Received for publication, November 25, 2002, and in revised form, January 15, 2003
The chemokine receptor CCR5 is constitutively
associated with the T cell co-receptor CD4 in plasma cell membranes,
but the physiological role of this interaction has not been elucidated. Here we show that detergent-solubilized, purified CCR5 can directly associate with purified soluble fragments of the extracellular portion
of CD4. We further demonstrate that the physical association of CCR5
and CD4 in membrane vesicles results in the formation of a receptor
complex that exhibits macrophage inflammatory protein 1
(MIP-1
)
binding properties that are distinct from CCR5. The affinity of the
CD4-CCR5 complex for MIP-1
was 3.5-fold lower than for CCR5, but the
interaction of CD4 and CCR5 resulted in a receptor complex that
exhibited enhanced G-protein signaling as compared with CCR5 alone.
MIP-1
-induced G-protein activation was further increased by
simultaneous stimulation of CD4 with its natural agonist,
interleukin-16. Thus, the physical association of CD4 and CCR5 results
in receptor cross-talk with allosteric CD4-dependent
regulation of the binding and signaling properties of CCR5. Although
the precise physiological role of the CD4 effects on CCR5-mediated
signaling remains unknown, one can speculate that the cross-talk is a
component of mechanisms involved in the fine tuning of immune system
cell responses.
 |
INTRODUCTION |
CD4 is a component of the molecular complex that facilitates the
interaction of the T cell receptor with major histocompatibility complex (MHC) class II molecules; it also serves as the primary receptor for attachment of the human immunodeficiency virus-1 (HIV-1)1 (1, 2). The
chemokine receptor CCR5 serves as the entry cofactor for
macrophage-tropic strains of HIV-1 (3-7). CD4 and CCR5 act in concert
for HIV-1 entry by a sequential, ordered, multistep mechanism. Both
receptors are expressed on lymphoid cells but belong to unrelated
receptor families. CCR5 is a member of the heptahelical
G-protein-coupled receptors (GPCR), whereas CD4 belongs to the
immunoglobulin superfamily of membrane receptors with a single
transmembrane segment that contributes to signal transduction through
its cytoplasmic association with the lymphocyte kinase Lck (8). GPCRs
have been known to associate with each other, but only recently has
hetero-oligomerization between unrelated receptors with direct coupling
been demonstrated in co-localized neurotransmitter systems (9-14).
Here, we present data that demonstrates that CD4 and CCR5, both of
which are involved in leukocyte activation and HIV-1 infectivity, form
a unique receptor complex that is distinct from CCR5 alone with respect
to affinity for its ligand, macrophage inflammatory protein-1
(MIP-1
), and for G-protein signaling.
We have shown previously that CD4 and CCR5 are physically associated
even in the absence of the gp120 glycoprotein (15). It has been
demonstrated that this interaction is unique to CD4 and CCR5 and is
mediated through the second extracellular loop of CCR5 and the first
two domains of CD4. To investigate the interaction between CCR5 and
CD4, we have now used human osteosarcoma
HOS-CD4+-CCR5+ and
HOS-CD4
-CCR5+ cells to study the
pharmacological and biochemical properties of these two HIV-1 receptor
molecules. Here we present data suggesting cross-talk between these molecules.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[125I]MIP-1
,
[35S]guanosine-5'-(
-thio)triphosphate
([35S]GTP
S) and soluble CD4 (sCD4) were
from PerkinElmer Life Sciences. MIP-1
was purchased from
Peprotech (Rocky Hill, NJ). The CD4 antiserum T4-4 was obtained from
the National Institutes of Health AIDS Research and Reference Reagent
Program. The polyclonal anti-CCR5 antibody CKR-5 was purchased from
Santa Cruz Biotechnology, and cyclohexyl-pentyl-
-D-maltoside (Cymal-5TM) was from
Anatrace (Maumee, OH). Recombinant two-fragment (first and second
domains) soluble CD4 (D1D2-sCD4) was a gift by Dr. Ed Berger.
Binding Assays--
HOS-CD4+-CCR5+ and
HOS-CD4
CCR5+ (5) were kindly provided by Dr.
Dan Littman. Cell membranes were prepared as described (16). Briefly,
cells were rinsed with phosphate-buffered saline, resuspended in lysis
buffer (50 mM Hepes, pH 7.4, 1 mM EGTA
containing protease inhibitor mixture (Sigma)), and then homogenized by
40 strokes with a tight pestle in a Dounce homogenizer. Nuclei and
unbroken cells were then pelleted by low speed centrifugation (800 × g for 10 min at 4 °C). The supernatant was centrifuged
at 45,000 × g for 30 min at 4 °C.The crude membrane
pellet was washed once and then resuspended in above buffer with the
aid of a Dounce homogenizer.
All binding studies, which are described in detail elsewhere (16), were
performed at 20 °C in 20 mM Hepes, pH 7.4, 1 mM CaCl2, 5 mM MgCl2,
and 1% bovine serum albumin in a final assay volume of 0.1-0.25 ml.
[125I]MIP-1
(72-272 pM) was incubated
with 0.048-0.17 mg/ml membrane protein for 60 min. Receptor-bound
radioligand was separated from unbound ligand by filtration through
Whatman GF/C filters. Filters were washed twice with 4 ml of ice-cold
incubation buffer containing 500 mM NaCl.
Solubilization and Purification of
CCR5--
Cf2Th/synCCR5 cells were a kind gift from Dr. Joseph
Sodroski. This canine thymocyte cell line stably expresses CCR5, which is expanded by the C9 tag TETSQVAPA (17). This tag is recognized by the
1D4 antibody, which was obtained from the National Cell Culture Center,
National Institutes of Health. Cells were grown to confluency in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum,
4 mM glutamate, 100 units/ml penicillin, 100 µg/ml
streptomycin, 0.5 mg/ml G418, 0.5 mg/ml zeocin, and 3 µg/ml
puromycin. Prior to harvesting, cells were incubated with 4 mM sodium butyrate for 40 h, washed with
phosphate-buffered saline, and detached by treatment with 5 mM EDTA. Cells were solubilized for 30 min with 3 ml of
solubilization buffer containing Cymal-5TM as described (17) and then
centrifuged for 30 min at 14,000 × g. The lysate was
incubated with 1D4-Sepharose beads at 4 °C for 10-12 h. The
Sepharose beads were then washed five times with 20 mM
Tris-HCl (pH 7.5), 100 mM
(NH4)2SO4, 10% glycerol, and 1% Cymal-5TM; the last wash included 500 mM
MgCl2. CCR5 was eluted from the Sepharose beads with
washing buffer containing 200 µM C9 peptide (TETSQVAPA)
and 500 mM MgCl2. The concentration of harvested CCR5 was estimated by Coomassie Blue staining of an SDS
polyacrylamide gel.
ELISA Assay for CCR5 Binding to CD4--
0.05-0.75 µg/ml of
D1D2-sCD4 or sCD4 was coated onto the ELISA plate in 50 mM
carbonate, pH 9.6. Free binding sites were blocked with 4% milk in
Tris-buffered saline. 1 µg/ml purified CCR5 was added in buffer
containing 1% Cymal-5TM and incubated for 12 h at 4 °C. Bound
CCR5 was detected with the goat anti-CCR5 antibody CKR5 and an alkaline
phosphatase-linked anti-goat antibody (Santa Cruz Biotechnology).
G-protein Activation--
[35S]GTP
S binding was
carried out in 50 mM triethanolamine (pH 7.4), 5 mM MgCl2, 1 mM EGTA, and 1 mM dithiothreitol containing 10 µM GDP
and 15 µg of membrane protein at 20 °C as described in detail
previously (18). The reaction was terminated by the addition of 4 ml of
ice cold 50 mM Tris-HCl, pH 7.4, 5 mM
MgCl2, and filtration through Whatman GF/C filters.
 |
RESULTS |
Detergent-solubilized Purified CCR5 Binds Specifically to Purified
Recombinant sCD4--
We have previously found that CD4 can be
coimmunoprecipitated by anti-CCR5 antibodies from cells coexpressing
the two molecules (15). To further study this interaction, we developed
a binding assay using purified receptor proteins. Increasing
concentrations of D1D2-sCD4 as well as full-length sCD4 were coated
onto ELISA plates. Incubation with purified CCR5 showed a
concentration-dependent binding of CCR5 to both D1D2-sCD4
and sCD4 (Fig. 1A). To further demonstrate the specificity of the CD4-CCR5 interaction, we used the
polyclonal anti-CD4 antiserum T4-4 and the monoclonal anti-CD4 antibody
OKT4 in these binding experiments. As illustrated in Fig.
1B, CCR5 bound specifically to D1D2-sCD4 as demonstrated by
inhibition of CCR5 binding to CD4 by the anti-CD4 antiserum T4-4. The
monoclonal anti-CD4 antibody OKT4, which was previously used to
coimmunoprecipitate CD4 and CCR5 (15, 19), had no effect on the
interaction of the two purified receptor molecules.

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Fig. 1.
Saturation binding of purified CCR5 to
D1D2-sCD4 and four-domain sCD4. A, increasing
concentrations (0.05-0.75 µg/ml) of D1D2-sCD4 (filled
column) or sCD4 (open column) were
coated on an ELISA plate and incubated with 1 µg/ml of purified CCR5
in a Cymal-5TM-containing buffer as described under "Experimental
Procedures." B, 0.5 µg/ml of D1D2sCD4 was coated on the
ELISA plate, and 1 µg/ml of purified CCR5 was added in the presence
or absence of increasing concentrations (0-15 µg/ml) of either T4-4
(filled column) or OKT4 (open
column) antibodies. Bound CCR5 was detected with the
goat-anti-CCR5 antibody CKR5.
|
|
Binding Properties of CCR5 Are Distinct from the CCR5-CD4
Complex--
We compared the ligand binding properties of CCR5 with
those of CCR5-CD4 complexes. Competition binding experiments with
125I-labeled MIP-1
to membranes from HOS cells
expressing either CCR5 or both CCR5 and CD4 are shown in Fig.
2A. Scatchard transformation revealed that the affinity of CCR5 for its ligand MIP-1
decreased by
3.5-fold when CD4 was coexpressed (Fig. 2B). We calculated by Scatchard analysis dissociation constants (KD) of 278 ± 20 pM and 997 ± 115 pM for
MIP-1
for HOS-CD4
-CCR5+ and
HOS-CD4+-CCR5+ cell membranes, respectively.
The CD4-CCR5 complex has therefore distinct pharmacological properties
from CCR5, with the coexpression of CD4 resulting in a partial
inhibitory effect on MIP-1
binding to CCR5 due to a decrease in the
affinity of CCR5 for its chemokine ligand. The CCR5 receptor density
was similar in the two membrane preparations. The
[125I]MIP-1
binding sites were 1.47 ± 0.1 pmol/mg and 1.88 ± 0.12 pmol/mg in
HOS-CD4
-CCR5+ and
HOS-CD4+-CCR5+ cell membranes, respectively.
The CD4 receptor density in our HOS-CD4+-CCR5+
cell membranes was in excess of that of the CCR5 receptors with 9.43 ± 0.4 pmol/mg as determined by
125I-gp120YU2 binding (data not shown).
Interestingly, pre-incubation of
HOS-CD4+-CCR5+ membranes with the T4-4
antiserum, which inhibits the association of CD4 and CCR5 (15),
stimulated [125I]MIP-1
binding to 140%, whereas OKT4
had no effect (Fig. 3).

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Fig. 2.
Binding properties of
[125I]MIP-1 to CCR5 and
CD4-CCR5. A, HOS-membranes expressing CCR5
(triangles) or coexpressing CD4 and CCR5
(squares) were incubated with 0.1 nM
[125I]MIP-1 in the presence of the indicated
concentrations of MIP-1 . Half-maximal inhibition (IC50)
occurred at 0.45 nM and 1.45 nM, respectively.
B, Scatchard transformation of displacement curves.
KD and Bmax values were
calculated by linear regression analysis. Coexpression of CD4
decreased the affinity of CCR5 for its ligand MIP-1 from 289 pM to 1.11 nM. The CCR5 density
(Bmax) was 1.5 pmol/mg and 1.9 pmol/mg in
HOS-CD4 -CCR5+ and
HOS-CD4+-CCR5+ cell membranes,
respectively.
|
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Fig. 3.
Binding of
[125I]MIP-1 to
HOS-CD4+-CCR5+ membranes. R5-tropic
gp120 (YU2 and ADA), X4-tropic gp120
(LAI) (100 nM each), IL-16 (5 µg/ml), the
anti-CD4 antibodies T4-4 (1:100) or OKT4 (10 µg/ml) were
pre-incubated with membranes for 1-6 h before the addition of
[125I]MIP-1 . The effect on
[125I]MIP-1 binding was assessed.
|
|
R5-tropic gp120, but Not X4-tropic gp120 or Interleukin-16 (IL-16),
Inhibits MIP-1
Binding to HOS-CD4+-CCR5+
Membranes--
Gp120 and IL-16 are the known ligands to CD4. A natural
ligand for CD4 IL-16 has been identified and characterized as having a
variety of biological effects similar to gp120 (20). R5-tropic gp120
has been shown to bind to CCR5 after interaction with CD4 and interfere
with MIP-1
binding to CCR5 (21, 22). We tested whether activation of
CD4 by IL-16 would affect the binding properties of the CD4-CCR5
complex for MIP-1
. As seen in Fig. 3, IL-16 did not affect the
interaction of the CD4-CCR5 complex with MIP-1
, confirming data
previously reported (23). However, R5 tropic gp120 (ADA and YU2)
interfered with MIP-1
binding to
HOS-CD4+-CCR5+membranes, whereas X4-tropic
gp120 had no effect.
CD4 Enhances CCR5-mediated G-protein Signaling in Co-transfected
Cells--
We next investigated whether there is a functional role for
the physical interaction of these two receptor molecules and whether the physiologic response of the CD4-CCR5 complex is distinct from that
of CCR5. For this purpose, we measured G-protein activation in
HOS-CD4
-CCR5+and
HOS-CD4+-CCR5+ membranes by assessing
[35S]GTP
S binding. GTP
S interacts with the
G-proteins with high affinity but is not hydrolyzed (24).
G-protein activation is classically studied in membrane preparations,
because guanine nucleotides cannot penetrate intact cells. As can be
seen in Fig. 4, MIP-1
(100 nM) accelerated the basal rate of
[35S]GTP
S binding in
HOS-CD4
-CCR5+ membranes, as expected for an
agonist to CCR5. In HOS-CD4+-CCR5+ membranes,
the addition of IL-16 further stimulated G-protein activation through
CCR5. The rate constants were calculated assuming a pseudo-first order
association as described (25) and are presented in Table
I. IL-16 alone had no effect on
CCR5+ or CD4+CCR5+ cell membranes
but doubled the rate constant in the presence of MIP-1
on
CD4+CCR5+ membranes. Therefore, simultaneous
occupation by the two agonists results in the most active signaling
form.

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Fig. 4.
Stimulation of [35S]GTP S
binding to HOS-CD4+-CCR5+ membranes.
Before the addition of [35S]GTP S, the membranes were
incubated in the absence (open circles) or
presence (filled squares) of 100 nM
MIP-1 or 100 nM MIP-1 plus 5 µg/ml IL-16
(filled circle). The reaction was terminated at
the indicated time.
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Table I
G-protein activation in membranes from HOS cells expressing CCR5 or
both CCR5 and CD4
[35S]GTP S binding to cell membranes was measured in the
absence (control) or presence of 100 nM MIP-1 , 5 µg/ml
IL-16, or 100 nM MIP-1 plus 5 µg/ml IL-16. The rate
constants were calculated assuming a pseudo-first order association and
are presented as mean from three independent experiments.
|
|
 |
DISCUSSION |
Our results demonstrate that CD4 and CCR5 functionally associate
on the plasma membrane and that co-receptors involved with immune
stimulation and HIV-1 entry, which are members of unrelated receptor
families, can interact. We provide biochemical and functional evidence
for direct CD4-CCR5 cross-talk; CD4-CCR5 interaction leads to synergy
such that CCR5 signaling is increased as a result of activation of CD4
by IL-16. The coexpression of CD4 itself had no effect on
MIP-1
-induced signaling of CCR5. Our data confirm a previous report
wherein no effect of CD4 coexpression was found in chemokine-induced
CCR5 internalization experiments (26) but also show that stimulation of
CD4 enhances CCR5 function. The CD4-CCR5 complex is also
pharmacologically distinct from CCR5 in that it is characterized by a
lower affinity for binding to MIP-1
. The association of CD4 and CCR5
at the cell membrane is reversible, because the T4-4 antiserum, which
inhibits this interaction, stimulated MIP-1
binding. Therefore, we
suggest a model in which CD4, by associating with CCR5, allosterically
modulates the binding properties of CCR5 for MIP-1
, exhibiting
decreased affinity for its ligand. Stimulation of CD4 with IL-16 does
not further affect the binding of MIP-1
to CCR5, but leads to
enhanced signaling of CCR5. Mueller et al. (26) reported
earlier a difference in MIP-1
-induced internalization of CCR5 in
CHO-CCR5+ and CHO-CCR5+-CD4+ cells.
The authors could demonstrate that this difference was attributable to the different levels of CCR5 expression in the two cell
lines (8-fold difference in CCR5 levels). In the cell line selected for
our experiments, CD4 coexpression did not significantly affect the
level of CCR5. We determined a similar MIP-1
binding site density in
HOS-CD4
-CCR5+and
HOS-CD4+-CCR5+ membranes (1.47 versus 1.88 pmol/mg). Furthermore, we have shown earlier
that, after uncoupling from the G-protein, CCR5 can no longer bind
MIP-1
(16). Therefore, the difference in affinity of CCR5 in
HOS-CD4
-CCR5+ and
HOS-CD4+-CCR5+ membranes cannot be attributed
to a difference in the level of CCR5 expression in the two cell lines,
because uncoupled CCR5 receptors do not exhibit detectable affinity for
CCR5 (16). We chose a cell line for our experiments that expressed CD4
in excess of CCR5 so that, presumably, the CCR5 receptors are
"saturated" with CD4. It will be interesting to determine whether
there is a reciprocal effect of CCR5 on the binding and signaling
properties of CD4.
It was previously reported that IL-16-induced chemotaxis is partially
inhibited by pertussis toxin, and it was suggested that if a direct
interaction of IL-16 with CCR5 exists, it could contribute to a
CD4-induced migratory signal (23). Our data could provide an
explanation for how CCR5 contributes to a CD4-induced signal.
Our findings may also have important implications for HIV-1 evolution
and immunopathogenesis, because it has been suggested by many that a
precursor of HIV-1 used CCR5 as the primary receptor (27-29) and that
the close physical association of CD4 and CCR5 may have permitted the
adaptation to CD4. HIV-1 has to react sequentially with its receptors
to gain entry into a susceptible cell, and the formation of complexes
between the HIV-1 receptors may make the entry process more efficient.
Finally, our data suggest a previously unknown signal transduction
mechanism for chemokine receptors and immunoreceptors. If receptor
interactions are a widespread phenomenon in cells of the immune system,
the array of receptor complexes could be immense. Association of
distinct receptor molecules would combine specificity with flexibility.
Specificity would guarantee binding of the ligand to its receptor, but
hetero-oligomerization would define a new level of functional
diversity, depending on which receptor(s) are expressed by a particular
cell and which ones form specific receptor complexes.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M212013200
The abbreviations used are:
HIV, human
immunodeficiency virus;
GPCR, G-protein coupled receptor;
MIP-1
, macrophage inflammatory protein-1
;
HOS, human osteosarcoma;
sCD4, soluble CD4;
ELISA, enzyme-linked immunosorbent assay;
IL-16, interleukin-16;
CHO, Chinese hamster ovary.
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