From the Laboratory for Molecular Pharmacology,
Department of Pharmacology, Panum Institute, University of Copenhagen,
DK-2200 Copenhagen, Denmark and the § PharmaBiotec Research
Center, Department of Medical Chemistry, Royal Danish School of
Pharmacy, DK-2100 Copenhagen, Denmark
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ABSTRACT |
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A number of CXC chemokines competed
with similar, nanomolar affinity against 125I-interleukin-8
(IL-8) binding to ORF-74, a constitutively active seven-transmembrane
receptor encoded by human herpesvirus 8. However, in competition
against 125I-labeled growth-related oncogene (GRO)- Chemokines are chemotactic cytokines that ensure that leukocytes
migrate to the right tissue or compartment at the right time. During
inflammation, chemokines are also involved, for example, in the
extravasation process and recruitment of the appropriate type of
leukocytes to the infected tissue. Furthermore, chemokines are involved
in cellular communication controlling processes such as angiogenesis
(1, 2). In a number of herpesviruses and poxviruses, genes coding for
homologs of chemokines as well as chemokine receptors have been found
(3, 4). Conceivably, these molecules have been obtained by the virus
through an ancient act of molecular piracy and subsequently are
structurally optimized for a particular pharmacological phenotype of
benefit to the virus. In the case of several of the virally encoded
chemokine ligands, their purpose appears to be rather obvious since
they act as broad spectrum chemokine antagonists, which could be used
by the virus to prevent the local recruitment of leukocytes (5, 6).
However, it is still rather unclear what the function of the virally
encoded chemokine receptors is. In general, these receptors are not
required for virus replication in vitro (7). Yet, gene
deletion experiments in both mouse and rat cytomegaloviruses have shown
that, for example, the UL33 receptor, which is homologous to the U12
receptor encoded by human herpesvirus 6 and 7, is essential for
targeting and/or replication of the virus in salivary glands (8, 9).
More important, the viral strains in which the UL33 receptor was
specifically knocked out were less virulent than wild-type
cytomegalovirus as judged from survival of infected animals (9).
ORF-74 is a CXC chemokine receptor encoded by many
In this study, we have tested a number of mammalian chemokines as well
as the viral chemokine antagonist vMIP-II to probe their ability to
affect the constitutive activity of ORF-74 from HHV8. We find that in
contrast to IL-8, other CXC chemokines such as GRO Materials--
The human chemokines were purchased from
Peprotech (IL-8, GRO Transfections and Tissue Culture--
COS-7 cells were grown at
10% CO2 and 37 °C in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, 2 mM
glutamine, and 0.01 mg/ml gentamicin. Transfection of the COS-7 cells
was performed by the calcium phosphate precipitation method (17).
NIH-3T3 cells (ATCC CRL 1658) were grown at 5% CO2 and
37 °C in Dulbecco's modified Eagle's medium supplemented with 4500 mg/liter glucose, 4 mM glutamine, 50 units/ml penicillin, 50 units/ml streptomycin, and 10% calf serum.
Binding Experiments--
COS-7 cells were transferred to culture
plates 1 day after transfection. The number of cells seeded per well
was determined by the apparent expression efficiency of the individual
clones; the goal was to obtain 5-10% specific binding of the added
radioactive ligand. Two days after transfection, cells were assayed by
competition binding performed on whole cells for 3 h at 4 °C
using 12 pM 125I-IL-8 or
125I-GRO Phosphatidylinositol Assay--
One day after transfection,
COS-7 cells (0.5 × 106/well) were incubated for
24 h with 5 µCi of myo-[3H]inositol in
1 ml/well inositol-free Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.01 mg/ml gentamicin. Cells were washed twice in 20 mM
Hepes buffer, pH 7.4, supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose, and 0.05%
(w/v) bovine serum albumin and were incubated in 1 ml of buffer
supplemented with 10 mM LiCl at 37 °C for 90 min in the
presence of various concentrations of chemokine. Cells were extracted
with 10% ice-cold perchloric acid, followed by incubation on ice for
30 min. The resulting supernatant was neutralized with KOH in Hepes
buffer, and the generated [3H]inositol phosphates were
purified on AG 1-X8 anion-exchange resin (18). Determinations were made
in duplicate.
Cell Proliferation Assay--
Receptor selection and
amplification technology assays (R-SATTM, Acadia
Pharmaceuticals Inc., San Diego, CA) were performed as described
previously (19). NIH-3T3 cells (7.5 × 105) were
plated 1 day before transfection into 10-cm Petri dishes and
transfected with 0.3 µg of receptor DNA (ORF-74 in the pTEJ8 vector)
and 4 µg of pSI- Calculations--
IC50 and EC50 values
were determined by nonlinear regression, and
Bmax values were calculated using Inplot Version
4.0 software (GraphPAD Software for Science, San Diego).
ORF-74 was cloned from a Kaposi's sarcoma skin lesion. Only a
single silent G-to-T nucleotide mutation was identified at position 907 in the coding region of ORF-74 as compared with the sequence deposited
in GenBankTM (accession number U24275). This represents a
very high degree of conservation in comparison with, for example, the
US28 receptor from human cytomegalovirus, which is widely divergent in
nucleotide and amino acid sequences among different viral strains
(21).
Binding Experiments--
Using 125I-IL-8 as tracer in
competition binding experiments in COS-7 cells transiently expressing
ORF-74, we could confirm that this virally encoded receptor binds a
number of human CXC chemokines with high and rather similar
affinity (Table I and Fig. 2,
A-C). Even the structurally rather distantly related
chemokines IP-10 and SDF-1
Previously, only IL-8 has been employed as a radioactive ligand in
binding experiments with ORF-74 (13). However, since GRO Signal Transduction Analysis--
The reported ability of ORF-74
to stimulate phosphatidylinositol turnover in a ligand-independent
manner was confirmed by gene dosage experiments in transiently
transfected COS-7 cells (Fig. 3) (13).
The inability of IL-8 to affect this constitutive signaling was also
confirmed (Fig. 4B) (13).
However, surprisingly, it was found that in contrast to IL-8, GRO Cell Transformation Assay--
Cotransfection of NIH-3T3 cells
with ORF-74 and the marker enzyme Inhibition of ORF-74 Signaling by "Non-peptide" Zinc
Ions--
Through the introduction of His residues, we have previously
both structurally and functionally exchanged a non-peptide
antagonist-binding site in the tachykinin NK1 receptor with
metal ion sites (22, 23). In contrast to binding sites for peptides and
non-peptide ligands, these metal ion sites can be transferred even to
distantly related seven-transmembrane receptors (24). To probe the
susceptibility of ORF-74 to inhibition by non-peptide inverse agonists,
two His residues were introduced at positions V:01 (Arg208)
and V:05 (Arg212), corresponding to two of the positions
previously tested in both the NK1 and This study indicates that the ORF-74 receptor encoded by HHV8 has
been optimized by the virus to recognize GRO peptides from its host as
agonistic modulators of its high constitutive activity. Inhibition of
this constitutive activity in wild-type ORF-74 by the chemokines IP-10
and SDF-1 Chemokine Ligands for ORF-74--
Although ORF-74 can be shown to
bind other CXC chemokines such as IL-8, NAP-2, and ENA-78
with nanomolar affinity in binding assays employing
125I-IL-8, these "non-GRO" chemokines are in fact at
physiological concentrations not able to interfere with either GRO
The ORF-74 gene product from Herpesvirus saimiri was
originally described as a viral IL-8 receptor (12). However, in fact, ECRF3 also binds GRO
The phenomenon that ligands (exemplified here by IL-8) can bind to a
seven-transmembrane receptor with high affinity without being able to
compete for binding against other ligands has been extensively studied,
especially in the tachykinin and opioid receptor systems (17, 26-29).
In the chemokine system, this phenomenon has previously been reported
in CXCR-2, where, for example, the affinity for NAP-2 can
vary up to 2000-fold depending upon whether it is measured against
125I-ENA-78 (0.5 nM) or against
125I-IL-8 (~1 µM) (30). The molecular or
cell biological correlate to this general phenomenon is, however, not
yet clear (29). The most simple explanation would be that the ligands
bind to two (or more) different sites on the receptor. This is,
however, hard to imagine with the relatively large chemokine ligands of this study, although it is not impossible. Another explanation could be
that ligands can bind to two (or more) distinct conformations of the
receptor that do not interchange readily (29). These conformations of
states could represent, for example, complexes with different G-protein
subtypes and/or monomeric versus dimeric forms of the
receptor. Whatever the molecular basis is, it is nevertheless a
phenomenon that has been exploited by viruses not only in the ORF-74
system, but also in other virally encoded chemokine receptors such as
US28 from human cytomegalovirus, possibly to obtain selective
recognition in systems where multiple ligands are
available.2
Modulation of Constitutive ORF-74 Signaling--
The observation
that ORF-74 is stimulated by GRO peptides and inhibited by IP-10 and
SDF-1
Due to its oncogenic properties and its effect on neovascularization,
ORF-74 appears to be an interesting drug target for the treatment of
HHV8-associated malignancies. Seven-transmembrane receptors are in
general excellent drug targets, and non-peptide antagonists have
accordingly been developed for a number of neuropeptide and peptide
hormone receptors (35, 36). The first high affinity non-peptide
antagonists for also chemokine receptors have recently been reported
(37-39). However, since we do not yet know the structural reason for
the high constitutive activity of the ORF-74 receptor, it is far from
evident that it should be possible to stop its signaling by binding of
a small non-peptide ligand to its extracellular surface. Although there
is general agreement that the binding sites for non-peptide ligands
usually are significantly different from those for the endogenous
peptide ligands (35, 40, 41), the molecular mechanism of action of
non-peptide ligands is the subject of debate. One opinion is that the
non-peptide antagonists act by blocking the binding of the agonist
through a space-filling process, although their binding sites may not
overlap directly (42). Another view is that the antagonist and agonist
bind to sites presented by different conformations and that the ligands compete for the whole receptor in an allosteric system (35). Thus, in
the original report on zinc site engineering, we suggested that
Zn2+ acted as an allosteric antagonist that stabilized an
inactive conformation, which thereby prevented the receptor from going into the active conformation (which would bind the agonist) rather than
actually directly interfering with the binding of the ligand (22). This
suggestion fits with the observations of the present study that the
metal ion binding at this location inhibits the ligand-independent signaling. From a drug development point
of view, the inhibition of ORF-74 signaling by Zn2+ in the
mutant receptor can be considered as proof of the concept that
non-peptide compounds that can block the constitutive signaling of this
viral oncogene can be developed, even though they target the
extracellular part of the receptor.
, the
ORF-74 receptor was highly selective for GRO peptides, with IL-8 being
10,000-fold less potent. The constitutive stimulating activity of
ORF-74 on phosphatidylinositol turnover was not influenced by, for
example, IL-8 binding. In contrast, GRO peptides acted as potent
agonists in stimulating ORF-74 signaling, whereas IP-10 and stromal
cell-derived factor-1
surprisingly acted as inverse agonists. These
peptides had similar pharmacological properties with regard to
enhancing or inhibiting, respectively, the stimulatory effect of ORF-74 on NIH-3T3 cell proliferation. Construction of a high affinity zinc
switch through introduction of two His residues at the extracellular end of transmembrane segment V enabled Zn2+ to act as a
prototype non-peptide inverse agonist, which eliminated the
constitutive signaling. It is concluded that ORF-74, which is believed
to be causally involved in the formation of highly vascularized tumors,
has been optimized for agonist and inverse agonist modulation by the
endogenous angiogenic GRO peptides and angiostatic IP-10 and stromal
cell-derived factor-1
, respectively. ORF-74 could serve as a target
for the development of non-peptide inverse agonist drugs as
demonstrated by the effect of Zn2+ on the metal ion
site-engineered receptor.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-herpesviruses, including the recently discovered human herpesvirus
8 (HHV8)1 or Kaposi's
sarcoma-associated herpesvirus (see Fig. 1) (10, 11). Initially, the
ORF-74 gene product from Herpesvirus saimiri, ECRF3, was
shown to bind interleukin-8 (IL-8) with high affinity even though it
structurally is only distantly related to the mammalian IL-8 receptors,
CXCR-1 and CXCR-2 (12). Interestingly, the ORF-74 receptor from HHV8 was found to be highly constitutively active (13).
Furthermore, although IL-8 binds with high affinity to ORF-74 from
HHV8, it does not affect the signaling of the receptor (13). In
contrast to other mammalian chemokine receptors, which preferentially
signal through the Gi pathway, ORF-74 activates the
phospholipase C pathway, leading to constitutively high turnover of
phosphatidylinositol, as well as signals through the c-Jun N-terminal
kinase and p38 mitogen-activated protein kinase, leading to the
production and secretion of vascular endothelial growth factor (14). As
a result of these activities, ORF-74 functions as an oncogene, leading
to cellular transformation and development of highly vascularized
tumors in nude and SCID mice (15). Consequently, it has been
proposed that ORF-74 could be causally involved in the development of
Kaposi's sarcoma lesions and lymphomas associated with HHV8
infection (14, 15). Since ORF-74 belongs to the class of rhodopsin-like
seven-transmembrane receptors, which classically are good drug targets,
it appears that it should be possible to develop antagonists or rather
inverse agonists for ORF-74 to be used in the treatment of
HHV8-associated malignancies.
are in
fact potent agonists on ORF-74; and, surprisingly we find that the
endogenous chemokines IP-10 and SDF-1
function as efficient inverse
agonists, which can block signaling of the viral oncogene. More
important, the susceptibility of ORF-74 to non-peptide antagonists is
probed by blockage of the constitutive signaling by Zn2+
after construction of a silent metal ion site in the viral receptor.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, and SDF-1
) or R&D Systems (GRO
, GRO
,
ENA-78 and IP-10) or were kindly provided by Timothy N. C. Wells
(Serono, Geneva, Switzerland; NAP-2 and vMIP-II) or Michael A. Luther
(Glaxo Wellcome; Met-SDF-1
). ORF-74 (GenBankTM accession
number U24275) was cloned from a biopsy taken from a Kaposi's sarcoma
skin lesion from an human immunodeficiency virus type 1-infected
patient (5). The cDNA was cloned into the eukaryotic expression
vector pTEJ8 (16). Monoiodinated 125I-IL-8,
125I-GRO
, and myo-[3H]inositol
(PT6-271) were purchased from Amersham International (Buckinghamshire,
United Kingdom). AG 1-X8 anion-exchange resin was from Bio-Rad.
plus variable amounts of unlabeled ligand in
0.5 ml of 50 mM Hepes buffer, pH 7.4, supplemented with 1 mM CaCl2, 5 mM MgCl2,
and 0.5% (w/v) bovine serum albumin. After incubation, cells were
washed quickly four times in 4 °C binding buffer supplemented with
0.5 M NaCl. Nonspecific binding was determined as the
binding in the presence of 0.1 µM unlabeled chemokine.
Determinations were made in duplicate.
-galactosidase DNA (Promega). One day after
transfection, the cells were split into 96-well plates, and ligands
were added the following day. After 24 h of exposure to ligand,
cell proliferation was quantified using a standard colorimetric assay
for
-galactosidase activity as described previously (19, 20).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(Fig. 1)
could displace 125I-IL-8 with single-digit nanomolar
affinity. In contrast, the CC chemokines MIP-1
, MIP-1
, MCP-1, and
RANTES (regulated on activation, normal T-cell expressed and secreted)
bound to the receptor with affinities (IC50) <10,000
nM (n = 3), whereas MCP-3 and
aminooxypentane (AOP)-RANTES had measurable affinities around 200 nM (n = 3) when tested against IL-8 as
radioactive ligand (data not shown).
Binding constants of selected CXC chemokines and the CC chemokine
vMIP-II for the HHV8-encoded ORF-74 receptor using different
radioactive ligands compared with biological activity measured by
phosphatidylinositol turnover
(Bmax = 44 ± 10 fmol/105 cell) are
shown as means ± S.E. of the indicated number of experiments
(n). The-fold difference in affinity using
125I-GRO
versus 125I-IL-8 as radioactive
ligand is listed. EC50 values for the individual chemokines in
either stimulating (S) or inhibiting (I) the basal constitutive
activity of ORF-74 as determined by phosphatidylinositol turnover are
shown as means ± S.E. of the indicated number of experiments
(n).
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Fig. 1.
Amino acid sequences of the ORF-74 receptor
and the employed chemokines. Shown is the alignment of GRO with
various human CXC chemokines (GRO
, GRO
, NAP-2, ENA-78,
IL-8, IP-10, and SDF-1
) and a virally encoded CC chemokine
(vMIP-II). Residues identical to those in GRO
are shown in
white on black. Asterisks indicate the conserved
Cys residues. Also shown is a serpentine diagram of the ORF-74 receptor
with residues conserved between this and human CXCR-2
indicated in black on gray. The histidine residues
introduced as a bis-His metal ion switch at the extracellular part of
transmembrane segment V are indicated in white on black. The
substituted residues are Arg208 and Arg212,
which both are also conserved residues.
displaced
125I-IL-8 with even higher affinity (IC50 = 0.23 nM) than unlabeled IL-8 itself (IC50 = 1.5 nM), we decided to probe ORF-74 with
125I-GRO
as well. As shown in Fig.
2 (D-F), the ligand binding
profiles displayed by 125I-GRO
were different from those
displayed by 125I-IL-8, in particular for the ligands
ENA-78, NAP-2, and IL-8 (Fig. 2, B versus
E). The competition binding curves for these three ligands
were shifted 16-, 23-, and 890-fold to the right, respectively, as
compared with the binding curves observed in competition with
125I-IL-8. In contrast, the competition binding curves for
GRO
, -
, and -
were shifted 2-5-fold to the left in
competition with 125I-GRO
as compared with
125I-IL-8 (Fig. 2, A versus
D). The binding curves for IP-10, SDF-1
, and vMIP-II were
rather similar in both assays (Fig. 2, C and F).
In conclusion, with 125I-GRO
as tracer, ORF-74 appears
to have >20 times higher affinity for the three GRO peptides than for
IP-10 and 100 to >1000 times higher affinity than for any other
chemokine ligand, with SDF-1
being the closest one. Thus, the
affinity of ORF-74 for the GRO peptides is ~10,000-fold higher than
the affinity for IL-8 in competition with radioactive GRO
(Table
I).
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Fig. 2.
Competition binding experiments with
ORF-74. Binding was performed on whole COS-7 cells transiently
expressing ORF-74 with either 125I-IL-8 (A-C)
or 125I-GRO (D-F) as radioactive ligand.
A and D, ligands that are agonists in signal
transduction assays: GRO
(
), GRO
(
), and GRO
(
).
B and E, ligands that are neutral ligands or
agonists with low potency: IL-8 (
), NAP-2 (
), and ENA-78 (
).
C and F, non-ELR (Glu-Leu-Arg located just prior
to the first Cys residue; see Fig. 1) CXC chemokines that
are inverse agonists in signal transduction assays: IP-10 (
),
SDF-1
(
), Met-SDF-1
(
), and vMIP-II (
).
and GRO
were able to stimulate ORF-74 signaling >2-fold and with
EC50 values of 1.1 and 2.8 nM, respectively
(Fig. 4A). GRO
also acted as an agonist, yet only with a
partial response as compared with GRO
, but with a similar
EC50 value of 3.1 nM. Like IL-8, NAP-2 and ENA-78 were unable to affect signaling through ORF-74 except at micromolar concentrations, where NAP-2 stimulated the
phosphatidylinositol turnover to ~50% of the maximal response
observed with GRO
(Fig. 4B). Most surprisingly IP-10,
SDF-1
, and Met-SDF-1
functioned as efficient inverse agonists on
ORF-74 with potencies very similar to their binding affinities measured
against GRO
as radioligand: EC50 = 3.3, 11, and 28 nM, respectively (Fig. 4C), versus
affinity = 2.3, 13, and 30 nM, respectively (Table I).
vMIP-II, which like ORF-74 is encoded by HHV8 and which acts as an
antagonist on multiple human chemokine receptors (5), could also block the constitutive signaling of ORF-74, albeit only with an
EC50 of 84 nM; however, this potency was
similar to the affinity (IC50 = 72 nM) for
vMIP-II measured against GRO
as radioligand. When ORF-74 signaling
was stimulated with 10 nM GRO
, the four inverse agonists
(IP-10, SDF-1
, Met-SDF-1
, and vMIP-II) acted as antagonists (Fig.
4F). Neither of the other chemokines had any effect on the stimulated activity (Fig. 4, D and E).
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Fig. 3.
Constitutive basal activity of wild-type
ORF-74 and R208H,R212H ORF-74. Measurements of
phosphatidylinositol (PI) turnover were performed on
transiently transfected COS-7 cells as described under "Experimental
Procedures." Shown are the results from gene dose experiments with
various concentrations of DNA from the empty expression vector pTEJ8
(white bars) (n = 3), wild-type ORF-74
(black bars) (n = 4), and R208H,R212H ORF-74
(hatched bars) (n = 4).
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Fig. 4.
Effect of selected chemokines on basal
(A-C) and GRO (10
8
M)-stimulated (D-F) phosphatidylinositol
turnover induced by ORF-74. Experiments were performed on
transiently transfected COS-7 cells as described in detail under
"Experimental Procedures." A and D, GRO
(
) (n = 9), GRO
(
) (n = 5),
and GRO
(
) (n = 4). B and
E, IL-8 (
) (n = 11), NAP-2 (
)
(n = 6), and ENA-78 (
) (n = 4).
C and F, IP-10 (
) (n =3), SDF-1
(
) (n = 5), Met-SDF-1
(
) (n = 5), and vMIP-II (
) (n = 6). PI,
phosphatidylinositol.
-galactosidase was used to measure
effects on cell transformation using the R-SATTM assay
(19). In agreement with previously published data showing an effect on
foci formation (13), ORF-74 was found to strongly stimulate cell
proliferation in a ligand-independent manner as determined by
-galactosidase activity in the transfected cells. The constitutive
activity of ORF-74 resulted in a
-galactosidase activity
(corresponding to degree of cell transformation) that was stronger than
observed with, for example, the muscarinic m1 receptor after maximal
ligand stimulation (data not shown). GRO
, IL-8, IP-10, and
Met-SDF-1
were selected for testing in the cell proliferation assay.
As shown in Fig. 5, GRO
stimulated the
transforming activity of ORF-74 with a potency of 3.0 nM,
but under the chosen assay conditions, only to ~25% above basal
activity as compared with the 100% stimulation observed in
phosphatidylinositol turnover (Fig. 4A). In contrast, both
IP-10 and Met-SDF-1
acted as inverse agonists also in the cell
proliferation assay, as they inhibited the basal activity to 30% under
the chosen assay conditions, with IC50 values of 36 and 350 nM, respectively (Fig. 5).
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Fig. 5.
Chemokine modulation of constitutive
ORF-74-induced cell transformation. NIH-3T3 cells were
cotransfected with the ORF-74 receptor and the marker gene
-galactosidase, and the assay was performed as described in detail
under "Experimental Procedures."
, GRO
;
, IP-10;
,
Met-SDF-1
.
-opioid receptors
(Fig. 1). Binding of 125I-IL-8 was eliminated in the
R208H,R212H ORF-74 mutant receptor. This is in accordance with the fact
that the two Arg residues, which were substituted with His residues,
are conserved between the ORF-74 and endogenous CXCR-1 and
CXCR-2 receptors, in which these two residues previously
have been shown to be involved in IL-8 binding (25). However, although
the mutant receptor did not bind IL-8, it was nevertheless expressed
well and still displayed a high degree of constitutive signaling (Fig.
3). In the R208H,R212H ORF-74 mutant receptor, Zn2+ acted
as a potent inverse agonist in blocking the constitutive signaling,
with an EC50 of ~1 µM (Fig.
6). Thus, the effect of this
"prototype" non-peptide antagonist (Zn2+; used here in
a zinc switch on the mutant receptor) indicates that it should be
possible to develop non-peptide inverse agonists targeted toward the
extracellular part of ORF-74.
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Fig. 6.
Inhibition by Zn2+ of
constitutive phosphatidylinositol turnover induced by ORF-74 with an
engineered bis-His metal ion switch. , wild-type ORF-74
(ORF74 wt; n = 5);
, R208H,R212H ORF-74
(n = 7). In the mutant receptor, His(V:01)
(His208) and His(V:05) (His212) are located in
i and i+4 positions, which in a helical
configuration is optimal for binding zinc ions (see Fig. 1).
PI, phosphatidylinositol.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and especially in the zinc site-engineered mutant receptor
by Zn2+ indicates that this viral oncogene is susceptible
to drug intervention by non-peptide inverse agonists.
binding or ORF-74 signaling. The only exceptions are IP-10 and
SDF-1
, which, at nanomolar concentrations, function as efficient
inverse agonists in ORF-74 signaling. It is interesting to note that
the affinities measured in competition against 125I-GRO
for all the chemokines appear to closely reflect their potency upon
receptor signaling (Table I; compare Fig. 2 (C-F) with Fig.
4 (A-C)). For the agonists, the EC50 values
were shifted ~10-fold to the right as compared with their "GRO
affinity." Thus, the GRO peptides show affinities around 0.1 nM in competition with GRO
, and they stimulate signaling
with an EC50 of ~1 nM. NAP-2 has an affinity
against GRO
binding of 82 nM and stimulates signaling
with an EC50 close to 1 µM. The potency of
the inverse agonists is almost identical to their GRO
affinity
(Table I). This would indicate that the 125I-GRO
binding
in fact displays an active or activable conformation of the receptor.
In contrast, there is no functional correlate to the single-digit
nanomolar affinity for IL-8, NAP-2, and ENA-78 as measured in
competition with 125I-IL-8. Apparently, at physiological
concentrations, these peptides merely function as neutral ligands on
the ORF-74 receptor.
preferentially as described here for ORF-74 from HHV8. There is a 50-fold difference in favor of GRO
versus IL-8 with respect to potency for stimulating calcium
mobilization through ECRF3. In contrast, on human CXCR-2,
there is 300-fold difference in favor of IL-8 versus GRO
(12). Thus, it appears that ORF-74 receptors from
-herpesvirus in
general have been optimized to recognize GRO peptides.
is interesting since these peptides are generally considered
to be angiogenic and angiostatic factors, respectively (31). In
addition to its cell transforming properties, ORF-74 has, through its
induction of expression of vascular endothelial growth factor, been
strongly implicated in the angiogenesis involved in the formation of
Kaposi's sarcoma (15). The angiogenic activity of GRO
and the
angiostatic activity of IP-10 have been demonstrated in several
systems, both in vitro (endothelial chemotaxis) and in
vivo (corneal neovascularization and tumor formation) (31-34). SDF-1
or rather its endogenous receptor (CXCR-4) was
recently, through gene knockout experiments, shown to be important for
the remodeling part of the neovascularization process, i.e.
the reorganization of small immature vessels into larger mature vessels
(2). Thus, according to the present data, the virally encoded ORF-74
receptor apparently exploits endogenous ligands, which normally are
involved in angiogenesis or neovascularization in the host organism,
for both positive and negative modulation of its high constitutive activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Acadia Pharmaceuticals Inc. for support in using the R-SATTM method. Furthermore, we thank Lisbet Elbak for excellent technical assistance.
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FOOTNOTES |
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* The work was supported by grants from the Danish Medical Research Council, the Carlsberg Foundation, and the Lundbeck Foundation.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: Lab. for Molecular Pharmacology, Panum Institute 18.6.12, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. Tel.: 45-3532-7603; Fax: 45-3135-2995; E-mail: schwartz{at}molpharm.dk.
The abbreviations used are:
HHV8, human
herpesvirus 8; CXCR, CXC receptor; IL-8, interleukin-8; vMIP-II, viral macrophage inflammatory protein II; GRO, growth-related oncogene; IP-10, interferon -inducible protein; SDF-1
, stromal cell-derived factor-1
; ENA-78, epithelial
cell-derived activating peptide-78; NAP-2, neutrophil-activating
peptide-2; MIP, macrophage inflammatory protein; MCP, monocyte
chemotactic protein.
2 Kledal, T. N., Rosenkilde, M. M., and Schwartz, T. W. (1999) FEBS Lett., in press.
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