THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 27, Issue of July 5, pp. 24515–24521, 2002
Printed in U.S.A.
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*
Received for publication, January 28, 2002, and in revised form, March 26, 2002
Published, JBC Papers in Press, March 28, 2002, DOI 10.1074/jbc.M200889200
Wen-bo Zhang‡§¶, Jean-Marc Navenot‡§储, Bodduluri Haribabu‡, Hirokazu Tamamura**,
Kenichi Hiramatu**, Akane Omagari**, Gang Pei‡‡, John P. Manfredi§§, Nobutaka Fujii**,
James R. Broach¶¶, and Stephen C. Peiper‡储 储储
From the ‡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
CXCR4 is a G protein-coupled receptor for stromalderived 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 devel* 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. This 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.
储 Present address: Dept. of Pathology, Medical College of Georgia,
Augusta, GA 30912.
储储 To whom all correspondence should be addressed. Tel.: 706-7212923; Fax: 706-721-2358; E-mail: [email protected].
1
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␥S, guanosine
5⬘-3-O-(thio)triphosphate; KSHV, Kaposi’s sarcoma herpesvirus.
This paper is available on line at http://www.jbc.org
opment (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␣ subunit activation and provide insight into the clinical
effects of AMD3100. Utilization of GPCR CAMs in pharmaceu-
24515
24516
Constitutive Activity of CXCR4
tical screening provides an efficient and powerful approach for
identification of novel antagonists.
EXPERIMENTAL PROCEDURES
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 ␤-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.
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 ␮g/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 ␮M 5-hydroxytryptamine as a control for Gi␣ activation in these cells. [35S]GTP␥S binding was performed using membrane pellets (7 ␮g) and 0.5 nM [35S]GTP␥S (⬎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 ␮M GTP␥S (Sigma). The percentage of stimulated
[35S]GTP␥S 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.
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.
RESULTS
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 pheromoneresponsive HIS3 or lacZ reporter genes were genetically engineered. CXCR4-CAMs were selected from pools of random mu-
tants 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 3
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 ␮M SDF-1, and CXCR4(N119K) was refractory to ligand
stimulation.
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␥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]GTP␥S 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]GTP␥S binding observed in CXCR4(N119S) transfectants (Fig. 1C).
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; MycCXCR4-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 Downmodulated—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)
Constitutive Activity of CXCR4
24517
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]GTP␥S binding was performed as described under “Experimental Procedures”. Utilization of Gi/o␣ subunits for SDF-1induced and CAM signaling was determined by preincubation of CHO transfectants expressing CXCR4-WT or CXCR4(N119S) with pertussis toxin
(C). [35S]GTP␥S 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.
24518
Constitutive Activity of CXCR4
TABLE I
CXCR4 binding affinity of SDF-1 and antagonists
CXCR4 (Myc/His)
Signaling
EC50 of SDF-1
IC50
T140
nM
Wild type
N119S
N119A
N119D
N119K
a
SDF-1
CAM
CAM
SDF-1
No
19.8 ⫾ 8.8
19.2 ⫾ 4.5
10.9 ⫾ 5.1
32.6 ⫾ 0.4
7.2 ⫾ 1.7
AMD3100
ALX40-4C
nM
16.0
27.6
13.4
16.6
8.4
106
3440
2270
81.7
66.9
717
4060
NDa
ND
ND
ND, not determined.
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.
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 incremen-
tal concentrations of ALX40-4C resulted in increased ␤-galactosidase levels at 1 ␮M (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.
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 ␮M ALX40-4C or AMD3100
increased [35S]GTP␥S binding (Fig. 3B). In contrast, exposure
to T140 resulted in a dramatic, dose-dependent decrease in
[35S]GTP␥S binding.
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 ␮M.
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 ␮M. Inhibition of SDF-1-induced signaling
mediated by the CAM was evident at AMD3100 concentrations
of 10 nM and complete at 1 ␮M.
Conformational Shifts in CXCR4-CAMs Preferentially Affect
AMD3100 and ALX40-4C Binding—The ability of T140,
Constitutive Activity of CXCR4
24519
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]GTP␥S 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.
24520
Constitutive Activity of CXCR4
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.
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 CXCR4CAMs was significantly decreased (Fig. 4 and Table I). Parallel
studies showed a similar effect with ALX40-4C (Table I).
DISCUSSION
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␣ 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.
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␣ subunits (20). It is predicted that Asn-119 is
located midway between the extracellular and cytoplasmic in-
terfaces 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.
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
Constitutive Activity of CXCR4
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␣ 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.
Acknowledgments—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.
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