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J. Biol. Chem., Vol. 277, Issue 27, 24515-24521, July 5, 2002
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From the
Received for publication, January 28, 2002, and in revised form, March 26, 2002
CXCR4 is a G protein-coupled receptor for
stromal-derived factor 1 (SDF-1) that plays a critical role in
leukocyte trafficking, metastasis of mammary carcinoma, and
human immunodeficiency virus type-1 infection. To elucidate the
mechanism for CXCR4 activation, a constitutively active mutant (CAM)
was derived by coupling the receptor to the pheromone response pathway
in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys,
conferred autonomous CXCR4 signaling in yeast and mammalian cells.
SDF-1 induced signaling in variants with substitution of Asn-119 to
Ser, Ala, or Asp, but not Lys. These variants had similar cell surface
expression and binding affinity for SDF-1. CXCR4-CAMs were
constitutively phosphorylated and present in cytosolic inclusions.
Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C
induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of
AMD3100 and ALX40-4C binding to CAMs was less than to wild type,
providing evidence of a conformational shift. These results illustrate
the importance of transmembrane helix 3 in CXCR4 signaling. Insight
into the mechanism for CXCR4 antagonists will allow for the development
of a new generation of agents that lack partial agonist activity that
may induce toxicities, as observed for AMD3100.
CXCR4 is the exclusive receptor for stromal-derived factor 1 (SDF-1),1 a CXC chemokine
that has been shown to play a critical role in directed cell migration
(1, 2), embryologic development (3-5), and the metastatic spread of
mammary carcinoma cells (6). In addition, T-tropic (X4) strains of
HIV-1 utilize CXCR4 for target cell entry (7), and this function is
blocked by SDF-1 (8, 9) and receptor antagonists (10-13). The clinical
significance of CXCR4 has led to intensive characterization of the
structural basis for its function and interaction with its physiologic
and pathologic ligands, SDF-1 and the gp120 subunit of X4 Env
glycoproteins, respectively. These studies have broadly defined the
domains of CXCR4 that play a role in HIV-1 infection, ligand binding,
and signaling. However, the molecular mechanisms of ligand-induced conformational changes and subsequent signaling remain unknown.
Three CXCR4 antagonists have been described that block infection by X4
strains of HIV-1 and SDF-1 binding, AMD3100 (10), ALX40-4C (12), and
T22/T140 (11, 13). Binding of AMD3100 to CXCR4 has been reported to
involve residues in transmembrane helix (TM) 4 and extracellular loop 3 (14) and possibly extracellular loop 2 (15). Administration of AMD3100
results in mobilization of hematopoietic stem cells (16), and clinical
trials in AIDS patients were discontinued because of cardiac
arrhythmias. The importance of CXCR4 as a potential molecular target in
HIV-1 infection and breast cancer provides a strong rationale for
characterizing the mechanism of the action of CXCR4 antagonists and
development of second generation antagonists with more favorable
pharmacological properties.
Here we have adopted a Saccharomyces cerevisiae expression
system to couple CXCR4 signaling to growth in the absence of histidine (17) and derived a CXCR4-CAM by random mutagenesis. The amino acid
substitution that conferred this phenotype involved Asn-119, which is
located in TM3, and further mutagenesis revealed substitutions that
stabilized inactive or active receptor conformations in yeast and
mammalian cells. Characterization of CXCR4 antagonists revealed that
T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak
partial agonists. Whereas binding studies failed to reveal evidence of
changes in extracellular domains in active or inactive variants, the
decreased affinity of CAMs for AMD3100 and ALX40-4C signifies a
conformational shift, most likely in the hydrophobic core of CXCR4.
Variants with autonomous signaling were chronically phosphorylated and
internalized. These findings illustrate the importance of TM3
conformation in the rearrangement in the hydrophobic core of CXCR4 that
leads to G Yeast Strains and Plasmids--
CXCR4 was functionally coupled
to the pheromone response pathway in the S. cerevisiae
strain CY12946 that has been described previously (17).
Yeast cells were transformed with CXCR4 constructs using the Frozen-EZ
Yeast Transformation-II kit (Zymo Research, Orange, CA). Site-directed
mutagenesis was performed using a QuikChange kit (Invitrogen).
Random Mutagenesis--
The open reading frame encoding CXCR4
was amplified in the presence of manganese and dITP by PCR to achieve a
final random mutation rate of 0.1-0.3%. This pool was cloned into
CP4258 and transformed into CY12946 yeast cells, which are
histidine auxotrophs. The yeast cell transformants were grown on medium
lacking histidine to select colonies with complementation of the
histidine auxotrophy by autonomous CXCR4 activation of the
pheromone-responsive FUS1-HIS3 reporter gene. The colonies were pooled
and grown, and plasmid DNA was extracted from these cultures. The
plasmid pool was transformed into CY12946 cells, and
colonies were selected for growth in medium lacking histidine. Plasmid
DNA from these colonies was sequenced.
Analysis of FUS1-HIS3 and FUS1-lacZ Reporter Genes--
Yeast
strain transformants expressing different CXCR4 constructs were tested
for expression of the pheromone-responsive FUS1-HIS3 reporter gene in medium lacking histidine. Recombinant SDF-1 was obtained from Leinco Technologies, Inc. (St. Louis, MO). Cell density
was determined from the absorbance at 600 nm. Expression of the
FUS1-lacZ reporter gene was determined using a fluorescent Derivation of Stable CHO Transfectants--
Stable CHO cell
transfectants expressing CXCR4 variants were prepared for each
construct as described previously (18). Transfectants expressing high
levels of each mutant were enriched from pools by magnetic sorting with
the anti-Myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Santa
Cruz, CA), and single cell clones with matched levels of cell surface
expression were isolated by immunofluorescent sorting with 12G5.
[125I]SDF-1 Binding and Displacement--
CHO
transfectants stably expressing CXCR4 variants were incubated with 0.1 nM [125I]SDF-1 (PerkinElmer Life Sciences) in
the presence or absence of cold inhibitors (SDF-1, T140, AMD3100, or
ALX40-4C) as described previously (18).
Signal Transduction--
For calcium flux experiments, cells
were loaded with Fura-2 acetoxymethyl ester (2 µg/ml)
(Molecular Probes) as described previously (18). The response to SDF-1,
T140, AMD3100, ALX40-4C, or 5-hydroxytryptamine (Sigma) was
recorded using a spectrofluorometer (F2500, Hitachi, San Jose, CA) as
described previously (18). The endogenous serotonin receptor expressed
by CHO cells was stimulated with 10 µM
5-hydroxytryptamine as a control for Gi CXCR4 Phosphorylation--
Phosphorylation of CXCR4 was
performed as described previously (19). CHO transfectants were
metabolically labeled with [32P]orthophosphate and then
stimulated with SDF-1 or phorbol ester. CXCR4 was immunoprecipitated
from detergent lysates via its N-terminal Myc tag with 9E10, and
labeled proteins were resolved by SDS-PAGE and visualized by autoradiography.
CXCR4 Internalization--
Stable CHO transfectants were exposed
to buffer or 100 nM SDF-1, permeabilized with 20%
methanol, and stained with 9E10 by indirect immunofluorescence.
Asn-119 Substitutions Regulate CXCR4 Activation of G Proteins in
Yeast and Mammalian Cells--
S. cerevisiae strains in
which activation of CXCR4 results in expression of pheromone-responsive
HIS3 or lacZ reporter genes were genetically
engineered. CXCR4-CAMs were selected from pools of random mutants of
the cDNA in this system. Screening of over 105
recombinant events with a mutational rate of 0.1-0.3% yielded clones
with different genotypes, all of which contained an A
The authenticity of the signaling phenotype of these CXCR4 mutants in
yeast was confirmed in stable CHO transfectants selected for matched
cell surface expression of the respective variants. Membrane fractions
from CXCR4(N119S) or CXCR4(N119A) transfectants exhibited basal
[35S]GTP
The ability of CXCR4 variants with substitutions of Asn-119 to
transduce SDF-1-mediated signaling in calcium mobilization experiments
is shown in Fig. 1D. CXCR4-WT transfectants demonstrated a
response to 0.03 nM SDF-1 that was of maximum intensity at
10 nM. In transfectants expressing CXCR4(N119S) or
CXCR4(N119A), a minimal response was detected following stimulation
with 0.3 nM SDF-1, and maximal calcium mobilization was
induced at 10 or 100 nM SDF-1, respectively. The magnitude
of peak calcium mobilization in CXCR4(N119S) or CXCR4(N119A)
transfectants was less than transduced by CXCR4-WT. The calcium
response to SDF-1 in CXCR4(N119D) transfectants was similar to that of
the WT receptor. In contrast, CXCR4(N119K) transduced a minimal
response following exposure to higher SDF-1 concentrations.
CXCR4 Variants with Asn-119 Substitutions Retain SDF-1 Binding
Properties of CXCR4-WT--
Binding experiments were performed to
determine whether the active conformation of CXCR4-CAMs affected
affinity for SDF-1. Parallel analysis of [125I]SDF-1
binding to stable CHO transfectants with matched levels of CXCR4-WT,
CXCR4(N119S), CXCR4(N119A), CXCR4(N119D), or CXCR4(N119K) expression
revealed that they bound ligand with similar IC50 values (Table I). These CXCR4 variants
had Myc and His6 epitope tags at the N and C termini,
respectively, and this was associated with a slight decrease in binding
affinity for SDF-1 (CXCR4, 2 nM;
Myc-CXCR4-His6, 20 nM). All CXCR4 variants with
Asn-119 substitutions had similar levels of 12G5 staining (data not
shown).
CXCR4-CAM Is Constitutively Phosphorylated and
Down-modulated--
Metabolic labeling experiments were performed to
determine whether the CXCR4-CAM is phosphorylated. Whereas CXCR4-WT was
not phosphorylated in resting cells, exposure to SDF-1 induced receptor phosphorylation (Fig. 2A).
Incubation of CXCR4-WT transfectants with phorbol ester also induced
receptor phosphorylation. In contrast, CXCR4(N119S) was phosphorylated
in resting cells, and the levels were not altered by exposure to SDF-1
or phorbol ester. The CXCR4(N119K) mutant showed minimal
phosphorylation induced by SDF-1 and had low level phosphorylation
following exposure to phorbol ester.
Immunofluorescence localization experiments were performed to determine
whether CXCR4-CAMs were internalized. CXCR4-WT was localized at the
surface of transfectants but was redistributed to cytoplasmic
inclusions following stimulation with SDF-1 (Fig. 2B). The
CXCR4-CAMs were localized to the cell surface and cytoplasmic inclusions in the absence and presence of SDF-1. CXCR4(N119K) was
present on the cell surface in the absence and presence of SDF-1.
Two Classes of CXCR4 Antagonists: T140 Is an Inverse Agonist, and
AMD3100 and ALX40-4C Are Weak Partial Agonists--
The effect of
CXCR4 antagonists on CXCR4-CAM signaling was determined in yeast and
mammalian cells. Exposure of yeast cells expressing CXCR4(N119S) to
incremental concentrations of ALX40-4C resulted in increased
The ability of these antagonists to influence CXCR4-CAM signaling was
tested in CHO cells stably expressing CXCR4(N119S). Incubation of
membrane fractions from CXCR4-CAM transfectants with 1 µM
ALX40-4C or AMD3100 increased [35S]GTP
The impact of CXCR4 antagonists on signal transduction by the CAM was
further characterized in calcium mobilization experiments (Fig.
3C). AMD3100 or ALX40-4C stimulated a low amplitude of
calcium mobilization in CXCR4-WT transfectants. In contrast, exposure of the same cells to T140 resulted in a brief decrease in free cytosolic calcium. All three antagonists inhibited subsequent responses
to SDF-1.
Parallel experiments were performed using CXCR4(N119S) transfectants to
characterize the effect of these antagonists (Fig. 3C).
Exposure to AMD3100 resulted in increased cytosolic calcium levels
similar to those elicited by 1 nM SDF-1. The transfectants were refractory to subsequent SDF-1 stimulation. Exposure of the transfectants to ALX40-4C also mobilized cytosolic calcium ions. Further exposure to SDF-1 induced a dampened response that was less
than that observed in naive cells. Incubation of CXCR4(N119S) transfectants with T140 induced a transient decrease in free cytosolic calcium and significantly reduced SDF-1-induced signaling. Identical experiments with CXCR4(N119A) transfectants revealed similar results (data not shown). The ability of SDF-1 or AMD3100 to induce signaling in a nontransfectant system was established in THP-1 cells, a primitive
monocytic cell line with endogenous CXCR4 expression. As shown in Fig.
3D, exposure to SDF-1 or AMD3100 resulted in calcium
mobilization responses similar to those observed in CHO cell
transfectants. Exposure to ALX40-4C did not induce a significant calcium flux (data not shown).
A dose response curve for the effect of AMD3100 on transfectants
expressing CXCR4-WT or CXCR4(N119S) is shown in Fig. 3 (E and F). A direct calcium mobilization response was first
evident in CXCR4-WT transfectants at 100 nM AMD3100 (Fig
3E). The response to stimulation with 1 nM SDF-1
was decreased by 100 nM AMD3100 and dramatically inhibited
at 10 µM.
Parallel analysis of CXCR4(N119S) CHO transfectants revealed that a
calcium flux response was detected following exposure to 1 nM AMD3100 (Fig. 3F). The magnitude of the
response was augmented by incremental doses of bicyclam to peak levels
at 1.0 µM. Inhibition of SDF-1-induced signaling mediated
by the CAM was evident at AMD3100 concentrations of 10 nM
and complete at 1 µM.
Conformational Shifts in CXCR4-CAMs Preferentially Affect AMD3100
and ALX40-4C Binding--
The ability of T140, AMD3100, and ALX40-4C
to displace [125I]SDF-1 binding was determined for
CXCR4-WT and the variants with Asn-119 substitutions.
[125I]SDF-1 displacement from stable transfectants failed
to reveal a difference in the IC50 for T140 binding to WT,
CAM, and inactive variants (Table I). Displacement of
[125I]SDF-1 binding by AMD3100 was similar in CXCR4-WT,
CXCR4(N119D), and CXCR4(N119K) (Fig. 4).
In contrast, AMD3100 displacement of [125I]SDF-1 binding
to the CXCR4-CAMs was significantly decreased (Fig. 4 and Table I).
Parallel studies showed a similar effect with ALX40-4C (Table I).
Here we demonstrate that Asn-119 plays a critical role in the
mechanism of CXCR4 signaling through its regulation of TM3
conformation. Conversion of Asn-119 to Ser or Ala was found to drive
the conformational equilibrium of CXCR4 to the active state, manifested
by autonomous signaling. In contrast, substitution of Lys for Asn-119
induced a conformation of TM3 that favors the inactive state, rendering CXCR4 unresponsive to SDF-1 binding, rendering CXCR4
unresponsive to SDF-1 without affecting the binding. The autonomous
coupling of the CXCR4-CAMs to Gi/o The availability of CXCR4-CAMs should provide a powerful tool for high
throughput screening for antagonists. Agents that inhibit the WT
receptor may increase or reduce the signaling of CAMs, depending on
whether the mechanism of action is that of a weak partial agonist, such
as AMD3100 and ALX40-4C, or an inverse agonist, such as T140. The
effects of AMD3100 and ALX40-4C on CXCR4 signaling were amplified in
the CAMs despite a significant decrease in the affinity, presumably
because the threshold for receptor activation was diminished in the variants.
Engagement of GPCR by ligand results in a conformational shift of the
hydrophobic core and cytoplasmic domains to a state permissive for the
formation of a high affinity ternary complex with G Constitutively active variants of several GPCR have been described,
either as a natural occurrence or as the product of genetic manipulations (22-29). This is the first report of a constitutively active variant of a chemokine receptor. Human herpesviruses encode chemokine receptor orthologs that exhibit autonomous signaling, the
Kaposi's sarcoma herpesvirus (KSHV) (30, 31) and human cytomegalovirus
(32). The GPCR encoded by the KSHV (KSHV-GPCR) is genetically related
to the chemokine receptor family (30, 31). This receptor displays
ligand-independent, constitutive activity that is mediated by the
phosphoinositide-specific phospholipase C signaling pathway (33). The
KSHV-GPCR binds multiple chemokines that may serve as agonists (34) or
inverse agonists (35). The precise mechanism for the autonomous
activity of this receptor is unclear, because it differs significantly
from even the most closely related genomically encoded receptor at the
level of primary structure. It has been shown that charged residues at
the interface of TM3 (Arg-143) and TM2 (Asp-83) with the adjacent
cytoplasmic interhelical loops influence the constitutive activity of
this receptor (36). Whereas CXCR4-CAM signaling in CHO cells was sensitive to pertussis toxin, KSHV-GPCR signaling in endothelial cells
involves multiple pathways, including pertussis toxin-sensitive and
-insensitive mechanisms (37).
The CXCR4-CAMs demonstrate evidence of chronic desensitization, as has
been observed in autonomously active variants of multiple GPCR (23,
25), that involves receptor phosphorylation and internalization. CXCR4
mutants with truncation of phosphorylation sites in the C-terminal tail
have been shown to exhibit increased signaling responses, indicative of
loss of negative regulatory control mechanisms (19). The finding that
CXCR4-CAMs were constitutively phosphorylated provides additional
evidence that the cytoplasmic aspect of these receptor variants mimics
the active conformation induced by ligand binding, which is recognized
by GPCR kinases. Whereas CXCR4 has been shown to be a substrate for
protein kinase C (19), the unresponsive CXCR4(N119K) variant appeared
to have decreased phosphorylation following ligand stimulation or
exposure to phorbol ester. These findings support the interpretation
that the architecture of the cytoplasmic aspect of the nonactivated receptor may be poorly recognized by GPCR kinases. The localization of
CXCR4-CAMs to perinuclear structures suggests that these receptors are
actively internalized in the absence of SDF-1 engagement, although the
formal possibility that this distribution is due to disruption of
intracellular trafficking cannot be fully excluded. However, CXCR4-CAMs
were targeted to the cell surface at levels similar to that of the WT
receptor, although a portion may be incompletely trafficked. A
transient decrease in cytosolic free calcium levels occurred following
exposure of CXCR4-CAM transfectants to T140, indicating a switch of the
receptor to the inactive conformation.
AMD3100, ALX40-4C, and T22/T140 have been reported to antagonize the
activation of CXCR4 by SDF-1 and to block infection by X4 strains of
HIV-1 in vitro (10-13). Whereas AMD3100 and ALX40-4C stimulated the signaling of the CXCR4-CAM in yeast and mammalian systems, T140 inhibited the autonomous coupling to G We thank Zi-xuan Wang for helpful advice and
suggestions, James Hoxie for providing 12G5, John Moore for supplying
AMD3100, and Krishna Prasad for help with phosphorylation experiments.
*
This work was supported by National Institutes of Health
Grant R01 AI41346 (to S. C. P.), by the Duggan Endowment for
Oncologic Research, and by Molecular Pathology Services of the Brown
Cancer Center.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.
§
These authors contributed equally to this work.
¶
Present address: Inst. of Biochemistry & Cell Biology, Chinese
Academy of Sciences, Shanghai 200031, China.
Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M200889200
The abbreviations used are:
SDF-1, stromal-derived factor 1;
CAM, constitutively active mutant;
GPCR, G
protein coupled receptor(s);
HIV-1, human immunodeficiency virus
type-1;
TM, transmembrane helix;
WT, wild type;
CHO, Chinese hamster
ovary;
GTP
A Point Mutation That Confers Constitutive Activity
to CXCR4 Reveals That T140 Is an Inverse Agonist and That AMD3100 and
ALX40-4C Are Weak Partial Agonists*
§¶,§
,,,
Henry Vogt Cancer Research Institute,
University of Louisville, Louisville, Kentucky 40202, the
** Graduate School of Pharmaceutical Sciences, Kyoto
University, Sakyo-ku, Kyoto 606-8501, Japan, the
Institute of Biochemistry & Cell Biology,
Chinese Academy of Sciences, Shanghai 200031, China,
§§ Myriad Genetics, Salt Lake City, Utah
84108, and the ¶¶ Department of Molecular Biology,
Princeton University, Princeton, New Jersey 08544
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit activation and provide insight into the clinical
effects of AMD3100. Utilization of GPCR CAMs in pharmaceutical
screening provides an efficient and powerful approach for
identification of novel antagonists.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase substrate (Molecular Probes, Eugene, OR). Enzymatic activity was determined using standard approaches. The experimental data were normalized using basal -galactosidase activity of
CXCR4-WT.
activation in
these cells. [35S]GTP
S binding was performed using
membrane pellets (7 µg) and 0.5 nM
[35S]GTPS (>1000 Ci/mmol; Amersham Biosciences) for
1 h at 37 °C. Bound isotope was separated by filtration with
GF/C membranes (Whatman) and measured by scintillation counting
of the washed, dried filters. Basal binding was determined in the
absence of agonists, and nonspecific binding was obtained in the
presence of 10 µM GTPS (Sigma). The percentage of
stimulated [35S]GTPS binding was calculated as
100 × (cpm/sample 
nonspecific cpm) 
(basal
cpm nonspecific cpm). Coupling to Gi/o subunits was determined by preincubating transfectants in the presence of
pertussis toxin (Calbiochem, La Jolla, CA) overnight.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
G
substitution resulting in the N119S mutation. Among 26 clones sequenced, no other mutation was found consistently. Expression of
CXCR4(N119S) in CY12946 yeast cells complemented the
histidine auxotrophy of this strain, indicating that this mutation is
sufficient to activate autonomous CXCR4 signaling. Substitution of
Asn-119 with neutral, acidic, or basic residues revealed that Ser and Ala, but not Asp or Lys, supported constitutive activation of the
pheromone-responsive expression of the FUS1-lacZ reporter gene (Fig. 1A). Exposure of
yeast cells expressing CXCR4(N119S) or CXCR4(N119A) to SDF-1
resulted in a small increase over respective unstimulated
-galactosidase levels (Fig. 1A). CXCR4(N119D)
demonstrated a minimal increase in -galactosidase activity to 1 µM SDF-1, and CXCR4(N119K) was refractory to ligand
stimulation.
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Fig. 1.
Substitution of Asn-119 in TM3 governs CXCR4
coupling to Gi/o subunits in yeast
and mammalian cells. CXCR4 Asn-119 mutants were tested for
autonomous and SDF-1-induced activation of the pheromone-responsive
FUS1-lacZ reporter gene in S. cerevisiae
(A). -Galactosidase activity from standardized cultures
of yeast strains expressing the CXCR4 variants was determined as
described under "Experimental Procedures." CXCR4 Asn-119 mutants
were tested for autonomous and SDF-1-induced coupling to G subunits
in stable CHO transfectants (B).
[35S]GTPS binding was performed as described
under "Experimental Procedures". Utilization of Gi/o
subunits for SDF-1-induced and CAM signaling was determined by
preincubation of CHO transfectants expressing CXCR4-WT or CXCR4(N119S)
with pertussis toxin (C). [35S]GTPS binding
was performed as described under "Experimental Procedures." The
values are the means ± S.E. of triplicate samples, and the
results are representative of three independent experiments
(A-C). The capacity of CXCR4 Asn-119 mutants to transduce
SDF-1 signaling was determined by calcium flux assay (D).
Stable CHO transfectants were loaded with Fura-2 and exposed to
incremental doses of SDF-1 as described under "Experimental
Procedures." The results are representative of two independent
experiments. PTX, pertussis toxin.
S binding that was >5-fold that of CXCR4-WT
transfectants (Fig. 1B). Exposure of each CAM to SDF-1
augmented G subunit activation. CXCR4(N119D) lacked autonomous
signaling but demonstrated SDF-1-induced [35S]GTPS
binding similar to that of CXCR4-WT. CXCR4(N119K) lacked signaling
activity. Exposure of the transfectants to pertussis toxin blocked the
response of CXCR4-WT to SDF-1 and extinguished the elevated basal
[35S]GTPS binding observed in CXCR4(N119S)
transfectants (Fig. 1C).
CXCR4 binding affinity of SDF-1 and antagonists
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Fig. 2.
CXCR4-CAMs are chronically desensitized.
Phosphorylation of CXCR4-WT and CXCR4 Asn-119 substitutions that confer
autonomous signaling (Ser) or inactivity (Lys) was determined by
metabolic labeling with [32P]orthophosphate and
immunoprecipitation (A). CHO transfectants with stable cell
surface expression of CXCR4-WT (lanes 1-3), CXCR4(N119K)
(lanes 4-6), or CXCR4(N119S) (lanes 7-9) at
matched levels were exposed to control buffer (lanes 1,
4, and 7), SDF-1 (lanes 2,
5, and 8), or phorbol ester (lanes 3,
6, and 9), and detergent lysates were
immunoprecipitated with 9E10, as described under "Experimental
Procedures." The results are representative of two independent
experiments. Down-modulation of CXCR4-WT (panels A and
B), CXCR4(N119S) (panels C and D), and
CXCR4(N119K) (panels E and F) in the absence
(panels A, C, and E) or presence
(panels B, D, and F) of SDF-1 was
determined by immunofluorescence as described under "Experimental
Procedures" (B). The results are representative of two
independent experiments.
-galactosidase levels at 1 µM (Fig.
3A). Incubation with AMD3100
resulted in significant increases in -galactosidase levels at 100 nM. In contrast, exposure to T140 decreased autonomous signaling in a dose-dependent manner.
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Fig. 3.
Classification of CXCR4 antagonists as weak
partial agonists (AMD3100 and ALX40-4C) or inverse agonists. The
effect of the known CXCR4 antagonists on autonomous signaling by
CXCR4(N119S) was determined from basal expression of the
pheromone-responsive FUS1-lacZ reporter gene (A).
Exposure of standardized yeast cultures to CXCR4 antagonists and
analysis of -galactosidase levels were determined as described under
"Experimental Procedures." The values are the means ± S.E. of
triplicate samples. The results are representative of three independent
experiments. The effect of the known CXCR4 antagonists on autonomous
coupling of CXCR4(N119S) to G subunits in mammalian cells was
determined by [35S]GTPS binding as described under
"Experimental Procedures" (B). The values are the
means ± S.E. of triplicate samples. The results are
representative of three independent experiments. The ability of CXCR4
antagonists to induce signaling and inhibit SDF-1-induced activation
was determined in calcium mobilization experiments (C).
Transfectants expressing matched levels of CXCR4-WT or CXCR4(N119S)
were loaded with Fura-2; exposed to control buffer, AMD3100, ALX40-4C,
or T140; and then exposed to SDF-1 (1 nM) as described
under "Experimental Procedures." The results are representative of
two independent experiments. The ability of SDF-1 or AMD3100 to induce
signaling in an hematopoietic cell line that expresses CXCR4 was
determined in THP-1 cells (D). THP-1 cells were loaded with
Fura-2 and exposed to SDF-1 or AMD3100 as described under
"Experimental Procedures." The results are representative of three
independent experiments. The dose-response effect of AMD3100 on SDF-1
(1 nM) stimulation of CXCR4-WT (E) and
CXCR4(N119S) (F) was determined as described under
"Experimental Procedures." The results are representative of two
independent experiments. 5-HT, 5-hydroxytryptamine;
AMD, AMD3100.
S binding (Fig.
3B). In contrast, exposure to T140 resulted in a dramatic,
dose-dependent decrease in [35S]GTPS binding.
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Fig. 4.
Conformational shift in CXCR4-CAMs decreases
the affinity of weak partial antagonist binding. CHO transfectants
with stable cell surface expression of CXCR4-WT or CXCR4 Asn-119
substitution mutants at matched levels were employed to analyze
displacement of [125I]SDF-1 binding by AMD3100. The
values are the means of duplicate samples. The results are
representative of two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits was
augmented by SDF binding, consistent with stabilization of an optimal
active conformation. AMD3100 and ALX40-4C were also found to increase
the signaling of CXCR4-CAMs and, to a lesser extent, that of CXCR4-WT,
indicating that they are weak partial agonists. In contrast, T140 was
found to be an inverse agonist that reversed the conformational
equilibrium induced by activating mutations to stabilize the inactive
form. Although there was no evidence for altered architecture of CXCR4 extracellular domains, significant decreases in the affinity of AMD3100
and ALX40-4C binding provided objective evidence for a conformational
shift in the hydrophobic core of the receptor.
subunits (20).
It is predicted that Asn-119 is located midway between the
extracellular and cytoplasmic interfaces of the TM3 helix of CXCR4 and
is oriented toward the center of the hydrophobic core based on
comparison with the rhodopsin crystal structure (21). The N(L/F)YSS
motif is highly conserved in TM3 of receptors for CXC chemokines and
may play a critical role as a switch that maintains the dynamic
conformational equilibrium that regulates coupling to G subunits.
AMD3100 binding has been reported to involve Asp-171, which is
predicted to reside in TM4 (14). This supports the interpretation that
the binding pocket for this partial agonist involves regions in the
hydrophobic core. Thus the decrease in binding affinity of CAMs
reflects a conformational shift in this region of CXCR4. The absence of
alterations in SDF-1, T140, and 12G5 binding by the CAMs provides
evidence that TM3 has sufficient intrinsic flexibility to permit
significant internal rearrangement of the hydrophobic core without
altering the architecture of extracellular domains.
subunits in
both. The maximum response of CXCR4-WT induced by high concentrations of AMD3100 or ALX40-4C was less than observed with SDF-1; thus these
agents are weak partial agonists. Because T140 functions as an inverse
agonist, it is likely that it interacts with CXCR4 through a mechanism
distinct from that employed by the partial agonists. The evidence that
coreceptor signaling by gp120 plays a critical role in viral
replication (38) raises the possibility that the weak partial CXCR4
agonist activity of AMD3100 and ALX40-4C could influence the infection
of target cells by R5 viruses or stimulate replication during latency
in vivo. This partial agonist activity would clearly
preclude the use of these agents as antagonists of metastatic behavior
in mammary carcinoma, because stimulation of CXCR4 on the surface of
tumor cells could increase dissemination. Patients receiving AMD3100
experienced cardiac conduction abnormalities, either because of the
weak partial agonist activity for CXCR4 or because of cross-reactivity
with another GPCR. The ability of AMD3100 to stimulate mobilization of
hematopoietic stem cells (16) mimics the effects of SDF-1. T140 was
found to suppress this process in mice (39), as expected for an inverse
agonist. The ability to discriminate between the biologic properties of T140 and those of AMD3100 and ALX40-4C illustrates the utility of CAMs
in the characterization of GPCR antagonists. This should provide a
powerful approach to the development of a new generation of agents to
block CXCR4 involvement in AIDS and the metastatic behavior of
malignant tumors.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Pathology, Medical College of
Georgia, Augusta, GA 30912.
To whom all correspondence should be addressed.
Tel.: 706-721-2923; Fax: 706-721-2358; E-mail:
speiper@mail.mcg.edu.
ABBREVIATIONS
S, guanosine 5'-3-O-(thio)triphosphate;
KSHV, Kaposi's sarcoma herpesvirus.
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