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J Biol Chem, Vol. 274, Issue 40, 28745-28750, October 1, 1999
From the Seven-transmembrane segment, G protein-coupled
receptors (GPCRs) play important roles in many biological processes in
which pharmaceutical intervention may be useful. High level expression and native purification of GPCRs are important steps in the biochemical and structural characterization of these molecules. Here, we describe enhanced mammalian cell expression and purification of a
codon-optimized variant of the chemokine receptor CCR5, a GPCR that
plays a central role in the entry of the human immunodeficiency virus-1
(HIV-1) into immune cells. CCR5 could be solubilized in its native
state as determined by its ability to be precipitated by 2D7, a
conformation-dependent anti-CCR5 antibody, and by the HIV-1
gp120 envelope glycoprotein. The 2D7 antibody recognized immature and
mature forms of CCR5 equally, whereas gp120 preferentially recognized
the mature form, a result that underscores a role for posttranslational
modification of CCR5 in its HIV-1 coreceptor function. The methods
described herein contribute to the analysis of CCR5 and are likely to
be applicable to many other GPCRs.
The entry of human immunodeficiency virus-1
(HIV-1)1 into host cells
usually requires the sequential interaction of the gp120 exterior
envelope glycoprotein with the CD4 glycoprotein and a chemokine
receptor on the cell membrane (1, 2). CD4 binding facilitates virus
attachment to the cell surface and mediates conformational changes in
gp120 that allow a high affinity interaction with the chemokine
receptor (3, 4). Chemokine receptor binding is believed to trigger
further conformational changes in the viral envelope glycoproteins that
allow the gp41 transmembrane envelope glycoprotein to fuse the viral
and host cell membranes (5, 6).
Chemokine receptors are members of the large family of
seven-transmembrane segment, G protein-coupled receptors (GPCRs). The The GPCRs play central roles in a wide variety of physiological,
neurological, and immunological processes and represent major targets
for current pharmaceutical therapies (26-28). Nonetheless, structural
information on this large family of proteins is very sparse. Generally
low levels of expression and the dependence of the native conformation
of these proteins on the hydrophobic, intramembrane environment have
complicated attempts to study GPCR structure. With the exception of the
light-sensitive opsins found in certain bacteria and in the retinae of
higher organisms, most GPCRs are naturally expressed at low levels (29,
30). No system has reproducibly resulted in levels of protein
expression suitable for the purification of most GPCRs (31). Bacterial,
yeast, or insect cell expression of GPCRs can result in protein
misfolding, aggregation, and heterogeneity (32). Furthermore, some
GPCRs, such as CCR5, require posttranslational modification, which may exhibit significant differences in nonmammalian cell types, for efficient function (33). Irreversible protein denaturation occurring during the solubilization of the cell membrane, a process required for
purification of the GPCR, represents another obstacle to biochemical, biophysical, and structural studies.
Here, we report the development of a system for efficient expression of
CCR5, and the identification of an approach that allows solubilization
and purification of CCR5 in its native state. We demonstrate a
cell-free association between CCR5 and the HIV-1 gp120 glycoprotein.
These methods should facilitate the identification of small molecules
that block the HIV-1-CCR5 association, the determination of CCR5
structure, and the study of other GPCRs.
Construction and Expression of Codon-optimized CCR5
(synCCR5)--
The analysis of codon usage for 45 GPCRs representing
different protein subfamilies was performed with GenBankTM
data and software developed by the University of Wisconsin Genome Sequence Group. The sequence encoding human CCR5 was optimized for
mammalian cell codon usage (28), utilizing the following codons:
alanine (GCC), arginine (CGC), asparagine (AAC), aspartic acid (GAC),
cysteine (TGC), glutamic acid (GAG), glutamine (CAG), glycine (GGC),
histidine (CAC), isoleucine (ATC), leucine (CTG), lysine (AAG),
methionine (ATG), phenylalanine (TTC), proline (CCC), serine (TCC),
threonine (ACC), tryptophan (TGG), tyrosine (TAC), and valine (GTG).
The 5' and 3' sequences flanking the CCR5 coding sequence were
modified. Following restriction sites for EcoRV, EcoRI and HindIII, the Kozak consensus
(GCCGCCACCATGG) was placed immediately 5' to the CCR5
reading frame. A sequence encoding a single glycine residue followed by
the bovine rhodopsin C9 peptide tag (TETSQVAPA) was introduced
immediately 5' to the natural stop codon of CCR5. At the 3' end of the
epitope-tagged CCR5 gene, XbaI, SalI, and
NotI restriction sites were introduced. Analogous constructs
were made for the wild-type human CCR5 gene and the bovine rhodopsin
gene, except that the codons were not altered and, in the latter case,
the C-terminal C9 sequence was naturally present.
A total of 34 oligonucleotides, each approximately 70 nucleotides in
length, corresponding to the complete sense and antisense strands of
the synCCR5 gene and flanking sequences, were constructed so that
approximately 50% of their sequences were complementary to those of
each of the two complementary oligonucleotides from the opposite
strand. Oligonucleotides were deprotected in pure ammonium hydroxide at
65 °C for 4 h, after which the ammonium hydroxide was
evaporated, and the oligonucleotides were dissolved in water at a final
concentration of 2 nM. For gene synthesis, the 34 oligonucleotides were separated into five groups (6 or 8 oligonucleotides per group) and 25 cycles of polymerase chain reaction
were performed using Pfu polymerase (Stratagene, La Jolla, CA) and a 3-fold molar excess of the 5' and 3' terminal
oligonucleotides in each group. This step generated five small segments
of the synCCR5 gene with complementary and overlapping ends.
Equal amounts of each polymerase chain reaction product were combined
with a 3-fold molar excess of the 5' and 3' terminal oligonucleotides of the complete synCCR5 sequence. A second round of 25 cycles of
polymerase chain reaction yielded the complete synCCR5 sequence. The product was sequenced to ensure that the sequence was correct.
The synCCR5, wild-type CCR5, and bovine rhodopsin sequences were cloned
into the following vectors: PMT4 (a gift from Dr. Reeves, Massachusetts
Institute of Technology), PACH (a gift from Dr. Velan, Israel Institute
for Biological Research), pcDNA 3.1(+) and pcDNA4/HisMax
(Invitrogen), and PND (a gift from Dr. Rhodes, University of
California, Davis). After cloning of the synCCR5 gene into the
pcDNA4/HisMax vector, the sequence encoding the N-terminal HisMax
region was removed by QuikChange mutagenesis (Stratagene). Different
cell lines were transfected with the synCCR5 and wild-type CCR5 genes
using the GenePorter transfection reagent (San Diego, CA). Following
transfection, cells expressing CCR5 were selected with 0.8 mg/ml of
neomycin (G418). Cells expressing the highest surface levels of CCR5
were selected by FACS after staining cells with the
R-phycoerythrin-conjugated anti-CCR5 antibody 2D7-PE (Pharmingen, San
Diego, CA). Among all tested cells (canine thymocytes
Cf2Th, human embryonic kidney cells HEK-293T, COS-1, and HeLa
(American Type Culture Collection)), the highest CCR5 expression levels
were observed in Cf2Th and HEK-293T cells transfected with
synCCR5 gene in the PACH vector. The highest synCCR5-expressing clones
were selected by FACS from a total of 76 clones of Cf2Th cells
and 62 clones of HEK-293T cells.
Radiolabeling and Immunoprecipitation of CCR5--
Approximately
4 × 106 CCR5-expressing Cf2Th or HEK-293T
cells grown to full confluency in 100-mm dishes were washed twice in PBS and starved for 1 h at 37 °C in Dulbecco's modified
Eagle's medium without cysteine and methionine (Sigma) or in
sulfate-free media (ICN, Costa Mesa, CA). The starvation medium was
removed and 200 µCi each of [35S]methionine and
[35S]cysteine or 500 µCi of [35S]sulfate
(NEN Life Science Products) in 4 ml of medium was added to the cells
for various times for pulse-chase experiments or overnight (12 h) in
all other cases. Cells were washed twice with PBS and lysed in 1 ml of
solubilization medium composed of 100 mM
(NH4)2SO4, 20 mM
Tris-HCl (pH 7.5), 10% glycerol, 1% (w/v) detergent (see below), and
Protease Inhibitor Mixture (one tablet of CompleteTM (Roche
Molecular Biochemicals) per 25 ml). The lysate was incubated at 4 °C
for 30 min on a rocking platform, and cell debris was removed by
centrifugation at 14,000 × g for 30 min. CCR5 was
precipitated with 20 µl of 1D4-Sepharose beads (38) overnight, after
which the beads were washed six times in the solubilization medium and pelleted. An equal volume of 2× SDS-sample buffer was added to the
beads, following by resuspension and incubation for 1 h at 55 °C. Samples were run on 11% SDS-polyacrylamide minigels, which were visualized by autoradiography or analyzed on a Molecular Dynamics
PhosphorImager SI (Sunnyvale, CA).
A total of 18 detergents were tested in the solubilization buffers. The
detergents, with abbreviations and critical micelle concentrations in
parentheses, were
n-octyl- Purification of CCR5--
Stable Cf2Th/PACH/synCCR5 cells
grown to full confluency in a 150-mm dish were incubated with medium
containing 4 mM sodium butyrate for 40 h, washed in
PBS, detached by treatment with 5 mM EDTA/PBS, pelleted,
and again washed in PBS. Cells were solubilized for 30 min with 3 ml of
the solubilization medium containing CymalTM-5 and
centrifuged for 30 min at 14,000 × g. The cell lysate
was incubated with 50 µl of 1D4-Sepharose beads on a rocking platform at 4 °C for 10-12 h. The Sepharose beads were washed five times with the washing buffer (100 mM
(NH4)2SO4, 20 mM
Tris-HCl (pH 7.5), 10% glycerol, and 1% CymalTM-5) and
once with washing buffer plus 500 mM MgCl2.
CCR5 was eluted from the beads by three successive washes with 50 µl
of medium containing 200 µM C9 peptide (TETSQVAPA), 500 mM MgCl2, 100 mM (NH4)2SO4, 20 mM
Tris-HCl (pH 7.5), 10% glycerol, and 0.5% CymalTM-5. The
total quantity of harvested CCR5 was estimated by Coomassie Blue
staining of an SDS-polyacrylamide gel run with standard quantities of
bovine serum albumin.
Binding of HIV-1 gp120 Envelope Glycoproteins to Solubilized
CCR5--
Approximately 4 × 106
Cf2Th/PACH/synCCR5 cells were labeled for 12 h with
[35S]Met/Cys and lysed in solubilization buffer
containing 1% CymalTM-5. One ml of cleared cell lysate was
incubated with 100-500 µl of the gp120-containing solutions. The
unlabeled JR-FL gp120 was produced in Drosophila cells
(3), and the ADA and 190/197 R/S gp120 glycoproteins were produced from
transiently transfected 293T cells that had been radiolabeled with
[35S]Met/Cys overnight.2 Except in the case
of the CD4-independent gp120 variant, 190/197 R/S, the gp120
glycoproteins (2-4 µg) were preincubated with sCD4 (2-4 µg) in 20 ml of PBS for 1 h at 22 °C prior to addition to the
CCR5-containing lysates. After 12 h at 4 °C, the gp120-CCR5 complexes were precipitated with either the C11 anti-gp120 antibody (kindly provided by Dr. James Robinson, Tulane University Medical School) or with the 1D4 antibody.
Expression of CCR5 in Mammalian Cells--
We compared the codon
usage for opsins, the only GPCRs that are naturally highly expressed,
with the codon usage for 45 other GPCRs representing a spectrum of
different GPCR subfamilies. Opsin codons are biased toward those shown
to be optimal for efficient translation in mammalian cells (34),
whereas other GPCRs, including CCR5, are associated with codons that
are more random and, in many cases, inefficiently translated (data not
shown). A codon-optimized CCR5 gene was designed, synthesized using the
polymerase chain reaction, and transiently expressed in several
different cell lines, using five different expression vectors
(pcDNA 3.1, PACH, PND, PMT4, and pcDNA4/HisMax). The level of
CCR5 expression directed by the codon-optimized gene was 2-5 times
that directed by the wild-type CCR5 gene (Fig.
1, A and B, and
data not shown). Among the cell lines tested, CCR5 expression was the
highest in Cf2Th canine thymocytes (data not shown), so these
cells were used to generate stable cell lines. The PACH vector was used
to express the codon-optimized gene encoding human CCR5 containing a
9-residue C-terminal epitope tag (the C9 tag) derived from bovine
rhodopsin. The presence of the C9 tag allows recognition of the CCR5
protein by the 1D4 antibody (35). CCR5 expression in the stable cell line, designated Cf2Th/PACH/synCCR5, could be enhanced
2-3-fold by treatment of the cells with sodium butyrate (Fig.
1C). Following this treatment, approximately 3-5 µg of
CCR5 of high purity could be isolated from 107
Cf2Th/PACH/synCCR5 cells, using techniques described below (Fig. 1D).
Precursor and Mature Forms of CCR5--
CCR5 synthesis and
turnover in Cf2Th cells were studied by pulse-chase analysis
(Fig. 2). A precursor of approximately 40 kDa chased into the mature form of CCR5, which migrated as a wide band
of approximately 43 kDa. The CCR5 precursor exhibited a half-life of
approximately 25 min (Fig. 2A). The half-life of the mature form of CCR5 was 11-14 h, regardless of whether CCR5 expression was
directed by the wild-type or codon-optimized CCR5 gene (Fig. 2,
B and C). The half-lives of the precursor and
mature forms of CCR5 in HEK-293 cells were similar to those in
Cf2Th cells (data not shown). In several different cell lines, a
lower molecular mass (approximately 36 kDa) form of CCR5 appeared in
parallel with the mature protein (Fig. 2, B and
C). This lower molecular mass form of CCR5 was expressed at
lower levels than the mature form of CCR5 and has not been completely
characterized. Its identity as a CCR5 isoform was confirmed by its
precipitation by the 1D4 antibody and the anti-CCR5 antibody 2D7 and by
mass spectrometry (Figs. 2 and
3A and data not shown).
Solubilization of Native CCR5--
Membrane protein purification
requires solubilization of the membranes, typically through the use of
detergents. A broad spectrum of conditions was studied to arrive at the
composition of the buffer that allowed solubilization and isolation of
native CCR5. This optimization was guided by a comparison of the amount
of solubilized CCR5 capable of being precipitated by the 2D7 antibody, which recognizes a conformation-dependent CCR5 epitope
(36), with that able to be precipitated by the 1D4 antibody directed against the linear C9 epitope tag. In this manner, the percentage of
solubilized CCR5 remaining in a native conformation could be estimated
(Fig. 3A). Eighteen detergents, most of which were designed specifically for the extraction and purification of membrane proteins, were studied. In terms of the quantity of isolated CCR5 protein, as
well as the percentage of protein in a conformation able to be
recognized by the 2D7 antibody, the most effective detergents were DDM,
CymalTM-5, and CymalTM-6 (Fig. 3B).
Of these detergents, CymalTM-5 exhibits the highest
critical micelle concentration (2.4 mM), facilitating
dialysis of the detergent from the protein solution for the purposes of
membrane reconstitution and/or crystallization. We also found that a
CCR5 conformation competent for binding HIV-1 gp120 was best preserved
in buffers containing CymalTM-5 (see below). Therefore,
CymalTM-5 was used for further refinement of the CCR5
solubilization/isolation protocol, examining a number of variables
(salt composition and concentration, pH, temperature, and minor
additives) known to influence the stability of solubilized proteins
(37). Ammonium sulfate and glycerol were found to prolong the existence
of a CCR5 conformation capable of being recognized by the 2D7 antibody (data not shown). The optimized CCR5 solubilization buffer was composed
of 100 mM (NH4)2SO4, 20 mM Tris-HCl (pH 7.5), 10% glycerol, and 1%
CymalTM-5.
Binding of Solubilized CCR5 to HIV-1 gp120--
To examine whether
the solubilized CCR5 was capable of binding the HIV-1 gp120
glycoprotein, coprecipitation experiments were conducted using three
different gp120 glycoproteins. The JR-FL and ADA gp120 glycoproteins,
like all characterized wild-type R5 HIV-1 envelope glycoproteins, bind
CCR5 efficiently only in the presence of CD4 (3, 4). By contrast, the
190/197 R/S variant of the ADA gp120 glycoprotein was derived from a
virus adapted in culture to replicate on CD4-negative, CCR5-positive cells,2 and it therefore binds CCR5 in the absence of CD4.
The mature CCR5 protein was precipitated from cell lysates by the C11
anti-gp120 antibody when unlabeled JR-FL gp120 and soluble CD4 (sCD4)
were added to the lysates (Fig.
4A), but not when either gp120
or sCD4 were left out of the mixture (data not shown). The C11 antibody precipitated the mature CCR5 protein when radiolabeled 190/197 R/S
gp120 was added to the lysates without sCD4 (Fig. 4B).
Conversely, the 1D4 antibody precipitated the labeled 190/197 R/S gp120
glycoprotein from these lysates. In the coprecipitation experiments
using the C11 anti-gp120 antibody, only the mature form of CCR5, and
not the 36-kDa CCR5 isoform, was precipitated (Fig. 4).
Sulfation of CCR5--
CCR5 has been shown to be
posttranslationally modified by O-linked carbohydrates and
by tyrosine sulfation (33). The latter modification has been suggested
to facilitate the efficiency with which CCR5 is utilized as an HIV-1
coreceptor (33). To examine the relationship of sulfation to the
observed conversion of the CCR5 precursor to the higher molecular mass,
mature form of the protein, a pulse-chase analysis of
Cf2Th/PACH/synCCR5 cells labeled with either
[35S]cysteine/methionine or
[35S]sulfate was performed. Sulfate label was
incorporated only into the mature form of CCR5 (Fig.
5).
The recognition of the precursor and mature, sulfated forms of CCR5 by
the 1D4 and 2D7 antibodies and by gp120-sCD4 complexes was examined
(Fig. 5). The conformation-dependent 2D7 antibody precipitated the CCR5 precursor at an efficiency approximately 20%
lower than that seen for the 1D4 antibody. Complexes of the ADA gp120
glycoprotein, sCD4, and the C11 antibody preferentially precipitated
the mature, sulfated form of CCR5; the precipitation of the CCR5
precursor by this complex was approximately 5-fold less efficient than
that of the mature CCR5 protein (Fig. 5). Similarly, the mature CCR5
protein was recognized more efficiently than the CCR5 precursor by a
complex of the C11 antibody and the 190/197 R/S gp120 glycoprotein, in
the absence of sCD4 (data not shown).
GPCRs serve as therapeutic targets for over 30% of the
pharmaceutical agents currently used in clinical practice (26).
Specific GPCRs, most of which are chemokine receptors, are necessary
cofactors for HIV-1 entry. Progress toward intervening in
virus-coreceptor interaction would be greatly expedited by the
development of screening assays in which relevant ligands bind
purified, native chemokine receptors. We provide herein an experimental
approach to the purification of human CCR5 that yields a native protein
capable of binding the HIV-1 gp120 envelope glycoprotein and a
conformation-dependent antibody. The approach should be
generally applicable to other GPCRs.
Achieving adequate levels of CCR5 in an expression system is essential
for attempts to establish screening assays and for structural analyses.
Codon optimization, empirical screening of plasmids and cell types, and
FACS screening yielded stable lines expressing a level of CCR5
approaching that achieved for bovine rhodopsin. In studies not shown,
we compared the synthesis, steady-state levels, and turnover of the
codon-optimized CCR5 with those of bovine rhodopsin transiently
expressed in the same cell type by the same vector and detected using
identical epitope tags. The results indicated that the synthesis and
steady-state levels of rhodopsin were approximately 5-fold higher than
those of CCR5, whereas the half-lives of the two proteins were
comparable. This difference was apparently compensated for by the
establishment and selection of the stable Cf2Th/PACH/synCCR5
cell lines. Scale-up of these cells could allow the purification of
milligram quantities of the CCR5 protein.
We evaluated the conformational integrity of CCR5 solubilized in
several different detergents, including DDM, which has been employed in
the study of some other GPCRs (38, 39). We show that
CymalTM-5 was at least as effective as DDM in solubilizing
CCR5 in a native conformation. Human CCR5 retained a native
conformation in CymalTM-5-containing buffers for a few days
at 4 °C, and bound HIV-1 gp120 more efficiently than CCR5
solubilized in DDM (data not shown). A further advantage of
CymalTM-5 is its critical micelle concentration, which is
14 times higher than that of DDM, the mild detergent commonly used for
membrane protein biochemistry. The high critical micelle concentration of CymalTM-5 should facilitate its removal during
reconstitution of the GPCR into membranes or for crystallization.
The C-terminal C9 peptide provides an easy and rapid means for
purification of CCR5 using the anti-peptide antibody 1D4. The presence
of the C9 epitope tag does not affect the ability of CCR5 to function
as an HIV-1 entry cofactor or as a receptor for chemokine ligands (data
not shown). The purified CCR5 can be gently eluted from the 1D4
antibody by using the free C9 peptide as a competitor (35, 38).
The synthesis and turnover of CCR5 were examined in the course of this
study. Approximately 80% of the 40-kDa CCR5 precursor is competent for
binding the conformation-dependent 2D7 antibody, indicating
that a native structure is achieved rapidly after synthesis of the
protein. The 40-kDa CCR5 precursor is rapidly converted into a more
slowly migrating form. This 43-kDa form of CCR5 is evident within 10 min of labeling the CCR5 precursor. Most of this shift in molecular
mass derives from the addition of O-linked carbohydrates to
the CCR5 protein (27). The single potential N-linked
glycosylation site on CCR5 is not utilized (27). Once synthesized, the
mature CCR5 protein exhibits a half-life of 11-14 h. The tyrosines in
the N terminus of the 43-kDa CCR5 form are modified by sulfation,
suggesting that sulfation occurs after O-glycosylation. The
incorporation of the sulfate label into the 43-kDa CCR5 protein
continuously increases over a 4-h labeling period. Because tyrosine
sulfation typically occurs in the trans-Golgi network, late
in the secretory pathway (15), some sulfate addition may be occurring
on CCR5 molecules that have been recycled from the cell surface back to
the Golgi. Our results indicate that the HIV-1 gp120 glycoprotein
preferentially recognizes the mature, sulfated form of CCR5, consistent
with the proposed role of sulfated tyrosines in gp120 binding and HIV-1
entry (33).
The availability of methods to purify adequate amounts of CCR5 should
expedite progress on understanding the structure and function of this
key HIV-1 coreceptor and facilitate the search for effective inhibitors
of virus-coreceptor interactions.
We thank Sheri Farnum and Yvette McLaughlin
for manuscript preparation and Minou Modabber for artwork. We
acknowledge the National Cell Culture Center for supplying the 1D4
antibody. We thank Drs. P. Reeves, B. Velan, G. Rhodes, and C. Rizzuto
for providing plasmids and for helpful discussions.
*
This work was supported by National Institutes of Health
Grant AI41851, by a Center for AIDS Research grant, and by the G. Harold and Leila Mathers Foundation, the Friends 10, William F. McCarty
Cooper, and Douglas and Judith Krupp.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.
**
To whom correspondence should be addressed: Dept. of Cancer
Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3371; Fax: 617-632-4338; E-mail: joseph_sodroski@dfci.harvard.edu.
2
P. Kolchinsky, T. Mirzabekov, M. Farzan, E. Kiprilov, M. Cayabyab, L. Mooney, H. Choe, and J. Sodroski, submitted
for publication.
The abbreviations used are:
HIV, human
immunodeficiency virus;
GPCR, G-protein coupled receptor;
DDM, n-dodecyl-
Enhanced Expression, Native Purification, and
Characterization of CCR5, a Principal HIV-1 Coreceptor*
§,§,§,§,§,§, and§
**
Department of Cancer Immunology and AIDS, Department of Immunology and
Infectious Diseases,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chemokine receptor CCR5 is the principal HIV-1 coreceptor used during natural infection (7-11). Individuals with genetic defects in
CCR5 expression are relatively resistant to HIV-1 infection (12, 13).
Some HIV-1 isolates can be adapted in tissue culture to replicate on
cells lacking CD4 (14, 16),2
but binding to either CCR5 or to the other common HIV-1 coreceptor, CXCR4 (17), is essential for the entry of these viruses. The necessity
of the HIV-1 gp120-chemokine receptor interaction to virus replication
makes an understanding of the structural basis of this binding a high
priority. Structures of unbound CD4 (18-20), an HIV-1 gp120 derivative
complexed with CD4 (21, 22), and segments of the HIV-1 gp41 ectodomain
(23-25) have been resolved by x-ray crystallography. Resolution of the
structure of CCR5, either alone or in a complex with HIV-1 gp120, would
provide information important for guiding attempts at intervention.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside (23.4 mM), n-decyl--D-maltoside (1.8 mM), n-dodecyl--D-maltoside (DDM)
(0.17 mM), cyclohexyl-butyl--D-maltoside
(CymalTM-4, 7.6 mM),
cyclohexyl-pentyl--D-maltoside (CymalTM-5,
2.4 mM), cyclohexyl-hexyl--D-maltoside
(CymalTM-6, 0.56 mM),
cyclohexyl-heptyl--D-maltoside (CymalTM-7,
0.19 mM),
cyclo-hexylpropanoyl-N-hydroxyethylglucamide (108 mM), cyclohexylbutanoyl-N-hydroxyethylglucamide
(35 mM),
cyclohexylpentanoyl-N-hydroxyethylglucamide (11.5 mM), N-octylphosphocholine
(Fos-CholineTM 8, 114 mM),
N-decylphosphocholine (Fos-CholineTM 10, 11 mM), N-dodecylphosphocholine
(Fos-CholineTM 12, 1.5 mM),
N-tetradecylphosphocholine (Fos-CholineTM 14, 0.12 mM), Triton X-100 (0.02 mM), CHAPS (8 mM), Nonidet P-40 (0.02 mM), and
diheptanoyl-phosphocholine (DHPC) (1.4 mM). All detergents
were purchased from Anatrace (Maumee, OH) except DHPC, which was
purchased from Avanti Polar Lipids (Alabaster, AL).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of CCR5 at the cell surface
(A) or in cell lysates (B) of
Cf2Th cells transfected by plasmids expressing wild-type
(Wt) and codon-optimized CCR5 (synCCR5
(Syn)). Forty-eight hours after transfection,
cells were suspended by treatment with 5 mM EDTA, washed in
PBS, stained with the 2D7-PE anti-CCR5 antibody, and analyzed by FACS
(A), or labeled with [35S]Met/Cys, lysed, and
precipitated with 1D4-Sepharose (B). C, increased
CCR5 expression following sodium butyrate treatment of cells.
Cf2Th/PACH/synCCR5 cells were incubated with sodium butyrate for
40 h, and CCR5 expression was estimated by FACS. D,
purified CCR5 isolated from cells. CCR5 was purified from
107 Cf2Th/PACH/synCCR5 cells grown in a 150-mm dish,
using 1D4-Sepharose. CCR5 was eluted with the C9 peptide, run on an
SDS-polyacrylamide gel, and stained with Coomassie Blue. In addition to
the monomeric CCR5 bands at 36-43 kDa, higher order CCR5 forms are
evident. The amount of CCR5 in the gel was estimated by comparison with
bovine serum albumin standards.
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Fig. 2.
A, precursor and mature forms of CCR5.
Cf2Th/PACH/synCCR5 cells were continuously labeled with
[35S]Met/Cys for the indicated times and then lysed and
used for precipitation by 1D4-Sepharose. The levels of the CCR5
precursor (circles) and mature forms (squares)
are shown, in arbitrary units. B and C, turnover
of CCR5 in Cf2Th cells transiently expressing CCR5. Forty-eight
hours after transfection with plasmids expressing the wild-type CCR5
gene (B) or codon-optimized, synthetic CCR5 (C),
Cf2Th cells were labeled with [35S]Met/Cys for
1 h, washed, and incubated for the indicated times in medium
without label. The cells were lysed and used for precipitation by
1D4-Sepharose. The precipitates were analyzed on SDS-polyacrylamide
gels. The gel in B was exposed to film for 48 h, and
the gel in C was exposed for 19 h. The levels of
expression of the 43-kDa (circles) and 36-kDa
(squares) CCR5 proteins were quantitated by
densitometry.
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Fig. 3.
Maintenance of native CCR5 conformation in
buffers containing different detergents. Approximately 4 × 106 [35S]Met/Cys-labeled
Cf2Th/PACH/synCCR5 cells were lysed in 1 ml of ice-cold
solubilization buffer (100 mM
(NH4)2SO4, 20 mM
Tris-HCl (pH 7.5), 10% glycerol) supplemented with a protease
inhibitor mixture and 1% (w/v) of different detergents. After 30 min
of solubilization and 30 min of centrifugation, the cleared cell
lysates were separated into two equal portions. One portion was
precipitated with 2D7 (a conformation-dependent antibody
against CCR5) and other portion with 1D4 (an antibody that recognizes
the linear C9 epitope tag). The precipitates were run on
SDS-polyacrylamide gels, and two parameters were examined: 1) the total
quantity of CCR5 precipitated by the 1D4 antibody, and 2) the ratio of
CCR5 precipitated by the 2D7 antibody relative to that precipitated by
the 1D4 antibody. A, precipitates from cells lysed in buffer
containing CymalTM-5, DHPC and
Fos-cholineTM-14. Similar levels of CCR5 were precipitated
by the 1D4 antibody from these lysates, but the percentage of
conformationally intact CCR5 varied (98% in CymalTM-5,
10% in DHPC, and 13% in Fos-CholineTM-14). The sample run
in the right-hand lane (asterisk) was the same as in the
lane labeled 1D4 but was boiled prior to running on the gel,
a procedure that results in the formation of high molecular weight
multimers of CCR5. B, the amounts of CCR5 precipitated by
the 1D4 ( 
) and 2D7 (
) antibodies from cell lysates containing
different detergents, over a range of pH values.
View larger version (67K):
[in a new window]
Fig. 4.
Coprecipitation of solubilized CCR5 and HIV-1
gp120. Approximately 4 × 106
Cf2Th/PACH/synCCR5 cells were labeled for 12 h with
[35S]Met/Cys and lysed in solubilization buffer
containing 1% CymalTM-5. The lysate was incubated either
with unlabeled JR-FL gp120 complexed with sCD4 (left four
lanes) or with [35S]Met/Cys-labeled 190/197 R/S
gp120 glycoprotein (right three lanes). The JR-FL gp120-sCD4
complex was formed by incubation of 2 µg of gp120 with 4 µg of sCD4
in 20 ml of PBS for 1 h at 22 °C. The mixtures were then
precipitated either with the C11 anti-gp120 antibody (as in
A) or with the 1D4 antibody directed against the C9 epitope
tag (as in B). Some of the lanes (as indicated) were exposed
to film for 12 h, but they are otherwise identical to the lanes
shown with the matching antibodies that were exposed to film for 30 min. Under these labeling conditions, the mature 43-kDa form and the
36-kDa form of CCR5, but not the 40-kDa CCR5 precursor, are
labeled.
View larger version (56K):
[in a new window]
Fig. 5.
Preferential binding of HIV-1 gp120 to the
sulfated, mature form of CCR5. Cf2Th/PACH/synCCR5 cells
were continuously labeled with [35S]Met/Cys (left
panel) or [35S]sulfate (middle panel) for
the indicated lengths of time. Cells were lysed in buffer containing
CymalTM-5 and precipitated with the 1D4 antibody
(left two panels). Under these conditions, 35 min of
labeling resulted in approximately one-half of the Cys/Met label being
incorporated into the 43-kDa glycosylated form of CCR5 and half into
the 40-kDa CCR5 precursor. [35S]Cys/Met-labeled cell
lysate from a 35-min labeling period was precipitated with either the
1D4 antibody (lane 1D4) (exposure time, 6 h), the 2D7
antibody (lane 2D7) (exposure time, 6 h), or ADA
gp120-sCD4 complexes and the C11 anti-gp120 antibody (lane gp120 + sCD4 + C11) (exposure time, 60 h). The ADA gp120
glycoprotein in the latter complex was metabolically
radiolabeled.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-D-maltoside;
FACS, fluorescence-activated cell sorter;
PBS, phosphate-buffered saline;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
DHPC, diheptanoyl-phosphocholine.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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