BRIEF DEFINITIVE REPORT:
Inhibition of Constitutive Signaling of Kaposi's
Sarcoma-associated Herpesvirus G Protein-Coupled
Receptor by Protein Kinases in Mammalian Cells in Culture
By
Elizabeth
Geras-Raaka,*
Leandros
Arvanitakis,
Carlos
Bais,§
Ethel
Cesarman,
Enrique A.
Mesri,§
and
Marvin C.
Gershengorn*
From the * Division of Molecular Medicine, the Department of Medicine, the
Department
of Pathology, and the § Division of Hematology-Oncology, the Department of Medicine, Cornell
University Medical College, New York 10021
 |
Abstract |
Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, which is consistently
present in tissues of patients with Kaposi's sarcoma and primary effusion lymphomas, contains a
gene that encodes a G protein-coupled receptor (KSHV-GPCR). We recently showed that
KSHV-GPCR exhibits constitutive signaling via activation of phosphoinositide-specific phospholipase C and stimulates cell proliferation and transformation. In this study, we determined whether normal cellular mechanisms could inhibit constitutive signaling by KSHV-GPCR and
thereby KSHV-GPCR-stimulated proliferation. We show that coexpression of GPCR-specific kinases (GRKs) and activation of protein kinase C inhibit constitutive signaling by KSHV-GPCR in COS-1 monkey kidney cells and in mouse NIH 3T3 cells. Moreover, GRK-5 but
not GRK-2 inhibits KSHV-GPCR-stimulated proliferation of rodent fibroblasts. These data
provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic
targets in human diseases involving constitutively active receptors.
 |
Introduction |
Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a gammaherpesvirus with homology to Herpesvirus saimiri and Epstein-Barr virus, which are
viruses that cause transformation of lymphocytes (1, 2). Accumulating evidence suggests that KSHV may be involved
in the pathogenesis of primary effusion (or body cavity-based) lymphomas (3) and Kaposi's sarcoma (KS) (4). KSHV
contains a gene that encodes a G protein-coupled receptor
(KSHV-GPCR; references 5, 6). We recently showed that
KSHV-GPCR exhibits constitutive signaling, that is, signaling in the absence of agonist, via activation of phosphoinositide-specific phospholipase C (PLC) (7). Moreover,
expression of KSHV-GPCR stimulates cell proliferation (7)
and causes transformation of mouse fibroblasts (8). KSHV-GPCR is homologous to a GPCR encoded by H. saimiri
(ECRF3) (9) and to human IL-8 receptors (human chemokine receptors CXCR1 and CXCR2; references 10, 11). However, ECRF3, CXCR1, and CXCR2 do not exhibit
constitutive signaling although they appear to signal via
PLC, like KSHV-GPCR. When activated by chemokines
in a monkey kidney (COS) cell line, CXCR1 and CXCR2 stimulate the formation of inositol phosphate second messenger molecules (IPs) when G protein
subunit G
16 is
coexpressed (12). In Xenopus laevis oocytes, chemokine activation of ECRF3 leads to calcium efflux that is likely
caused by PLC-mediated generation of inositol-1,4,5-trisphosphate (13).
GPCRs in general, and chemokine GPCRs specifically,
may be desensitized by GPCR-specific kinases (GRKs) and
by second messenger-activated protein kinases such as protein kinase C (PKC) (14). For phosphorylation and desensitization by GRKs, GPCRs must be in an active state. Desensitization occurs because GPCRs phosphorylated by
GRKs bind arrestin proteins and this complexation inhibits
GPCR coupling to G proteins. In general, GPCRs must be
occupied by an agonist to be activated and, therefore, most
unoccupied GPCRs are not phosphorylated by GRKs and
are not desensitized. Constitutively active GPCRs, however, are GRK substrates and may be desensitized even in
the absence of agonist (15, 16). GRKs themselves are activated by several mechanisms including binding to activated GPCRs, interacting with phospholipids, and binding to G
protein 
subunits (14). These processes allow translocation of the GRK to the cell surface membrane, but phosphorylation is limited by accessibility to activated GPCRs.
In contrast to the mechanism of desensitization mediated
by GRKs, second messenger-activated protein kinases must
be activated and then may phosphorylate, and thereby desensitize, inactive or active GPCRs. The mechanism whereby
second messenger-activated protein kinases inhibit coupling of receptors to G proteins has not been determined.
Chemokine receptors are substrates for these kinases and
may be desensitized by GRKs and PKC. For example, the
monocyte chemoattractant protein-1 receptor (CCR2B)
that is a GPCR for CC chemokines has been shown to be
desensitized by GRK3 but not by GRK1 or 2 (17).
CXCR1 and CXCR2 appear to undergo GRK-induced
desensitization in neutrophils (18). In fact, CXCR1 appears
to be desensitized by both GRKs and PKC (19). In this report, we show that increases in GRK and PKC activities
lead to inhibition (desensitization) of constitutive signaling
by KSHV-GPCR.
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Materials and Methods |
COS-1 cells were transfected by the DEAE-dextran method
with plasmid encoding KSHV-GPCR (pcKSHV-GPCR) as described (7). In different experiments, plasmids encoding GRK2,
4, 5, or 6, CXCR2, or G
16 were transfected. All transfection cocktails were made to contain equal amounts of plasmid DNA by the
addition of appropriate amounts of vector. 24 h after transfection,
cells were harvested and reseeded in 12-well plates in Dulbecco's
modified Eagle's medium with 10% NuSerum and 1 µCi/ml myo-
[3H]inositol (where indicated), and maintained in culture in 5% CO2 at 37°C for an additional 24 h. After removing the culture medium and washing with buffer, cells were incubated in Hank's balanced salt solution containing 25 mM Hepes, pH 7.4, and 10 mM LiCl. Cells were harvested after 90 min and IP accumulation was measured using ion exchange chromatography as described
previously (20). IP accumulation was measured as the 3H-radioactivity in IPs as a fraction of the 3H-radioactivity in inositol lipids
plus IPs (lipids + IPs).
Competition binding experiments were performed as described
(7). In brief, COS-1 cells were transfected with plasmid encoding KSHV-GPCR (10 µg/ml) in the absence or presence of plasmid
encoding GRK5 (3 µg/ml) and tested for IL-8 binding 48 h after
transfection. Approximately 100,000 cells/well were washed with
Hank's balanced salt solution containing 25 mM Hepes, pH 7.4. Between 0.3 and 0.5 nM 125I-IL-8 in the absence or presence of
various concentrations of unlabeled IL-8 was added and binding
was allowed to proceed at 4°C for 2 h. The binding buffer contained bovine serum albumin (1 mg/ml), bacitracin (1 mg/ml),
and phenyl methyl sulfonyl fluoride (1 mM). Assays were terminated by aspirating the binding buffer, washing three times with
chilled buffer, and solubilizing the cells with 0.4 N NaOH. An
aliquot of this solution was counted in a gamma counter and the
data were expressed as cpm bound. Binding curves were analyzed
using Prism software (GraphPad, Inc., San Diego, CA).
For experiments with NIH 3T3 cells or NRK cells, the cDNA
encoding KSHV-GPCR was subcloned into plasmid pCEFL (a
gift from Dr. Silvio Gutkind, National Institute of Dental Research,
National Institutes of Health). NIH 3T3 cells and NRK cells were
transfected by the calcium phosphate method with plasmid encoding KSHV-GPCR (pCEFLKSHV-GPCR) or with pCEFL
without or with plasmids encoding GRK2, 4, 5, or 6. On the day
after transfection, the medium was removed and the cells were
incubated in Dulbecco's modified Eagle's medium with 10% calf
serum and 0.75 mg/ml Geneticin. Cell populations were maintained in medium containing Geneticin. Experiments were performed 5-9 d after transfection.
Materials.
Plasmids encoding bovine GRK2, human GRK4,
bovine GRK5, and human GRK6 were provided by Dr. Robert
J. Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC). Plasmid encoding G
16 was
provided by Dr. Melvin I. Simon (California Institute of Technology, Pasadena, CA). Plasmid encoding CXCR2 was provided
by Dr. Lijun Wu (LeukoSite, Inc., Cambridge, MA). COS-1 and
NIH 3T3 cells were from the American Type Culture Collection (Rockville, MD). myo-[3H]inositol and 125I-IL-8 were purchased
from DuPont New England Nuclear (Boston, MA). Dulbecco's
modified Eagle's medium, Geneticin, calf serum, and Hank's balanced salt solution were from GIBCO BRL (Gaithersburg, MD).
Nu-Serum was from Collaborative Biomedical Products (Bedford, MA). Recombinant human IL-8 was from R&D Sys. Inc.
(Minneapolis, MN). All other chemicals were from Sigma Chemical
Co. (St. Louis, MO).
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Results and Discussion |
To determine whether constitutive signaling by KSHV-GPCRs could be inhibited by GRKs, we coexpressed
KSHV-GPCR and GRK2, 4, 5, or 6 in COS-1 cells. We
measured formation of IPs to assess signaling. Expression of
GRK2, 4, 5, or 6 alone had no effect on IP formation (data
not shown). Fig. 1 a illustrates that GRKs 4, 5, and 6, but
not GRK2, inhibited KSHV-GPCR stimulation of IP formation by 41-63%. The lack of effect of GRK2 on KSHV-GPCR signaling was not due to lack of its expression because GRK2 inhibited agonist-stimulated signaling by the
thyrotropin-releasing hormone receptor by 20 ± 9.4% in
parallel incubations. Of note, GRKs 4, 5, and 6 appear to
comprise a distinct subfamily of receptor-specific kinases (14).
Specificity of desensitization by different GRKs has been
observed (21, 22). Fig. 1 b confirms that GRK5 inhibits KSHV-GPCR signaling and shows that the extent of inhibition of IP formation by GRK5 was similar, ~53%, at all
levels of KSHV-GPCR expression. This level of inhibition
is similar to that found for inhibition of agonist-stimulated
IP second messenger formation when
1B-adrenergic receptors and GRKs were coexpressed in COS-7 cells (22).

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Fig. 1.
Effect of coexpression of KSHV-GPCR and GRKs on IP
formation in COS-1 cells. (a) Comparison of the effects of GRK2, 4, 5, and 6. COS-1 cells were cotransfected with plasmids encoding KSHV-GPCR (3 µg/ml) and one of the GRKs (3 µg/ml), and IP accumulation
was measured after 90 min. IP accumulation in cells expressing KSHV-GPCRs (Control) is from 4-10-fold higher than in untransfected or
"mock-transfected" (cells transfected with pcDNA3.1(+) alone) COS-1
cells, which was 0.038 ± 0.0046 disintegrations per minute (dpm) in IPs/
(dpm in IPs + dpm in lipids) per 90 min. Data represent the mean ± SEM of four experiments. (b) Effect of expressing various levels of
KSHV-GPCR on inhibition of IP accumulation by GRK5. COS-1 cells were cotransfected with various amounts of plasmid encoding KSHV-GPCR (from 0.1 to 3 µg/ml) in the presence or absence of plasmid encoding GRK5 (3 µg/ml). Data represent the mean ± SD of triplicate determinations in one of two experiments.
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To determine whether the effect of GRK5 to inhibit IP
formation was general or whether it may be specific for
KSHV-GPCR, we coexpressed GRK5 and CXCR2. Because chemokine signaling via CXCR2 in COS cells requires G
16 (or G
14 or G
15) (12), we coexpressed G
16
also. Coexpression of G
16 does not enhance KSHV-GPCR
signaling in COS-1 cells (7). We reasoned that it may be
necessary to activate CXCR2 before measuring the effect
of the GRK on agonist-stimulated signaling so as to have
activated CXCR2 as a substrate for GRK-mediated phosphorylation. Therefore, we measured the effects of GRK5
on IL-8 stimulation of IP formation via CXCR2 when IL-8
was added acutely or added for 1 or 48 h before IP measurement. As we showed previously (7), IL-8 stimulated IP
formation via CXCR2 to a level only 55-65% of that stimulated constitutively by KSHV-GPCR under these conditions. In this series of experiments, KSHV-GPCR stimulation of IP formation was inhibited 63% by coexpression of
GRK5 (Fig. 2). In contrast, GRK5 had no effect on IL-8
stimulation of IP formation via CXCR2 whether IL-8 was
present only during the period of IP measurement or chronically. Thus, it appears that GRK5 does not desensitize all
GPCRs under these conditions, but there is specificity to
the effect of GRK5 on KSHV-GPCR signaling.

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Fig. 2.
Effect of expressing GRK5 on IP accumulation in COS-1
cells expressing human CXCR2. COS-1 cells were cotransfected with
plasmid encoding KSHV-GPCR (3 µg/ml) or with plasmids encoding
human CXCR2 (3 µg/ml), G 16 (2 µg/ml), and a plasmid encoding
GRK5 (3 µg/ml). CXCR2 expressing cells were treated with IL-8 (0.1 µM)
chronically (for 1 or 48 h) or acutely (during measurement of IP accumulation). There was no difference between cells exposed to IL-8 for 1 or 48 h.
Human recombinant IL-8 was added to a final concentration of 0.25 µg/ml. Data represent the mean ± SEM of three experiments.
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It was recently proposed that GRKs (and arrestin) are involved in GPCR internalization (23). Because enhanced
rates of internalization can lead to increased rates of receptor degradation (24), it was possible that the decreased signaling observed when KSHV-GPCR and GRK5 were coexpressed was secondary to decreased receptor levels due to
increased receptor turnover. Decreased receptor expression
might have occurred by other mechanisms also. We found that the levels (and affinities, equilibrium inhibitory constants of 35 ± 12 nM) of KSHV-GPCRs were not different
in COS-1 cells expressing KSHV-GPCR alone or KSHV-GPCR and GRK5 (data not shown). Thus, GRK inhibition of KSHV-GPCR signaling occurred without a change
in KSHV-GPCR expression.
We considered the possibility that KSHV-GPCR signaling could be inhibited by PKC also. Fig. 3 illustrates that
phorbol 12-myristate 13-acetate (PMA) causes a dose-dependent inhibition of IP formation by KSHV-GPCR. Only active phorbol esters, PMA (0.1 µM, to 36 ± 4.7% of control), and phorbol 12,13-didecanoate (0.1 µM, to 66 ± 5.4%
of control), but not an inactive phorbol ester, 4
-phorbol 12,13-didecanoate (0.1 µM, to 96 ± 5.5% of control), inhibited KSHV-GPCR-stimulated IP formation. These effects occur exclusively via activation of PKC because they
are prevented by a specific PKC inhibitor, calphostin C
(25; data not shown). As was found for CXCR1 (19), we
found that PMA inhibited IL-8 stimulation of IP formation
via CXCR2 (data not shown). Thus, KSHV-GPCR signaling, like signaling by CXCR1 and CXCR2, is inhibited
by PKC activation.

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Fig. 3.
Inhibition of KSHV-GPCR stimulation of IP formation by
PMA. COS-1 cells were transfected with plasmid encoding KSHV-GPCR (3 µg/ml) in the absence or presence of plasmid encoding GRK5
(3 µg/ml) and IP accumulation was measured after 90 min. PMA was added 20 min before the experimental incubation at the final concentrations shown. Data represent the mean ± SD of triplicate determinations in one of two experiments.
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Fig. 4 illustrates that the effects of expression of GRK4,
5, or 6 and PKC activation on constitutive IP formation
stimulated by KSHV-GPCR are additive. GRK2 had no
effect on KSHV-GPCR-stimulated IP formation and did
not affect inhibiton by PMA. In this series of experiments,
GRK4, 5, or 6 inhibited IP formation by 56-69%, PMA
inhibited IP formation by 65%, and the combination of expression of GRK4, 5, or 6 and activation of PKC by PMA
inhibited IP formation by 85-90%.

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Fig. 4.
Additive effects of GRK expression and PMA on IP formation stimulated by KSHV-GPCR. COS-1 cells were transfected with plasmid encoding KSHV-GPCR (3 µg/ml) in the absence or presence of
plasmids encoding GRK2, 4, 5, or 6 (3 µg/ml). PMA was added 20 min
before the experimental incubation and IP accumulation was measured
for the subsequent 90 min. Data represent the mean ± SEM of two experiments.
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To show that GRK-mediated inhibition of KSHV-GPCR
signaling was not peculiar to COS cells in which proteins
may be markedly overexpressed, and that these protein kinases would inhibit an important cellular response to KSHV-GPCR, we constructed NIH 3T3 and NRK cell populations expressing KSHV-GPCRs, KSHV-GPCRs and GRK2,
or KSHV-GPCRs and GRK5. Mouse NIH 3T3 cells and
rat NRK cells are good models because proliferating fibroblasts are typically present in KS lesions (4) and KSHV-like
viruses have recently been found in retroperitoneal fibromatosis tissues in monkeys (26). Fig. 5 shows that, as in
COS-1 cells, expression of GRK5 inhibited IP accumulation in NIH 3T3 cells; GRK2 had no effect. More importantly, expression of GRK5, but not GRK2, inhibited proliferation of NIH 3T3 cells (Fig. 5). GRK4, 5, and 6, but
not GRK2, inhibited KSHV-GPCR-stimulated proliferation of NRK cells by >75% (data not shown). Thus, inhibition of KSHV-GPCR signaling by expression of GRK5
was found in COS-1 and NIH 3T3 cells and of KSHV-GPCR stimulation of proliferation in NIH 3T3 and NRK
cells. We recently showed that expression of KSHV-GPCR induces transformation of NIH 3T3 cells and that
transformation is inhibited by coexpression of GRK5 but
not of GRK2 (8).

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Fig. 5.
Effect of expressing GRK2 or GRK5 on KSHV-GPCR-
stimulated IP accumulation and proliferation in NIH 3T3 cells. NIH 3T3
cells expressing KSHV-GPCRs (Control), KSHV-GPCRs and GRK2
(+GRK2), or KSHV-GPCRs and GRK5 (+GRK5) were used 8 or 9 d
after transfection. For IP experiments, cells were labeled with [3H]myo-inositol for 48 h and IP accumulation was measured during a 90 min incubation. IP accumulation in cells expressing KSHV-GPCR alone was
three- to fivefold greater than in mock-transfected cells (cells transfected
with pCEFL). Data represent the mean ± SD of triplicate determinations
in two experiments. For proliferation experiments, cells were harvested
and seeded 5 d after transfection and cells were counted 4 d later. Data
represent the mean ± SD of triplicate determinations in two experiments.
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Constitutive signaling activity has been exhibited by
some native GPCRs (27). More commonly, however,
agonist-independent signaling has been observed with mutated GPCRs including a number that have been associated
with human diseases (30), including nonmalignant tumors
(31, 32). Moreover, GPCRs have been found to be oncogenic under some circumstances (33). Although Herpesvirus saimiri is an oncogenic virus (1), it is not known whether the GPCR encoded within the genome of this virus is involved in the transformation process. As KSHV is found in
primary effusion lymphomas and KS tissues (3, 4, 34) and
KSHV-GPCR stimulates proliferation of fibroblasts in tissue culture (7), it is possible that KSHV-GPCR is causally
involved in tumorigenesis in these diseases. However, this
has not yet been shown.
We (7) had previously suggested that if KSHV-GPCR
were shown to be involved in the pathogenesis of primary
effusion lymphoma or KS it would be a target for drug
therapy using a negative antagonist (or inverse agonist). An
inverse agonist is an antagonist that inactivates constitutively active receptors even in the absence of an agonist
(35). Other investigators have suggested that the protein kinases involved in desensitization of GPCRs may be drug
targets (36). In this report, we have shown that coexpression of GRK4, 5, or 6 as well as direct activation of PKC
inhibit constitutive signaling by KSHV-GPCR. Moreover,
inhibition of KSHV-GPCR signaling by GRKs causes inhibition of KSHV-GPCR-stimulated cell proliferation (Fig.
5) and transformation (8). Thus, drug-mediated activation of GRK4, 5, or 6 or of PKC might inhibit tumorigenesis.
The GRKs would be the preferred targets as their activation would presumably lead to receptor-specific effects.
In conclusion, to our knowledge, this is the first demonstration of GRK and PKC inhibition of constitutive signaling by a GPCR associated with human disease. Although
the molecular mechanism(s) of KSHV-GPCR desensitization was not proved, these data provide evidence that cell
regulatory pathways of receptor desensitization may be therapeutic targets in diseases involving constitutively active receptors. Based on the findings reported here, we suggest that these protein kinases, along with the KSHV-GPCR itself, may be therapeutic targets for pleural effusion lymphomas and KS if KSHV-GPCR is shown to be involved in
the pathogenesis of these diseases.
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Footnotes |
Received for publication 27 October 1997 and in revised form 5 December 1997.
We thank Bianca Santomasso for excellent technical assistance.
This work was supported by National Institutes of Health grants DK-43036 (to M.C. Gershengorn), CA-73531 (to E. Cesarman) and AI-39192 (to E.A. Mesri).
Address correspondence to Marvin C. Gershengorn, Cornell University Medical College, 1300 York Ave.,
Rm A328, New York, NY 10021. Phone: 212-746-6275; Fax: 212-746-6289; E-mail: mcgersh{at}mail.med.cornell.edu
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