Báo cáo Y học: Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes pot

Functional reconstitution of the HIV receptors CCR5 and CD4
in liposomes
Franc¸ois Devesa, Vida Chams, Premkumar Dinadayala, Alexandre Stella, Aude Ragas, Henri Auboiroux,
Toon Stegmann and Yannick Poquet
Institut de Pharmacologie et de Biologie Structurale; CNRS UMR 5089, Toulouse, France
Reconstitution of membrane proteins allows their study in a
membrane environment that can be manipulated at will.
Because membrane proteins have diverse biophysical pro-
perties, reconstitution methods have so far been developed
for individual proteins on an ad hoc basis. We developed a
postinsertion reconstitution method for CCR5, a G protein
coupled receptor, with seven transmembrane a helices and
small ecto- and endodomains. A His
6
-tagged version of
CCR5 was expressed in mammalian cells, purified using the
detergent N-dodecyl-b-
D
-maltoside (DDM) and reconstitu-
ted into preformed liposomal membranes saturated with
DDM, removing the detergent with hydrophobic polysty-
rene beads. We then attempted to incorporate CD4, a protein
with a single transmembrane helix and a large hydrophilic
ectodomain into liposomal membranes, together with
CCR5. Surprisingly, reconstitution of this protein was also
achieved by the method. Both proteins were found to be
present together in individual liposomes. The reconstituted
CCR5 was recognized by several monoclonal antibodies,
recognized its natural ligand, and CD4 bound a soluble form
of gp120, a subunit of the HIV fusion protein that uses CD4
as a receptor. Moreover, cells expressing the entire fusion

protein of HIV bound to the liposomes, indicating that the
proteins were intact and that most of them were oriented
right side out. Thus, functional coreconstitution of two
widely different proteins can be achieved by this method,
suggesting that it might be useful for other proteins.
Keywords: membrane protein; reconstitution; liposome.
Reconstitution of membrane proteins allows the study of
their behaviour in a membrane in the absence of other
proteins, and the manipulation of their concentration and
environment. Unfortunately, no single reconstitution
method is applicable to all membrane proteins, most likely
because their biophysical character varies. The most
frequently used method is coinsertion reconstitution, in
which detergent is removed from a mixture of detergent-
solubilized protein and lipid [1,2], a method which has led to
the successful reconstitution of membrane proteins with
diverse structures [3–6]. However, no common conditions
are so far known that would allow a standard operating
procedure for reconstitution by this method to be esta-
blished. Postinsertion reconstitution is a fundamentally
different method based on the removal of detergent from
detergent-solubilized protein added to a preformed lipo-
some incubated with a detergent at a concentration almost
leading to the onset of membrane solubilization [1].
Although the physical basis for this type of reconstitution
is not clear, a number of proteins have now been reconsti-
tuted by this method [2,7–14].
The abundant proteins belonging to the superfamily of
G-protein coupled receptors (GPCR) that possess seven
transmembrane ahelices, have important roles in eukary-

otic signalling. Few GPCR have been reconstituted, by a
variety of coinsertion or postinsertion protocols, with
variable results [4,15–17]. The b-chemokines RANTES,
MIP-1a,andMIP-1b are recognized by the GPCR CCR5
[18–21]. Through binding to the chemokine, this receptor
plays a crucial role in inflammatory processes. CCR5 is also
used as a receptor by primary strains of HIV-1 [21–24].
When infecting a host cell, the membrane protein complex
of HIV-1, gp120/gp41, first binds to CD4 [25,26], an
integral membrane protein with a single transmembrane
a-helix. Binding induces a conformational change in gp120/
gp41 [27,28], which leads to an increased affinity for CCR5
present in the same host cell membrane [29–31]. Further
conformational changes induced by the latter interaction
then induce fusion between the viral and the cellular
membrane, allowing the virus to enter its host cell [32].
In many cell membranes, CCR5 and CD4 are associated,
and this association may be important to HIV infection
[33].
In this paper, we describe a postinsertion reconstitution
method that allows simultaneous incorporation of both
CCR5 and CD4, isolated from a mammalian cell in which
they were expressed together, into a single proteoliposomal
membrane. Proteoliposomal membranes containing both
proteins recognized the natural ligands for CCR5, a
conformation-specific antibody recognized CCR5, the recon-
stituted CD4 bound HIV gp120, and the proteoliposomes
Correspondence to Y. Poquet, Institut de Pharmacologie et de
Biologie Structurale; CNRS UMR 5089, 205 Route de Narbonne,
31077 Toulouse Cedex, France. Fax: + 33 5 61 17 59 94,

Tel.: + 33 5 61 17 54 64, E-mail:
Abbreviations: Cmc, critical micelle concentration; DDM, N-dodecyl-
b-
D
-maltopyranoside; Egg-PtdEth, egg phosphatidylethanolamine;
Egg-PtdCho, egg phosphatidylcholine; N-NBD-PtdEth, N-(7-nitro-
2,3,1-benzoxadiazol-4-yl)-phosphatidylethanolamine; N-Rh-PtdEth,
N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine;
SM, egg sphingomyelin; GPCR, G-protein coupled receptor;
DMEM, Dulbecco’s modified Eagle’s MEM.
(Received 14 June 2002, revised 13 August 2002,
accepted 29 August 2002)
Eur. J. Biochem. 269, 5163–5174 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03213.x
bound strongly to cells expressing the HIV fusion protein.
Therefore, functional reconstitution of different types of
membrane proteins was achieved by our method.
MATERIALS AND METHODS
Materials
Egg phosphatidylcholine (egg-PtdCho), egg phosphati-
dylethanolamine (egg-PtdEth), egg-sphingomyelin (SM),
cholesterol, N-(lissamine rhodamine B sulfonyl)-phosphati-
dylethanolamine (N-Rh-PtdEth), and N-(7-nitro-2,3,1-ben-
zoxadiazol-4-yl)-phosphatidylethanolamine (N-NBD-PtdEth)
were purchased from Avanti Polar Lipids (Birmingham,
Ala.). Detergents were obtained from Sigma, the chemo-
kines MIP-1a and RANTES from R & D Systems
(Wiesbaden, Germany), and
125
I-labelled MIP-1a from
NEN Life Science Products (Paris, France). Anti-myc mAb

9E10, fluorescein isothiocyanate (FITC) conjugated goat
anti-mouse Ig, anti-CD4 Q4120 and the anti-His
6
Ig His1
were purchased from Sigma, horse-radish peroxidase con-
jugated goat anti-mouse Ig from Biorad (Marnes-la-Co-
quette, France),
125
I-labelled sheep anti-mouse Ig from
Amersham Pharmacia Biotech (Saclay, France), anti-CCR5
mAb 2D7 from PharMingen Becton-Dickinson (Le Pont
des Claix, France), and anti-CCR5 mAbs 181 (Mab181)
and 182 (Mab182) from R & D Systems. Paramagnetic
beads coupled to anti-mouse Ig or anti-CD4 Igs were
purchased from Dynal (Compie
`
gne, France). NIH
3T3.T4.CCR5 cells and cells expressing a soluble version
of the gp120/gp41 of strain JR-FL (CHO JR-FL gp160,
clone A19) were obtained from the NIH AIDS Research
and Reference Reagent Program, Division of AIDS,
NIAID, NIH through D. R. Littman and J. Arthos,
respectively. HeLa cells expressing the gp120/gp41 from
strain ADA was the kind gift of E. Bahraoui, Universite
´
Paul Sabatier, Toulouse, France.
Construction and stable expression of HIV receptor
vectors
Plasmid DNA containing the CCR5 gene was obtained
from the NIH AIDS Research and Reference Reagent

Program, Division of AIDS, NIAID, NIH from N. Landau
(ref no. 3325). For the construction of a tagged version of
CCR5, an XhoI restriction site was introduced upstream of
the start codon of CCR5 and an ApaI site upstream of the
stop codon by PCR and expanded in pBSK1 plasmid. The
resulting XhoI/ApaI fragment was cloned into the corres-
ponding sites in the polylinker region of the pcDNA3.1-
myc-His expression vector (Invitrogen, Groningen, the
Netherlands) in order to obtain a recombinant protein
containing the C-myc epitope followed by six histidines at
the C-terminus. A plasmid containing the wild type version
of CCR5 was constructed by introducing the coding
sequence of CCR5 between the ApaIandHindIII restric-
tion sites of a pRC-CMV vector (Invitrogen).
For protein expression, CHO cells grown in Dulbecco’s
modified Eagle’s Medium (DMEM), containing 10% foetal
calf serum (Life Technologies, Cergy-Pontoise, France)
were transfected using LipofectinÒ Reagent (Life Technol-
ogies). After 3 days of culture, 500 lgÆmL
)1
of neomycin
(G418, Life Technologies) was added, and resistant cells,
obtained after two weeks of selection, were cloned by
limiting dilution. The resulting clones were tested for CCR5
cell surface expression by FACS. In order to coexpress
CCR5 and CD4 on CHO cells, clones expressing CCR5
were also transfected with pcDNA3 containing the CD4
gene. This plasmid was made by removing the CD4 cDNA
from the pT4b plasmid (obtained from the NIH AIDS
Research and Reference Reagent Program, Division of

AIDS, NIAID, NIH) between the EcoRI and XbaIsites,
and inserting the gene between the corresponding sites into
the polylinker of pcDNA3. CD4-his was made by cutting a
CD4–His
6
construct (kindly donated by M. Marsh, Medical
Research Council Laboratory, University College, London,
UK) between two BamHI sites from pSG5 and recloning
the gene into the polylinker of pcDNA3. After 2 weeks of
culture in the presence of G418, CD4 positive cells were
isolated using magnetic beads bearing an anti-CD4 Ig
(DynabeadsÒ M-450 CD4, Dynal). After two rounds of
selection, cells were cloned by limiting dilution and the
clones analysed by FACS for coexpression of CCR5 and
CD4.
Western blot analysis and
35
S-labelled CCR5-myc-his
detection
CCR5-myc-his containing samples were mixed 1 : 1 with
Laemmli sample buffer and loaded on SDS/PAGE gels
without heating. After migration, proteins were transferred
to nitrocellulose membranes, probed with the anti-myc
mAb 9E10 (Sigma) and revealed with horseradish peroxi-
dase-conjugated goat anti-mouse IgG and the NEN signal
chemiluminescence kit (NEN Life Science Products). For
35
S-labelled CCR5-myc-his detection, SDS/PAGE gels were
dried in a gel dryer, and exposed for 5–20 h on an
Molecular Dynamics (Bondoufle, France) phosphor

screen. The screen was scanned with a STORM
Ò
device
anddatawereanalysedwith
IQNT
software (Molecular
Dynamics).
CCR5-myc-his labelling and purification
CHO cells (grown to 90% confluence in 140 mm dishes)
were washed twice with NaCl/P
i
and incubated with
DMEM without methionine and cysteine plus 4% dialysed
foetal calf serum for 6 h. A
35
S-labelled methionine/cysteine
mixture (Easytag, New England Nuclear, Zaventem,
Belgium) was added (250 lCi per dish). After overnight
incubation at 37 °C, about 200 millions cells were removed
from Petri dishes with NaCl/P
i
, containing 10 m
M
EDTA,
andwashedwithNaCl/P
i
. After centrifugation, the pellet
was suspended in lysis buffer [NaCl/P
i
supplemented with

1% Triton X-100, 1000 UÆmL
)1
DNAse I, 1 m
M
MgCl
2
,
and the protease inhibitors Chymostatin, Leupeptin,
Antipaı
¨
n, Pepstatin (Sigma), all at 1 lgÆmL
)1
]. After
45 min at 4 °C, the preparation was centrifuged at
25 000 g for 15 min. CCR5-myc-his was purified from the
supernatant by immobilized metal ion affinity chromatogra-
phy (Ni-nitrilotriacetic acid, Qiagen, Courtaboeuf, France)
or anti-polyhistidine Igs immobilized on agarose beads
(Sigma). In both cases, N-dodecyl-b-
D
-maltopyranoside
(DDM) was used at 0.3 m
M
final concentration in all the
buffers needed for the various purification steps, thereby
replacing the Triton X-100 of the lysis buffer in the purified
5164 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
product. For purification on the Ni-nitrilotriacetic acid
column, the supernatant was diluted with an equal volume of
binding buffer (Tris 20 m

M
pH 8, NaCl 500 m
M
,imidazole
5m
M
, DDM 0.3 m
M
), and passed twice through the column.
The column was then washed with Tris 20 m
M
,NaCl
500 m
M
,imidazole25m
M
, DDM 0.3 m
M
,pH8andeluted
with a similar buffer containing 200 m
M
imidazole. For
purification on anti-polyhistidine agarose, the supernatant
was passed through the column which was washed with
Hepes 2.5 m
M
pH 7.4, NaCl 145 m
M
,DDM0.3m
M

.
CCR5-myc-his was eluted with the same buffer, to which
imidazole (200 m
M
) was added.
Liposome preparation and CCR5-myc-his reconstitution
Large unilamellar liposomes (0.1 lm in diameter) were
prepared from dry lipid films containing egg-PtdCho
(55 mol%), egg-PtdEth (27 mol%), SM (8.2 mol%),
cholesterol (8.2 mol%), N-Rh-PtdEth (0.82 mol%),
N-NBD-PtdEth (0.82 mol%) as described previously
[34]. For CCR5-myc-his reconstitution, these liposomes
(66 l
M
of phospholipids) were first incubated with DDM
at 0.2 m
M
for 30 min at 4 °C. Purified fractions of
CCR5-myc-his in DDM (0.3 m
M
)werethenmixedwith
detergent saturated liposomes at a 1 : 3 vol. ratio of
protein preparation to liposomes. After a 30-min incu-
bation, detergent was removed by the addition of
Biobeads SM-2 (Bio-Rad). A 4-h incubation with
0.5 mg of beads was followed by two 1-h incubations
with an additional 10 mg of beads with continuous
agitation at 4 °C. After detergent extraction, the resulting
proteoliposomes were pelleted three times at 100 000 g
for 30 min. All steps were at 4 °C.

MIP-1a binding experiments
CHO cells (1 · 10
6
cells in 50 lL) were suspended in Hepes
50 m
M
pH 7.4, MgCl
2
5m
M
,CaCl
2
1m
M
, BSA 0.5% at
25 °C for 1 h in the presence of 0.55 n
M
[
125
I]MIP-1a. Non–
specific interactions were measured by adding an excess of
unlabeled MIP-1a (500-fold). For MIP-1a binding to
reconstituted CCR5-myc-his, freshly prepared proteolipo-
somes (3 nmol of phospholipids) were incubated for 45 min
at 25 °C(Hepes2.5 m
M
, NaCl 145 m
M
,BSA0.1mgÆmL
)1

)
in the presence of different concentrations of [
125
I]MIP-1a
andcoldMIP-1a or RANTES (1000 and 500-fold
[
125
I]MIP-1a quantities). Unbound ligand was removed by
filtration on BSA-presaturated GFB-filters (Whatman), and
the filters were washed twice with Hepes 2.5 m
M
,NaCl
150 m
M
,BSA0.1 mgÆmL
)1
. The filters were then counted in
a c-counter (Packard).
Anti-CCR5 binding experiments
Freshly prepared proteoliposomes (8 nmol of phospho-
lipids) were incubated for 45 min at 23 °C in Hepes 2.5 m
M
pH 7.4, NaCl 145 m
M
,BSA0.1mgÆmL
)1
containing
10 lgÆmL of anti-CCR5 mAb 2D7 (Pharmingen), 181 or
182 (R & D). The proteoliposomes were separated from
unbound antibody by ultracentrifugation (100 000 g,

30 min), the pellet was resuspended in the same
buffer, and incubated with
125
I-labelled anti-mouse Ig
(3 lCiÆmL
)1
)for30minat4°C. The proteoliposomes
were then washed with the same buffer and pelleted by
ultracentrifugation.
Protein orientation
Proteoliposomes containing CD4, approximately 20 nmol,
wereincubatedinNaCl/P
i
for 20 min at 37 °C, with or
without trypsin (0.5 gÆL
)1
). The proteoliposomes were
washed once by ultracentrifugation to remove the protease,
and the pellet was resuspended in gel loading buffer for
Western blot analysis. The blot was revealed by the anti-
CD4 mAb Q4120.
Co-immunoprecipitation of proteoliposomes
4 · 10
6
paramagnetic beads with or without anti-CD4 Igs
were incubated overnight at 4 °C with 20 nmol of CD4-
CCR5 proteoliposomes, in Hepes 2.5 m
M
,NaCl145m
M

,
and BSA 1 mgÆmL
)1
. The beads were then washed in the
same buffer five times, and once in the same buffer without
BSA. Finally the beads were resuspended in sample buffer
SDS/PAGE for Western blot analysis.
Cell-cell fusion experiments
8 · 10
4
cells expressing the gp120/gp41 of strain ADA were
seeded per well (1.55 cm diameter) in 24-well plates. The
following day 18 · 10
4
CHO cells were added and after
3–5 h at 37 °C, a fusion percentage FI was calculated as
FI ¼ [1 ) (number of cells/number of nuclei)] · 100. A
minimum of 500 nuclei were counted for each assay.
Binding of a soluble version of the JR-FL gp120/gp41
to proteoliposomes
A
35
S-labelled preparation of this protein, secreted by CHO
JR-FL gp160, clone A19, cells was prepared by radiolabel-
ling as described above for CCR5-myc-his. Tissue culture
supernatant was passed 5–8 times through a 1-mL column
of Galanthus nivalis lectin coupled to Sepharose beads
equilibrated with Hepes 2.5 m
M
pH 7.4, NaCl 145 m

M
buffer, at approximately 1 mLÆmin
)1
.Thecolumnwasthen
washed with 40 vol. of Hepes 2.5 m
M
pH 7.4, NaCl
145 m
M
, and the protein was eluted with 10 vol. of the
same buffer containing 250 m
M
a-methyl-mannopyrano-
side. The protein was then concentrated by size filtration
(Centricon-30, Qiagen). For binding experiments, the
labelled protein (10 n
M
) was added to proteoliposomes
(100 l
M
phospholipid) in Hepes 2.5 m
M
pH 7.4, NaCl
145 m
M
,BSA0.1%,CaCl
2
1 l
M
,MgCl

2
1m
M
.
Binding of proteoliposomes to gp120/gp41 expressing
cells
200 · 10
3
ADA cells per well were seeded in 24-well plates.
After two days of growth, the cells were washed twice with
cold HBSS (Hank’s Balanced Saline Solution, Life Tech-
nologies, Cergy-Pontoise, France) and kept for 30 min at
4 °C. About 15 nmol of proteoliposomes were diluted in
200 lL of HBSS, and incubated with the cells for 30 min at
4 °C. The cells were then washed with HBSS to remove
unbound proteoliposomes, and incubated for 30 min at
37 °C. After this step, the cells were scraped from the wells
Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5165
in NaCl/P
i
and the fluorescence associated with the cells was
analysed by FACS.
RESULTS
Functional expression of a Myc-his tagged version
of CCR5, and a his-tagged version of CD4
Because the post-translational modifications of CCR5, such
as tyrosine sulfation appear to be important for its function
[35], we produced the protein in mammalian cells. In order
to facilitate the proteins’ purification, a recombinant version
was made containing a C-terminal myc tag followed by a

His
6
sequence, by cloning the CCR5 gene into a pcDNA3.1
myc-his vector, and transfecting CHO cells with this
construct. After G418 selection, clones expressing CCR5-
myc-his were obtained by limiting dilution and analysed by
FACS using the antibody 2D7 (Fig. 1), which recognizes
CCR5 [36]. Western blot analysis of the cloned protein from
a postnuclear supernatant of lysed cells was carried out
using the 9E10 mAb directed against the myc epitope (none
of the commercially available anti-CCR5 mAbs were able to
recognize wild type- or CCR5-myc-his on blots). Two
proteins were detected by 9E10, one with a molecular mass
of around 38 kDa, close to the estimated molecular mass of
CCR5-myc-his, and a second of 33 kDa (Fig. 1). The latter
protein was also detected in untransfected cells. Thus, the
tagged version of CCR5 was present on the surface of
transfected cells, and had the expected molecular mass.
To test whether the presence of the myc-his tag at the
C-terminus of CCR5 would interfere with the protein’s
ability to function as a chemokine receptor or HIV
coreceptor, we first determined the binding of MIP-1a to
the CCR5-myc-his expressing cells. Cells expressing CCR5
or CCR5-myc-his were incubated with [
125
I]MIP-1a alone
or in the presence of a large excess of cold MIP-1a. Specific
binding of the ligand to both cell types was observed
(Fig. 2A). Some binding of radioactive MIP-1a to non-
transfected CHO cells, even in the presence of excess cold

MIP-1a was consistently observed, and probably reflects low
affinity binding to heparan-like glycosaminoglycans [37].
As the myc-his tag of the protein was present at the
C-terminus of the receptor which is located on the inside
of the cell, we then determined if signal transduction by
the protein was affected by this modification. One of the
consequences of signalling is a down-modulation of the
expression of CCR5 [38]. CCR5-myc-his or wtCCR5
expressing cells were incubated at 37 °Cfor30minwith
10 n
M
of MIP-1a and then the CCR5 present on the plasma
membrane was quantified by FACS analysis using the 2D7
antibody (Fig. 2B). It was found that the 60–70% of the
myc-his tagged protein was internalized, as much as for
wild-type CCR5 in CHO cells [39].
To test the ability of CCR5-myc-his to function as a
coreceptor for gp120/gp41 induced fusion, CHO clones
coexpressing CCR5-myc-his or wt CCR5 were transfected
with pcDNA3.1 containing a His
6
-tagged version of CD4,
CD4-his, as described in Materials and methods. After
cloning and selection by FACS analysis (not shown) of cells
coexpressing CCR5 and CD4,sincytium formation with cells
expressing gp120/gp41 from strain ADA was tested. The
level of fusion obtained with cells expressing tagged or
untagged proteins was comparable (Fig. 2C). Thus, these
results indicated that CCR5-myc-his and CD4-his are
functional HIV coreceptor and receptors, respectively.

Purification of CCR5-myc-his
For the purification of CCR5-myc-his we tested two
different methods based on the presence of the six histidines
at the C-terminus of the protein. The first involved
immobilized metal ion affinity chromatography. Cells were
lysed with a buffer containing Triton X-100 and spun at
25 000 g for 15 min. The supernatant was applied to a
Nickel-nitrilotriacetic acid (Ni-nitrilotriacetic acid) column.
The column was washed with different buffers to remove
contaminant proteins and then eluted with a buffer
containing 200 m
M
imidazole and the detergent, as des-
cribed in Materials and methods. The protein was followed
throughout the different purification steps by Western
blot, using the anti-myc Ig, and by SDS/PAGE of
35
S-labelled protein (Fig. 3A). CCR5 was quantitatively
retained on the column, and could be eluted with imidazole.
The protein was unidentifiable by autoradiography in cell
lysates, but appeared as the predominant band after
purification, although a background of contamination by
other proteins was still present. The second method involved
His1 anti-His
6
Igs coupled to Sepharose beads. A column
made of this material also retained CCR5 quantitatively,
and elution with 200 m
M
imidazole resulted in a more

highly purified protein than purification on Ni-nitrilo-
triacetic acid columns (Fig. 3). Ten percent of the total
radioactivity present in the lane containing CCR5-myc-his
purified on Ni-nitrilotriacetic acid columns was associated
with the protein, whereas 36% of the total radioactivity was
associated with CCR5-myc-his after purification on an
antihistidine column.
Reconstitution of CCR5-myc-his
A limited number of protocols for the membrane reconsti-
tution of GPCR exist, and there does not seem to be a
Fig. 1. CCR5-myc-his expression in CHO cells. CHO cells were
transfected with pcDNA3 containing the ccr5-myc-his gene. Expres-
sion was measured by FACS, using the 2D7 anti-CCR5 Ig [36].
Western blot analysis was carried out using an anti-myc Ig. Trans-
fected cells are marked with +, nontransfected cells that were used as a
negative control, with The approximate position of molecular mass
markers (in kDa) is shown on the right.
5166 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
generally applicable method. Several coinsertion reconsti-
tution methods, involving the preparation of a mixture of
detergent, lipids and protein, followed by removal of
detergent, were first tried for CCR5. CCR5-myc-his
expressing cells were lysed with Triton X-100, and the
protein was immobilized on Ni-nitrilotriacetic acid columns.
To exchange the Triton X-100 for other detergents, the
column was then washed with buffer containing another
detergent, imidazole elution was performed in this detergent
and the eluate applied to dry lipid films. Several detergents
with a high critical micelle concentration (cmc), such as
b-D-octylglucoside and 3-[(3-cholamidopropyl)-dimethyl-

ammonio]-1-propanesulfonate (CHAPS) were tested, and
detergent removal methods tested involved rapid dilution,
dialysis or gel filtration. The low cmc detergents octaethye-
leneglycol-mono-N-dodecylether (C
12
E
8
) and Triton X-100
were also tested. In these cases, detergent was removed by
hydrophobic polystyrene beads (Biobeads SM2). We were
unable to reconstitute the CCR5-myc-his in a lipid bilayer
by any of these methods.
Postinsertion reconstitution was therefore attempted
next. This involves adding the detergent-solubilized protein
to preformed membranes, followed by detergent removal.
Although the physicochemical basis of this type of recon-
stitution is not clear, saturating the membranes with
detergent before protein addition was found to promote
reconstitution in a number of cases [1]. One detergent, which
has been used frequently for postinsertion reconstitution, is
DDM. This detergent, like other alkyl-glucoside detergents,
also seems to conserve the structure of solubilized CCR5
[40]. We first determined how much DDM could be added
to liposomes without solubilizing them. To this end,
liposomes containing the lipids egg-PtdCho (55 mol%),
egg-PtdEth (27 mol%), SM (8.2 mol%), cholesterol
(8.2 mol%), and the fluorescent phospholipid analogues
N-NBD-PtdEth and N-Rh-PtdEth (0.82 mol% each) were
produced. Resonance energy transfer (RET) between these
probes depends on their membrane concentration; insertion

of detergent into the membrane should result in a gradual
decrease of RET [41,42], and lysis of the liposomes in abrupt
abolition of RET. A decrease in RET results in an increase
in NBD fluorescence [43]. Addition of DDM at concentra-
tions between 0.1 and 0.4 m
M
to 1.5 mL of a 66-l
M
(lipid
concentration) solution of liposomes at 4 °C led to gradual
increases in NBD fluorescence, which reached stable levels
after about 30 min (Fig. 4A). In contrast, addition of Triton
X-100 (0.5% w/v) at the end of the experiment led to an
immediate jump in fluorescence, indicating the lysis of the
membranes. The cmc of DDM is around 0.25 m
M
.Thirty-
five percent fluorescence dequenching was obtained at
0.2 m
M
DDM. Assuming that DDM occupies about half as
much volume in the membrane as a phospholipid and that
detergent is only incorporated in the outer leaflet, the
Fig. 2. The tagged version of CCR5 binds MIP-1a, is down-regulated
and can serve as a coreceptor for fusion. A: CHO cells expressing
CCR5-myc-his, wt CCR5 or CHO cells transfected with pcDNA3
without the ccr5-myc-his gene as a negative control were incubated
with 0.55 n
M
[

125
I]MIP-1a or 0.55 n
M
[
125
I]MIP-1a in the presence of
250 n
M
of cold MIP-1a in Hepes 50 m
M
pH 7.4, MgCl
2
5m
M
,CaCl
2
1m
M
, BSA 0.5% at room temperature. The cells were washed twice
with Hepes 10 m
M
pH 7.4, NaCl 0.5
M
, BSA 0.5%. Specific binding
was then calculated by subtracting the cell-associated counts in the
presence of cold MIP-1a from those obtained with radioactive MIP-1a
only. B: Downregulation of CCR5 after MIP-1a stimulation. Cells
were incubated with (open bars) or without (closed bars) 10 n
M
MIP-

1a for 30 min at 37 °C, and then CCR5 expression on the cell surface
was measured by flow cytometry using 5 lgÆmL
)1
of antibody 2D7
and an FITC-labelled secondary antibody. C: Fusion between cells
expressing different versions of CCR5, or nontransfected cells (CHO)
with HeLa cells expressing gp120/gp41 from strain ADA. 8 · 10
5
ADA cells were plated on 35 mm Petri dishes, overlaid with 1.6 · 10
6
CHO cells the following day and incubated for 3 h at 37 °C. A fusion
index was calculated as described before [55].
Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5167
detergent to phospholipid ratio in this leaflet is 1 : 2, close to
the optimal conditions for reconstitution [1,44].
To reconstitute CCR5, we then incubated liposomes
(66 l
M
) with 0.2 m
M
of detergent. After reaching a stable
level of fluorescence, one-quarter volume of CCR5-myc-his,
purified on a Ni-nitrilotriacetic acid column in 0.3 m
M
of
DDM, the minimum concentration required to solubilize
the protein, was added (Fig. 4B). After 30 min of coincu-
bation at 4 °C, detergent was removed. In postinsertion
reconstitution, usually some detergent is slowly removed
first, with a low amount of hydrophobic biobeads SM2, and

then the rest is removed rapidly by incubation with larger
amounts of beads [1]. For initial slow removal, several initial
bead concentrations (from 0.25 to 1.5 mg per mL) and
various incubation times (from 2.5 to 5 h) were tried. For
the second step, two additions of 2.5–20 mg per mL for
45 min to 1 h were tested. The final protocol involved a 4-h
incubation with 0.5 mg of beads, followed by the addition
of 5 mg of beads, per mL of reconstitution solution. After
1 h, these beads were removed from the mixture, and 10 mg
of fresh beads was added for another hour of incubation at
4 °C. The mixture was stirred continuously. The removal of
detergent was followed by monitoring the NBD fluores-
cence (Fig. 4B). The resulting proteoliposomes were pelleted
by ultracentrifugation, resuspended in detergent free buffer,
pelleted again, and this step was then repeated. Using this
protocol, approximately 12% of the purified CCR5
(approximately 3% of the total detergent-solubilized
CCR5) was found in the final pellet, whereas unincorpo-
rated protein was found in the first supernatant (Fig. 5).
Under these conditions, more than 65% of the total proteins
incorporated in the proteoliposomes was CCR5. If deter-
gent was removed from protein in the absence of liposomes
by this protocol, no protein was pelleted. Thus, the proteins
present in the pellet were associated with the liposomes.
Moreover, as judged by the fluorescence quenching of
N-NBD-PtdEth by N-Rh-PtdEth in the pelleted proteo-
liposomes, the first centrifugation step also removed the
remaining detergent from the proteoliposomes (Fig. 4B).
Similar results were obtained with CCR5-myc-his protein
Fig. 3. CCR5-myc-his purification. CCR5-myc-his was purified by

immobilized metal ion affinity (A) or immunoaffinity chromatography
using anti-histidine Igs (B). Results were analysed by Western blot as in
Fig. 1, or by autoradiography of
35
S-labelled proteins.
Fig. 4. Reconstitution of CCR5-myc-his. A: Saturation of liposomes
with DDM. DDM, at final concentrations between 0.1 and 0.4 m
M
was added to liposomes (66 l
M
of lipid) containing N-NBD-PtdEth
and N-Rh-PtdEth at time 0, and the fluorescence of N-NBD-PtdEth
was measured and normalized to a scale where 0% representing the
initial residual fluorescence of N-NBD-PtdEth before the addition of
detergent, and 100% represents the fluorescence of completely
dequenched N-NBD-PtdEth, obtained by addition of Triton X-100
(0.5% w/v), corrected for the quenching of NBD by Triton [43]
(marked with ÔTXÕ). Note the rapid increase after addition of Triton
X-100, and the lack of interaction at DDM concentrations below its
cmc. B: Reconstitution. At time 0, 0.2 m
M
of DDM was added to
liposomes as described above, then protein was added in 0.3 m
M
of
DDM (P), and detergent was removed by addition of 0.5 mg Biobeads
SM-2, followed by a second addition of 10 mg of beads. These beads
were then removed from the mixture, and 10 mg of fresh beads were
added (indicated by Ô0.5Õ, Ô10Õ and Ô10Õ). All incubations were at 4 °C.
Pellet denotes the fluorescence of the same quantity of lipid after

purification by ultracentrifugation as described in the text.
5168 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
purified on antihistidine coupled agarose beads. In conclu-
sion, postinsertion reconstitution produced CCR5 contain-
ing liposomes without residual detergent.
Characterization of reconstituted CCR5-myc-his
To characterize the membrane orientation and functionality
of the reconstituted protein, we first measured the binding of
antibody 2D7, 181 or 182, which recognize the extracellular
loops of CCR5 [36,45], to the proteoliposomes. The
proteoliposomes were incubated with the anti-CCR5 anti-
body, pelleted by ultracentrifugation, resuspended, incuba-
ted with a
125
I-labelled secondary antibody, and washed by
ultracentrifugation. Specific binding of monoclonal anti-
CCR5 to CCR5-myc-his proteoliposomes could be demon-
strated (Fig. 6). These data demonstrated that at least some
of the CCR5 is oriented with its N-terminus toward the
outside of the liposomes and the binding of antibody 2D7,
known to recognize a conformational epitope on CCR5
[36], indicated that the conformation of the receptor was
maintained in the liposomal membrane.
Binding of [
125
I]MIP-1a to the proteoliposomes was then
measured. Proteoliposomes were incubated with the ligand
for 45 min at room temperature, and subsequently filtered
on BSA-presaturated GF-B filters, which were repeatedly
washed, after which the filters were counted. Specific

binding of [
125
I]MIP-1a to CCR5-myc-his liposomes was
demonstrated. However, when we attempted to measure the
number of binding sites per proteoliposome by adding
mixtures of [
125
I]MIP-1a and increasing concentrations of
cold MIP-1a, a strange phenomenon was observed; addi-
tion of cold MIP-1a strongly increased the binding of
[
125
I]MIP-1a, in fairly linear manner (Fig. 7). Although we
have no explanation for this behaviour, nonclassical binding
of MIP-1a has been described before [16]. We therefore
attempted to use another ligand, RANTES, to compete
with MIP-1a for specific binding sites on proteoliposomes;
partial competition by RANTES and concentration-
dependent specific radioactive MIP-1a binding to proteo-
liposomes were observed (Fig. 8). However, at the highest
concentration of radioactive MIP-1a attainable (4 n
M
), we
did not reach saturation, which could indicate the presence
of a high concentration of CCR5 in the membranes. In
conclusion, these experiments demonstrate that at least part
of the CCR5 present in proteoliposomes is correctly
oriented and still capable of recognizing its natural ligand.
Co-reconstitution of CD4 and CCR5 in the same
membrane

To preserve potential associations between CD4-his and
CCR5-myc-his, these two proteins were then copurified
from the same cell line on a Ni-nitrilotriacetic acid column;
as they carried the same hexahistidine tags, optimal
conditions for their elution were nearly identical. We then
attempted to reconstitute the two proteins together in the
Fig. 7. Non-classical behaviour of MIP-1a observed with CCR5 pro-
teoliposomes. Proteoliposomes (6 nmol of phospholipids) were incu-
batedwith[
125
I]MIP-1a (open circles) or a mixture of radioactive plus
a 500-fold excess of cold MIP-1a (closed squares) in Hepes 2.5 m
M
pH 7.4, NaCl 145 m
M
,BSA0.1mgÆmL
)1
buffer for 45 min at room
temperature, washed on GFB-Whatman filters with the same buffer
and then the filters were counted.
Fig. 6. Specific binding of anti-CCR5 antibody to proteoliposomes.
Proteoliposomes (8 nmol of phospholipids) were incubated for 30 min
with 10 lg/mL of antibody 2D7, 181 or 182, washed once with Hepes
2.5 m
M
pH 7.4, NaCl 145 m
M
,BSA0.1 mgÆmL
)1
, and then incubated

with
125
I-labelled sheep anti-mouse IgG (5 lCiÆmL
)1
)for30minat
room temperature. The proteoliposomes were then washed twice in the
same buffer and bound radioactivity was determined. The negative
control consists of incubating the proteoliposomes with the labelled
secondary antibody only.
Fig. 5. Analysis of CCR5-myc-his reconstituted into liposomes. CCR5-
myc-his proteoliposomes were washed three times by ultracentrifu-
gation. The consecutive supernatants S1, S2 and S3 and the final
proteoliposome pellet (PL) were analysed by Western blot and auto-
radiography, and show successful reconstitution of the protein, as well
as the absence of unincorporated protein in the second and third
supernatant.
Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5169
same liposomal membrane, simply by applying the protocol
worked out for CCR5-myc-his, in spite of the biochemical
differences between the two proteins. To our surprise,
Western blot analysis readily demonstrated the presence of
CD4-his and CCR5-myc-his in the proteoliposomal pellet,
indicating that CD4-his could be reconstituted using a
protocol optimized for a RCPG (Fig. 9, Panel A). However,
the proteins could still be present in individual liposomes.
To determine whether CD4 and CCR5 were present
together in individual or separate liposomes, these were
immunoprecipitated with paramagnetic beads bearing anti-
CD4 Igs. The precipitate analysed by Western blot with an
anti-myc Ig recognizing the myc-his tagged CCR5. As

showninFig.9(PanelB),CCR5-myc-his(the38kDa
band) was found to be present in these proteoliposomes.
Thus, CD4 and CCR5 were present together in individual
liposomal membranes. The relative stoechiometry of the
two incorporated proteins was estimated by Western blot
analysis. If we assume that the anti-His
6
Ig used (anti-tetra-
his Ig, QIAGEN) recognizes both His
6
-tagged proteins with
similar affinities, quantifying the relative intensities of each
bandwouldleadtoanestimateratioof5:1forthe
CD4-CCR5.
In order to determine the orientation of CD4 with respect
to the proteoliposomal membranes, proteoliposomes were
digested with trypsin, after which the trypsin was removed,
and the CD4 quantified by Western blot relative to
Fig. 9. Co-reconstitution of CD4 and CCR5. A: Western blot showing
the presence of CD4 and CCR5, after purification by immobilized
metal ion affinity from a lysate obtained from CHO cells, coexpressing
CD4-his and CCR5-myc-his and reconstitution by the protocol
worked out for CCR5-myc-his. S1, S2 and S3 represent the three
consecutive supernatants obtained after ultracentrifugation, and PL
the final pellet (cf. Fig. 5). The Q4120 antibody (2 lgÆmL
)1
)wasused
for detection of CD4, and an anti-myc Ig for CCR5, as described in the
legend of Fig. 1. B: 20 nmol of proteoliposomes containing CD4 and
CCR5 were incubated with 4 · 10

6
paramagnetic beads with (lane A)
or without (lane B) the anti-CD4 Ig. After washing the beads they were
resuspended in gel electrophoresis sample buffer and analysed by
Western blot with the anti-myc Ig. Besides the 38 kDa band corres-
ponding to CCR5-myc-his which is present in lane a, the heavy and
light chains of the anti-CD4 Ig (50 and 25 kDa, respectively, lane a) are
also revealed by the anti-mouse IgG secondary antibody, while no
CCR5isdetectedinlaneb.
Fig. 8. MIP-1a binds specifically to CCR5 proteoliposomes. CCR5-
myc-his proteoliposomes (6 nmol of phospholipids) were incubated
with [
125
I]MIP-1a in Hepes 2.5 m
M
pH 7.4, NaCl 145 m
M
,BSA
0.1 mgÆmL
)1
for 45 min at room temperature, washed on GFB-
Whatman filters with the same buffer and then the filters were counted.
A: Binding in the presence of radioactive MIP-1a only, or in the
presence of a 500-fold excess of nonradioactive RANTES. B: Specific
binding at different concentrations of MIP-1a to CCR5-myc-his
liposomes. The proteoliposome-associated counts in the presence of a
500-fold excess of cold RANTES were subtracted.
5170 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
untreated controls. After digestion, no more CD4 was
detected by Western blot using the anti-CD4 mAb Q4120,

suggesting that the vast majority of CD4 was oriented right-
side out, as the ectodomain of the protein is recognized by
the antibody.
In order to test the biological activity of CD4, a
35
S-labelled soluble construct, containing the whole ectodo-
main of the gp120/gp41 of strain JR-FL was produced and
purified over a lectin column as described in Materials and
methods. It was found that this protein bound specifically to
CCR5/CD4 liposomes. As previously described for the
binding of gp120/gp41 to CXCR4-paramagnetic proteo-
liposomes [46], approximately 30% of binding was dis-
placed by a 200-fold excess of cold gp120/gp41 (Fig. 10,
Panel A). To determine whether the proteoliposomes could
recognize the complete gp120/gp41 protein, they were
incubated with cells expressing this glycoprotein complex
and the fluorescence of cell-associated proteoliposomes was
quantified by FACS analysis (Fig. 10, Panel B). The gp120/
gp41 expressing cells were found to bind proteoliposomes
containing CCR5 and CD4 specifically. These data suggest
that CD4 was also inserted right side out into proteolipo-
somal membranes, and that CD4 reconstituted in those
model membranes remains functional.
DISCUSSION
Postinsertion or coinsertion protocols have been used for the
reconstitution of membrane proteins [1,47]. Co-insertion
involves the removal of detergent from mixtures of free and
micellar detergent and mixed protein/detergent and lipid/
detergent micelles. Upon removal of detergent, the protein
will likely become insoluble and either self-aggregate, or

associate with lipids if these also become insoluble at this
point. In the latter case, a membrane containing the protein
may be formed. However, protein aggregates may be
formed on the one hand and lipid membranes with little
incorporated protein on the other [48]. The success of this
method thus probably depends on the relative affinities of
interaction of the protein and lipid with the detergent. As it is
not always possible to predict these parameters, a variety of
detergents and detergent removal protocols have to be tested
for every individual protein, and successful reconstitutions
by these methods have involved a variety of lipid/detergent
combinations and detergent removal protocols [3,5,6].
Postinsertion reconstitution involves mixing detergent-
solubilized protein with detergent-saturated liposomes,
followed by detergent removal. The physicochemical basis
of postinsertion reconstitution is not clear, but whether
proteins will aggregate or associate with the lipid bilayer
probably also depends on the relative affinities of the
protein for a membrane or itself. Most successful reconsti-
tutions by this method were with transporter proteins, over-
expressed in bacteria, containing 6–12 transmembrane
a helices [2,9–11,49]. These proteins are robust and have
large hydrophobic domains, facilitating reconstitution, and
their production can easily be scaled up, making the
efficiency of reconstitution less critical.
No GPCR over-expressed in mammalian cells had
previously been reconstituted by postinsertion protocols,
although some had been successfully reconstituted by
coinsertion methods [4,15,17,50]. CCR5 and the related
CXCR4, isolated from mammalian cells in which they were

expressed at relatively high levels, were recently reconstitu-
ted by an innovative method involving paramagnetic beads
[16,46]. After extraction with a detergent, the proteins were
immunoprecipitated with antibodies coupled to the beads
and then lipid membranes were formed around these. An
advantage of this method is that the antibodies can orient
the GPCR. However, the immobilization on beads may
affect the protein’s behaviour.
In order to develop a postinsertion reconstitution proto-
col, the choice of the detergent is crucial. DDM,
b-D-octylglucoside or Triton X-100 are among the deter-
gents which seems most suited for this type of reconstitution
[11,49]. Interestingly, both CD4 and CCR5 are associated
with membrane raft microdomains [51,52], known to be
solubilized preferentially by alkyl-glucoside type detergents
[53]. As the native conformation of CCR5 was well
preserved in DDM [40], probably because of its structural
Fig. 10. Binding of soluble gp 120 or cells expressing gp120/gp41
to proteoliposomes containing CD4 and CCR5. A: Binding of the
35
S-labelled soluble version of gp120/gp41, strain JR-FL to CD4-
CCR5 or only CCR5 containing proteoliposomes containing CD4 and
CCR5, in the absence or presence of a 200-fold excess (Ô200xÕ)ofcold
protein, as described in Materials and methods. Error bars are one
standard deviation. B: FACS analysis of cells expressing gp120/gp41
(ADA strain), incubated with about 15 nmol of proteoliposomes
(PL, thick line) or liposomes (L, thick line). The thin line corresponds
to the auto-fluorescence of the cells.
Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5171
resemblance to raft glycolipids, this detergent was chosen.

Efficient reconstitution is strongly dependent on detergent
concentration for a given lipid/protein ratio [44]. The
optimal detergent concentration was frequently found to be
close to that at the onset of liposome solubilization [2], and
the most critical steps in postinsertion usually are the
(stepwise) addition of the detergent to the membranes
without causing their solubilization, followed by detergent
removal [1]. Saturation of liposomes with detergent is
mostly measured by techniques [1,54] that require high lipid
concentrations and do not give quantitative information on
the concentration of detergent present in the membrane. As
expression of proteins in mammalian cells leads to the
isolation of lg rather than mg of receptors, small quantities
of liposomes can be produced only. In this paper, detergent
insertion was therefore followed by resonance energy
transfer measurements using fluorescent phospholipid ana-
logues present in the liposomal membrane [41,42]. Satura-
tion with 0.2 m
M
of DDM was found to suffice to
destabilize membranes for reconstitution, leading to a bulk
solution lipid/detergent molecular ratio of about 3 : 1 in
solution. Usually, at higher lipid concentrations, a 1 : 1
ratio is attained [10]. Besides differences in the nature of the
detergent, this could be a consequence of the lipid concen-
tration, because the detergent partitions between the
solution, protein and membrane, but only the concentration
in the membrane determines the outcome of reconstitution.
Therefore, an advantage of the resonance energy transfer
method is that the membrane concentration of the detergent

can be estimated. At less than 0.2 m
M
DDM, poor
reconstitution of proteins was observed, and at higher
concentrations liposomes were solubilized. In the latter case,
if detergent was removed with beads, no reconstitution of
protein was observed at all. The resonance energy transfer
assay also allowed direct observation of the removal of
detergent from the membrane in real time, and therefore the
efficient comparison of a number of protocols. Initial slow
detergent removal and biobeads quantity were found to be
crucial for the recovery of high concentrations of functional
proteins in proteoliposomes. An initial four-hour incuba-
tion time with 0.25 mg of biobeads per ml led to optimal
protein incorporation. Studies of molecular interactions
between CD4 and CCR5 reconstituted in proteoliposomes
could lead to an understanding of the constitutive associ-
ation of CCR5 and CD4 in vivo, recently suggested to be
important for HIV entry [33].
In conclusion, two rather different proteins, one largely
hydrophobic with seven transmembrane helices and a small
ecto- and endodomain (CCR5), and one with a large
hydrophilic ectodomain and a single transmembrane seg-
ment (CD4), were reconstituted into proteoliposomal
membranes by the same protocol, using the same detergent,
DDM. These data suggest that the protocol might be
applicable to membrane proteins in general. Moreover, it
might be useful for the reconstitution of other membrane
proteins usually available only in small quantities from
mammalian cells.

ACKNOWLEDGEMENTS
This research was supported by the Re
´
gion Midi-Pyre
´
ne
´
es, the Agence
Nationale pour la Recherche sur le SIDA (ANRS), the Fondation pour
la Recherche Me
´
dicale (FRM), the Association pour la Recherche sur
le Cancer (ARC), and the comite
´
scientifique SIDACTION of the
FRM.
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