From the Institute of Interdisciplinary Research,
** Service de Génétique Médicale, and
§§ Laboratoire de Cytologie et de
Cancérologie Experimentale, Université Libre de
Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Brussels,
Belgium, the ¶ Department of Microbiology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, and the
Serono Pharmaceutical Research Institute,
Geneva 1228, Switzerland
Received for publication, June 8, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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CCR5 is a functional receptor for
various inflammatory CC-chemokines, including macrophage inflammatory
protein (MIP)-1 Chemokines are a family of small proteins (8-12 kDa) that play a
crucial role in the development of immune response by organizing the
recruitment and the trafficking of immune cell populations throughout
the body, under both physiological and pathological conditions (1, 2).
They mediate their biological activities by signaling through G
protein-coupled receptors (2). CCR5 is a functional receptor for the
CC-chemokines MIP-1 Chemokines share a similar monomeric fold, characterized by a
disordered amino-terminal domain, followed by a conserved core region,
consisting of the so called "N-loop," three anti-parallel In this study, we have determined the binding and functional properties
of chemokines and chemokine variants onto CCR5 point mutants. We have
found that several residues in CCR5 extracellular domains are able to
discriminate between the core region of MIP-1 CCR5 Mutants--
Plasmids encoding the CCR5 mutants were
constructed by site-directed mutagenesis, using the QuikChange method
(Stratagene). Following sequencing of the constructs, the mutated
coding sequences were subcloned into the bicistronic expression vector
pEFIN3 as previously described (23). All constructs were verified by
sequencing before transfection.
Chemokines--
RANTES, the MIP/RANTES chimera, and
RANTES-(8-68) were produced as previously described (16, 24). The
proteins were subjected to Edman degradation and electrospray mass
spectroscopy for sequence verification. MIP-1 Expression of Mutant Receptors in CHO-K1 Cells--
CHO-K1 cells
were cultured in Ham's F-12 medium supplemented with 10% fetal calf
serum (Invitrogen), 100 units/ml penicillin, and 100 µg/ml
streptomycin (Invitrogen). Constructs encoding wild-type or mutant CCR5
in the pEFIN3 bicistronic vector were transfected using Fugene 6 in a
CHO-K1 cell line expressing an apoaequorin variant targeted to
mitochondria, as previously described (25). Selection of transfected
cells was made for 14 days with 400 µg/ml G418 (Invitrogen), and the
population of mixed cell clones expressing wild-type or mutant
receptors was used for binding and functional studies. Cell surface
expression of the receptor variants was measured by flow cytometry
using mAbs recognizing distinct extracellular epitopes of the receptor.
The phycoerythrin-conjugated 2D7 and 3A9 mAbs were purchased from
Pharmingen. mAbs 531, 523, and CTC5 were purchased from R&D Systems.
mAbs MC-1, MC-4, MC-5, and MC-6 were kindly provided by Mathias
Mack (University of Munich, Munich, Germany). The epitope
mapping of these mAbs has been described previously (26, 27).
Binding Assays--
CHO-K1 cells expressing wild-type or mutant
CCR5 were collected from plates with Ca2+- and
Mg2+-free phosphate-buffered saline supplemented with 5 mM EDTA, gently pelleted for 2 min at 1000 × g, and resuspended in binding buffer (50 mM
Hepes, pH 7.4, 1 mM CaCl2, 5 mM
MgCl2, 0.5% bovine serum albumin). Competition binding
assays were performed in Minisorb tubes (Nunc), using 0.1 nM [125I]MIP-1 Functional Assays--
The functional response to chemokines was
analyzed by measuring the luminescence of aequorin as described (28,
29). Briefly, cells were collected from plates in Ca2+- and
Mg2+-free Dulbecco's modified Eagle's medium supplemented
with 5 mM EDTA, pelleted for 2 min at 1000 × g, resuspended in Dulbecco's modified Eagle's medium at a
density of 5 × 106 cells/ml, and incubated for 2 h in the dark in the presence of 5 µM coelenterazine H
(Molecular Probes). Cells were diluted 5-fold before use. The cell
suspension (50,000 cells in 50 µl of Dulbecco's modified Eagle's
medium) was added to agonists placed in the wells or microtiter plates
in 50 µl of the same medium, and luminescence was recorded for
30 s in a Berthold luminometer.
Effects of CCR5 Extracellular Loop Mutations on the Functional and
Binding Properties of RANTES, MIP-1
All mutants were expressed at levels similar to that of wtCCR5 (Fig.
2A). For most mutants, there
was a fairly good correlation in the relative fluorescence obtained
with the different mAbs. Differences in the staining pattern by the
various mAbs were, however, found for mutants E172A and D276A. The
substitution of Glu172 by Ala strongly affected the
labeling by some ECL2 and multidomain mAbs (Fig. 2B),
whereas recognition by other mAbs (MC-1, MC-4, MC-5, etc.) was not
altered (see Fig. 2B and Refs. 26 and 27), suggesting that
these mutations affected the epitopes recognized by some mAbs but did
not alter receptor expression. A slight increase in relative staining
was observed for D276A, using the antibodies directed at amino-terminal
epitopes, such as MC-4, MC-5, or CTC5 (Fig. 2C).
We first measured the ability of the mutant receptors to activate
intracellular cascades in response to MIP-1
To determine whether the reduction in the functional response of the
mutants to some CCR5 ligands is the consequence of a reduced affinity
for these chemokines, we performed competition binding assays on the
mutants, using the best agonist as a tracer. In agreement with the
functional assay, wtCCR5 displayed a higher binding affinity for
LD78
From this series of experiments, we can conclude that
mutants affecting charged residues in CCR5 extracellular domains
(mostly in ECL2) affected differently the binding and, as a
consequence, the functional properties of RANTES and MIP-1 Binding and Functional Properties of MIP/RANTES and
LD78
The abilities of MIP-1
The contribution of binding parameters to the observed reduction of
functional response to some chemokine variants was investigated whenever possible by competition binding assays, using either [125I]RANTES or [125I]MIP-1
From this second set of data, we can conclude that most mutations of
aromatic residues located in TM2 and TM3 of CCR5 differentially affect
the functional response to MIP-1 A number of studies have identified residues in chemokines or
chemokine receptors that are important for binding and receptor activation, although the precise molecular mechanisms by which chemokines interact with their receptors are largely unknown. By using
chimeras between CCR5 and its closest homologue, CCR2b, we have
previously identified the second extracellular loop (ECL2) of CCR5 as
the main determinant of ligand selectivity (23). The importance of the
CCR5 amino-terminal domain for chemokine binding was also demonstrated,
in particular structural determinants consisting of negatively charged
and aromatic residues (34). A number of tyrosines in the amino-terminal
domain of CCR5 are sulfated, and this post-translational modification
has been proposed to provide a larger, potentially more flexible and
negatively charged surface for ligand binding (35, 36). Some CCR5
mutations, including in extracellular domains, affect the
pharmacological profile of the receptor, with a loss of functional
response to specific chemokines but not others. This observation
suggests that different chemokines interact with different residues
within the extracellular domains of a given receptor (30). Such mutants are particularly interesting because they may help to test hypotheses concerning the precise way chemokines bind and activate their receptors. In this study, we have used CCR5 mutants that discriminate between MIP-1 The Globular Core of the Chemokine Interacts with the Extracellular
Domain of CCR5--
Using different chemokines as tracers, we found
that a number of CCR5 mutants, in which charged residues in
extracellular domains were substituted for alanine, continued to bind
RANTES with high affinity, but not MIP-1
We and others have identified in different CCR5 ligands conserved or
chemokine-specific residues that are important for receptor binding. A
conserved aromatic residue located in the N-loop of MIP-1
Motifs of acidic and hydrophobic residues located in the amino-terminal
domain of chemokine receptors represent a common binding motif for
chemokines (20, 34). It is therefore tempting to speculate that the
conserved binding sites identified in chemokines and in chemokine
receptors interact with one another. This common interaction surface might account for the promiscuous character of
CC-chemokine binding to their receptors. In addition to this conserved binding site in the N-terminal domain, CCR5-specific residues, in particular those in ECL2, are involved in the selective binding of a chemokine subset. As discussed above, the MIP-1 The Amino Terminus of the Chemokine Interacts with the TM Domain of
CCR5--
Activation of G protein-coupled receptors is thought to
involve conformational changes within transmembrane helices, which are
either induced or stabilized upon agonist binding and which allow the
receptor to trigger signaling through G proteins (39). We have recently
identified a key motif (TXP) in CCR5 transmembrane helix 2 that plays a major role in chemokine-induced receptor activation (32).
We found that mutations of this TXP motif induce a profound
alteration of CCR5 activation, although this functional defect was
strongly chemokine-selective. RANTES responses were the least affected,
whereas MCP-2 effects were highly dependent on the integrity of this
motif. Molecular dynamics simulations predicted that the Pro residue of
this structural determinant would orient the extracellular part of TM2
toward TM3, and not toward TM1 as seen in the structure of rhodopsin,
suggesting a direct interaction between TM2 and TM3 at this level. To
analyze further the residues involved in the activation switch of CCR5, we studied the role of a cluster of aromatic residues located at close
proximity of the TXP motif in TM helices 2 and 3. The substitution of these aromatic residues in CCR5 by their CCR2b counterparts resulted, for some of the mutants, in a profound alteration of their ability to functionally respond to chemokines, while retaining their ability to bind them with high affinity. As for
TXP mutants, the functional alteration induced by mutations of aromatic residues was chemokine-selective, some mutants presenting a
clear difference in their respective response to MIP-1
The most widely accepted model for G protein-coupled
receptor activation is the ternary complex model (39). According to this model, the receptor exists in a equilibrium between an inactive conformation (R) and an active conformation (R*). Agonists are predicted to bind with high affinity to the R* conformation, and due to
the mass action law, to increase the proportion of R*. It is somewhat
surprising that the mutation of residues expected to interact with the
N-terminal domain of chemokines does not result in a significant
decrease in the measured binding affinity. It is therefore likely that
the interaction responsible for receptor activation does not contribute
much to the overall stability of the chemokine-receptor complex, in
which the contacts between the core of the chemokine and the
extracellular domains of the receptor play the major role. Mutations
strongly impairing activation without significantly affecting ligand
binding have been described for other G protein-coupled receptors,
including the C5a receptor (40) and the thyrotropin receptor (41).
Several structural differences between the amino-terminal
domain of the various chemokines might account for the differences in
their ability to activate receptors mutated in the aromatic cluster.
The N terminus of RANTES is shorter (9 residues) than that of other
CCR5 ligands (10 residues). RANTES, like LD78
In summary, we can now propose a more detailed scheme for the
interaction between CCR5 and its ligands (Fig.
7). The core domain of chemokines
mediates high affinity receptor binding through interactions with
various residues located in CCR5 extracellular domains, whereas their
amino-terminal domain interacts with TM residues and mediates receptor
activation. Further studies will be needed to understand more precisely
the molecular details of this process.
and RANTES (regulated on activation normal T
cell expressed and secreted), and is the main coreceptor of human
immunodeficiency viruses. The second extracellular loop and
amino-terminal domain of CCR5 are critical for chemokine binding,
whereas the transmembrane helix bundle is involved in receptor
activation. Chemokine domains and residues important for CCR5 binding
and/or activation have also been identified. However, the precise way
by which chemokines interact with and activate CCR5 is presently
unknown. In this study, we have compared the binding and functional
properties of chemokine variants onto wild-type CCR5 and CCR5 point
mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1
binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the
MIP-1
N terminus and the RANTES core, bound to these mutants with an
affinity similar to that of RANTES. Several CCR5 mutants affecting
transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F)
reduced the potency of MIP-1
by 10-100 fold with little effect on
activation by RANTES. However, the MIP/RANTES chimera activated these
mutants with a potency similar to that of MIP-1
. In contrast,
LD78
, a natural MIP-1
variant, which, like RANTES, contains a
proline at position 2, activated these mutants as well as RANTES.
Altogether, these results suggest that the core domains of MIP-1
and
RANTES bind distinct residues in CCR5 extracellular domains, whereas
the N terminus of chemokines mediates receptor activation by
interacting with the transmembrane helix bundle.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
(CCL3), MIP-1
(CCL4), RANTES (CCL5), MCP-2 (CCL8), LD78
,
and HCC-1-(9-78) (CCL14-(9-78)) and is expressed on memory T
cells, macrophages, dendritic cells, and migroglia (2-6). CCR5 is also the principal coreceptor of human immunodeficiency viruses that, in
concert with CD4, mediates the binding of the viral envelope protein to
the cell surface, allowing subsequent entry into target cells (7). The
key role played by CCR5 in human immunodeficiency virus pathogenesis
has been demonstrated by the almost complete resistance to human
immunodeficiency virus-1 infection of individuals homozygous for a
32-base pair deletion in the coding sequence of the receptor, which
results in the absence of functional coreceptor at the cell surface (8,
9). The absence of pathological phenotype in individuals lacking
functional CCR5, together with the potent human immunodeficiency
virus-suppressive activity of CCR5 antagonists, makes this receptor an
attractive candidate for pharmacological intervention (10). Moreover,
CCR5 appears to be involved in a much broader range of human immune
diseases, including multiple sclerosis, rheumatoid arthritis, and renal allograft rejection, suggesting that blocking CCR5 function might be
beneficial in these diseases as well (11, 12). For these reasons,
understanding at the molecular level how CCR5 interacts with chemokines
and gp120 and how CCR5 activates G protein signaling as a consequence
of chemokine binding might help in the rational design of CCR5 blocking agents.
-strands, and a carboxyl-terminal
-helix (13). The currently prevailing model for chemokine-chemokine receptor interaction postulates a two-step mechanism, in which the core of the chemokine interacts first with a binding site formed by the extracellular domains
of the receptor, while another interaction is required between the
chemokine N terminus and a second binding site on the receptor in order
to trigger receptor activation. Such a two-step model is analogous to
the interaction of C5a, a chemoattractant protein with a size similar
to that of chemokines, with its cognate G protein-coupled receptor
(14). In agreement with this model, amino-terminal truncations of
various CC-chemokines, including CCR5 ligands such as MIP-1
and
RANTES, result in a profound reduction of their biological activity,
although they retain most of their binding capability (13, 15-17).
Amino-terminally truncated CC-chemokines act therefore as partial
agonists or full antagonists. On the other hand, we and others have
recently identified various residues located in the core domain of
CC-chemokines that contribute to their high affinity binding to
receptors (17-22). There is, however, no direct experimental
demonstration that the N-terminal and core domains of chemokines
interact with structurally and functionally independent sites on their
cognate receptors.
and RANTES. We have
also identified several residues in CCR5 transmembrane helices 2 and 3 that are crucial for chemokine-induced receptor activation. Their
substitution affects differentially the functional response of MIP-1
and RANTES, and we show that the N-terminal domain of chemokines is
involved in this specificity.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, LD-78
, and MCP-2
were purchased from R&D Systems.
or 0.05 nM
[125I]RANTES (2000 Ci/mmol; Amersham Biosciences) as
tracer, variable concentrations of competitors, and 40,000 cells in a
final volume of 0.1 ml. Total binding was measured in the absence of
competitor, and nonspecific binding was measured with a 100-fold excess
of unlabeled ligand. Samples were incubated for 90 min at 27 °C, and
then bound tracer was separated by filtration through GF/B filters
presoaked in 1% bovine serum albumin for [125I]MIP-1
or 0.5% polyethylenimine (Sigma) for [125I]RANTES.
Filters were counted in a
-scintillation counter. Binding parameters
were determined with the Prism software (GraphPad Software) using
nonlinear regression applied to a one-site competition model.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, LD78
, and
MIP/RANTES--
We have previously shown that various CCR5
agonists display differential sensitivity to substitutions of residues
located in the extracellular domains of the receptor, suggesting that the binding site of these agonists is partially nonoverlapping (30). We
have therefore investigated mutants of all charged amino acids present
in the CCR5 extracellular domain for their ability to bind
[125I]MIP-1
, MIP
1
, or RANTES (data not
shown). Several mutants, affecting residues located in ECL2 (R168A,
E172A, K191A) or ECL3 (D276A) (Fig.
1A), discriminated between
MIP-1
and RANTES in a binding assay. These mutants and wild type
CCR5 were stably expressed in CHO-K1 cell lines coexpressing
apoaequorin, and cell surface expression of the receptor was assayed by
flow cytometry using a panel of mAbs recognizing different linear as
well as conformation-sensitive amino-terminal epitopes (MC-4, MC-5,
CTC5), conformation-sensitive epitopes in ECL2 (MC-1, 2D7), or a
multidomain epitope (MC-6) (26, 27).
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Fig. 1.
CCR5 mutants and chemokine variants.
A, CCR5 mutants. The putative transmembrane organization of
CCR5 is represented, as well as the location and the nature of the
amino acid substitutions (in black) analyzed in this study.
Extracellular loops (ECL), intracellular loops
(ICL), and transmembrane domains (TM) are
numbered. Disulfide bonds linking together CCR5 extracellular domains
are shown (Cys20-Cys269 and
Cys101-Cys178). B, sequence
alignment of the various CCR5 agonists used in this study.
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Fig. 2.
Surface expression and receptor conformation
of CCR5 mutants. Cell surface expression of wtCCR5 and the
different mutants was analyzed by fluorescence-activated cell sorting
using 2D7-PE and MC-5 monoclonal antibodies. Mean channel fluorescence
was obtained for all mutants using the 2D7 and MC-5 mAb. A typical
experiment out of three performed independently is represented.
Staining of untransfected cells with mAb was used as a negative
control. Receptor conformation of wtCCR5 and the different mutants was
analyzed by fluorescence-activated cell sorting using mAbs of various
classes.
, RANTES, LD78
(a
natural variant of MIP-1
containing, like RANTES, a Pro in position
2), and a MIP/RANTES chimera, by using a calcium reporter assay based
on the coexpression of apoaequorin (28). The MIP/RANTES chimera, which
contains the amino-terminal domain of MIP-1
and the RANTES core, was
designed to investigate which domain of the chemokines is involved in
their receptor binding and activation properties. Earlier work has
shown that the proline in position 2 of LD78
contributes greatly to
the high affinity of this chemokine for CCR5 (5). As described
previously (5, 24, 30), LD78
appeared as the most potent ligand for
wtCCR5, with an EC50 of 0.71 nM, followed by
RANTES (EC50 of 3.2 nM), MIP-1
(EC50 of 3.2 nM), and MIP/RANTES
(EC50 of 6.7 nM; Fig.
3A and Table
I). On R168A-expressing cells, LD78
,
RANTES, and MIP/RANTES elicited a much stronger functional response
(EC50 of 14, 14, and 45 nM, respectively) than
MIP-1
, for which a minute signal could be detected only at the
highest concentration tested (EC50
300 nM; Fig. 3B). The E172A mutant responded to RANTES with a
potency similar to that of wtCCR5 (EC50 = 3.4 nM); its response to LD78
(EC50 = 4.1 nM) and MIP/RANTES (EC50 = 27 nM)
was moderately affected, whereas that to MIP-1
was strongly reduced
both in terms of potency and efficacy (Fig. 3C). K191A
behaved grossly in a similar way (EC50 of 14 and 45 nM for RANTES and MIP/RANTES, respectively), except that no
functional response was observed up to 300 nM of MIP-1
(Fig. 3D). The response of D276A (mutation located in ECL3) to the three agonists tested was affected (EC50 of 9.8, 72, and 104 nM for RANTES, MIP/RANTES, and MIP-1
,
respectively) (Fig. 3E). Interestingly, whereas RANTES was
usually more efficient than MIP-1
on the various mutants affecting
CCR5 extracellular domains, MIP
1
elicited the strongest functional
response in D276A-expressing cells (EC50 = 29 nM, Emax twice as high as for RANTES; data not
shown).
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Fig. 3.
Functional response of CCR5 extracellular
domain mutants. The functional response of the cell lines
co-expressing apoaequorin and CCR5 extracellular domain mutants was
tested following the addition of MIP-1 , RANTES, LD78
,
and the MIP/RANTES chimera. A, wtCCR5; B,
R168A; C, E172A; D, K191A, E, D276A.
The luminescent signal resulting from the activation of the
apoaequorin-coelenterazine complex was recorded for 30 s in a
luminometer. Results were analyzed by nonlinear regression using the
Graphpad Prism software. The data were normalized for basal (0%) and
maximal luminescence (100%). All points were run in triplicate
(error bars represent S.E.). The displayed curves
represent a typical experiment out of three performed
independently.
Binding and functional parameters for extracellular domain mutants
than for RANTES, MIP-1
, or MIP/RANTES, both in
homologous and heterologous assays (Fig.
4A and Table I). Using
[125I]RANTES as a tracer, LD78
, RANTES, and MIP/RANTES
bound the R168A mutant with similar potency (IC50 of 0.46, 0.47, and 2.4 nM, respectively), whereas MIP-1
hardly
competed for RANTES binding at the highest concentrations tested
(IC50 > 1 µM; Fig. 4B). E172A bound RANTES with an affinity similar to that of wtCCR5
(IC50 = 0.15 nM) but displayed a reduced
affinity for LD78
(IC50 of 4.1 nM) and
MIP/RANTES (IC50 of 25 nM) and, to a much
greater extent, for MIP-1
(IC50 > 1 µM;
Fig. 4C). As shown in Fig. 4D, RANTES and
MIP/RANTES competed with a similar potency for binding to D276A, using
[125I]MIP-1
as a tracer (IC50 = 0.27 and
0.15 nM, respectively), whereas MIP-1
was 20-fold less
potent (IC50 = 5.9 nM). Specific binding to
K191A-expressing cells was below the limit of detection, even using
higher concentrations of [125I]RANTES (data not shown),
making it impossible to determine chemokine binding affinities for this
mutant.
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Fig. 4.
Chemokine binding to CCR5 extracellular loop
mutants. Competition binding curves were performed on CHO-K1 cell
lines expressing wtCCR5 (A), R168A (B), E172A
(C), or D276A (D) mutants using 0.05 nM [125I]RANTES or 0.1 nM
[125I]MIP-1 as tracer. Results were analyzed by the
Graphpad Prism software, using a single site model, and the data were
normalized for nonspecific (0%) and specific binding in the absence of
competitor (100%). All points were run in triplicate (error
bars represent S.E.). Data are representative of three independent
experiments.
. The
MIP/RANTES chimera behaved essentially as RANTES, suggesting that these
CCR5 extracellular residues constitute binding sites for the core
domain of chemokines. The behavior of LD78
, which keeps a relatively high affinity for the mutant receptors, suggests a strong contribution of its N-terminal domain in the overall stability of the interaction with CCR5.
onto Mutants Affecting CCR5 Transmembrane Segments--
We
have recently observed that mutations in CCR5 transmembrane (TM) helix
2 differentially affected the functional response to various high
affinity ligands without significantly affecting their binding affinity
(32). In our effort to understand the molecular mechanisms involved in
CCR5 activation, we have investigated further the role played in this
process by aromatic residues located in TM2 and TM3. As a result, we
found that a number of additional amino acid substitutions within TM2
and TM3 also differentially affected the functional response to CCR5
ligands (33). We have selected here three mutants that do not affect
the binding affinity to MIP-1
and RANTES (L104F, L104F/F109H/F112Y,
and F85L-L104F) while exhibiting different patterns of functional
responses to these chemokines (Fig. 1A). Combining a set of
different chemokines with these mutants allowed us to determine which
part of the chemokines account for this selective reduction of agonist potency.
and RANTES to activate the mutant receptors
were compared with that of MIP/RANTES (which contains MIP-1
amino
terminus), LD78
, and RANTES- (8-68) (a variant lacking the first
seven amino acids). All transmembrane mutant receptors were
expressed in apoaequorin-expressing CHO-K1 cell lines. Their apparent
expression level at the cell surface, as determined by fluorescence-activated cell sorting analysis using mAbs directed either
at conformational or linear epitopes, was similar to that of wt CCR5
(data not shown). As described above (Fig.
5A and Table II), LD78
was the most potent agonist
for wtCCR5 (EC50 of 0.71 nM). RANTES-(8-68)
resulted only in a partial activation of CCR5 at high concentrations
(EC50 > 300 nM). Some CCR5 mutants affecting aromatic residues in TM2 and/or TM3 (L104F, L104F/F109H/F112Y, F85L/L104F) were characterized by a general reduction of their functional response to chemokines, but the potency and efficacy of
RANTES were generally less affected than those of MIP-1
(Fig. 5,
A-D, and Table II). For some mutants, such as F85L/L104F,
RANTES potency was decreased by half a log, whereas MIP-1
potency
was decreased by more than 1 order of magnitude (Fig. 5C).
For other mutants, such as L104F/F109H/F112Y, RANTES potency was
not affected (EC50 of 2.3 nM), whereas MIP-1
potency was reduced by about 1 order of magnitude (Fig. 5D).
For all of these mutants, the functional response to LD78
was
similar to that of RANTES, whereas the MIP/RANTES chimera behaved
similarly to MIP-1
(Fig. 5, A-D, and Table II).
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Fig. 5.
Functional response of CCR5 transmembrane
helix mutants. The functional response of cell lines co-expressing
apoaequorin and CCR5 transmembrane mutants was tested following the
addition of MIP-1 , RANTES, MIP/RANTES, and LD78
. A,
wtCCR5; B, L104F; C, L104F/F109H/F112Y;
D, F85L/L104F. The results were analyzed and normalized as
described for Fig. 4. All points were run in triplicate (error
bars represent S.E.). The displayed curves represent a typical
experiment out of three performed independently.
Binding and functional parameters for transmembrane domain mutants
as tracers.
As previously described, LD78
appeared as the natural ligand
displaying the highest affinity for wtCCR5 (IC50; 0.067 nM using [125I]MIP-1
as tracer), followed
by RANTES (IC50 = 0.23 nM), MIP/RANTES (IC50 = 0.36 nM) and MIP-1
(IC50 = 0.38 nM). In agreement with previous reports,
amino-terminal truncation of RANTES resulted in a moderate decrease of
its affinity for wtCCR5 (IC50 = 0.94 nM; Fig.
6A). For all three
transmembrane CCR5 mutants, the affinity for the various ligands,
including RANTES-(8-68) did not change significantly as compared with
wtCCR5 (Fig. 6, A-D, and Table II).
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Fig. 6.
Chemokines binding to CCR5 transmembrane
helix mutants. Competition binding curves were performed on CHO-K1
cell lines expressing wtCCR5 (A) or the L104F
(B), L104F/F109H/F112Y (C), or F85L/L104F
(D) mutants using 0.05 nM
[125I]RANTES or 0.1 nM
[125I]MIP-1 as tracer. The results were analyzed and
normalized as described for Fig. 3. All points were run in triplicate
(error bars represent S.E.). Data are
representative of three independent experiments.
and RANTES, without significantly altering their binding affinity. A functional defect on these mutants
was observed only for chemokine variants containing the N terminus of
MIP-1
(i.e. MIP-1
and MIP/RANTES). The Pro in position
2 of LD-78
was sufficient to recover loss of activity of MIP-1
.
These observations suggest strongly that the N terminus of chemokines
triggers receptor activation through an interaction with the
transmembrane helix bundle.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and RANTES, to determine which chemokine domains interact with specific CCR5 sites and the role of these interactions in
determining binding affinity and receptor activation.
. These mutations were
largely located in ECL2. We determined, by using a chimeric chemokine (MIP/RANTES) containing the amino-terminal domain of MIP-1
and the
RANTES core, which chemokine region was responsible for this specific
binding deficit. MIP/RANTES bound and activated most receptor mutants
(R168A, K191A, and D276A) with affinities and potencies similar to
RANTES. These results strongly suggest that important determinants
involved in the differential binding of MIP-1
and RANTES are located
in CCR5 ECL2 and that these determinants interact with the core domain
of these chemokines. The affinity of MIP/RANTES for E172A was reduced
as compared with RANTES, although much less severely than that of
MIP-1
. This might be the consequence of a conformational change of
this mutant receptor, as suggested by the lower fluorescence-activated
cell sorting signal obtained with several conformation-sensitive mAbs
(26). However, we cannot exclude the possibility that this region of
ECL2 might interact both with the chemokine core and with the
amino-terminal domain.
(Phe13) plays a critical role in CCR5 binding (17). The
same residue is also important for the binding of other CC-chemokines,
including RANTES, to their respective receptors (15, 20, 21, 37, 38).
Conserved basic residues located in the N-loop (Arg18,
Lys19), 310-helix (Arg22), and 40s
loop (Arg46-Lys48) of MIP-1
also contribute
to CCR5 binding (18, 22), and homologous residues of MCP-1 and eotaxin
play a similar role for CCR2b and CCR3 binding, respectively (20, 21).
This suggests that a common binding surface of CC-chemokines to their
receptors involves patches of basic residues separated by an
hydrophobic groove. With the exception of Lys33, single
substitutions of basic residues in RANTES do not result in a
significant decrease of affinity for CCR5, although combinations of
these mutations result in a progressive reduction of binding affinity
(19).2 The functional
response of CCR5 to these chemokine mutants correlates well with their
binding affinity, suggesting that these residues are involved in
binding but not directly in the activation process (18, 19, 22).
residues involved in ECL2 binding are expected to be charged amino acids located in the core domain of the protein. Although these residues are presently unknown, the alignment of MIP-1
and RANTES sequences (Fig. 1B) shows that MIP-1
is much more acidic
than RANTES. It exhibits several clusters of acidic residues,
especially in the 30s and 50s loops and in the carboxyl-terminal
-helix, but also a cluster of basic residues in the 40s loop.
Identifying which of these residues interacts with charged amino acids
of CCR5 extracellular domain will require further investigation.
or RANTES (33). Because the amino-terminal domain of chemokines is known to be
important for receptor activation, it is tempting to hypothesize that
this part of the ligand interacts with a CCR5 domain involved in
receptor activation. This hypothesis was tested by measuring the
biological activity of chemokines, differing in their amino-terminal domain, onto these CCR5 mutants. The binding and functional properties of amino-terminally truncated chemokines were not affected by mutations
of the aromatic cluster of CCR5. In contrast to what was found for
extracellular mutants, the chimeric chemokine MIP/RANTES displayed a
biological activity similar to that of MIP-1
on CCR5 aromatic
cluster mutants, while retaining a normal binding affinity. LD78
activated these mutants as well as RANTES, which can probably be
attributed to the presence of a Pro residue in position 2 (5), as found
in RANTES. These results strongly argue for an interaction between the
chemokine N terminus and a site in the helix bundle of the receptor,
necessary for triggering the activation process.
and MIP-1
, have a
Pro in position 2, whereas MIP-1
has a Ser. This Pro residue
certainly plays an important role in chemokine function, since the P2A
substitution significantly decreases the biological activity of RANTES
(38). This role is, however, highly context-dependent, since cleavage of RANTES by the CD26 peptidase, removing the Pro and
generating RANTES-(3-68), does not affect its activity (42). Mutational analyses of different chemokines has shown that high sequence variability in the N-terminal domain is well tolerated for
normal function, suggesting that interactions between this domain and
the receptor might involve primarily backbone atoms (13, 15, 31,
43).
View larger version (50K):
[in a new window]
Fig. 7.
Schematic representation of the
proposed receptor-chemokine interaction. The receptor is depicted
in blue with the transmembrane helices shown as
solid tubes. The chemokine is in
orange, with the C-terminal helix shown as a
solid tube and the three -strands as
flat ribbons. In the proposed mode of binding,
the core of the chemokine would interact mainly with the extracellular
loops (ECL) and in particular with the N terminus and the
second part of ECL2. The N-terminal portion of the ligand would
interact specifically with residues inside the transmembrane bundle.
The displayed conformations of the extracellular loops are not relevant
per se and are shown for illustration purposes of the
binding mode. In particular, no attempt was made to predict secondary
structure elements. The exact relative orientations of the receptor and
its ligand have also been chosen arbitrarily for this purpose. The
intracellular loops are not displayed.
![]() |
ACKNOWLEDGEMENT |
---|
Expert technical assistance was provided by M. J. Simons.
![]() |
FOOTNOTES |
---|
* This work was supported by the Actions de Recherche Concertées of the Communauté Française de Belgique; the French Agence Nationale de Recherche sur le Syndrome d'Immunodéficience Acquise; the Centre de Recherche Inter-universitaire en Vaccinologie; the Belgian program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming; the Fonds de la Recherche Scientifique Médicale of Belgium, Télévie; and the Fondation Médicale Reine Elisabeth (to M. P.).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.
§ Aspirant of the Belgian Fonds National de la Recherche Scientifique.
Recipient of a FRIA fellowship.
¶¶ To whom correspondence should be addressed: IRIBHN, ULB Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium. Tel.: 32-2-5554171; Fax: 32-2-5554655; E-mail: mparment@ulb.ac.be.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M205684200
2 C. Blanpain, B. J. Doranz, A. Bondue, C. Govaerts, A. De Leener, G. Vassart, R. W. Doms, A. Proudfoot, and M. Parmentier, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; mAb, monoclonal antibody; PBS, phosphate-buffered saline; wtCCR5, wild type CCR5; TM, transmembrane.
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