
Following agonist treatment at 37 °C, cells were fixed for 1 h
at 4 °C with 1% paraformaldehyde in PBS and then permeabilized in
100% methanol at
Immunofluorescence microscopy was used to follow internalization and
recovery of cell surface receptors. Transfected Rat1 fibroblasts were
plated on glass coverslips (22 × 22 mm) and treated as described
below for cell surface ELISA. Following fixation with paraformaldehyde,
cells were incubated with fluorescein-conjugated goat anti-mouse
secondary antibody for 1 h at 25 °C. Cells were then washed
four times with PBS and once with SlowFade equilibration buffer, and then one drop of SlowFade reagent was added to
each coverslip before mounting. Images were acquired using a Nikon Microphot-FXA fluorescence microscope fitted with a PlanApo 100× oil
objective (Nikon Corp.).
Cell Surface ELISA--
To follow the cohort of cell surface
receptors, transfected Rat1 fibroblasts plated in 24-well culture
dishes (Falcon) were incubated with 3 µg/ml M1 anti-FLAG antibody
diluted in DMEM/bovine serum albumin/HEPES for 1 h at 4 °C.
Cells were washed, warmed to 37 °C, and then exposed to agonists in
media containing 10 µM cycloheximide. This concentration
of cycloheximide was previously shown to inhibit new receptor synthesis
in transfected Rat1 cells (19). Following various treatments, cells
were fixed in 4% paraformaldehyde for 5 min at 4 °C and then washed
twice with PBS. Cells were then incubated with horseradish
peroxidase-conjugated goat anti-mouse antibody (1:1000) for 1 h at
25 °C, washed three times in PBS, and incubated with the horseradish
peroxidase substrate 1-Step ABTS (2, 2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid) (Pierce). After
10-15 min, an aliquot was removed, and the optical density was read at
405 nm using a Molecular Devices microplate spectrophotometer.
Phosphoinositide Hydrolysis--
Cells plated in 12-well dishes
(Falcon) were labeled overnight with 2 µCi/ml
[3H]myo-inositol in DMEM containing 1 mg/ml bovine serum
albumin. Cells were washed and treated as described in the legend to
Fig. 6, and the formation of inositol phosphates was assayed as
reported previously (28).
Degradation of Wild-type and Chimeric Receptors--
To identify
the domain(s) that specify the distinct intracellular sorting fates of
activated PAR1 and SPR (Fig.
1A), we made chimeras in which
the C-tails of these receptors were exchanged carboxyl to their
putative palmitoylation sites. PAR1 bearing the SPR C-tail is
designated "P/S chimera" and SPR bearing the PAR1 C-tail is
designated "S/P chimera" (Fig. 1B). Both wild-type and
chimeric receptors displayed the same FLAG epitope at their extracellular amino termini and were stably expressed in Rat1 fibroblasts.
To determine whether wild-type or chimeric receptors were sorted to a
degradative pathway upon activation, we incubated receptor-expressing cell lines in the presence or absence of agonist for 90 min and then
assessed the amount of receptor protein in cell lysates by immunoblot
using anti-FLAG antibodies. Immunoblot of lysates from untreated
PAR1-expressing cells revealed one major
transfection-dependent band migrating at 68 kDa (Fig.
2A). Incubation with PAR1
agonist peptide SFLLRN caused a striking decrease in the intensity of this band; this loss was even more evident when new receptor synthesis was blocked with cycloheximide (Fig. 2A). Immunoblot of
lysates prepared from SPR-expressing cells showed a
transfection-dependent band migrating at ~60 kDa.
Exposure of cells to substance P caused a decrease in mobility of this
band, probably due to receptor phosphorylation (Fig. 2F),
but had no significant effect on the level of receptor protein even
when cycloheximide was included (Fig. 2B). Strikingly, PAR1
bearing the SPR C-tail (P/S chimera) behaved very much like wild-type
SPR in this assay. The PAR1 agonist peptide SFLLRN caused a decrease in
the mobility of the P/S chimera but little or no loss of P/S receptor
protein (Fig. 2C). Immunoblot of lysates prepared from cells
expressing a SPR bearing the C-tail of PAR1 (S/P chimera) revealed
three transfection-dependent bands (Fig. 2D).
Exposure of S/P expressing cells to trypsin at 4 °C caused a
decrease in the intensity of the intermediate ~50-kDa band but not
the other bands (data not shown), suggesting that only the 50-kDa form
of the S/P chimera was expressed on the cell surface and cleaved by
extracellular protease. Moreover, only the 50-kDa form was
phosphorylated upon exposing cells to substance P (Fig. 2H).
These data are consistent with the hypothesis that the 50-kDa band
represents a properly processed and activatable form of this receptor
chimera. Interestingly, the 50-kDa band was the only form markedly
reduced in cells incubated with substance P (Fig. 2D),
consistent with the notion that it undergoes
agonist-dependent sorting to a degradation pathway (see
below).
The structural basis for misfolding or improper processing of the other
species of S/P protein is unknown. Of note, when analogous chimeras
were made between PAR1 and Phosphorylation of Wild-type and Chimeric Receptors--
PAR1 was
phosphorylated within 3 min of exposure to agonist peptide SFLLRN, but
no phosphorylated receptor was detected in PAR1-expressing cells after
prolonged exposure to agonist (Fig. 2E). Only the middle
band representing the S/P chimera showed phosphorylation upon addition
of ligand, again consistent with this band representing functional
chimeric receptor expressed at the cell surface (Fig. 2H).
As with wild-type PAR1, little or no detectable phosphorylated S/P
chimera was detected after 90 min of exposure to agonist. These
findings are consistent with the nearly complete degradation of PAR1
after 90 min of agonist treatment (Fig. 2A). Wild-type SPR
was also rapidly phosphorylated upon exposure to agonist, but in
contrast to PAR1, phosphorylated SPR was still detected after prolonged
exposure of cells to substance P (Fig. 2F). SPR
phosphorylation was reversible within 30 min of agonist withdrawal
(data not shown), and new receptor synthesis was blocked in these
studies, thus detection of phosphorylated SPR after 90 min of constant
exposure to agonist is consistent with ongoing activation and
phosphorylation of recycled receptors. The P/S chimera behaved like SPR
in this regard, showing continued phosphorylation in the presence of
agonist peptide SFLLRN and reversible phosphorylation upon removal of
peptide agonist (Fig. 2G and data not shown).
Sorting of Wild-type and Chimeric Receptors to Lysosomes--
We
next examined the effect of lysosomal inhibitors on agonist-induced
changes in the half-lives of wild-type and chimeric receptors in these
cell lines as described under "Experimental Procedures." Incubation
with agonist decreased the half-life of wild-type PAR1 by a remarkable
18-fold (Table I). Similarly, the S/P
chimera 50-kDa band showed a marked ~7-fold decrease in half-life in
the presence of agonist. By contrast, wild-type SPR and P/S chimera
showed only modest decreases in half-life of ~0.5- and ~2-fold,
respectively, upon exposure to agonist. To test whether agonist-promoted degradation of PAR1 and the S/P chimera required active lysosomal hydrolases, we incubated cells with chloroquine or
NH4Cl and then assayed the amount of receptor protein
remaining after incubation in the presence or absence of agonist.
Chloroquine or NH4Cl did not block internalization of PAR1
or SPR (data not shown). However, the striking agonist-induced
decreases in cellular PAR1 and S/P chimera protein levels were
substantially attenuated by these lysosomal hydrolase inhibitors (Table
I). These data are consistent with the hypothesis that PAR1 and the
50-kDa form of the S/P chimera are degraded in lysosomes after
activation and internalization.
To directly test whether the activated receptors and receptor chimeras
sorted to lysosomes, we used confocal microscopy to determine whether
these receptors co-localized with a lysosomal marker in an
agonist-dependent manner. Cells were incubated in the
presence or absence of agonist for 30 min, fixed, immunostained for
receptor protein (Fig. 3,
green) and the lysosomal integral membrane protein lgp120
(Fig. 3, red), and then imaged by confocal microscopy. In
the absence of agonist, little co-localization (Fig. 3,
yellow) of receptor and lgp120 was seen in cells expressing either wild-type or chimeric receptors. After incubation with agonist,
co-localization (Fig. 3, yellow) of both PAR1 and the S/P
chimera with lgp120 was easily detected (Fig. 3, A and
D). These data are consistent with at least a fraction of
the S/P chimera (presumably the functional 50-kDa form) undergoing
agonist-triggered internalization and sorting to lysosomes. At face
value, these data suggest that information specifying the sorting of
activated PAR1 to lysosomes resides in its C-tail. In contrast to
activated PAR1 and S/P chimera, wild-type SPR and P/S chimera failed to show any co-localization with the lysosomal marker lgp120 following 30 min of exposure to agonist. Taken together with the half-life and
lysosomal hydrolase inhibitor data cited above, this observation suggests that replacing the C-tail of PAR1 with that of the SPR prevents its sorting to lysosomes and its agonist-dependent
degradation.
Internalization and Recycling of Wild-type and Chimeric
Receptors--
To determine whether replacing the C-tail of PAR1 with
that of the SPR conferred recycling of the activated chimeric receptor, we followed internalization and recycling of receptor-bound antibody (Fig. 4). Rat1 fibroblasts stably
transfected with wild-type or chimeric receptors bearing a FLAG epitope
at their amino termini were incubated with the
calcium-dependent M1 FLAG antibody for 60 min at 4 °C
(Fig. 4, t0); under these conditions, antibody was
bound to cell surface receptor but did not internalize. Cells were then
washed to remove unbound antibody and incubated at 37 °C for 90 min
in the presence or absence of agonist (Fig. 4, t1).
After this incubation, surface-bound antibody was removed by washing
cells briefly with PBS/EDTA, agonist was removed, and the reappearance
of previously internalized receptor-bound antibody was followed over
the next 60 min. The amount of antibody on the cell surface at various
times was quantitated by cell surface ELISA. Antibody binding in these
studies was dependent on transfection with cDNAs encoding the
FLAG-tagged receptors. In cells expressing PAR1 (in which the FLAG
epitope is cleaved from the receptor by thrombin), antibody binding was
ablated by thrombin treatment (17). M1 antibody bound to the same FLAG
epitope displayed at the extracellular amino-terminal of PAR1, SPR, or
the chimeras showed distinct recycling properties depending upon the
receptor to which it was bound. Studies examining internalization of
receptor-bound antibody have yielded results concordant with studies of
loss of receptor from the cell surface (21). Lastly, when
receptor-bound antibody internalized and then returned to the cell
surface, it could be reinternalized by again exposing the cells to
agonist (data not shown). These data strongly suggest that antibody
detected on the cell surface in these studies represents receptor-bound antibody and that such antibody does not interfere with either agonist-induced receptor internalization or recycling.
In PAR1-transfected cells not exposed to agonist during the 90-min
incubation, the amount of receptor-bound antibody on the cell surface
declined to a new steady state approximately two-thirds of the initial
level (Fig. 4A, t1). It is unlikely that this decrease in surface-bound antibody was due to antibody
dissociation or antibody-induced internalization of receptor, because
parallel experiments with cells expressing wild-type SPR bearing the
same amino-terminal epitope showed little loss of surface-bound
antibody (Fig. 4B, t1). Rather, these
findings are consistent with tonic internalization of cell surface PAR1
and equilibration with an intracellular pool as previously reported
(19, 21).
Exposure to the PAR1 agonist peptide SFLLRN during the 90-min
incubation caused a 90% decrease in the level of PAR1-bound antibody
on the cell surface (Fig. 4A, t1)
consistent with agonist-triggered internalization and a lack of
receptor recycling. By contrast, the addition of substance P caused a
more modest decrease in SPR cell surface levels consistent with
internalization and recycling of this receptor (Fig. 4B,
t1).
After this initial incubation in the presence or absence of agonist,
antibody was stripped from the cell surface with PBS/EDTA, and recovery
of internalized antibody-receptor complexes was followed. In
PAR1-expressing cells, little recovery of antibody was seen in cells
that had not been preincubated with agonist. Similarly, in cells
exposed to agonist, there was little recovery of internalized antibody
despite the large cohort of receptor-bound antibody that had been
previously internalized (Fig. 4A). These data are consistent with the hypothesis that activated PAR1 is internalized and sorted to
lysosomes with the bound antibody. In SPR-expressing cells, there was
relatively little recovery of antibody in cells that had not seen
agonist. However, in striking contrast to PAR1-expressing cells,
SPR-expressing cells that had been pretreated with agonist showed
substantial recovery of antibody on the cell surface with time (Fig.
4B). Re-addition of substance P caused rapid internalization of this surface-bound antibody (data not shown); thus recovered antibody likely reflects the reappearance of SPR on the cell surface. These data are consistent with agonist-dependent
sequestration of SPR and its recycling to the cell surface after
removal of agonist, known properties of this classic GPCR (23, 24).
Remarkably, the P/S chimera behaved very much like the wild-type SPR in
this assay; cells expressing the P/S chimera clearly exhibited the
phenomenon of an agonist-dependent recoverable pool of
receptor-bound antibody (Fig. 4C). Taken together with the results shown in Figs. 2 and 3, these data suggest that the P/S chimera
undergoes agonist-dependent internalization but then
recycles back to the cell surface instead of sorting to lysosomes like wild-type PAR1. By contrast, the S/P chimera behaved like PAR1 in this
assay, with a profound agonist-dependent loss of surface receptor-bound antibody and no agonist-dependent
recoverable pool (Fig. 4D). These observations suggest that
the S/P chimera fails to recycle after agonist-triggered
internalization and are consistent with its sorting to lysosomes (see
Fig. 3D).
Studies using fluorescence microscopy supported the ELISA results in
Fig. 4. Treatment of PAR1-expressing cells with SFLLRN caused almost
complete internalization of PAR1-bound antibody from the cell surface
(Fig. 5A,
t1). After this initial internalization little
return of antibody to the cell surface was detected in either untreated
or agonist-treated cells (Fig. 5A). The S/P chimera behaved
like wild-type PAR1 (Fig. 5D), consistent with lysosomal
sorting of internalized antibody-receptor complexes. Untreated
SPR-expressing cells also showed little recovery of receptor-bound
antibody over time (Fig. 5B). Strikingly, however, a
substantial recovery of SPR-bound antibody was observed in
SPR-expressing cells that had been treated with agonist (Fig.
5B). The P/S chimera also displayed a robust agonist
pretreatment-dependent recovery of receptor-bound antibody
on the cell surface (Fig. 5C). These observations are
consistent with agonist-induced internalization and recycling of SPR
and the P/S chimeric receptor to the cell surface.
Signaling by Wild-type and Chimeric Receptors--
The data
presented above reveal substantial down-regulation of PAR1 protein by
agonist consistent with lysosomal sorting of activated PAR1. By
contrast, the P/S chimeric receptor was long lived in the presence of
agonist consistent with its recycling. To determine whether these
differences in down-regulation of receptor protein had a functional
correlate, we examined the ability of cells expressing PAR1 or the P/S
chimera to respond to agonist after a prolonged agonist exposure. In
these experiments, transfected Rat1 fibroblasts labeled with
[3H]inositol were incubated in the presence or absence of
agonist in media without LiCl. Without LiCl, receptor activation does trigger phosphoinositide hydrolysis, but the released inositol phosphates are rapidly metabolized and fail to accumulate. After such
pretreatment, agonist was washed out, LiCl was added to the medium,
cells were again incubated in the presence or absence of agonist, and
inositol phosphate accumulation was measured. Without prior agonist
treatment, SFLLRN cause an approximately 8-fold increase in
phosphoinositide hydrolysis in cells expressing PAR1. Agonist
pretreated cells showed only a 3-fold response, a 62% decrease in
signaling response (Fig. 6A).
Cells expressing the S/P chimera also showed a 68% decrease in
signaling after agonist exposure (Fig. 6D). By contrast,
cells expressing the substance P receptor (Fig. 6B) and the
P/S chimera (Fig. 6C) showed little decrement in their
ability to respond to agonist. Taken together these data suggest that,
unlike PAR1 itself, the P/S chimera recycles efficiently after
activation and is capable of responding to agonist again after such
recycling.
The magnitudes of the signaling responses in these cells are also
noteworthy. Cells expressing the P/S chimera reliably showed greater
absolute levels of phosphoinositide hydrolysis than wild-type PAR1,
whereas the S/P chimera showed less than substance P receptor. It is
possible that the apparent gain-of-function of the P/S chimera is
another manifestation of its ability to recycle and avoid
down-regulation during the stimulation period. Conversely, the
decreased signaling by the S/P chimera may be a manifestation of its
gaining the ability to down-regulate. It is certainly also possible
that intrinsic differences in the efficiency of G protein coupling
contribute to these phenomena.
These studies demonstrate that the cytoplasmic carboxyl tails of two G
protein-coupled receptors, PAR1 and SPR, specify their distinct
intracellular sorting patterns following activation. SPR bearing the
C-tail of PAR1 (S/P chimera) internalized and sorted to lysosomes upon
activation like wild-type PAR1. By contrast, PAR1 bearing the SPR
C-tail (P/S chimera) internalized upon activation but recycled back to
the plasma membrane like wild-type SPR. These distinct sorting fates
correlated with the extent to which agonists down-regulated receptor
protein and signaling responses over time. Thus the cytoplasmic tails
of these GPCRs specify distinct intracellular sorting fates that
dictate the extent to which a cell down-regulates or maintains its
ability to respond to their cognate ligands. The ability to confer
distinct trafficking and down-regulation patterns by exchanging
cytoplasmic tails may provide a useful tool for defining the importance
of receptor down-regulation in various responses in transgenic mouse
models. Whether exchanging the cytoplasmic tails of other GPCRs will
provide such a clean change in agonist-dependent
trafficking phenotype is not known.
The P/S chimera also provided an opportunity to evaluate the importance
of lysosomal sorting for termination of thrombin signaling. One might
predict that the irreversible proteolytic mechanism by which PAR1 is
activated necessitates that PAR1 not recycle if temporal fidelity of
signaling is to be maintained. We recently showed that
SFLLRN-stimulated phosphoinositide hydrolysis in P/S chimera-expressing
cells rapidly ceased upon removal of the SFLLRN peptide. However,
thrombin-stimulated phosphoinositide hydrolysis in these cells
persisted long after removal of thrombin, in marked contrast to the
efficient shut off seen for wild-type PAR1 (29). These data suggest
that lysosomal sorting of PAR1 is indeed critical for temporal fidelity
of signaling to thrombin; such fidelity is presumably important in
endothelial cells, fibroblasts, and other cells that may need to
respond to thrombin appropriately over time (29).
As noted above, the cytoplasmic tails of PAR1 and SPR appear to dictate
their specific intracellular sorting patterns. Recycling is generally
thought to be the default pathway for internalized membrane proteins,
and specific information is believed to be required for their sorting
to lysosomes. If this is the case for GPCRs, the cytoplasmic tail of
PAR1 presumably contains such information. Tyrosine- and
di-leucine-based motifs have been implicated in the sorting of
internalized membrane proteins to lysosomes (30, 31). Sequences highly
homologous to such motifs are not obvious in the cytoplasmic tail of
PAR1. We have mutated the two tyrosines (Y397A,Y420A) as well as the
di-leucine (L423A,L424A) in the cytoplasmic tail of PAR1 to
alanines without a dramatic effect on PAR1 down-regulation (data not
shown). Interestingly, ubiquitination of the cytoplasmic tail of the
yeast
INTRODUCTION
Top
Abstract
Introduction
References
2-adrenergic receptor has served as a prototype for
the molecular events responsible for GPCR desensitization and
resensitization (Refs. 11-13; reviewed in Ref. 14). Most activated
GPCRs are desensitized initially by rapid phosphorylation of the
agonist-occupied form of the receptor. Phosphorylated receptor then
binds arrestin, which prevents receptor interaction with G proteins,
thereby uncoupling it from effectors. Arrestin may also mediate the
interaction of
2-adrenergic receptor with
clathrin-coated pits, thereby promoting internalization of activated
receptors (15, 16). Within endosomes the
2-adrenergic
receptor dissociates from its ligand, is dephosphorylated, and recycles
back to the cell surface competent to signal again. In this case
trafficking serves to remove activated receptor from the cell surface
and to return the receptor to the surface in a off state, ready to
respond again to ligand.
2-adrenergic receptor, PAR1 is rapidly
phosphorylated and uncoupled from signaling after activation (17, 18). PAR1 is also internalized after activation (19-21). However, unlike classical GPCRs, activated PAR1 is sorted predominantly to lysosomes after internalization (19, 22). To begin to understand the function of
intracellular trafficking of PAR1 in regulation of signaling, we first
sought to identify the domain(s) that specifies sorting of activated
PAR1 to lysosomes. We constructed chimeras between PAR1 and the classic
GPCR for substance P (SPR) by exchanging their cytoplasmic carboxyl
tails. SPR, also known as the neurokinin-1 receptor, behaves like the
2-adrenergic receptor and other classic GPCRs; it is
activated reversibly by the peptide substance P, internalized, and then
recycled back to the plasma membrane (23, 24). Remarkably, exchanging
the cytoplasmic carboxyl tails (C-tails) of PAR1 and SPR switched their
trafficking behaviors after activation. SPR bearing the C-tail of PAR1
(S/P chimera) internalized upon activation and sorted to lysosomes like
wild-type PAR1. Conversely, PAR1 bearing the C-tail of SPR (P/S
chimera) internalized upon activation but recycled back to the plasma
membrane like wild-type SPR. Recycling versus lysosomal
sorting of activated receptors correlated with the extent to which
signaling was down-regulated over time. Cells expressing the wild-type
substance P receptor and P/S chimera were capable of responding to
their cognate agonists even after prolonged agonist exposure. By
contrast, signaling by wild-type PAR1 and the S/P chimera was
significantly down-regulated. Taken together, these data strongly
suggest that the cytoplasmic tails of PAR1 and SPR specify their
distinct intracellular sorting fates after activation. Moreover, the
P/S and S/P chimeras provide reagents for identifying the molecular
mechanisms by which PAR1 is sorted to lysosomes and for determining the
importance of this process for terminating PAR1 signaling.
EXPERIMENTAL PROCEDURES
20 °C for 30 s. Cells were then washed
three times with PBS containing 1% nonfat dry milk, 150 mM
sodium acetate, pH 7.0, followed by another three washes with PBS
containing only 1% nonfat dry milk (blocking buffer). Following
washes, cells were incubated with the lgp120 antibody GM10 for 1 h
at 25 °C, washed, and then incubated with Texas Red-X goat
anti-mouse and fluorescein goat anti-rabbit secondary antibodies for
1 h at 25 °C. Finally, cells were washed four times with PBS then once with Molecular Probes SlowFade equilibration
buffer. One drop of SlowFade anti-fade reagent was added to
each slide before mounting the coverslip. Confocal images were
collected using a Nikon PCM 2000 laser scanning confocal system
configured with an Eclipse E800 microscope fitted with a CFI Plan
Apochromat 60× oil objective (Nikon, Corp.). Fluorescein and Texas
Red-X images were captured sequentially at 1024 × 1024 resolution
with 2× optical zoom in 2 s and processed using C·IMAGING 1280 system (Compix, Inc.). The final image composite was created using
Adobe Photoshop 4.0.
RESULTS AND DISCUSSION
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Fig. 1.
Model of intracellular trafficking patterns
of PAR1 and SPR. A, in the absence of agonist, PAR1
tonically cycles between the plasma membrane (parallel
lines) and an intracellular compartment (hatched oval).
Upon activation PAR1 is phosphorylated, rapidly internalized, and
sorted predominantly to lysosomes (open oval) where it is
degraded. By contrast, SPR resides largely at the plasma membrane in
the absence of agonist. Upon activation, SPR is phosphorylated,
internalized into an early endosomal compartment (speckled
oval), and then recycled back to the cell surface. B,
FLAG epitope-tagged PAR1 (solid) and SPR (open)
receptor chimeras were generated by exchanging their cytoplasmic tails
carboxyl to their putative palmitoylation sites. PAR1 bearing the SPR
C-tail is designated P/S chimera, and SPR bearing the PAR1
C-tail is designated S/P chimera.
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Fig. 2.
Agonist-induced degradation of PAR1 and S/P
chimera correlate with the loss of phosphorylated receptor.
Immunoblot of lysates from stably transfected Rat1 cells expressing
similar levels of surface PAR1 (A), SPR (B), P/S
chimera (C), or S/P chimera (D). Cells were
incubated in the presence or absence (Ctrl) of PAR1 agonist
SFLLRN (100 µM) or substance P (100 nM) as
indicated for 90 min at 37 °C. Where indicated, 10 µM
cycloheximide was included during the incubation period
(+CHX). Cell lysates were then analyzed by
SDS-polyacrylamide gel electrophoresis and immunoblotting using M1
anti-FLAG antibody; each lane represents an equivalent amount of cell
lysate. Similar findings were observed in three separate experiments.
Phosphorylation of PAR1 (E), SPR (F), P/S chimera
(G), or the S/P chimera (H) in stably transfected
Rat1 fibroblasts is shown. Cells were labeled with
[32P]orthophosphate and then incubated with 100 µM SFLLRN, 100 nM substance P, or media alone
(Ctrl) for 3 or 90 min at 37 °C. Cycloheximide (10 µM) was included during the incubation periods. Cell
lysates from an equivalent number of cells were prepared, and receptors
were immunoprecipitated as described under "Experimental
Procedures." Receptor immunoprecipitates were resolved by
SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography.
No phosphorylated proteins were detected in immunoprecipitates from
untransfected Rat1 fibroblasts (Rat1 lane in G
and H). Similar results were observed in two independent
experiments.
2-adrenergic receptor, no functional chimeric receptors were obtained (data not shown). It is
possible that in such chimeras, interactions between the C-tail and the
body of the receptor led to misfolding or misprocessing or that
conflicting trafficking signals interfered with their biogenesis. By
contrast, generation of chimeras between PAR1 and SPR did yield
receptors capable of mediating signaling, and we have chosen to
focus on the agonist-dependent trafficking behaviors of
these chimeras in the current study to avoid events involving improperly processed receptors.
Effect of agonist and lysosomal hydrolase inhibitors on receptor
degradation
, without
agonist.
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Fig. 3.
Co-localization of wild-type and chimeric
receptors with lysosomes. Laser scanning confocal microscopy was
used to examine sorting of wild-type and chimeric receptors to
lysosomes. Rat1 fibroblasts expressing PAR1 (A), SPR
(B), P/S chimera (C), or the S/P chimera
(D) were incubated in the absence (Control) or
presence of their respective agonists (100 µM SFLLRN or
100 nM substance P) for 30 min at 37 °C.
Co-immunostaining for receptors (green) and the lysosomal
membrane protein lgp120 (red) was performed as described
under "Experimental Procedures." Note the agonist-induced
co-localization (yellow) of PAR1 and S/P chimera with
lgp120.
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Fig. 4.
Agonist-induced internalization and recovery
of wild-type and chimeric receptors. Internalization and recovery
of receptors following exposure to agonist was measured by a cell
surface ELISA as described under "Experimental Procedures." Rat1
cells expressing similar amounts of surface PAR1 (A), SPR
(B), P/S chimera (C), or the S/P chimera
(D) were incubated with M1 anti-FLAG antibody at 4 °C.
Unbound antibody was removed and cells were incubated in the absence
( ) or presence (
) of the indicated agonist (100 µM
SFLLRN or 100 nM substance P) for 90 min at 37 °C.
Cycloheximide (10 µM) was included during incubations.
Surface-bound antibody was then removed with PBS/4% EDTA, and the
amount of receptor-bound antibody reappearing at the cell surface was
measured after 0, 30, and 60 min of recovery time. Surface-bound
antibody was quantitated after the initial 4 °C incubation
(t0), after the 37 °C incubation in the presence
or absence of agonist (t1) and after the indicated
times of recovery. Antibody binding to untransfected Rat1 fibroblasts
("nonspecific binding") was less than 5% of total binding to
transfected cells in all cases and was subtracted from total binding to
obtain the values shown. Data are expressed as (surface-bound antibody
at the indicated time point)/(surface-bound antibody at
t0). Each point represents the mean ± S.D.
(n = 3); where error bars are not seen they are smaller
than the representative symbol. Similar results were obtained in five
separate experiments. Note the agonist
pretreatment-dependent recovery of receptor-bound antibody
at the cell surface for SPR (B) and P/S chimera
(C).
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Fig. 5.
Internalization and recovery of wild-type and
chimeric receptors examined by immunofluorescence microscopy.
Stably transfected Rat1 fibroblasts expressing PAR1 (A), SPR
(B), P/S chimera (C), or the S/P chimera
(D) were treated as described in the legend to Fig. 4 and
examined by fluorescence microscopy. Images of cells preincubated with
M1-FLAG antibody that were either left untreated (Control)
or treated with agonists (100 µM SFLLRN or 100 nM substance P) for 90 min are indicated by
t1. Following agonist treatment, remaining
surface-bound antibody was removed with PBS/EDTA, images are indicated
by tR = 0 min. Fluorescent image (left)
shows substantial decrease in antibody binding, the adjacent
phase-contrast image (right) is of the identical field. At
60 min, recovery of receptor-bound antibody to the cell surface was
then examined, images are indicated by tR = 60 min.
Constant exposure times of 20 s for PAR1 (A), 30 s
for SPR (B), 30 s for P/S chimera (C), and
30 s for S/P chimera (D) were used to collect
fluorescent images. The amount of fluorescent staining in
tR = 0 min was similar to that observed in untransfected Rat1 cells (data not shown).
Note the remarkable agonist-dependent recovery of
receptor-bound antibody for SPR (B) and the P/S chimera
(C) expressing cell lines. The images shown are
representative of many cells examined, and these results are similar to
those observed in two separate experiments.
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Fig. 6.
Down-regulation of signaling by wild-type and
chimeric receptors. Transfected Rat1 fibroblasts expressing PAR1
(A), SPR (B), P/S chimera (C), or the
S/P chimera (D) labeled with [3H]myo-inositol
were left untreated (Control) or pretreated with agonists
(100 µM SFLLRN or 100 nM substance P) for 90 min in the absence of LiCl. Cells were washed and agonists were
re-added with 20 mM LiCl for 2 h, and then the total
amount of [3H]inositol phosphates accumulated was
determined. Cycloheximide (10 µM) was included during all
incubations. The data shown are the mean ± S.D.
(n = 3) of [3H]inositol phosphate
formation. Agonist-treated untransfected Rat1 cells showed no
significant increase in [3H]inositol phosphate formation
(A and B). PAR1 and S/P chimera showed
substantial down-regulation of signaling following agonist
pretreatment, 62 and 68%, respectively (A and
D). By contrast, SPR and P/S chimera showed only a small
decrease in signaling following agonist pretreatment (B and
C). These results are representative of five separate
experiments. The initial amount of receptor expressed on the surface of
wild-type and chimeric receptor expressing cell lines in this
experiment was 0.8 for PAR1, 0.5 for SPR, 1.0 for P/S chimera, 0.5 for
S/P chimera, and 0.1 for untransfected Rat1 fibroblasts.
-factor receptor Ste2p, a GPCR, is required for ligand-induced
endocytosis and degradation by the yeast vacuole (32). Attempts to
detect ubiquitination of PAR1 have been negative thus far and mutation
of the four lysines (K389R,K407R,K421R,K422R) to arginine to remove
potential ubiquitination sites from in the cytoplasmic tail of PAR1 had
little effect on PAR1 down-regulation (data not shown). The cytoplasmic
tail of PAR1 might thus serve as a probe for identifying new molecules
that recognize and sort this receptor to lysosomes.
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ACKNOWLEDGEMENTS |
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We thank Drs. Henry Bourne, Mark von Zastrow, and Harold S. Bernstein for critical review of this manuscript.
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FOOTNOTES |
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* This work was supported by HL44907 and HL59202 (to S. R. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an American Heart Association Minority Scientist Career Development Award.
** To whom correspondence should be addressed: University of California, San Francisco, HSE-1300, Box 0130, 505 Parnassus Ave., San Francisco, CA 94143-0130. Tel.: 415-476-6174; Fax: 415-476-8173; E-mail: shaun_coughlin{at}quickmail.ucsf.edu.
The abbreviations used are: PAR1, protease-activated receptor-1; GPCR, G protein-coupled receptor; SPR, substance P receptor; C-tail, cytoplasmic carboxyl tail; DMEM, Dulbecco's modified Eagle's media; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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