From the Department of Pharmacology, Yale
University School of Medicine, New Haven, Connecticut 06520, § Cadus Pharmaceutical Corp., Tarrytown, New York 10591, and the ** Department of Chemistry, Yale University,
New Haven, Connecticut 06520
Received for publication, May 13, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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The chemokine receptor CXCR4 is a co-receptor for
T-tropic strains of HIV-1. A number of small molecule antagonists of
CXCR4 are in development but all are likely to lead to adverse effects due to the physiological function of CXCR4. To prevent these
complications, allosteric agonists may be therapeutically useful as
adjuvant therapy in combination with small molecule antagonists. A
synthetic cDNA library coding for 160,000 different SDF-based
peptides was screened for CXCR4 agonist activity in a yeast strain
expressing a functional receptor. Peptides that activated CXCR4 in an
autocrine manner induced colony formation. Two peptides, designated
RSVM and ASLW, were identified as novel agonists that are insensitive to the CXCR4 antagonist AMD3100. In chemotaxis assays using the acute
lymphoblastic leukemia cell line CCRF-CEM, RSVM behaves as a partial
agonist and ASLW as a superagonist. The superagonist activity of ASLW
may be related to its inability to induce receptor internalization. In
CCRF-CEM cells, the two peptides are also not inhibited by another
CXCR4 antagonist, T140, or the neutralizing monoclonal antibodies 12G5
and 44717.111. These results suggest that alternative agonist-binding
sites are present on CXCR4 that could be screened to develop molecules
for therapeutic use.
CXCR4 is a member of the chemokine receptor family of G
protein-coupled receptors. A number of mechanisms for receptor
activation of G protein-coupled receptors have been proposed (1, 2). For example, small molecule agonists bind within the transmembrane helices and cause activation. Larger molecules bind to specific sites
on the extracellular surface of the receptor leading to a
conformational change that is transmitted to an intracellular G SDF-1 The first 7 or 8 N-terminal residues of SDF-1 A number of mammalian receptors have been successfully expressed in
Saccharomyces cerevisiae and shown to couple to the native or modified versions of G proteins leading to activation of the pheromone response pathway (14-22). To identify allosteric agonists or
modulators of CXCR4 activity, we adapted a yeast expression system that
produces functional CXCR4. S. cerevisiae is known to possess
one of two G protein-coupled receptors, Ste2p and Ste3p, for each
haploid cell type. In yeast G Vector Construction and Strains--
The initial S. cerevisiae strain was CY12946 (MAT Media--
All basic media were prepared essentially as
described (24). In cases where plasmid selection was necessary, media
that lacked uracil, leucine, or tryptophan or an appropriate
combination of these nutrients were employed. Library screening was
performed in plates that lacked histidine and contained
3-amino-1,2,4-triazole (Sigma), which suppresses the basal level of
histidine biosynthesis due to the activity of the
fus1 promoter.
Library Construction and Transformation--
An oligonucleotide
library coding for 17-mer peptides, in which the first four codons were
randomized, was chemically synthesized by the Yale University Keck
facility. The library was made with oligonucleotides that used the
triplet NNK (N is any nucleotide and K is either G or T) to encode the
first four amino acids (25). Codons 5-17 were from the wild-type
N-terminal sequence of SDF-1 Peptide and Protein Production--
Peptides were synthesized
and purified by the Keck Peptide Synthesis Facility at Yale.
Me2SO was used when necessary to facilitate dissolution of the lyophilized peptides. SDF-1 Antibodies and CXCR4 Antagonists--
Neutralizing monoclonal
antibodies 12G5 and 44717.111 were obtained from the National
Institutes of Health AIDS Research Program and R & D Systems,
respectively. The FITC-linked rat anti-mouse IgG2a and the
isotype-matched control antibodies were obtained by Pharmingen. T140
was a kind gift of Dr. Nobutaka Fujii of Kyoto University. A
modification of the original method was used to synthesize AMD3100
(30). NMR and mass spectrometry were used to verify the synthesis.
Chemotaxis Assay--
The CCRF-CEM acute lymphoblastic leukemia
cell line was shown previously to express CXCR4 and to migrate in
response to SDF-1 Flow Cytometry--
Flow cytometric analysis of cell
subpopulations was performed using a FACS Vantage flow cytometer (BD
Biosciences) for detection of CXCR4 surface expression with the 12G5
monoclonal antibody. An indirect staining protocol was used. Briefly,
after a 90-min incubation with various agents, the cells were washed
and resuspended in phosphate-buffered saline. Nonspecific (Fc-mediated)
binding was blocked using purified rat IgG. The primary antibody (12G5, a mouse IgG2a antibody) was added at 1 µg/ml for 45 min. After centrifugation the cells were resuspended in phosphate-buffered saline,
and the secondary antibody (FITC-linked rat anti-mouse IgG2a) was added
at 10 µg/ml for 45 min. The cells were fixed with 2% formaldehyde
(in phosphate-buffered saline) and used within 2 days for data
acquisition. Mouse IgG2a was used as an isotype control. For the
fluorescence measurements the cells were excited at 488 nm. The FITC
fluorescence was collected through a 530/30 nm bandpass filter. A
minimum of 15,000 cells was examined for each sample. Analysis of data
was performed using the CellQuest software (BD Biosciences).
Functional Expression of Human CXCR4 in Yeast--
To identify
allosteric peptide agonists or modulators of CXCR4, it was necessary to
establish an assay that could isolate molecules with the desired
properties from a large peptide library. The screen that was used
consisted of two steps. The first step was to isolate peptide sequences
from a synthetic cDNA library that would allow cells to grow only
after activation of CXCR4. A second step was required that would
segregate agonists that bind to the native active site from those that
bind to allosteric sites. This step involved an agonist assay in the
presence of a receptor antagonist. A number of genetic modifications
made to S. cerevisiae allowed it to be used for monitoring
the interactions of peptides or proteins with CXCR4 (Fig.
1). First, the endogenous
A number of genes for biosynthetic enzymes have been mutated to allow
selection of the corresponding marker on a plasmid transformed into the
yeast strain. The genomic HIS3 gene, coding for an enzyme essential for the biosynthesis of histidine, has been disrupted by an
insertion. The wild-type HIS3 gene has been placed under the
control of the pheromone-responsive FUS1 promoter; as a
result activation of CXCR4 enables cells to grow in media lacking
histidine. To reduce false positives due to any basal activity of
FUS1-HIS3, 3-amino-1,2,4-triazole, an inhibitor of the
enzyme coded by HIS3, is present at low levels in the media
during agonist selection. Endogenously expressed SDF-1 Library Screening and Agonist Isolation--
The cDNA library
encodes a 17-mer peptide with random amino acids at positions 1-4. At
these positions the wild-type sequence is KPVS. The amino acid sequence
for the remaining peptide (positions 5-17) retains the SDF-1 CXCR4 Dependence of RSVM and ASLW Effect--
Initial experiments
to test the effect of each exogenous peptide on the Peptide Assays--
To verify that yeast screening provided
peptides that possessed biological activity on mammalian cells,
chemotaxis of the human leukemic T cell line CCRF-CEM was measured.
Fig. 4, A and B,
display the concentration-response curves for the RSVM and ASLW
peptides, respectively. The RSVM peptide has an apparent EC50 of greater than 100 µM (Fig.
4A). Due to poor solubility of the peptides we have not been
able to test concentrations higher than 200 µM in order
to observe the typical bell-shaped concentration-response curve of most
chemoattractants. The second peptide, ASLW, has an apparent
EC50 of greater than 60 µM (Fig.
4B) and displays superagonist activity, with a chemotactic
index higher than the maximum observed with SDF-Fc. (SDF-Fc is a fusion
protein between SDF-1 Effect of AMD3100, T140, 12G5, and 44717.111--
The chemotactic
activity of SDF-Fc or the wild-type 17-mer is sensitive to AMD3100
(Fig. 5A). However, neither
RSVM (Fig. 5B) nor ASLW (Fig. 5C) show any
reduction in activity in the presence of AMD3100. The data suggest that
AMD3100 and the peptides bind to different sites on the receptor. The
unique agonist-binding site, however, continues to activate CXCR4.
Strong resistance to inhibition remains even at very high AMD3100
concentrations (100 µg/ml, ~115 µM). This fact
essentially excludes the possibility of a competitive interaction with
the antagonist.
T140 is a 14-residue analogue of polyphemusin II from American
horseshoe crabs that is also an antagonist of CXCR4 (35). The presence
of this antagonist also does not inhibit CCRF-CEM chemotaxis in
response to RSVM (Fig. 5D) or ASLW (Fig. 5E).
Again, no significant reduction of the peptide activities is seen, even at T140 concentrations comparable with those of the agonists. Because
AMD3100 and T134, a 14-residue analogue of T140, do not compete for
binding to CXCR4 (36), it is reasonable to expect that AMD3100 and T140
also do not compete with each other and therefore bind to different
sites on CXCR4. These sites must sufficiently overlap with SDF-1
12G5 and 44717.111 are monoclonal antibodies to CXCR4 that prevent
SDF-1 Full-length Mutants--
To determine the effect of the mutations
in the context of the full-length SDF, we overexpressed and purified
the two mutant SDFs from mammalian cells. The [ASLW]SDF-Fc mutant was
inactive in chemotactic assays on CCRF-CEM cells and did not inhibit
the activity of SDF-Fc on these cells at the concentrations tested (up
to 2 µM) (data not shown). However, the [RSVM]SDF-Fc
mutant displayed chemotactic activity on CCRF-CEM cells at ~500
nM-1 µM (Fig.
7A). The efficacy of this
full-length mutant was substantially lower than wild type. Furthermore,
this activity was completely inhibited by AMD3100, T140, 12G5, and
44717.111 (Fig. 7, B-E) indicating that the full-length
mutant has a similar binding site to CXCR4 as the wild-type
protein.
Flow Cytometry--
After CXCR4 activation, SDF-1 CXCR4 Activation--
The activation of CXCR4 and most other
chemokine receptors is believed to involve a two-step process. The
first step involves the binding of SDF-1 Allosteric CXCR4 Agonists--
The phenomenon of allosteric
agonism is relatively uncommon in the literature for G protein-coupled
receptors. Most of the available allosteric ligands are either
antagonists or modulators/enhancers of the activity of the natural
ligands without having intrinsic agonist activity. It has been
suggested that the relative lack of allosteric agonists in the ligand
repertoire of the G protein-coupled receptors may reflect the bias of
most screens with radioligand binding assays for the native
(orthosteric) site (45). With the advent of functional screens, the
identification of allosteric agonists may become more widespread and
thus lead to novel therapeutic agents.
Based on the differences of IC50 values of AMD3100 on
chemotactic and CXCR4 binding assays of SDF-1
When the same mutations are introduced into the full-length SDF-1 Implications for Drug Development--
Although our studies
present proof of principle for allosteric agonism on CXCR4 that may
allow the screening of non-peptide libraries for this activity, the
RSVM and ASLW peptides will need to be modified if they are to be
transformed into clinically useful compounds. The potency and chemical
stability of these molecules must be increased, and their size must be
reduced. There are a number of approaches to accomplish these goals.
One of them consists of identifying the minimal peptide sequence that
retains allosteric agonist activity followed by further
randomization to improve potency. The yeast CXCR4 expression system can
be used to pursue these goals. The chemical stability of the peptide
ligands can be increased by synthesis of peptidomimetics based on the
most potent sequences.
Therapeutic Implications--
Current therapy for HIV infection
involves a combination of agents that act at the reverse transcriptase
and proteolytic steps of the HIV-1 life cycle. This therapy is termed
highly active antiretroviral therapy. These agents did not show any
serious toxicity in clinical trials. However, after long term and
widespread use in HIV patients, lipodystrophy and other metabolic
complications were reported (49).
The chemokine receptors CXCR4 and CCR5 are the two major co-receptors
used by HIV-1 to infect cells after the HIV-1 surface protein gp120
engages CD4 (50). This induces a conformational change that leads to
the insertion of the viral protein gp41 into the cell membrane. This
results in the release of virion contents into the cell. The initial
infection by HIV-1 usually involves M-tropic strains that utilize the
chemokine receptor CCR5 and are associated with the asymptomatic stage
of the disease. Individuals lacking functional CCR5 due to a 32-bp
deletion in both alleles are highly resistant to infection by HIV-1
(51). The evolution from M-tropic strains to T-tropic strains involves
mutations mainly in the V3 loop of gp120, which changes the co-receptor
specificity from CCR5 to CXCR4 (52). The change to T-tropic strains
leads to a decrease in T-cells and all the symptoms associated with full-blown AIDS. Agents that can prevent the transition or induce the
reversion to CCR5-utilizing strains may decrease the morbidity and
mortality of this infectious disease. However, therapeutic use of CXCR4
antagonists can be expected to have adverse effects due to the
CXCR4-mediated trafficking of lymphocytes, monocytes, and hematopoietic
stem cells (53, 54). The CXCR4 antagonists AMD3100 and ALX40-4C have
entered clinical trials for short term studies
(55).3 Further development of
AMD3100 was discontinued due to cardiac toxicity. This toxicity is not
general for all CXCR4 antagonists as no major adverse effects were
reported in the case of ALX40-4C administration (55). It should be
noted that any CXCR4 antagonist that is approved for clinical use would
be used chronically. Adverse effects that are not observed during
clinical trials may be manifested over long term use as in the case of
highly active antiretroviral therapy.
T-20 is a synthetic peptide corresponding to a region of HIV-1 gp41
that blocks viral fusion to cell membranes (56). It is currently in
phase III clinical trials without evidence of any serious systemic
toxicity.2 Interestingly, strong in vitro
synergy has been observed between T-20 and AMD3100 (26) suggesting that
a possible combination of gp41 and CXCR4 inhibitors may provide
significant therapeutic advantages in HIV treatment. This possibility
further establishes the need for an approach that minimizes the toxic
effects of AMD3100 and other CXCR4 antagonists. In this context,
allosteric CXCR4 agonists could maintain essential receptor function
and allow the clinical combination of CXCR4 antagonists with other
anti-HIV agents that have a different mechanism of action.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
complex. This results in an exchange of GTP for GDP in the G
protein, dissociation of G
from the G
complex, and
activation of downstream signal transduction pathways. Competitive
receptor antagonists bind to the same or overlapping agonist site and
prevent the physiological agonist from activating the receptor. In
principle, allosteric modulators that bind to different sites on G
protein-coupled receptors should have no effect on activity except in
the presence of receptor agonists or antagonists (3). Allosteric
agonists or antagonists could also bind at different sites and induce
biological activity.
is the sole physiological agonist for the receptor CXCR4.
Deletion of either of the SDF or CXCR4 genes in
mice is lethal, with developmental defects in the cerebellum, the
heart, the gastrointestinal tract, and hematopoiesis (4-6). In adults,
SDF-1
is constitutively secreted from virtually all cell types and
is involved in the migration and development of hematopoietic cells.
CXCR4 is an important target for
HIV-11 drug discovery due to
its additional role as an HIV-1 co-receptor (7). However, inhibition of
the HIV-1-binding site, which corresponds to the agonist-binding site,
is likely to lead to severe adverse effects. Allosteric agonists or
modulators of CXCR4 activity would be therapeutically useful but have
not yet been identified. Such agents could preserve the function of
CXCR4 and minimize adverse effects expected from the use of competitive
receptor antagonists in HIV-1 therapy.
that precede the first
of four cysteines, which define the chemokine superfamily, are not
evident in the three-dimensional structure (8, 9). In virtually all
chemokine structures, the N-terminal sequence preceding the first
cysteine is not observable in either NMR or x-ray structures and is
presumed to be flexible. The flexibility of the N-terminal sequence in
over a dozen known chemokine structures suggests that this mobility is
important for the biological function of these proteins (10). Mutation
or deletion of the residues at the N-terminal sequence of chemokines
usually leads to a functional change from receptor agonism to
antagonism (11, 12). Despite a large number of studies on chemokine
structure and function, how these proteins interact with their G
protein-coupled receptors remains to be elucidated. A two-step process
has been proposed involving the binding of a chemokine to its receptor
followed by placement of the N-terminal flexible loop of the chemokine on another site of the receptor, leading to its activation. In this
model, the N-loop, consisting of the first 20-30 residues of
chemokines, provides most of the specificity for the receptor. For
SDF-1
, N-terminal peptides of 9-17 amino acids are weak agonists of
CXCR4 (13, 57).
activates a mitogen-activated protein kinase pathway resulting in transcription of genes containing a
pheromone-responsive element in their promoter, growth arrest, and
mating of the two different haploid yeast strains. We describe a
genetically modified yeast strain that allowed us to identify both
normal and allosteric agonists. We focus on the characterization of two
peptide agonists, designated RSVM and ASLW, that are resistant to the
small molecule CXCR4 antagonists AMD3100 and T140 and the neutralizing
antibodies 12G5 and 44717.111. These peptides may serve as lead
compounds for development of drugs that could be used in conjunction
with antagonists for anti-HIV therapy. The data also illustrate the
presence of alternative agonist-binding sites for screening non-peptide
allosteric agonists.
MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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FUS1p-HIS3
GPA1G
i2(5) can1 far1
1442 his3 leu2 sst2
2
ste14::trp1::LYS2 ste3
1156
tbt1-1 trp1 ura3) (23). Except for the specific chimeric G
subunit expressed, CY12946 is genetically similar to CY1141, reported
in Klein et al. (18), with two differences. First, the two
strains express different G
subunits. Specifically, CY12946 expresses a chimeric G
subunit (GPA1G
i2(5)) in which
the C-terminal 5 amino acids of the yeast G
subunit, GPA1, are
replaced by the C-terminal 5 residues of G
i2. In
contrast, as described by Klein et al. (18), CY1141
expresses a chimeric G
subunit
(GPA1(41)G
i2) in which the N-terminal 33 residues of G
i2 are replaced by the 41 N-terminal
residues of GPA1. We have used CY12946 in the current report because
GPA1G
i2(5) is more efficient than
GPA1(41)G
i2 in coupling CXCR4 to the
pheromone response pathway. The use of GPA1G
i2(5)
necessitated deletion of SST2, which down-regulates the
pheromone response pathway by accelerating the GTPase activity of GPA1.
Thus, the second difference between CY12946 and CY1141 is the presence
of an sst2 deletion allele in CY12946. All plasmids were
2 µm-derived and contained a REP3 element for
autonomous replication in S. cerevisiae and AmpR
for selection in Escherichia coli. The plasmid Cp4181
contains the CXCR4 gene under the control of the
constitutive phosphoglycerate kinase (PGK1) promoter and a
LEU2 selectable marker. Plasmid Cp1584 possesses the
FUS1-lacZ construct that allows production of
-galactosidase when the pheromone pathway is stimulated. It also
contains the TRP1 selectable marker. Cp6160 expresses
full-length wild-type SDF-1
in yeast under the control of the
constitutive alcohol dehydrogenase promoter. The
-factor signal
sequence is cloned at the 5'-end of the SDF sequence to allow secretion
of the mature polypeptide. The vector also has the URA3
selectable marker allowing transformed cells to grow in media deficient
in uracil. For expression of the 17-mer peptide library, Cp6293 was
used. This URA3+ plasmid was derived from Cp6160
and contains a frameshift in the SDF-1
sequence so that no
functional protein is expressed unless an insert is appropriately
cloned. The cDNA coding for 17-mer peptides was cloned using the
restriction sites HindIII and Acc65I, which also
results in the secretion of the mature peptide. S. cerevisiae strains YAS1, YAS2, and YAS3 were constructed by
transforming CY12946 with Cp1584, Cp1584/Cp4181, and
Cp1584/Cp4181/Cp6160, respectively.
. The modified genetic code used for
randomization allows a more balanced representation of all amino acids
while at the same time limiting the number of possible stop codons to
one. The double-stranded oligonucleotides containing the random
nucleotides were constructed so that the new sequence is in-frame with
the prepro-
signal sequence after ligation. The sequences of the two
primers are as follows:
5'-GCCGTCAGTAAAGCTTGCTTAAGCGTNNKNNKNNKNNKTTGTCTTACAGATGTCCATGTAGATTCTTCGAATCTCACTGAGGTACCAGTCTGTGACGC-3' and 5'-GCGTCACAGACTGGTACCT-3', where N is an equimolar mixture of A, G, C, and T; K represents an equimolar mixture of G and
T, and sequences in boldface are either HindIII or
Acc65I sites. Formation of the semi-randomized gene was
performed as described previously (25). Briefly, 1 nmol each of the two
single-stranded oligonucleotides was annealed by combining in 200 µl
of 40 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 0.1 mg/ml of bovine serum albumin, and 0.5 mM dithiothreitol, heating the mixture to 65 °C for 20 min, and then slowly cooling to 37 °C. The partially double-stranded primers were filled in by adding 5 µl of 10 mM
deoxynucleoside triphosphates and 3 µl of Sequenase (13 units/ml,
United States Biochemical Corp.) followed by successive incubations at
37 °C for 30 min and 65 °C for 20 min. The DNA product was
digested with HindIII and Acc65I and
gel-purified. The Cp6293 vector was similarly
HindIII/Acc65I-digested and gel-purified.
Different insert/vector ratios were used in overnight ligations at
14 °C followed by transformation of E. coli XL-10
ultracompetent cells (Stratagene). Aliquots were removed and plated in
LB-ampicillin plates to estimate library size; the remaining bacteria
were added to 500 ml of LB-ampicillin for overnight growth at 37 °C
for isolation of library DNA. Optimization of the insert:plasmid ratio
resulted in 108 ampicillin-resistant clones that should
contain all of the possible nucleotide sequences. The quality of the
library was evaluated by sequencing the peptide-encoding inserts in
plasmids recovered randomly from bacterial clones. All inserts encoded
heptadecapeptides, and the frequencies of amino acids at the randomized
positions were not different from those predicted by chance alone.
Yeast cells were transformed with the library DNA by using a
modification of the lithium acetate
method.2
-Galactosidase Assay--
The liquid
-galactosidase assays
were performed essentially as described previously (24, 27) with the
following modifications. After growth in the appropriate medium, 5 ml
of cells displaying the agonist phenotype were centrifuged (2500 rpm
for 5 min) and resuspended in 0.5 ml as described (28).
and its enantiomer were kind gifts of Gryphon Sciences (29). For expression of SDF-1
and its mutants, a mammalian vector pcDNA3.1 that coded for a
fusion protein between SDF-1
and the Fc domain of antibodies was
kindly provided by Drs. Qing Ma and Timothy Springer (Harvard University). HEK-293 cells were transformed by the calcium phosphate method; the media were collected; iodoacetamide was added to prevent aggregation from the Fc portion, and the protein was purified using
protein A-Sepharose affinity chromatography. For construction of
mutants in the full-length protein, the Quikchange (Stratagene) site-directed mutagenesis kit was used, and each mutation was confirmed
by DNA sequencing.
(31). The procedure involved cells that were
~70-80% confluent and were centrifuged at 1000 rpm for 5 min and
resuspended at a concentration of ~107 cells/ml. The
transwell system (Corning-Costar®) was used for quantifying the
chemotactic response. Six hundred µl of solution with different
concentrations of a CXCR4 agonist were added to the bottom of the wells
of 24-well culture plates. One hundred µl of the cell suspension was
aliquoted in the transwell cups and inserted in the wells. The bottom
of the cups is layered with a membrane that has a 5-µm pore size and
allows cell movement toward the higher concentration of
chemoattractant. The cells were incubated for 2 h at 37 °C,
after which the migrated cells were collected and counted with an
electronic particle counter (Coulter®). In cases where the effect of
neutralization by CXCR4 monoclonal antibodies or antagonists was
examined, the inhibitor was added to the upper compartment along with
the cells.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-factor G
protein-coupled receptor was genetically disrupted. CXCR4 expression
was achieved by its presence on an episomal plasmid. Second, the yeast
G
protein was modified such that its five C-terminal residues were
replaced with those from human G
i2, the G
subunit known to mediate signaling by CXCR4. The resulting chimeric G
subunit effectively couples CXCR4 to the yeast pheromone response pathway. Third, FAR1 was deleted, since its gene product
mediates the growth arrest that normally results from activation of the pheromone response pathway (32). As a result, deletion of
FAR1 dissociated pathway stimulation from growth arrest
while still allowing CXCR4-dependent induction of
pheromone-responsive genes. Fourth, SST2, a negative
regulator of GPA1 (the yeast G
subunit) was deleted in order to
enhance responsiveness to activated CXCR4. Finally, STE14
was deleted to dampen background activity of the pheromone response
pathway.
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Fig. 1.
A, the normal pheromone pathway in
S. cerevisiae. B, genetic modifications created to
identify new agonists. Genes that are inactivated are indicated by a
×. Expression of HIS3 and lacZ is under the
control of the fus1 promoter, which contains a
pheromone-response element (PRE).
stimulates
the production of histidine and allows the strain expressing CXCR4 to
grow in the absence of this amino acid due to the FUS1-HIS3
gene (Fig. 2A). In a strain where FUS1-lacZ is also present, endogenous or exogenous
SDF-1
induces an increase of
-galactosidase activity (Fig.
2B). Similar effects were observed in response to a peptide
agonist composed of the first 14 amino acids (1-14) of the SDF-1
sequence that had been shown in mammalian cells to act as a weak
partial agonist (33). To show specificity, we tested the activity of
the enantiomer of the chemokine, D-SDF-1
, which showed no
effect on the
-galactosidase assay (Fig. 2B).
Furthermore, the CXCR4 antagonist AMD3100 (34) inhibited YAS2 colony
formation (data not shown) and
-galactosidase activity in response
to SDF-1
(Fig. 2C). The effects of SDF-1
are dependent
on the expression of CXCR4, as a strain without the receptor (YAS1)
does not respond to SDF-1
or the 14-mer peptide (data not
shown).
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Fig. 2.
Functional expression of CXCR4 in yeast.
A, comparison of colony induction in histidine-deficient
plates as a function of 3-amino-1,2,4-triazole concentration by
HIS3 /CXCR4+/SDF+ yeast strains
(
) versus parental HIS3
strains (
).
Growth is due to stimulation of the pheromone pathway through
activation of CXCR4 by SDF-1
. B, induction of
-galactosidase activity of HIS3
/CXCR4+
yeast cells by SDF-1
(100 nM), the enantiomeric
D-SDF-1
as a negative control (100 nM), and
the 14-mer N-terminal peptide (1 µM). C, the
-galactosidase activity induced by 100 nM SDF-1
is
inhibited by AMD3100 (1 µM), a known CXCR4 antagonist.
AMD3100 does not affect the basal activity of the
pheromone-responsive fus1 promoter.
wild-type sequence. Transformation of strain YAS2 with the plasmid
library yielded ~106 transformants. Each possible peptide
is expected to be represented by an average of approximately six times.
The transformants were replica-plated en masse to
histidine-deficient medium containing 1 mM
3-amino-1,2,4-triazole. Originally ~120
Ura+/His+ clones grew on these plates. The
-galactosidase activity of these colonies was measured and compared
with strain YAS2, which expresses only CXCR4, or YAS3, which expresses
SDF-1
with CXCR4. Forty one of these colonies displayed significant
-galactosidase activity. The active library plasmids were isolated,
amplified in E. coli, and re-introduced into the YAS2 strain
to confirm the CXCR4-mediated effects. From the 41 clones, 13 presented
-galactosidase activity that was plasmid-dependent. Two
of these clones, predicted to express peptides with RSVM and ASLW
sequences at their N termini, showed resistance to AMD3100 in
-galactosidase assays and were selected for further characterization
using chemically synthesized peptides.
-galactosidase
activity of CXCR4+ yeast cells that contained the
pheromone-responsive FUS1-lacZ construct were hampered by
the toxicity of the peptides during the overnight exposure at the high
concentrations (
50 µM) required for testing. Therefore,
we tested the CXCR4 dependence by the in vivo expression of
the peptides in two different yeast strains, one that contained CXCR4
and another one that lacked this receptor. The results of these
experiments are shown in Fig. 3.
Endogenous expression of the peptides leads to a significant increase
of
-galactosidase activity only in the strain that co-expresses CXCR4. Consequently, CXCR4 is required for activation of the pheromone pathway by the RSVM and ASLW peptides in yeast.
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Fig. 3.
CXCR4 dependence of each peptide activity
established in yeast. The background strain (1st bar)
expresses only the receptor and reporter plasmids. Its
-galactosidase activity is significantly increased when it is
transformed with the RSVM- (A) or ASLW-expressing plasmid
(2nd bar) (B). The presence of the reporter and
peptide-expressing plasmids does not induce this effect in the absence
of CXCR4 (3rd bar).
and the Fc region of an immunoglobulin that
has similar agonist effects and is sometimes used instead of SDF-1
due to its stability.) Chemotactic effects induced by simultaneous
stimulation of more than one agonist vary depending on which two
agonists are used. The co-stimulation by SDF-Fc and RSVM leads to a
chemotactic index that is about 30% lower than SDF-Fc alone (Fig.
4C). Thus, RSVM behaves as a weak partial agonist. However,
the activities of SDF-Fc and ASLW are approximately additive (Fig.
4D). Chemotaxis in the presence of the two peptide agonists
ASLW and RSVM is also additive (Fig. 4E). The relative
chemotactic activities of all different CXCR4 agonists used in this
study are shown in Fig. 4F at their optimal
concentrations.
View larger version (32K):
[in a new window]
Fig. 4.
Chemotactic properties of agonists on
CCRF-CEM acute lymphoblastic leukemia cells.
Concentration-response chemotactic activity of RSVM (A) and
ASLW (B). A plateau in the chemotactic index of either
peptide could not be observed due to insolubility of both peptides
above the indicated concentrations. C, chemotactic activity
of RSVM in the presence of SDF-Fc indicates that RSVM has properties of
a partial agonist. Mixtures of agonists ASLW and SDF-Fc (D)
or ASLW and RSVM (E) have additive chemotactic effects.
F, chemotactic activities of the two mutant peptides, RSVM
(200 µM) and ASLW (100 µM), compared with
the activities of the wild-type agonists SDF-Fc (100 nM),
SDF-1 (5 nM), and N-terminal 17-mer peptide (20 µM). All ligands were used at their optimal
concentration. B = background chemotaxis.
View larger version (27K):
[in a new window]
Fig. 5.
Effect of various antagonists on the
chemotactic activity of CXCR4 agonists on CCRF-CEM cells. A,
sensitivity of SDF-Fc and the wild-type 17-mer peptide to AMD3100.
Resistance of RSVM (B) and ASLW (C) agonist
activity to inhibition by AMD3100. Resistance of RSVM (D)
and ASLW (E) to inhibition by the CXCR4 antagonist
T140.
or
HIV-1 gp120 in order to exert their antagonist effects. The experiments
here indicate that RSVM and ASLW bind to sites that differ from and do
not overlap with the AMD3100 and T140 sites.
-mediated effects and HIV-1 infection of T-tropic cells (37,
38). We examined the effect of co-incubation of each antibody with
CCRF-CEM cells on the chemotactic activity of the peptides. Fig.
6A illustrates a substantial
decrease in the chemotactic index for SDF-Fc in the presence of 12G5
but a very small effect of the antibody on RSVM. The same is observed for the ASLW peptide mutant (Fig. 6B). 12G5 binds an epitope
on the first and second extracellular loop (39, 40) that appears to
overlap the AMD3100-binding site on the second extracellular loop and
the adjacent transmembrane segment TM4 (41). The epitope for 44717.111 has not yet been determined. However, as can be seen in Fig. 6,
C and D, neither RSVM- nor ASLW-mediated
chemotactic activity is significantly affected by the presence of
44717.111, even at a concentration (50 µg/ml) 10-fold higher than the
one that almost completely abolishes the activity of SDF-Fc.
View larger version (35K):
[in a new window]
Fig. 6.
Effect of CXCR4-specific monoclonal
antibodies on the chemotactic activity of the peptides. Lack of
neutralization of RSVM (A) and ASLW (B) activity
by 12G5. 44717.111 shows the same lack of effect on the RSVM
(C) and ASLW (D) peptide activities on CCRF-CEM
cells.
View larger version (22K):
[in a new window]
Fig. 7.
Effects of full-length mutant
[RSVM]SDF-Fc. A, chemotactic activity of full-length,
mutant [RSVM]SDF-Fc protein on CCRF-CEM cells. Sensitivity of
chemotactic activity of [RSVM]SDF-Fc to the CXCR4 antagonists AMD3100
(B), T140 (C), and the CXCR4 neutralizing
monoclonal antibodies 12G5 (D) and 44717.111 (E).
induces
receptor internalization (42). In order to gain insight on the
differences in potency between the peptides and SDF-1
, we examined
their effect on surface levels of CXCR4 as detected using flow
cytometry (and a FITC/12G5-based indirect staining procedure). As can
be seen in Fig. 8A, SDF-Fc (1 µM) caused a clear reduction of the CXCR4 levels on the
cell surface. The RSVM peptide had a similar effect (Fig.
8B), whereas ASLW caused only a minor decrease (Fig.
8C). The effect of RSVM was
concentration-dependent (Fig. 8D). No
significant concentration-dependent response was observed
for ASLW (Fig. 8E). These data show that RSVM causes
down-regulation of CXCR4 in a manner similar to SDF-Fc. On the
contrary, ASLW does not induce a marked reduction in CXCR4 surface
levels after cell activation, which may partially explain its
superagonist activity.
View larger version (27K):
[in a new window]
Fig. 8.
Effect of SDF-Fc and the RSVM and ASLW
peptides on CXCR4 surface levels detected by flow cytometry. FL1-H
on the x axis represents fluorescence intensity.
A-C, the basal levels are indicated by heavy
lines, the agent-treated cells by dashed lines, and the
isotype and secondary antibody controls by dotted and
straight lines, respectively. A, SDF-Fc (1 µM) causes a clear decrease of the basal CXCR4 surface
levels. B, RSVM (200 µM) causes a similar
effect. C, the effect is not shared by ASLW (100 µM). D, RSVM has a
concentration-dependent response. E, the lack of
a significant response for ASLW is evident at different
concentrations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
to the CXCR4 N terminus
followed by activation of the receptor through interactions with the
second extracellular loop (43). Each extracellular region of chemokine
receptors possesses a cysteine. The four cysteines form two disulfides
that are believed to bring the extracellular regions together into a
compact structure with a stable binding site (44). Accordingly, the
binding and activation sites are likely to be in close proximity. The
activation of the receptor by the N-terminal region of SDF-1
results
in an intracellular conformational change leading to an exchange of GDP
for GTP in G proteins and dissociation of the G proteins from CXCR4 to
initiate downstream signal transduction mechanisms.
as well as the
biphasic binding behavior of 125I-SDF-1
on
CXCR4-expressing cell lines, it has been proposed that AMD3100 and
SDF-1
can bind to CXCR4 simultaneously (46). Mutagenesis experiments
suggest that the second extracellular loop, transmembrane IV, and
transmembrane VI of CXCR4 are critical for its interaction with AMD3100
(47, 48). In principle, this allows novel agonists to bind to the N
terminus or to other regions of the receptor that are not necessary for
antagonist(s) interactions. Our results suggest that other binding
sites exist for non-physiological agonists. These agonists were
identified by screening a semi-randomized 17-mer library in a yeast
strain expressing a functional CXCR4 receptor. The original, wild-type
17-mer has the same sequence as the first 17 amino acids of SDF-1
and has been shown previously to activate CXCR4 (13). Activation of
CXCR4 in this yeast strain induces the pheromone-response pathway, with
the exception that growth arrest is replaced with cell proliferation
due to a number of genetic modifications. As a result of these changes,
only cells that secrete an agonist peptide can grow on the appropriate
selective media. In contrast to the wild-type 17-mer peptide and
SDF-1
, two agonist peptides were identified that are insensitive to
the CXCR4 small molecule antagonists AMD3100 and T140 and to the
neutralizing anti-CXCR4 monoclonal antibodies 12G5 and 44717.111 in
CCRF-CEM cells. One peptide with a sequence of RSVM for the first four amino acids behaves as a partial agonist. The other peptide, ASLW, is a
superagonist, displaying a chemotactic index that is greater than the
maximum observed with SDF-Fc or SDF-1
. SDF-Fc and RSVM peptide
display a typical down-regulation of surface CXCR4 (Fig. 8,
A, B, and D). However, ASLW does not
induce CXCR4 internalization (Fig. 8, C and E),
which may explain the superagonist activity of this peptide due to
continuous activation of the receptor. Although experiments with yeast
strains clearly indicate that RSVM and ASLW activities are mediated by
CXCR4 (Fig. 3), an alternative explanation in CCRF-CEM cells is that
ASLW-induced chemotaxis may be mediated by another mechanism. This
possibility remains to be further investigated.
protein, one mutant, [ASLW]SDF-Fc, is inactive and the other,
[RSVM]SDF-Fc, displays properties of a partial agonist that is
sensitive to antibodies and small molecule antagonists (Fig. 7). The
activity and pharmacological behavior of the [RSVM]SDF-Fc mutant
indicates that it binds to the natural agonist site. Furthermore, the
EC50 increases from 1 to 10 nM for SDF-1
(31) to >1 µM for the wild-type 17-mer peptide (13).
Taken together these data indicate that the rest of the protein
contributes substantially to receptor binding. The results also suggest
that the mutations at the N-terminal region of the two peptides alter
the binding site on the receptor.
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ACKNOWLEDGEMENTS |
---|
We thank Jim Broach, Qing Ma, and Timothy Springer for providing reagents. We acknowledge technical support from Rocco Carbone of the Yale Cancer Center Flow Cytometry Shared Resource. We thank Cathy Berlot for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM35208 (to A. H.) and AI43838 (to E. L.).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.
¶ Present address: Cellular Genomics, Branford, CT 06405.
Present address: Myriad Pharmaceuticals, Salt Lake
City, UT 84108.
Present address: Dept. of Biochemistry and Biophysics,
University of North Carolina, Chapel Hill, NC 27599.
§§ To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8066. Tel.: 203-785-6233; Fax: 203-737-2027; E-mail: elias.lolis@yale.edu.
Published, JBC Papers in Press, November 3, 2002, DOI 10.1074/jbc.M204667200
2 R. Agatep, R, Kirkpatrick, D. Parchaliuk, R., Woods, and R. Gietz, Technical Tips Online (tto.trends.com).
3 C. D. Pilcher (University of North Carolina, Center for AIDS Research, Chapel Hill, NC), www.natap.org/2002/9retro/day28.htm.
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ABBREVIATIONS |
---|
The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; FITC, fluorescein isothiocyanate.
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REFERENCES |
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