(Received for publication, September 29, 1994; and in revised form, November 18, 1994)
From the
We have compared the desensitization of two receptors, the
thrombin receptor which displays dual coupling to both pertussis
toxin-sensitive (G) and -insensitive (G
)
proteins and the serotonin type 2 (5-HT
) receptor which
selectively couples to G
. In the case of the thrombin
receptor, cleavage induces activation and irreversible receptor
modification followed by rapid (T
= 3
min) and extensive desensitization of the receptor's ability to
modulate phospholipase C (G
). 5-HT-induced desensitization
of its receptor is markedly slower (T
=
10 min) and by 60 min only 50% of the phospholipase C response is lost.
This effect occurs with a parallel disappearance of 5-HT receptors from
the cell surface. Whole cell phosphorylation studies showed that the
thrombin receptor is rapidly phosphorylated upon activation. In
contrast, the 5-HT
receptor displays a low basal level of
phosphorylation which is not increased upon agonist treatment. The
cytoplasmic tail of the 5-HT
receptor which contains
several protein kinase consensus sequences was found not to be involved
in receptor activation or desensitization. However, a chimeric receptor
having the core of the 5-HT
receptor and the cytoplasmic
tail of the thrombin receptor was able to undergo 5-HT-induced
desensitization and phosphorylation. These results indicate that (i)
both 5-HT
and thrombin receptors have unique shut-off
mechanisms, and (ii) that sequences in the carboxyl terminus of the
thrombin receptor are sufficient to trigger rapid uncoupling of the
receptor from its G protein(s) and downstream effector(s).
Loss of the biological response to a ligand despite its constant
presence is termed desensitization. Mechanisms involved in
desensitization of G protein-coupled receptors have been elucidated in
part using the 2-adrenergic receptor as a prototype. It has been
shown that at least three different phenomena underlie the
desensitization process (reviewed in (1) ). (i) Following
activation, the receptor is phosphorylated by either a
2-adrenergic receptor kinase which belongs to the G protein
receptor kinase family (2) or by the cAMP-dependent protein
kinase A (termed heterologous phosphorylation) as a consequence of
receptor-mediated stimulation of cAMP formation. In the case of
receptors coupled to phospholipase C activation, the kinase implicated
in heterologous phosphorylation is protein kinase C. Receptor
phosphorylation is a rapid event, since it can be detected as early as
1 min after agonist treatment. Phosphorylated receptors are
subsequently recognized by an arrestin-like protein (1) which
binds to the receptor and inhibits G protein interaction. (ii) The
receptor is internalized/sequestered in an intracellular compartment
where it is inaccessible to ligand. This phenomenon also occurs within
minutes to hours of agonist exposure and induces an impairment of
receptor G protein coupling. Recently, a consensus sequence for
sequestration of the
2-adrenergic receptor has been
identified(3) . This sequence, which encompasses a tyrosine
residue highly conserved among members of the G protein-coupled
receptor family, is located in close proximity to the seventh
transmembrane domain and is present in both the 5-HT
and
thrombin receptors. Recent observations of Yu et al.(4) demonstrate that serine and threonine residues in the
carboxyl-terminal tail of the
2-adrenergic receptor are important
for sequestration. In fact, the sequestration pathway has been proposed
to be an important mechanism by which cells re-establish their normal
responsiveness to agonists following removal of the
stimulus(4, 5) . (iii) Finally, after prolonged
agonist exposure, the receptor is down-regulated, a phenomenon which
leads to a dramatic decrease in the total cellular receptor level.
Although the mechanisms which result in down-regulation are not
precisely defined, the presence of tyrosine residues in the
carboxyl-terminal tail appears to be necessary.
To study the
desensitization process of G protein-coupled receptors involved in
mitogenic signaling, we selected two receptors, 5-HT and
thrombin receptors, which have been shown to stimulate growth in a line
of hamster fibroblasts (CCL39 cells) (for review, see (6) ).
cDNA clones corresponding to both receptors have previously been
isolated in the laboratory. In CCL39 cells, the 5-HT
receptor is coupled to the activation of phospholipase C via a
G
protein(7, 8) . The thrombin receptor is
coupled to both activation of phospholipase C and inhibition of
adenylate cyclase, implicating at least two G proteins (G
and
G
)(9, 10, 11, 12) .
It is known from previous reports that the 5-HT receptor
undergoes ligand-induced desensitization in P11 cells and transfected
Swiss 3T3 cells(13, 14) . However, in these studies,
only long term desensitization was addressed since the effect of 5-HT
was examined from 1 to 8 h following agonist addition. In the present
study, we have examined rapid ligand-dependent 5-HT
receptor desensitization. In vivo labeling experiments
were performed to determine the possible role of phosphorylation in
desensitization of this receptor. Further, the role of the receptor
carboxyl-terminal extension was analyzed using a truncated receptor
mutant.
Thrombin is a serine protease with multiple cellular
effects, including growth stimulation in CCL39 hamster fibroblasts,
reviewed in Refs. 15 and 16. Thrombin activates its receptor by a
mechanism which involves cleavage of the amino-terminal ectodomain of
the receptor to expose a new amino-terminal sequence which functions as
a built-in ligand(17) . This irreversible modification of the
receptor by thrombin is accompanied by rapid attenuation of signaling
in cells. In CCL39 fibroblasts, it has previously been demonstrated
that thrombin induces homologous desensitization of phosphoinositide
breakdown(9) . Studies on the premegakaryocytic HEL cell line
indicate that desensitization of the receptor involves both proteolysis
of the receptor and phosphorylation(18) . More recently, it has
been shown that rapid internalization and recycling of thrombin
receptors occurs in HEL cells subsequent to stimulation by thrombin or
thrombin receptor peptide agonist. Interestingly, the recycled
receptors are refractory to thrombin since they are already
cleaved(19, 20) . Finally, Ishii et al.(21) demonstrated that the inhibition of thrombin receptor
signaling can be induced by co-expression of the receptor and the
-adrenergic receptor kinase 2 in a Xenopus oocyte
expression system(21) . They further showed that removal of the
thrombin receptor carboxyl-terminal tail, containing several potential
serine and threonine phosphorylation sites, prevented
adrenergic
receptor kinase 2-mediated desensitization of the thrombin-stimulated
Ca
response.
The present studies were designed to
explore the distinct regulatory mechanisms that exist between different
serpentine receptors. Whereas thrombin receptor desensitization is
rapid and nearly complete, desensitization of the 5-HT receptor is slower, and only 50% of the response is lost. We show
for the first time in a thrombin-responsive cell line that
ligand-dependent desensitization of the thrombin receptor correlates
well with ligand-induced phosphorylation of the receptor. By contrast,
phosphorylation of the 5-HT
is not stimulated by ligand
treatment. Rather, loss of 5-HT responsiveness parallels receptor
sequestration. Finally, since the last 72 amino acids of the 5-HT
receptor are not required for G protein coupling or
desensitization, we used a chimeric receptor composed of the truncated
5-HT
receptor and the cytoplasmic tail of the thrombin
receptor to examine the role of the thrombin receptor cytoplasmic tail
in the desensitization process.
Since specific anti-receptor antibodies are required to
assess the role of phosphorylation in desensitization and antibodies
which immunoprecipitate 5-HT and thrombin receptors were
not available, we added an epitope tag to each receptor sequence. In
the case of the 5-HT
receptor, the amino-terminal 11 amino
acids of the receptor were exchanged with 11 residues encoding an
immunodominant peptide from influenza hemagglutinin (HA). For the
thrombin receptor, we added an epitope tag corresponding to a sequence
from the vesicular stomatitis virus glycoprotein to the carboxyl
terminus of the full-length receptor. This position was chosen for two
reasons. First, a signal peptide sequence is present at the extreme
amino terminus of the receptor sequence. Second, and more important,
the receptor is cleaved after residue Arg-41 by thrombin upon
activation. Schematic representations of the HA epitope-tagged
5-HT
receptor, the VSVG epitope-tagged thrombin receptor,
and the chimeric 5-HT
/Thr receptor, which encodes both
epitopes, are shown in Fig. 1. The residues in the cytoplasmic
tails of the receptors which are potential phosphorylation sites for G
protein receptor kinases are indicated. Consensus phosphorylation sites
for PKC are also present in both receptors. One putative PKC
phosphorylation site is present in the third intracellular loop of the
thrombin receptor, and five sites were identified in the 5-HT
receptor by inspection of the sequence (two in the third
intracellular loop and three in the carboxyl tail). No consensus
protein kinase A phosphorylation sites exist in the 5-HT
receptor sequence, whereas two are present in the third
intracellular loop of the thrombin receptor(28) .
Figure 1:
Representation of the epitope-tagged
5-HT, thrombin, and chimeric 5-HT
/Thr
receptors. The PstI restriction site was used to construct the
truncated 5-HT
receptor. Potential phosphorylation sites
for G protein receptor kinases (circles) and protein kinase C (triangles) are indicated. Potential sites of palmitoylation
are shown.
It has
been reported for many G protein-coupled receptors, including the
thrombin receptor(21) , that the carboxyl-terminal receptor
tail is involved in the desensitization process. To elucidate the role
of the 5-HT receptor's cytoplasmic tail in mediating
this response, we have constructed a truncated receptor (using a PstI restriction enzyme site in the cDNA denoted in Fig. 1) in which 72 out of 86 carboxyl-terminal amino acids were
removed. In this mutant, the potential G protein-coupled receptor
kinase phosphorylation sites present in the cytoplasmic tail of the
receptor and three of the PKC consensus sites are eliminated; two
possible PKC sites remain in the intracellular loops.
Figure 2:
Functional expression of the 5-HT and epitope-tagged 5-HT
receptors in PS200 cells. A, PS200 cells were transfected with plasmids encoding the
5-HT
R (
), HA-5-HT
R (
), and
HA-5-HT
R
1244 (
) receptors. The dose-dependent
activation of phospholipase C by 5-HT in each of the transfected clones
is presented. Values are expressed as a percent of maximal stimulation.
Minimal and maximal values are 500-4468, 663-10,353, and
636-13,260 cpm for 5-HT
R (
),
HA-5-HT
R (
), and HA-5-HT
R
1244
(
), respectively. Error bars reflect the variation
between duplicate determinations of inositol phosphate formation over
the 10-min period of stimulation. B, flow cytometric analysis
of cell surface staining with anti-HA antibodies. Nontransfected
(PS200) and transfected cells are shown. The fluorescence intensity
determined for 3000 cells is represented as a function of cell number.
Mean fluorescence values are 4.2 for PS200 and 5-HT
R cells,
13.1 for HA-5-HT
R
1244 cells, and 16.1 for
HA-5-HT
R cells.
Figure 3:
Desensitization rates of agonist-induced
phosphoinositide breakdown in transfected cells. A, quiescent
[H]inositol-labeled cells stably expressing the
indicated receptor were stimulated with 5-HT (10
M) for various times, then 20 mM LiCl was added
and total inositol phosphate formation was determined over a 10-min
period. Values are expressed as a percentage of maximal stimulation.
Minimal and maximal values are 225-1810, 356-3672, and 347-1939 for
5-HT
R (
), HA-5-HT
R (
), and
HA-5-HT
R
1244 (
), respectively. B,
quiescent 5-HT
R transfected cells were pretreated with 1
unit/ml thrombin (Thr) for the indicated times. Then, 20 mM LiCl was added, and total inositol phosphates were measured after
10 min as described under ``Experimental Procedures.'' Error bars reflect variation between duplicate
determinations.
Thrombin receptor desensitization was assessed by measuring
phospholipase C activity following different times of thrombin
treatment in the clone 5-HTR which expresses the wild type
5-HT
receptor. As shown in Fig. 3B, the
ability of the thrombin receptor to stimulate phospholipase C is
rapidly attenuated following receptor activation (T
= 3 min). After 1 h of thrombin pretreatment, more than
80% of the response is lost. These results are consistent with
previously reported studies of thrombin-induced desensitization of
phosphoinositide breakdown carried out in parental CCL39
cells(9) . It is noteworthy that the thrombin receptor peptide
agonist induces receptor desensitization with the same time course, and
to the same extent, as thrombin thereby indicating that receptor
cleavage is not responsible for this effect(11) . Thus, we show
that, whereas desensitization of thrombin receptor is rapid and
extensive, desensitization of the 5-HT
receptor in the same
cells displays a markedly slower time course and reduced maximal
effect. The difference in desensitization of two G protein-coupled
receptors which activate at least one common effector suggests the
presence of different regulatory mechanisms.
Figure 4:
Comparison of the rate of desensitization
of agonist-induced phosphoinositide breakdown in pretreated or
nontreated transfected PS200 cells. A, HA-5-HT cells; B, HA-5-HT
1244 cells. Quiescent
[
H]inositol-labeled cells were pretreated with
the GF 109203X compound, 5 min before agonist addition (open
triangles in A and open circles in B)
or with 100 ng/ml PDBu for 24 h before agonist addition (filled
symbols). Then, 20 mM LiCl was added and production of
total inositol phosphates was determined after 10 min. Points represent
the mean of duplicate values.
The role of PKC in
thrombin-induced desensitization in CCL39 cells has been addressed
previously in the laboratory. It was concluded from these studies that
desensitization of polyphosphoinositide breakdown induced by thrombin
is independent of PKC(9) . The absence of a PKC effect on
thrombin receptor desensitization in CCL39 cells has also been observed
using the GF 109203X inhibitor in similar experiments as described
above. ()
The finding that PKC is not a major contributor
to 5-HT or thrombin receptor desensitization is further
confirmed by the absence of heterologous desensitization of these two
receptors which both activate PKC (9) and results not shown.
As shown in Fig. 5A (left
panel), expression of both the full-length and truncated
5-HT receptors can be detected in 293 cells by Western blot
analysis following transient transfection. The HA-5-HT
receptor migrates as a band of approximately 80,000 Da whereas
the truncated receptor migrates with an apparent molecular mass of
50,000 Da. We also observe the presence of higher molecular mass bands
in immunoblots of the truncated or full-length tagged receptors, which
may correspond to dimeric and trimeric forms, as judged by their size.
The formation of these larger molecular species presumably occurs
following lysis of the cells, and not in intact cells, since the
intensity of their labeling increases with prolonged incubation of cell
lysates with Sepharose beads.
Figure 5:
A, Expression of the transfected
5-HT and 5-HT
1244 receptors in transfected
cells. Left, 293 cells transiently transfected with 8 µg
of vector alone or 5-HT
receptor constructs were lysed in
300 µl of Triton X-100 lysis buffer, and wheat germ-enriched
fractions were subjected to immunoblotting as described under
``Experimental Procedures.'' Right, 100 µl of
P-labeled cell lysates were purified on wheat
germ-Sepharose and subjected to immunoblotting analysis. B,
phosphorylation of 5-HT
receptors. 293 cells transiently
transfected with 5-HT
receptor constructs were labeled with
[
P]orthophosphate (200 µCi/ml) for 3.5 h,
and 10
M 5-HT was added during the last 10
min. The cells were lysed and immunoprecipitated with 12Ca5 antibody
coupled to Protein A-Sepharose. Immune complexes were washed and
proteins were analyzed by SDS-PAGE in 10% acrylamide gels. Positions of
molecular mass markers are shown.
For phosphorylation experiments
following transient transfection of 293 cells, the receptor level in
each P-labeled cell lysate was followed by Western
analysis (Fig. 5A, right panel). It can be
seen for the experiment presented in Fig. 5that the same
quantity of receptor was present in lysates from control and
5-HT-stimulated cells. The effect of 5-HT on phosphorylation of
tagged-5-HT
receptors is shown in Fig. 5B.
Basal phosphorylation of the wild type 5-HT
receptor was
observed in some experiments; however, no significant increase in
phosphorylation of the tagged-5-HT
receptor could be
detected following a 10-min incubation of cells with 5-HT. Additional
time points also failed to reveal 5-HT-stimulated phosphorylation of
the receptor (data not shown). We did detect a labeled band of
approximately 80,000 Da in cell lysates; however, we don't
believe that this band corresponds to the phosphorylated 5-HT
receptor since it is also present in cells which express the
truncated receptor form. Concerning the truncated receptor, we were
unable to detect its phosphorylation in either control or stimulated
cells, despite its presence in the labeled cell lysates (Fig. 5A, right). These results suggest that
the residues of the wild type receptor which are phosphorylated in
nonstimulated cells are located carboxyl to the site of truncation.
Since the T
for desensitization of
phosphoinositide breakdown is 10 min, and no stimulation of receptor
phosphorylation could be detected within 10 min of 5-HT treatment, it
appears that receptor phosphorylation is not a key mechanism involved
in desensitization of the 5-HT
receptor.
To analyze
phosphorylation of the thrombin receptor, experiments were carried out
in both transiently transfected 293 cells and in 293 cells stably
expressing the epitope-tagged thrombin receptor, designated 293TRV2J. ()Both stably and transiently transfected cells express high
levels of thrombin receptor which can be immunoprecipitated with the
anti-VSVG antibody. The thrombin receptor migrates as a broad band with
an apparent molecular weight ranging from 62,000-72,000 following
cleavage with thrombin, and 72,000-86,000 when cells are
stimulated with thrombin receptor agonist peptide or PDBu (Fig. 6). Whereas we were unable to detect receptor
phosphorylation in nontreated cells, thrombin treatment was found to
stimulate receptor phosphorylation, as shown in Fig. 6.
Phosphorylation was observed as early as 1 min following thrombin
addition. Therefore, phosphorylation does correlate well with
desensitization of the thrombin receptor since similar time courses are
observed for both phosphorylation and desensitization of the
phospholipase C response.
Figure 6:
Phosphorylation of the epitope-tagged
thrombin receptor. Stably transfected 293 cells which express the
thrombin receptor (TRV2J cells) were labeled with 200 µCi/ml
[P]orthophosphate for 3.5 h. At the end of the
labeling incubation, thrombin (1 unit/ml), thrombin receptor peptide
agonist (TRP) (30 µM), or PDBu (100 ng/ml) was
added for 10 min. Cells were lysed and immunoprecipitated with P4D5
antibody coupled to Protein A-Sepharose. Immune complexes were washed
and proteins were analyzed by SDS-PAGE in 10% acrylamide gels. Brackets on the right side of the autoradiogram
indicate the migration of the receptor from thrombin receptor peptide
agonist/PDBu (upper) or Thr (lower) stimulated cells.
Positions of molecular mass markers are
shown.
Figure 7:
Desensitization rates of agonist-induced
phosphoinositide breakdown in 5-HT/Thr transfected cells.
Quiescent [
H]inositol-labeled cells stably
expressing the chimeric receptor were stimulated with 5-HT
(10
M) for various times, then 20 mM LiCl was added and total inositol phosphate formation was
determined over a 10-min period. Values are expressed as percentage of
maximal stimulation. Minimal and maximal values are 86-445 cpm.
Points represent the mean of four independent
experiments.
Figure 8:
Phosphorylation of the chimeric
5-HT/Thr receptor. 293 cells transiently transfected with
the chimeric 5-HT
/Thr receptor construct were labeled with
[
P]orthophosphate (200 µCi/ml) for 3.5 h and
10
M 5-HT was added during the last 10 min.
The cells were lysed and immunoprecipitated with 12Ca5 antibody coupled
to Protein A-Sepharose. Immune complexes were washed as described under
``Experimental Procedures,'' and proteins were analyzed by
SDS-PAGE in 10% acrylamide gels. Positions of molecular mass markers
are shown.
The study of G protein receptors and their role in growth
stimulation is an area of intense investigation. Although the precise
events leading to DNA synthesis stimulation by this family of receptors
remain to be elucidated, certain G protein-coupled signaling pathways,
notably one or more G-mediated
pathways(7, 30, 31) , have been implicated in
mitogenic stimulation of cells by a number of different receptors. The
CCL39 hamster fibroblast line has proven to be a particularly useful
model for these studies (for reviews see Refs. 32 and 33) since these
cells express receptors for 5-HT and thrombin coupled to both G
and G
proteins. cDNAs encoding both 5-HT
and thrombin receptors have been cloned from CCL39
cells(34, 35) , and the intracellular signaling
pathways activated by their respective ligands have been extensively
characterized. It has been proposed that the duration of signal
generation by growth factors is a key element in determining cell cycle
re-entry(27, 36, 37) ; therefore, it is
necessary to characterize the attenuation of cellular responses as well
as their activation. In the present paper we have focused on the
desensitization of 5-HT and thrombin receptors in an attempt to
identify the molecular mechanisms involved in this phenomenon. We found
that both the rate and extent of desensitization of these two receptors
is quite different. Whereas rapid ligand-induced desensitization of the
thrombin receptor correlates well with receptor phosphorylation, the
5-HT
receptor does not undergo ligand-induced
phosphorylation. The relatively slow loss of 5-HT responsiveness occurs
concomitantly with receptor disappearance from the cell surface.
Inspection of the primary sequence of these two receptors indicates
that the thrombin receptor may be a good substrate for G protein
receptor kinases, since several serine/threonine residues in an acidic
environment are present in its cytoplasmic tail(38) . In
addition, one consensus phosphorylation site for PKC can be identified
in the third cytoplasmic loop of the thrombin receptor(28) . By
contrast, the cytoplasmic tail of the 5-HT receptor
contains individual rather than clustered serine/threonine residues and
thus is not such a good substrate for G protein receptor kinases.
However, at least five PKC phosphorylation sites (K/RXXS*/T*
or K/RXS*/T* (39) can be found in the 5-HT
receptor, including three in the cytoplasmic extension.
Analysis of ligand-stimulated phosphorylation of these receptors
revealed that the thrombin receptor is rapidly phosphorylated in
response to either thrombin, the thrombin receptor agonist peptide, or
the phorbol ester, PDBu. These results obtained on fibroblasts are in
agreement with a recent study by Ishii et al.(21) ,
who also reported that the G protein receptor kinase -adrenergic
receptor kinase 2 is capable of blunting thrombin receptor signaling
when expressed together with the receptor in Xenopus oocytes.
The analysis of receptor mutants lacking serine and threonine residues
in the intracellular loops or in the cytoplasmic tail led the authors
to conclude that the kinase inhibits receptor signaling by
phosphorylating serine/threonine residues in the carboxyl tail, rather
than in the intracellular loops, as expected by the localization of
putative G protein receptor kinase consensus sequences.
In the case
of the 5-HT receptor, we were not able to detect any
increase in phosphorylation following stimulation with 5-HT. The same
results were obtained with the truncated 5-HT
receptor in
which the potential phosphorylation sites present in the cytoplasmic
tail have been removed. These findings support the proposal that the
5-HT
receptor is a poor G protein receptor kinase substrate
and, therefore, is not subject to short-term desensitization mediated
by a member of this kinase family. Indeed, recent studies confirm the
presence of G protein receptor kinase phosphorylation sites in G
protein-coupled receptors that display rapid agonist-dependent
phosphorylation and functional desensitization. For example, the
2-adrenergic receptor, which contains these sites, is
phosphorylated and desensitized following exposure to agonist, whereas
the
3-adrenergic receptor is not(40) . Further, a chimeric
receptor with the core of the
3-adrenergic receptor and the
cytoplasmic tail of the
2 receptor is phosphorylated in response
to agonist and can undergo desensitization. In the present study, we
demonstrate that the carboxyl tail of the thrombin receptor, which is a
G protein receptor kinase substrate, can signal rapid desensitization
of a chimeric 5-HT
receptor. More detailed mutagenesis
analyses should help to further define residues involved in this
function.
The phorbol ester PDBu is also able to induce thrombin
receptor phosphorylation, albeit with a slower time course and a lower
maximal effect than that induced by thrombin ( Fig. 6and (21) ). The functional consequence of PDBu-stimulated receptor
phosphorylation is not clear in light of the findings in CCL39-derived
fibroblasts that PKC activation has no significant effect on
desensitization at the receptor level. Rather, inhibitory effects of
the phorbol ester analog on phosphatidylinositol turnover stimulated by
thrombin and 5-HT (not shown and (9) ) are presumably due to
downstream inhibition of phospholipase C by the kinase. Tobin and
Nahorski (41) have reported that the M3 muscarinic receptor is
phosphorylated in response to 12-O-tetradecanoylphorbol
13-acetate. However, a PKC inhibitor blocks this phosphorylation
without affecting carbachol-stimulated receptor
phosphorylation(41) . Thus, receptors coupled to phospholipase
C activation may be substrates of PKC; however, the role of PKC in the
regulation of each of these receptors remains to be elucidated.
PKCmediated desensitization of 5-HT receptors was addressed
in the present study since this receptor displays five potential PKC
phosphorylation consensus sites. We found in our CCL39-derived clones
that PKC inhibition did not change the time course of 5-HT
receptor desensitization. It has been hypothesized that
stimulation of PKC is responsible for acute desensitization of the 5-HT
response in platelets(42) . The authors of that report proposed
that the PKC activation may desensitize the 5-HT-stimulated IP
accumulation and Ca
mobilization based on
results obtained using a PKC inhibitor (H7 at 300 µM).
However, it is not clear whether PKC activation acts at the receptor
level or rather distal to the receptor. Since blocking PKC activation
in our system (with a specific inhibitor and by down-regulation) was
without effect on receptor desensitization and, since we were unable to
detect phosphorylation of the 5-HT
receptor upon
agonist-induced PKC stimulation, we believe that PKC is not involved in
desensitization of the 5-HT
receptor. Consistent with this,
we did not observe any difference in ligand-induced desensitization of
the truncated receptor in which three of the five PKC phosphorylation
consensus sites were removed.
Even though the 5-HT receptor is not phosphorylated in response to 5-HT, we did
observe receptor internalization by flow cytometric analysis. Since
this procedure does not distinguish between rapid receptor
sequestration, presumably occurring at or near the cell surface, and
translocation of receptors to intracellular compartments, we use the
term internalization for both processes. A recent report describes a
consensus sequence for internalization (NPXXY) of
2-adrenergic receptors which is present in nearly all G
protein-coupled receptors(3) . Mutation of the tyrosine residue
completely abolishes agonist-mediated sequestration without affecting G
protein coupling, receptor phosphorylation, or down-regulation.
Interestingly, this sequence is present in both the thrombin and the
5-HT
receptors, as well as in the truncated 5-HT
receptor mutant which internalizes as efficiently as the
full-length receptor. The role of this tyrosine-containing sequence in
sequestration of these two receptors awaits evaluation by mutational
analysis. In addition to the presence of this sequence, many reports
indicate the importance of serine/threonine residues in either the
third intracellular loop or in the receptor's cytoplasmic tail
for internalization(4, 43, 44) .
Strikingly different time courses of receptor internalization are
displayed by 5-HT and thrombin receptors. In CCL39 cells,
the thrombin receptor undergoes very rapid surface redistribution
(
5 min) and nearly complete internalization within minutes (
15
min) of agonist treatment. (
)Similar findings have been
reported in other cells(19) . Disappearance of the 5-HT
receptor occurs more slowly, with maximal effects corresponding
to approximately 50% receptor loss, observed 30 min following agonist
addition. Many reports indicate that sequestration is an obligatory
step for receptor recycling. This could be the case for the
serotoninergic receptor, but not for the thrombin receptor, since,
following cleavage, de novo receptor synthesis is required for
its recognition by thrombin and subsequent activation.
Experiments
with the chimeric receptor described in this study have allowed us to
clearly demonstrate that the carboxyl-terminal extension of the
thrombin receptor is a substrate for one or more protein kinases
involved in desensitization. Sequences within this tail appear to be
selective, since Ishii et al.(21) have reported that
-adrenergic receptor kinase 2 (considerably more efficiently than
-adrenergic receptor kinase 1) blocks thrombin receptor signaling.
The 5-HT
/Thr receptor should prove to be useful in
identifying the specific G protein receptor kinases which mediate in vivo regulation of thrombin receptors, since its use
circumvents the problem of endogenous thrombin receptor activation.
Further studies should help to define regulatory sites of the receptor
involved in recognition of the appropriate kinases.