From the Department of Physiology and Pharmacology, Sackler Faculty
of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel and the
Department of Physiology and § Division of
Molecular Medicine, Weill Medical College of Cornell University,
New York, New York 10021
Received for publication, July 18, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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We coexpressed Kaposi's sarcoma-associated
herpesvirus G protein-coupled receptors (KSHV-GPCRs) with
thyrotropin-releasing hormone (TRH) receptors or
m1-muscarinic-cholinergic receptors in Xenopus oocytes and
in mammalian cells. In oocytes, KSHV-GPCR expression resulted in
pronounced (81%) inhibition (heterologous desensitization) of
Ca2+-activated chloride current responses to TRH and
acetylcholine. Similar inhibitions of cytoplasmic free Ca2+
responses to TRH were observed in human embryonic kidney HEK 293 EM
cells and in mouse pituitary AtT20 cells. Further study of oocytes
showed that this inhibition was partially reversed by
interferon- KSHV-GPCR,1 the gene
product of ORF74 in the KSHV (human herpesvirus 8) genome, is a
GPCR homologous to human chemokine CXC receptor 2 (1, 2). Our group
demonstrated that rodent fibroblasts expressing KSHV-GPCRs formed
tumors in mice (3). Recently, transgenic mice in which the KSHV-GPCR
gene was regulated by the human CD2 enhancer/promoter and locus control
region were shown to develop angioproliferative lesions that resembled
the lesions of KS (4). These reports make the mechanism of action of
KSHV-GPCR interesting in terms of tumorigenesis. We reported that
KSHV-GPCR exhibited marked constitutive, agonist-independent activity
in mammalian cells (2). We also demonstrated that the constitutive activity of the viral GPCR could be inhibited by IP-10, acting as an
inverse agonist (5). These results were later confirmed by Rosenkilde
et al. (6). Last, we showed that signaling by KSHV-GPCR
could be inhibited by GPCR-specific protein kinases and suggested that
this may represent a form of homologous desensitization of this viral
receptor (7). However, the effect of the constitutive activity of
KSHV-GPCR on signaling by other receptors (heterologous desensitization) has not been previously investigated.
To investigate the effect of constitutive signaling by KSHV-GPCR on
other receptors that utilize the phosphoinositide/calcium signaling
pathway, we studied the behavior of this receptor in Xenopus
oocytes and in mammalian cells. Our results show that KSHV-GPCR causes
marked desensitization of responses mediated by TRH-Rs and m1-Rs by
inhibiting InsP3-stimulated mobilization of
Ca2+.
Xenopus laevis Oocytes--
Defolliculated oocytes were obtained
from mature Xenopus females, essentially as previously
described (8). In vitro transcribed cRNAs for
wild type TRH-Rs, m1-Rs (1 ng/oocyte), gastrin-releasing peptide (GRP)
receptors (0.23 ng/oocyte), and KSHV-GPCRs (1-3 ng/oocyte) were
injected 48 h before the assay as described previously (9-11).
Electrophysiology--
Two-electrode voltage clamp measurements
were performed at VH = 45Ca2+ Efflux
Measurements--
45Ca2+ efflux studies were
performed essentially as described previously (10), with the following
modifications. Oocytes were either loaded with
45Ca2+ by overnight incubation in a small
volume of ND96 (CaCl2 concentration reduced to 0.6 mM) in the presence of 2 mCi/ml of
45CaCl2 or injected with ~120,000 cpm of
carrier-free 45CaCl2. At various times after
injection, oocytes were rinsed in ND96 and incubated each in 10 µl of
ND96. At the desired time intervals, a sample of 5 µl was withdrawn
and replaced with 5 µl of ND96 containing the desired additions,
whenever required. At the end of the experiment, the residual
radioactivity in each oocyte was determined. All results were presented
as fractional efflux rates (percentage of residual label/min).
Calcium Mobilization in Mammalian Cells--
Calcium
mobilization was assayed in HEK 293 EM cells and AtT20 pituitary cells
by ratiometric determination of [Ca2+]i using
fura-2 as previously described (14, 15). HEK 293 EM cells were
transiently transfected with TRH receptor cDNA (0.25-1 µg/ml) or
cotransfected with TRH-R (0.25-1 µg/ml) and KSHV-GPCR (1 µg/ml)
cDNAs using the calcium phosphate method as described (16). After
48 h, cells were used in binding and in calcium mobilization
experiments. [3H]MeTRH binding was performed in 24-well
dishes as described (17). Cells expressing similar numbers of TRH-Rs
were studied. Cells expressing TRH-Rs and KSHV-GPCRs expressed TRH-Rs
at levels that were 103 ± 8.8% those in cells expressing TRH-Rs
alone. For calcium measurements, cells grown to confluence (2 days
after plating) on 22-mm square standard glass coverslips (number 1) and
maintained in Dulbecco's modified Eagle's medium containing 5% fetal
bovine serum at 37 °C, 5% CO2 were loaded with the
membrane-permeant form of the calcium indicator fura-2 acetoxymethyl
ester (1 µM) for 20-30 min at 37 °C. After loading
with the dye, cells were rinsed with HBSS, and the coverslip with the
fura-2-loaded cells was attached to the bottom of a chamber and mounted
on the stage of an inverted epifluorescence microscope (Nikon Diaphot).
The cells in the chamber were bathed in HBSS (200 µl) and maintained at room temperature. Cells were visualized under transmitted light with
a Nikon CF Fluor oil immersion objective (40×/1.3 NA) before starting
the fluorescence measurements. After monitoring the basal 340/380
fluorescence signal in the cells in the field of view, TRH was added in
a 100-µl volume to the 200-µl chamber volume to yield a final
concentration of ~1.0 µM. In certain experiments, calibration of the emitted fura-2 fluorescence signal from each cell in
the field was performed in the presence of the Ca2+
ionophore, ionomycin (10 µM), and either HBSS or HBSS
with 10 mM EGTA. Intracellular Ca2+ levels were
then calculated as described by Grynkiewicz et al. (18).
Each cell in the field of view was calibrated with its own
corresponding Rmin and
Rmax as previously described (15). Individual
vials (50 µg) of the acetoxymethyl derivative of fura-2 were stored
dry at 0 °C and reconstituted in dimethyl sulfoxide, at a
concentration of 1 mM, for each experiment. The basic
components of the experimental apparatus have been described previously
(15, 19). Briefly, the imaging work station was controlled with the Metafluor software package (Universal Imaging, Westchester, PA). Image
pairs were obtained every 0.1 s for the duration of the experiment
at 340- and 380-nm excitation with emission at 510 nm. The fluorescence
excitation was shuttered off except during the brief periods required
to record an image. To check for interference from intrinsic
autofluorescence and background, images were obtained on unloaded cells
using the same exposure time and filter combinations used for the
experiments and found to be an insignificant component of the
fluorescence signal. To determine the effect of KSHV-GPCR expression on
TRH responses in another mammalian cell line, AtT20 cells stably
expressing TRH-Rs were transiently cotransfected with two vectors:
pcDNA3.1(+), with KSHV-GPCR coding DNA inserted between
XbaI and EcoRI restriction sites and pIT (20)
coding for GFP mutant Topaz®. The Topaz® GFP mutant has
excitation-emission (514/527 nm) characteristics that do not interfere
with fura-2 imaging at 340/380-nm excitation and 515-nm emission. GFP
fluorescence was visualized using standard fluorescein isothiocyanate
block. The two vectors were transfected using LipofectAMINE(+),
according to the instructions of the manufacturer. Briefly, number 1 22-mm diameter round coverslips were seeded with AtT20 cells to low density (<30% confluence) and transfected in 1 ml of medium with the
two vectors (0.8 µg of KSHV-GPCR and 0.2 µg of GFP DNAs/well). The
coverslips were analyzed for calcium mobilization 48-72 h post-transfection, using the Life Sciences Miracal® system at 1-2
s/frame pair acquisition rate. Responses to 5 µM
TRH were recorded at 37 °C.
Chemicals and Solutions--
TRH, Acetylcholine, Gro Statistics--
All experiments were performed on a large number
of oocytes (n) from a number of donors (N). In
calcium imaging experiments, several coverslips were assayed for each
condition (N), and the number of cells was denoted by
n. Results were presented as mean ± S.E. All results
were analyzed by unpaired Student's t test with
p < 0.05 considered as a significant difference
between two populations.
Expression of KSHV-GPCRs in Xenopus oocytes did not
affect whole cell chloride currents or membrane potential in the
absence of agonist. Moreover, there was no current response when the
oocytes were exposed to several chemokine agonists (data not shown).
However, expression of KSHV-GPCRs caused marked inhibition of
Ca2+-activated chloride current responses to the cognate
agonists of other coexpressed GPCRs. When 1-3 ng of KSHV-GPCR RNA was
injected, the response to 1 µM TRH was inhibited from
2980 ± 148 to 576 ± 168 nA (81% inhibition,
n = 145, N = 14) and the response to 10 µM acetylcholine (mediated by m1-Rs) was inhibited from
2067 ± 117 to 390 ± 44 (81% inhibition, n = 224, N = 22), and in a separate experiment in oocytes
expressing the receptor for GRP, the response decreased from 2211 ± 426 to 675 ± 251 nA (70% inhibition, n = 11, N = 1). Representative responses to TRH are shown in
Fig. 1a and mean amplitudes in
Fig. 1b. The extent of inhibition was dose-dependent on the amount of KSHV-GPCR RNA injected
(Fig. 2).
-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of 45Ca2+ efflux
in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control
oocytes, and IP-10 rapidly inhibited increased 45Ca2+ efflux. In the absence of IP-10,
growth-related oncogene
caused a further 2-fold increase in
45Ca2+ efflux. In KSHV-GPCR-expressing oocytes,
responses to microinjected inositol 1,4,5-trisphosphate were inhibited
by 74%, and this effect was partially reversed by
interferon-
-inducible protein 10. Treatment with thapsigargin
suggested that the pool of calcium available for mobilization by TRH
was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest
that constitutive signaling by KSHV-GPCR causes heterologous
desensitization of responses mediated by other receptors, which signal
via the phosphoinositide/calcium pathway, which is caused by depletion
of intracellular calcium pools.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 mV, as previously
described (10, 12). Chloride currents were continuously recorded.
Intracellular injections of InsP3 were performed using a
Drummond microinjector (volume 4.6-9.2 nl/oocyte) as previously
described (13).
,
InsP3, and IP-10 were purchased from Sigma (Israel); GRP
was from Bachem. 45CaCl2 was purchased from
Amersham Pharmacia Biotech. Thapsigargin was purchased from Alomone
Laboratories, Jerusalem. GFP Topaz® was from Packard. LipofectAMINE(+)
was purchased from Life Technologies. Fura-2/AM was a product of
Teflabs or Molecular Probes, Inc. (Eugene, OR). Ionomycin was from
Calbiochem. All other chemicals were of analytical grade. ND96 solution
was used in all short term incubations, and NDE solution was
used for culturing of oocytes (21).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of KSHV-GPCR inhibits responses to
agonists of other GPCRs in oocytes. Oocytes expressing TRH-Rs,
m1-Rs, or GRP receptors, either alone or together with KSHV-GPCR, were
assayed for responses to cognate agonists: TRH (1 µM),
acetylcholine (10 µM), or GRP (0.1 µM).
A, representative tracings of a response to TRH in oocytes
expressing TRH-Rs alone (left) or coexpressing TRH-Rs and
KSHV-GPCRs (right). The arrows indicate the time
of the addition of TRH. B, mean amplitudes of
responses.
View larger version (10K):
[in a new window]
Fig. 2.
Inhibition of the response to TRH as a
function of the amount of KSHV-GPCR RNA. Oocytes injected with RNA
coding for TRH-Rs (1 ng/oocyte) alone or together with increasing
amounts of RNA coding for KSHV-GPCRs (0.3-3.0 ng/oocyte) were assayed
for responses to 1 µM TRH. A representative experiment
(n = 15) is shown.
We tested whether the apparent heterologous desensitization of
responses to TRH in oocytes could be observed in mammalian cells. In
human embryonic kidney HEK 293 EM cells, we included in our analysis
only cells that were shown to respond to TRH. This most likely biased
the analysis against showing an effect of coexpression of KSHV-GPCR,
because 60% of the cells (n = 474, N = 5) in the population expressing TRH-Rs responded to TRH, whereas 47%
of the cells (n = 430, N = 5) in the
population expressing TRH-Rs and KSHV-GPCRs responded to TRH. It is
likely, therefore, that some cells in the population expressing TRH-Rs
and KSHV-GPCRs had no measurable response to TRH although they
expressed TRH-Rs; i.e. KSHV-GPCR totally inhibited the
response to TRH in these cells. These cells were not included in the
analysis. HEK 293 EM cells transiently expressing TRH-Rs or TRH-Rs and
KSHV-GPCRs exhibited similar "basal" [Ca2+]i;
in TRH-R-expressing cells, basal [Ca2+]i was
82 ± 3 nM (n = 247, N = 3), and in TRH-R- and KSHV-GPCR-expressing cells, basal
[Ca2+]i was 87 ± 4 nM
(n = 201, N = 3). Typical responses to
TRH are illustrated in Fig. 3. In
TRH-R-expressing cells, there was a rapid increase in
[Ca2+]i that peaked 12 s after TRH addition
and declined rapidly, whereas in cells expressing TRH-Rs and KSHV-GPCRs
there was a slower increase to a lower maximum followed by a slower
decline. These time courses and amplitudes of responses are similar to those observed with control and desensitized TRH-Rs in HEK 293 cells
(22). The peak increase in the fura signal was inhibited by 53 ± 2% in cells expressing TRH-Rs and KSHV-GPCRs compared with cells
expressing TRH-Rs, corresponding to estimated increases in
[Ca2+]i of approximately 400 and 200 nM, respectively.
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AtT20 pituitary cells stably expressing TRH-Rs (11) were transiently cotransfected with DNAs coding for KSHV-GPCR and for the Topaz mutant of GFP (see "Experimenal Procedures"). We assumed that cells transfected with GFP were likely to be also cotransfected with the KSHV-GPCR vector. Neighboring cells that displayed little or no GFP fluorescence were used as internal controls. In parallel, cells from the same batch, transfected with 0.2 µg of GFP-containing vector alone and exhibiting GFP fluorescence, were assayed as negative controls, reflecting the effect of GFP expression alone. In control cells, the net increase in [Ca2+]i was 646 ± 68 nM (9.2 ± 0.8-fold basal, n = 22, N = 5), while in cells expressing the viral receptor, the net increase in [Ca2+]i was only 301 ± 70 nM (4.9 ± 1.0-fold basal, n = 14, N = 5), i.e. 53% inhibition. By comparison, the response in cells transfected with GFP DNA alone was 504 ± 31 nM (9.4 ± 1.0-fold basal, n = 13, N = 3).
We previously showed that a constitutively active TRH-R mutant causes
homologous and heterologous desensitization of responses assayed in
Xenopus oocytes. This desensitization was fully or partially
abolished by chlordiazepoxide, an inverse agonist of TRH-R (14). The
heterologous desensitization caused by coexpression of KSHV-GPCRs with
other receptors may therefore be attributed to the constitutive
activity of the viral receptor. To test this hypothesis, we used IP-10,
which was shown to act as an inverse agonist of KSHV-GPCRs in mammalian
cells in culture (5), to inhibit basal signaling. Incubation of oocytes
coexpressing TRH-Rs and KSHV-GPCRs with 1 µM IP-10
resulted in a rapid increase in responses to TRH, i.e.
resensitization of the response. Fig. 4 illustrates the time course of resensitization of the response to TRH
in a representative experiment. The results showed a rapid increase
(within 10 min of the addition of IP-10), exhibiting a plateau around
8-9-fold the initial response at 40-80 min. This represented a
resensitization of the response to TRH, from 6% of control in
untreated oocytes to ~35% at 60-80 min of incubation with IP-10.
Similar resensitization was observed when oocytes coexpressing m1-Rs
and KSHV-GPCRs were treated with IP-10 (not shown).
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While the degree of potentiation of the desensitized response (for either TRH-Rs or m1-Rs) following incubation with IP-10 was large (a 6.2 ± 2.3-fold increase, N = 11), the degree of resensitization varied among different experiments. In some experiments, only a moderate decrease in desensitization was observed, while in some a complete reversal was seen. Overall, incubation with 1 µM IP-10 for 30-80 min resulted in resensitization to a level of 49 ± 15% of control, compared with 18 ± 16% in oocytes not treated with the inverse agonist (n = 11).
The results described here were consistent with the idea that
KSHV-GPCR-induced desensitization may be caused by a high level of
constitutive signaling by this receptor in Xenopus oocytes. It was therefore likely that persistent production of InsP3
in oocytes resulted in constitutive Ca2+ mobilization
affecting InsP3-sensitive Ca2+ stores. Indeed,
oocytes coexpressing KSHV-GPCRs with TRH-Rs exhibited significantly
higher rates of basal 45Ca2+ efflux than
oocytes expressing TRH-Rs alone (Fig. 5).
Expression of TRH-Rs did not affect the rates of basal
45Ca2+ efflux either in control oocytes or in
those coexpressing KSHV-GPCRs (data not shown). Moreover, the rate of
entry of microinjected 45Ca2+ into a pool
available for basal and agonist-induced release appeared to be much
more rapid. In oocytes expressing KSHV-GPCRs, the maximal 45Ca2+ release rate was reached within 42 ± 11 min after the injection of the radioactive tracer, while in
control oocytes the maximal rate was achieved at ~80 min (Fig. 5),
confirming our previous results (13). The maximal fractional rate of
45Ca2+ efflux in oocytes (either naive or
expressing TRH-Rs) was 0.19 ± 0.03% of residual label/min, and
in those expressing KSHV-GPCRs it was 0.83 ± 0.12% of residual
label/min (n = 22-25, N = 6) at 45-60
min after the injection of the label. IP-10 virtually abolished the
enhanced basal 45Ca2+ efflux, thereby
confirming its link to KSHV-GPCRs constitutive signaling activity. The
addition of IP-10 abolished the enhanced efflux of
45Ca2+ within 20 min (Fig.
6).
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Oocytes expressing KSHV-GPCR responded to a challenge with the cytokine
agonist Gro by a slow increase in 45Ca2+
efflux. By 20 min of exposure to 1 µM of Gro
, the
fractional rate of efflux reached 1.14 ± 0.14, while the basal
rate was 0.6 ± 0.06% of residual label/min (n = 18-19, N = 3).
The increased rate of basal 45Ca2+ efflux
caused by KSHV-GPCR was accompanied by a decrease in TRH-induced efflux
in oocytes that coexpressed KSHV-GPCRs and TRH-Rs. While in oocytes
expressing TRH-Rs alone, the net efflux of
45Ca2+ induced by 15-min incubation with 10 µM TRH was 31 ± 5% of total label, coexpression of
KSHV-GPCRs resulted in an efflux of 12 ± 4%, i.e.
61% inhibition of the TRH response (Fig.
7). This was also true for the initial
phase of efflux within the first minute (data not shown), which is more
directly related to the rapid, transient electrophysiological response
to TRH.
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To investigate the mechanism of heterologous inhibition of TRH-R- and m1-R-mediated responses, we studied the effect of KSHV-GPCR expression on InsP3-evoked Ca2+ release by monitoring chloride current responses to microinjected InsP3. The injection of 0.92 pmol of InsP3 into control oocytes (injected with RNAs coding for TRH-Rs or m1-Rs) resulted in a response of 229 ± 16 nA (n = 103, N = 8), whereas responses of 91 ± 9 nA (n = 98, N = 8) were obtained in oocytes coexpressing KSHV-GPCRs. Hence, the expression of KSHV-GPCRs caused a 60% inhibition of the response to InsP3. When responses to agonists were compared with the responses to microinjected InsP3 in the same batches of oocytes, the responses to InsP3 were inhibited by 62% and those to agonists by 86%. These results suggest that the inhibition of InsP3-evoked calcium mobilization explains most of the marked inhibition of responses mediated by TRH-Rs or m1-Rs coexpressed with KSHV-GPCRs.
IP-10 partially reversed the inhibition of responses to microinjected
InsP3 (Fig. 8A).
Incubation of oocytes coexpressing TRH-Rs and KSHV-GPCRs with 1 µM IP-10 for 30-60 min increased the response to
InsP3 from 26 to 51% of control. The time course of the
action of IP-10 on InsP3-evoked responses (Fig.
8B) was similar to that on the TRH response (see Fig. 4) and
on the enhanced efflux of 45Ca2+ (Fig. 6).
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To test the hypothesis that the constitutive signaling of KSHV-GPCR
causes depletion of the calcium pool, we used thapsigargin, which
inhibits the endoplasmic reticulum calcium ATPase and, consequently, empties the pool. Oocytes labeled with 45Ca2+
were exposed to 2 µM thapsigargin. In control oocytes,
the basal efflux was low, resulting in a cumulative loss of 16.6 ± 1.0% of total radiolabel at 150 min of incubation. The addition of thapsigargin at 30 min resulted in a major increase in efflux, leading
to a loss of 83.5 ± 2.0% of total radiolabel. In contrast, oocytes expressing KSHV-GPCR exhibited a much higher efflux (loss of
75.0 ± 3.1% of total radiolabel at 150 min of incubation). Treatment with thapsigargin led to a small, statistically insignificant increase of cumulative efflux. After 120 min with thapsigargin, the
KSHV-GPCR-expressing oocytes lost 76.0 ± 3.1% of total label. These results are shown in Fig.
9A. The effect of KSHV-GPCR's constitutive signaling on calcium pools was more apparent when fractional efflux rates were calculated (Fig. 9B). Before
the addition of thapsigargin, basal fractional efflux rates were
0.17 ± 0.03 and 0.76 ± 0.07% of residual label/min for
control or KSHV-GPCR-expressing oocytes, respectively. Upon the
addition of thapsigargin, the fractional efflux rate in control oocytes
increased rapidly, to 1.43 ± 0.16% of residual label/min 30 min
after the addition of the drug. Thereafter, the rate decreased slightly
till the end of the incubation. In oocytes expressing KSHV-GPCRs, 20 min of incubation with thapsigargin accelerated the fractional efflux rate to a maximum of 1.84 ± 0.24% of residual label/min. Further incubation with thapsigargin led to a major decrease in fractional efflux rate, indicating that the depleted pools could not support the
high rate of efflux.
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DISCUSSION |
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We previously showed that a constitutively signaling TRH-R mutant caused homologous (of TRH-R itself) and heterologous (of other coexpressed GPCRs) desensitization in Xenopus oocytes (11, 14). This desensitization was partially or fully abolished by short term incubation with chlordiazepoxide, an inverse agonist of TRH-R. KSHV-GPCR is known to signal constitutively in mammalian cells in culture, but its signaling properties in Xenopus oocytes have not been reported. The present study was designed to answer five questions relating to the biology of KSHV-GPCRs: Does KSHV-GPCR signal constitutively through the phosphoinositide-InsP3-calcium pathway in Xenopus oocytes? Can the signaling be abolished by an inverse agonist of KSHV-GPCR? Does KSHV-GPCR cause heterologous desensitization of other GPCRs when coexpressed in the same oocyte? What is the mechanism of desensitization? Does expression of KSHV-GPCR in mammalian cells result in heterologous desensitization?
The data presented herein show that KSHV-GPCRs continuously stimulate
calcium flux in oocytes. This finding is consistent with the idea that
these viral receptors continuously stimulate InsP3
generation and chronically affect cellular Ca2+ homeostasis
and, therefore, signal constitutively in oocytes. This conclusion is
supported by the finding that the specific inverse agonist IP-10
decreases calcium flux. Nevertheless, we observed no increased chloride
current in oocytes expressing the viral receptor. This represents, most
likely, a strong homologous desensitization of the response. The
sluggish increase in 45Ca2+ efflux upon
challenge with the KSHV-GPCR agonist, Gro, is compatible with the
suggestion that the viral receptor's response is homologously desensitized. The same phenomenon was found, albeit to a lower level of
stimulation, in oocytes and in HEK 293 EM cells (Fig. 3), mouse
pituitary AtT20 cells expressing a constitutively active mutant of
TRH-R, C335Stop (14).2
Our data clearly indicate that expression of KSHV-GPCRs causes pronounced (50-80%) desensitization of responses mediated by other GPCRs (TRH-Rs, m1-Rs, or GRP receptors) coexpressed in the same cell. In some experiments in oocytes, response inhibition greater than 99% was observed. This may have been true in HEK 293 EM cells also, because we found a smaller fraction of the cell population responding to TRH in populations expressing TRH-Rs and KSHV-GPCRs than in populations expressing TRH-Rs. Hence, the degree of heterologous desensitization attributable to KSHV-GPCRs greatly exceeded that caused by a constitutively active mutant TRH-R (14). To correlate the desensitization with the constitutive activity of KSHV-GPCR, we studied both basal and agonist-induced 45Ca2+ efflux as a reporter of cellular Ca2+ mobilization. Indeed, the basal fractional rate of 45Ca2+ efflux was much greater in oocytes expressing KSHV-GPCRs than in naive oocytes or those expressing other GPCRs. This enhancement of efflux was reflected both in the maximal fractional rates achieved (0.83 versus 0.19% of residual label/min) and in the time course of efflux development (reaching plateau twice as rapidly as in control oocytes). These results strongly suggest that the expression of KSHV-GPCRs is accompanied by a very rapid flux of Ca2+ through the releasable stores. Indeed, there is a direct correlation between the degree of heterologous desensitization caused by KSHV-GPCR (Fig. 1B) and the C335Stop mutant TRH-R (14) in Xenopus oocytes and the level of constitutive signaling activity observed in mammalian cells (2, 23).
Oocytes expressing KSHV-GPCRs exhibited impaired responses to InsP3 injection, suggesting that the viral receptor interferes with Ca2+ mobilization from the endoplasmic reticulum pool. The degree of inhibition of InsP3-evoked responses corresponded well with the degree of heterologous desensitization. The results observed in our system are consistent with depletion of InsP3-releasable Ca2+ pools. Indeed, experiments with thapsigargin clearly indicated that the size of releasable Ca2+ pool was much smaller in oocytes expressing KSHV-GPCRs. The finding that TRH-evoked 45Ca2+ efflux is strongly inhibited in oocytes coexpressing TRH-Rs and KSHV-GPCRs is compatible with the heterologous desensitization of the electrophysiological response and with the data that suggest major depletion of calcium pool(s).
Coexpression of KSHV-GPCRs and TRH-Rs in HEK 293 EM cells and AtT20 cells in culture resulted in 50% inhibition of TRH-evoked cytosolic calcium elevation, indicating that the viral receptor causes heterologous desensitization in mammalian cells as well.
The heterologous desensitization in oocytes could have been a result of decreased expression of TRH-Rs (or, for that matter, any other GPCRs) due to coexpression of KSHV-GPCRs. There are two findings that argue against this interpretation. First, in the Xenopus oocyte system, coexpression of two GPCRs does not result in decreased expression and mutual response inhibition but rather in potentiation of responses mediated by either receptor (11). Second, in mammalian cells, where receptor expression is easily quantified, there was no effect of cotransfection of KSHV-GPCR on the level of TRH-Rs expression under the conditions of these experiments.
The findings presented here, together with our previous findings concerning a constitutively active TRH-R mutant (11, 14, 23), strongly suggest that constitutively active receptors cause heterologous desensitization either in Xenopus oocytes or in mammalian cells. Considering the existence of pathologies resulting from mutations that cause constitutive activity and the findings that many wild type receptors exhibit some degree of constitutive activity, these novel desensitization phenomena should be further investigated.
Last, our findings may have important implications for KSHV biology in
infected cells expressing KSHV-GPCRs. Expression of KSHV-GPCRs leads to
continuous stimulation of its signaling pathways while suppressing
responses mediated by other GPCRs. Thus, in cells expressing
KSHV-GPCRs, regulation of cell function has been coopted by a viral
receptor, while the strong desensitization partially removes the
infected cell from normal control by extracellular regulatory factors.
These changes may create a cellular environment that is optimal for
viral survival.
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FOOTNOTES |
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* 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.
¶ To whom correspondence should be addressed.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M006359200
2 M. Lupu-Meiri and Y. Oron, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
KSHV-GPCR, Kaposi's
sarcoma-associated herpesvirus G protein-coupled receptor;
IP-10, interferon--inducible protein 10;
TRH, thyrotropin-releasing
hormone;
TRH-R, TRH receptor;
m1-R, m1-muscarinic-cholinergic receptor;
InsP3, inositol 1,4,5-trisphosphate;
GFP, green
fluorescent protein;
Gro
, growth-related oncogene
;
GRP, gastrin-releasing peptide;
HBSS, Hanks' balanced salt solution.
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