www.elsevier.com/locate/yexcr
Experimental Cell Resear
Cysteine residues are critical for chemokine receptor CXCR2
functional properties
Cristina Limatolaa,b,T,1, Sabrina Di Bartolomeoa,1, Myriam Catalanoa, Flavia Trettela,
Sergio Fucilea, Loriana Castellanic, Fabrizio Eusebia,d
aIstituto Pasteur-Fondazione Cenci Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Centro di Eccellenza BEMM,
Universita La Sapienza, I-00185 Roma, ItalybNeuromed I.R.C.C.S., Via Atinese 18, I86077 Isernia, Italy
cDipartimento di Scienze Motorie e della Salute, Universita di Cassino, ItalydFondazione Santa Lucia, via Ardeatina 306, I-00179 Roma, Italy
Received 7 September 2004, revised version received 22 February 2005
Available online 24 March 2005
Abstract
We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization,
signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants
transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in
the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor
expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve
TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions.
We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys–Cys bonds are not the
exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface,
indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2,
including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling,
suggesting that some structural requirement is likely fulfilled by Cys presence.
D 2005 Elsevier Inc. All rights reserved.
Keywords: CXCR2; Cysteines; Chemotaxis; Signal transduction; Receptor dimerization
Introduction
Chemokines are a family of low molecular weight
proteins that regulate leukocyte recruitment to inflammatory
sites by binding to, and activating, specific G-protein-
coupled receptors (GPCRs) expressed on target cells.
0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2005.02.020
Abbreviations: GPCR(s), G-protein coupled receptor(s); Cys, cysteine;
HEK, human embryonic kidney 293 cells; ERK1/2, extracellular signal-
regulated kinase 1/2; mAb, monoclonal antibody; TM, transmembrane;
DTT, dithiothreitol; PBS, phosphate buffered saline.
T Corresponding author. Istituto Fisiologia Umana, Citta Universitaria,
Piazzale Aldo Moro 5, 00185 Roma, Italy. Fax: +39 06 49910851.
E-mail address: [email protected] (C. Limatola).1 These authors equally contributed.
CXCR2 is a GPCR that is activated, with different potency,
by at least seven different chemokines to induce cell
chemotaxis, intracellular signaling, and receptor internal-
ization [1–4]. CXCR2, though widely expressed in the
immune system, is also expressed throughout the nervous
system where it plays a neuromodulatory role on neuro-
transmission [5–10]. Despite the great interest in CXCR2
biological properties, little is known about the relationships
between its structure and function, with important excep-
tions for the C-terminal tail, where regions involved in
receptor internalization, signaling, and chemotaxis have
been mapped in good details. Such regions contain either
specific serine–threonine phosphorylation residues or leu-
cine-rich sequences involved in physical interaction with
ch 307 (2005) 65–75
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–7566
adaptor proteins, like Hsc/Hsp70 interacting protein, adaptin
2, and the phosphatase 2A core enzyme [11–14]. Moreover,
CXCR2 physically interacts with the AMPA-type glutamate
receptors playing modulatory effects on nerve cell migration
and glutamatergic neurotransmission; and the CXCR2
regions involved in such interaction have been mapped
between the amino acids 49–142 [9,15,16].
A growing body of evidence shows that two highly
conserved Cys residues in the first two extracellular regions
of most GPCRs are important for correct receptor folding
which, in turn, is necessary both for proper ligand binding
and receptor signaling. These Cys are conserved in most
eukaryotic GPCRs and are linked by a disulfide bond [17].
Disulfide-linkages formation has been also shown as one of
the mechanisms implicated in dimerization of several
GPCRs: y and n opioid receptors homo- and heterodimers
[18,19], metabotropic glutamate receptors [20], and extrac-
ellular Ca2+ receptor [21,22] homodimers, are all at least
partially maintained throughout the formation of intermo-
lecular Cys–Cys bonds. Recently, we have reported that
CXCR2 forms dimers and oligomers in both a reconstituted
cell system and cerebellar neurons [16], but the role of Cys
residues in CXCR2 dimerization has not been exploited. It is
known that chemokine receptors possess several conserved
and other more unique Cys residues in their sequence, and
that these Cys play relevant functional roles. For instance, a
Cys cluster in the C-terminal tail of CCR5 results crucial for
surface receptor expression [23], and the four extracellular
Cys of CCR6 are differently involved in receptor expression,
agonist binding, and Ca2+ signaling [24].
In addition to the conserved extracellular Cys119 and
Cys196 residues, CXCR2 presents two further Cys in the N-
terminal region (Cys39) and in the third extracellular loop
(Cys286), and five additional Cys residues in the trans-
membrane (TM) helices TM3-7 (Cys139, Cys166, Cys230,
Cys263, Cys308; Fig. 1). To address the functional signifi-
cance of single Cys residues in CXCR2, we replaced the nine
Fig. 1. Map of Cys residues in CXCR2. Cys indicated were substituted by
site-directed mutagenesis as described in Materials and methods. Numbers
and black dots indicate the relative positions of Cys residues (C) in CXCR2
sequence (Swiss Prot P25025).
Cys residues present in CXCR2 sequence, one at a time, with
Ser or Leu, expressed them in HEK cells and examined a
variety of biological events such as membrane insertion,
dimerization at cell surface, agonist-binding affinity, receptor
internalization, intracellular signaling, and chemotaxis. It was
found that: (i) elimination of Cys residues in the extracellular
loops or in the TM7 abolished agonist binding; (ii) none of the
Cys residues present in CXCR2 is essential for the expression
and translocation at the cell surface of monomeric and
dimeric forms; (iii) all Cys mutants, regardless of their ability
to bind CXCL8, display severely compromised ability to
respond to the agonist by either intracellular signaling
(ERK1/2 phosphorylation and cytosolic Ca2+ elevation) or
cell chemotaxis; (iv) for the Cysmutants that bind the agonist,
this impairment is not due to major differences in receptor
internalization with wtCXCR2.
Materials and methods
Materials
Polyclonal antibodies (Abs) to CXCR2 (C19), mono-
clonal Abs (mAbs) to CXCR2 (E2), and anti-ERK2 were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA); anti-phosphoERK1/2 was from Cell Signaling Tech-
nology (Hertfordshire, UK); recombinant human CXCL8
was from Peprotech Inc. (London, UK); transwell cell culture
inserts were from Becton Dickinson Labware (Franklin
Lakes, NJ); Gene Tailor Site-Directed mutagenesis system
and lipofectamine 2000 plus reagent were from Invitrogen
(Paisley, UK). EZ-Link Sulfo-NHS-Biotin and Immuno Pure
Streptavidin agarose were from Pierce (Rockford, IL). [125I]
CXCL8 (2000 Ci/mmol) was from Amersham Biosciences
(U.K.). Anti-actin, other drugs and chemicals were from
SigmaAldrich Italia (Milan, Italy); and all culturemedia were
from Life Technology Italia (Milan, Italy).
Site-directed mutagenesis
The cDNA encoding for human CXCR2, cloned in pCEP4
(Invitrogen), was kindly provided by Dr. Massimo Locati
(University of Brescia, Italy). Point mutations in CXCR2
were generated with the Gene Tailor Site-Directed Muta-
genesis System, following the manufacturer’s instructions.
Briefly, the cDNA encoding for human wild type (wt)
CXCR2 (Swiss Prot P25025) was subcloned in pc-
DNA3.1D/V5-His-TOPO (Invitrogen) to obtain a PCR
template suitable for the mutagenesis reactions. wtCXCR2
in pcDNA3.1D/V5-His-TOPO was then methylated on
cytosine residues, for 1 h at 378C. Methylated plasmids were
amplified in the mutagenesis PCR reactions, with two
overlapping primers, one of which contained the target
mutation. The following pairs of primers were used: 5V-CTAGATGCCGCCCCATCTGAACCAGAATCC-3V and 5V-TGGGGCGGCATCTAGTAGAAAAGGGGGCAG-3V for
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–75 67
CXCR2-C39S; 5V-TTTGGCACATTCCTGTTAAAGGT-CGTCTCA-3V and 5V-CAGGAATGTGCCAAAAATCCAG-CCATTCAC-3V for CXCR2-C119L; 5V-ATCCTGCTACTGQGCCTTGATCAGTGTGGAC-3V and 5V-GGCCAGTAG-CAGGATGCCACTATAGAAGTT-3V for CXCR2-C139L;
5V-CTTGGTCAAATTCATAAGTCTCAGCATCTG-3V and
5V-TATGAATTTGACCAAGTAGCGCTTCTGGGT-3V for
CXCR2-C166S; 5V-AATGTTAGCCCAGCCCTCTATGAG-GACATG-3V and 5V-GGCTGGGCTAACATTGGATGAG-TAGACGGT-3V for CXCR2-C196L; 5V-CTGCTGATCATGQCTGTTCAGCTACGGATTC-3V and 5V-GAACAGCATGAQTCAGCAGTGGCACGATGAA-3V for CXCR2-C230S; 5V-GTCCTCATCTTCCTGCTTAGCTGGCTGCCCTAC-3Vand 5V-AAGCAGGAAGATGAGGACGACAGCAAAGAT-3V for CXCR2-C263S; 5V-GTGATCCAGGAGACCAGTG-AGCGCCGCAAT-3V and 5V-GGTCTCCTGGATCAC-
CTGGGTCCTCATGAG-3V for CXCR2-C286S; 5V-GCATCQCTTCACAGCTTACTCAACCCCCTCA-3V and 5V-GC-TGTGAAGGATGCCCAGAATCTCGGTGGCA-3V for
CXCR2-C308L. PCR products were then transformed into
DH5a-T1 E. coli, where mutated DNA is circularized and
methylated wtCXCR2 is digested by the host endonucleases.
Once purified, CXCR2 mutants were all subcloned in pCEP4
in order to have comparable expression system between
mutants and wt receptors. CXCR2 mutants were completely
sequenced to check for the introduction of nonspecific
mutations. DNA sequencing was performed at Centro di
Ricerca Interdipartimentale per le Biotecnologie Innovative
(CRIBI, Padova University, Italy).
Cell transfection and ERK signaling
HEK 293 (HEK) cells were routinely grown in Dulbec-
co’s modified Eagle’s medium (DMEM) plus 10% fetal
bovine serum. Before transfection, HEK cells were plated on
poly-l-lysine-coated 35-mm or 60-mm dishes (350,000 and
700,000 cells/dish, respectively) and transfected 24 h later
with lipofectamine. Cells were used for experiments 48 h
after transfection; briefly, cells were serum-starved for 16 h,
incubated in Locke’s buffer (containing: 154 mM NaCl, 5.6
mM KCl, 3.6 mM NaHCO3, 2.3 mM CaCl2, 1 mM MgCl2,
5.6 mM glucose, buffered with 5 mM HEPES, pH 7.4) for
additional 2 h and stimulated with 120 nM CXCL8 in the
same buffer. After 10 min, cells were washed with
phosphate-buffered saline (PBS), lysed in a modified RIPA
buffer, and protein concentration was determined by BCA
assay; the same amounts of cellular proteins (10–20 Ag) wereanalyzed by SDS-PAGE and Western blot analysis with Abs
specific for phospho-ERK1/2 and ERK2. Specific bands on
chemiluminescence films were quantified by densitometry
with Sigma Gel Software (Jandel Scientific).
Expression of mutant CXCR2 proteins
HEK cells, transiently transfected with CXCR2 con-
structs, were analyzed for protein expression by Western blot
and Fluorescence-Activated Cell Sorter (FACS) analysis.
Cells were lysed by addition of hot SDS-Laemmli buffer in
the presence or in the absence of DTT (50 mM) or by using a
Triton X-100-containing lysis buffer, as previously described
[16]. The same amounts of proteins were analyzed by
Western blot with two different CXCR2 Abs (E2 and C19),
and specific bands were quantified by densitometry. FACS
analysis was performed to test the membrane expression of
CXCR2 mutants. Briefly, transfected cells (~106) were
collected, washed in serum-free DMEM and incubated with
mAb directed to the N-terminal CXCR2 region (E2) for 45
min on ice. After washing, cells were incubated with a FITC-
conjugated anti-mouse Ab for 30 min on ice. Washed cells
were then suspended in PBS and analyzed for immuno-
fluorescence using a FACScalibur flow cytometer (Becton
Dickinson, San Jose, CA). In each experiment, cells were
analyzed for auto-fluorescence, and mock-transfected
(pCEP) cells were used to verify Ab specificity. Nonspecific
binding of FITC conjugated secondary Ab (1.68% of total
events) was always subtracted from all points. As a control
for variations of cell transfection, immunofluorescence
analysis was performed on permeabilized HEK cells
transiently transfected with wt- and Cys-mutated CXCR2.
Surface biotinylation
48 h after transfection, HEK cells were detached in PBS
containing 0.1 mM EDTA, washed in PBS with 1 mM
MgCl2 and 0.1 mM CaCl2, and incubated with 0.5 mg/ml
of EZ-Link Sulfo-NHS-Biotin at RT for 10 min. Reaction
was blocked with NH4Cl (50 mM, for 15 min on ice); cells
were gently washed twice with PBS to remove Biotin in
excess and lysed in buffer containing 50 mM Tris–HCl, pH
8, 20 mM EDTA, 1% Nonidet P40, 10 Ag/ml leupeptin, 10
Ag/ml aprotinin, 1 mM PMSF, and 10 mM iodoacetamide.
Cell lysates were centrifuged at 15,000 � g (30 min, 48C)to remove debris; biotinylated proteins were affinity
purified with ImmunoPure immobilized streptavidin
(Pierce) and analyzed by Western blot with mAbs specific
for CXCR2 (E2) or for actin, as negative control for
specific protein surface biotinylation.
Chemokine binding assay
For binding assays, 1 � 106 per ml of transfected HEK
cells were plated on polylysine pre-coated 96-multiwells.
After 24 h, cells were washed once with phosphate-buffered
saline (PBS, with Ca2+ and Mg2+); once with BSA medium
(50 mM HEPES pH 7.2, BSA 0.5%, 5 mM MgCl2, 1 mM
CaCl2); and incubated in this same medium with [125I]-
labeled CXCL8 (0.1 nM, specific activity, 2000 Ci/mmol),
and varying concentrations of unlabeled ligand at 48C for 2 h.
After three washes, cells were lysed with 0.1 M NaOH and
counted for radioactivity determination. Experiments were
performed in triplicate, and nonspecific binding was deter-
mined in the presence of 1 AM of unlabeled ligand. The
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–7568
binding data were fitted to a Hill equation with the Sigma Plot
Software (Jandel Scientific) to determine the agonist affinity
(Kd).
Chemotactic assay
In vitro chemotactic assays were performed as described
previously [16]. Briefly, HEK cells, transfected with wt or
mutated CXCR2, were transferred in chemotaxis medium
(serum-free DMEM plus 0.1% bovine serum albumin and
25 mM HEPES, pH 7.4) and plated (500,000 cells/well) on
collagen pre-coated 12-mm Transwell cell culture inserts
with 12-Am pore size. The lower chambers of the Transwell
system contained chemotaxis medium supplemented with
vehicle (water) or 6 nM CXCL8. After 2 h of incubation at
378C, cells were washed in PBS and treated with trichloro-
acetic acid (10%) on ice for 10 min. Cells adhering to the
upper side of the chamber were scraped off and cells on the
lower side were stained with a solution containing 50%
isopropyl alcohol, 1% formic acid, and 0.5% Coomassie
Brilliant Blue R-250. Stained cells were counted in at least
20 fields using a 20� objective. The chemotactic index was
obtained as the ratio between the number of migrated cells
in chemokine-treated versus untreated sample for each
transfection.
Fluorescence measurements
Fluorescence determinations for the measurement of
Ca2+ transients were made using a conventional fluores-
cence microscopy system composed by an upright micro-
scope (Axioskop 2, Zeiss, Germany), a digital 12-bit cooled
camera (SensiCam, PCO, Germany), and a monochromator
(Till, Germany). The system was driven by Axon Imaging
Workbench software (Axon Instruments, CA, USA).
Images were acquired and stored on a PC (Dell, USA),
then analyzed off-line. Measurements of fluorescence over
time had a resolution of 1 Hz. Cells were incubated with
fura-2 AM (3 AM, Molecular Probes, USA) for 45 min,
then extensively washed with external solution. The level
of intracellular free Ca2+ concentration ([Ca2+]i) was
estimated from the ratio between the digital images
obtained with 340 and 380 nm excitation wavelength.
Emission was monitored at 510 nm (optical filters and
dichroic beamsplitter were from Chroma, USA).
Receptor internalization
Receptor internalization upon agonist binding was
investigated for wt- and Cys-mutated CXCR2. Transfected
HEK cells were starved in Locke’s buffer for 30 min and
then incubated in the absence or in the presence of CXCL8
(600 nM) for 1 h at 378C. Cells were then washed in PBS
and fixed with PFA 4% for 15 min, permeabilized with
PBS/Triton X-100 0.2% for 5 min, then incubated with
mAb anti-CXCR2 for 1 h at room temperature. After
washing, cells were incubated with TRITC-conjugated
secondary Ab and analyzed by confocal-microscopy with
a Leica NBT system, equipped with 40 � 1.00–0.5 and
100 � 1.3–0.6 oil-immersion lenses.
Results
Disulfide bonds are involved in CXCR2 dimerization
Recently, we reported that CXCR2 forms dimers and
oligomers in HEK cells and in cultured rat cerebellar
granule neurons [16]. To test the possible involvement of
disulfide bonds in CXCR2 dimerization, lysates of CXCR2-
expressing HEK cells were analyzed by Western blot in
either the presence or the absence of the reducing agent
DTT. Fig. 2A shows that, under non-reducing (�DTT)
conditions, the dimeric form of CXCR2 becomes much
more evident, with an increase of the dimer/monomer ratio
for wt from 0.16 (+DTT) to 0.48 (�DTT), suggesting that
disulphide bonds might play some structural role on
CXCR2 dimerization.
We have previously demonstrated that drastically
truncated CXCR2, lacking the first 142 N-terminal
residues (143D), and a CXCR2 fragment containing only
the first N-terminal 142 amino acids (V142), are not able
to form dimers in the presence of DTT, in contrast with
what observed with wtCXCR2 and with a variety of
other, less drastic, CXCR2 deletion mutants [16]. In this
work, we analyzed the ability of 143D- and V142-
CXCR2 to oligomerize in the absence of DTT: Figs. 2B–
C show that specific bands, identified as dimeric
truncated receptors by molecular weight analysis,
appeared when DTT was absent, while, again, they were
completely absent under reducing conditions (+DTT); for
these deletion mutants, the increase of the dimer/mono-
mer ratio is even more evident, since in the two
experimental conditions, the two molecular forms of
CXCR2 are almost exclusive. Both the monomeric and
dimeric forms of wt and deleted CXCR2 (indicated by
arrows in Figs. 2A–C) were specific since they were
absent in mock-transfected (pCEP) HEK cells equally
under reducing and non-reducing conditions (Fig. 2D).
Considered together, these results demonstrate that the
disulfide bonds contributed to CXCR2 dimer formation.
To exclude the possibility that the dimers of wt and
deleted CXCR2 could be intracellularly trapped misfolded
forms shaped as a consequence of receptor over-expres-
sion, wt and deleted CXCR2 were transfected in HEK
cells, and the plasma membrane proteins were biotiny-
lated with the cell impermeable EZ-Link Sulfo-NHS-
Biotin reagent. Biotinylated proteins were then recovered
by affinity precipitation with ImmunoPure streptavidin
agarose, and analyzed by Western blot to detect the
presence of CXCR2 dimers on cell surface. Results,
shown in Figs. 2E–H, indicate that the dimeric forms of
Fig. 3. Effects of Cys mutations on CXCR2 surface expression. HEK cells
were transiently transfected with wt and Cys-mutated CXCR2. 48 h after
transfection, cells were incubated with an N-terminal anti-CXCR2 antibody
(E2) and then stained with a FITC anti-mouse antibody as described in
Materials and methods. The level of receptors expressed on cell surface was
measured by flow cytometry. Results are expressed as the percentage of
wtCXCR2 expression. Columns represent means F SEM of 4 experiments.
Note significant (Student’s t test, *P V 0.1; **P V 0.01) reduction of
surface expression for C39S and C286S.
Fig. 2. Immunoblot analysis of CXCR2 under reducing and non-reducing conditions, in total cell lysates and after cell surface protein biotinylation. HEK293
cells were transiently transfected with wtCXCR2 (A,E), 143D (B,F), V142 (C,G), or pCEP (D,H). 48 h after transfection, cells were: (A–D) lysed in Laemmli
buffer; (E–H) surface biotinylated and proteins recovered by affinity precipitation, in the presence (+) or in the absence (�) of DTT (50 mM). After 5 min
boiling, samples were analyzed by Western blot for CXCR2 immunoreactivity using a mAb directed to the N-terminus of the protein (E2, A,C,D,E,G,H) or a
polyclonal Ab recognizing the C-terminus (C19) in (B,F). Arrows indicate the position of monomers (m) and dimers (d) for each couple of samples. Note the
increase of the dimeric form for all the CXCR2 proteins in the absence of DTT.
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–75 69
CXCR2 were expressed on cell surface, for both wt and
truncated CXCR2. Again, dimers of mutated CXCR2
were only detectable under non-reducing conditions
(�DTT).
Cys mutation does not impair CXCR2 cell surface
expression
As illustrated in Fig. 1, CXCR2 sequence contains
nine Cys residues, highly conserved in all chemokine
receptors. We replaced these Cys (C) residues with Ser
(S) or Leu (L), generating the following CXCR2 mutants:
C39S, C119L, C139L, C166S, C196L, C230S, C263S,
C286S, C308L. To investigate the plasma membrane
expression of the Cys mutants CXCR2 generated, FACS
analysis was performed staining transiently transfected
cells with mAb E2, directed against the extracellular N-
terminal region of CXCR2. Results show that the CXCR2
mutants C39S and C286S, where Cys residues were
substituted, respectively, in either the N-terminal domain
or the third extracellular loop, exhibited a reduction of
about 35% of plasma membrane expression in comparison
with wtCXCR2 (Fig. 3). These differences were signifi-
cant, since when the transfection efficiencies were
analyzed by immunofluorescence on transfected permea-
bilized cells, variations between different transfections
randomly oscillated between 15 and 20% of total cell
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–7570
number (data not shown), suggesting alterations at the
level of receptor trafficking more than synthesis. Cell
surface expression of the remaining extracellular (C119L
and C196L) and of two of the TM mutants (C166S and
C308L) was about 20% less than wtCXCR2, while for
the other TM mutants (C139L, C230S, and C263S), the
plasma membrane expression was fully comparable with
wtCXCR2.
Cys-mutated CXCR2 dimers are expressed at the plasma
membrane
To study whether Cys mutations of CXCR2 impair dimer
translocation at the cell surface, wt- and Cys-mutated
CXCR2 were transiently transfected in HEK cells, and
analyzed as described above for plasma membrane protein
biotinylation. Results, illustrated in Fig. 4, indicate that both
wt- and Cys-mutated receptors could be expressed as dimers
at the plasma membrane, suggesting that Cys mutations did
not impair dimer surface expression. Interestingly, when
affinity-precipitated proteins were eluted with SDS buffer
under non-reducing conditions (Fig. 4, �DTT), we recov-
ered both dimeric and monomeric forms of CXCR2; never-
theless, when DTT was present in the SDS buffer, only the
monomeric forms of CXCR2 appeared (Fig. 4, +DTT). To
check for nonspecific biotinylation of intracellular proteins,
possibly due to cell damage during biotinylation handling,
the blots were re-probed with anti-actin or anti-ERK2 Abs;
no immunoreactivity was detected in affinity-purified
samples (data not shown), indicating that biotinylated
CXCR2 dimers were actually expressed at the cell surface.
These results point to the expression of CXCR2 at the cell
surface both as monomer and dimers, leaving open the
question of whether both molecular forms are active.
Fig. 4. Surface expression of Cys mutants CXCR2 dimers. HEK cells were
transfected with wt and the indicated Cys-mutant CXCR2; plasma
membrane biotinylated proteins were affinity purified and analyzed by
Western blot for CXCR2 immunoreactivity under non-reducing (�DTT)
and reducing (+DTT) conditions. Results are representative of five different
experiments. Dimeric forms are indicated for each gel by the arrow (d),
while the bracket (m) indicates monomeric forms.
Cys substitution alters CXCR2 binding affinity for the
agonist
Another issue we were interested to investigate was
whether the Cys mutants CXCR2 displayed any differ-
ences in agonist-binding affinity. For this reason, homol-
ogous competitive binding experiments were performed for
wt- and all the Cys-mutated CXCR2, using [125I] CXCL8
(0.1 nM) and various doses of cold CXCL8, ranging from
0.05 to 300 nM. Results, reported in Fig. 5, indicate that
three extracellular Cys mutants, C119L, C196L, C286S,
and the seventh TM Cys-mutant C308L, all failed to bind
the agonist at the tested concentrations. In contrast, the
remaining mutants still bound CXCL8. Specifically, as
shown both in Fig. 5 and in Table 1, C39S CXCR2
showed an increased agonist affinity, while the other
mutants displayed a general reduced affinity.
Cys residues are involved in CXCL8-induced chemotaxis
To investigate the possible role of Cys residues in receptor
function, we tested the ability of Cys-mutated CXCR2 to
migrate in response to a CXCL8 gradient. Chemotactic
assays were performed with HEK cells transfected with
either wt- or Cys-mutated CXCR2. As expected, the four
Cys-mutated CXCR2, C119L, C196L, C286S and C308L,
which did not bind the agonist, displayed no chemotactic
activity towards CXCL8. Nevertheless, we also found that,
despite the ability to bind the agonist, all the other Cys
mutants displayed a severely reduced chemotaxis toward
CXCL8 (6 nM), indicating that each Cys residue is
determinant for proper chemotactic function (Fig. 6). All
these data, considered together, indicate that the correct
protein folding is necessary for proper chemotactic activity
of CXCR2-expressing cells in response to CXCL8, and
compromised in Cys-mutated CXCR2.
Cys substitution alters CXCL8-induced signaling
We have previously reported that CXCR2-transfected
HEK cells respond to CXCL8 with a sustained phosphor-
ylation of ERK1/2 [16]. Cys-mutated CXCR2 receptors,
transiently expressed in HEK cells, were therefore analyzed
for ERK1/2 phosphorylation and the results compared with
those obtained with wtCXCR2. As shown in Fig. 7, CXCL8-
induced ERK1/2 phosphorylation was drastically reduced in
all Cys mutants analyzed, with the exception of C166S,
which fully retained its signaling properties, similarly to
wtCXCR2. Although greatly reduced, C263S also retained a
partial ability to phosphorylate ERK1/2. To further evaluate
the signaling potential of all these mutants, we also analyzed
Ca2+ transients induced by CXCL8 stimulation. Results
obtained indicated that, in addition to wtCXCR2, only C39S
and C166S CXCR2 mutants responded to agonist with Ca2+
transients (Fig. 8). Interestingly, the Ca2+ transients from
these mutants were significantly larger (P V 0.001, Student’s
Fig. 5. Effects of Cys mutations on CXCL8 binding. Homologous competitive binding experiments were performed on CXCR2 Cys mutants expressed in HEK
cells. Competitive binding curves for transmembrane (A), and extracellular (B) Cys-CXCR2 mutants, in comparison with wtCXCR2. Symbols represent
different Cys mutants as indicated in the insets; each point represents the mean of six experiments; bars represent standard error means (SEM).
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–75 71
t test) than those from wtCXCR2, showing the same delayed
rising phase after CXCL8 application. Our findings indicate
that even in the presence of agonist binding, Cys substitution
greatly impairs CXCR2 coupling to intracellular signaling,
similarly to what was observed for chemotactic activity,
further indicating that these structural alterations deeply
modify receptor function.
To further investigate the observation that some mutants
could signal in the absence of chemotaxis (as described for
C166S and, partially, for C39S and C263S), we compared
the internalization capabilities of wtCXCR2 with those of
Cys-mutant CXCR2. Results, obtained by confocal analysis,
indicate that C166S CXCR2 was efficiently internalized, not
differently from wtCXCR2, upon cell stimulation with
CXCL8 (600 nM; 1 h treatment, see Fig. 9). Same results
were obtained with C39S and C263S while, as expected,
under the same experimental conditions, C119L, C196L,
C286S and C308L, which do not bind the agonist, did not
internalize (Fig. 9). Also, C139L and C230S were effi-
ciently internalized upon agonist stimulation (Fig. 9). These
results indicate that the phenotype described for some
Table 1
Kd values of wt and Cys-mutant CXCR2 for CXCL8, measured with
homologous competitive binding experiments
CXCR2 Kd F SE
wt 5.3 F 0.2
C39S 2.7 F 0.5TC119L nba
C139L 13.0 F 1.5TC166S 13.5 F 0.7TC196L nb
C230S 7.4 F 0.7TC263S 12.2 F 0.3TC286S nb
C308L nb
a nb: Not binding.
T Significantly different from wt ( P b 0.01).
CXCR2 Cys mutants, which diverges for signaling and
chemotaxis, is not related to gross differences in ligand-
induced receptor internalization.
Discussion
GPCRs, initially believed to function only as monomers
on the plasma membrane, show the capability to homo- and
Fig. 6. Chemotactic function impaired in CXCR2 Cys mutants. HEK cells
were transiently transfected as indicated and used in a chemotactic assay. 48 h
after transfection, cells were plated on top of collagen-coated polycarbonate
filters (12-Am pores) of a Transwell system. 6 nM CXCL8 or vehicle (water)
was added to the lower chamber. After 2 h, cells adhering to the lower side of
the filter (migrated cells) were counted as described inMaterials andmethods.
The ratio between the numbers of cells migrated in CXCL8-treated
(stimulated) and vehicle-treated cells (control) represents the chemotactic
index (Ic). wtCXCR2: Ic = 3 F 0.4, n = 4. Columns represent the ratio (in
percentage) between Ic for each different transfection vs. Ic for wtCXCR2.
Note the drastic, always highly significant (Student’s t test, **P V 0.01),
reduction of chemotactic activity for all Cys mutants.
Fig. 8. CXCL8-induced Ca2+ mobilization in wt and Cys-mutated CXCR2.
(A) Typical examples of Ca2+ transients elicited by CXCL8 (100 nM) in
HEK cells, transiently transfected with wt, C39S, or C166S CXCR2.
Horizontal open bar, CXCL8 application, as indicated. (B) Mean
amplitudes of Ca2+ transients elicited by CXCL8 (100 nM) in HEK cells
transfected with wt- and Cys-mutated CXCR2.
Fig. 7. CXCL8-mediated ERK1/2 phosphorylation modified by Cys CXCR2
mutations. HEK cells were transiently transfected as indicated and stimulated
with CXCL8 (120 nM) for 10 min. The same amounts (20 Ag of total protein)of the corresponding cell lysate were analyzed by Western blot for
phosphoERK1/2 immunoreactivity. Data are reported as the percentage of
ERK1/2 phosphorylation in each CXCR2mutant vs.wtCXCR2 and refer the
means (FSEM) of 4–6 experiments. Note significant reduction for all but
C166S mutant. Student’s t test, **P V 0.01.
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–7572
heterodimerize, as was proven for adrenergic, sphingosine-1
phosphate, dopamine, somatostatin, GABA, vasopressin,
Ca2+, opioid, mGlu, chemokine receptors, and others [25].
Several recent reviews resume the complex picture emerg-
ing from studies on the functional consequences of receptor
assembly: GPCR homo- and heterodimerization, in fact,
play key roles in processes like receptor trafficking to the
cell membrane, ligand specificity, binding, and signal
transduction [25–28]. In a previous work, we demonstrated
that CXCR2 forms dimers in an agonist-independent
manner during receptor biosynthesis [16]. By using drasti-
cally truncated forms of CXCR2, both at the C- and N-
terminal regions, we concluded that a region including the
TM3 and the adjacent extra- and intracellular loops could be
crucial for CXCR2 dimerization. In this paper, we analyzed
the role of Cys in CXCR2 dimerization and function.
The first observation here reported is that the analysis of
CXCR2 dimerization under non-reducing conditions shows
an increased dimer/monomer ratio both for the wt and the
truncated CXCR2 mutants in comparison with what was
obtained under reducing conditions. Interestingly, we show
that CXCR2 truncated at the end of the TM3 (generating the
C-terminal deleted V142 and N-terminal deleted 143D
mutants), which do not dimerize under reducing conditions,
acquired the ability to form homodimers when analyzed
under non-reducing conditions. These results indicate that,
in addition to the hydrophobic interactions between TM3
regions we previously hypothesized [16], disulfide bonds
are in part responsible for CXCR2 dimerization, similarly to
what already shown for Ca2+ and mGlu receptors, where
both covalent and non-covalent interactions underlie recep-
tor dimerization [22,29]. Interestingly, this dimerization is
likely not due to intracellular aggregate formation, since
truncated dimers are also expressed at the cell plasma
membrane, as assessed by surface protein biotinylation.
To dip inside the molecular mechanisms leading to
CXCR2 dimerization and oligomerization, we created and
expressed CXCR2 mutants where the nine Cys present in
the aminoacidic sequence were individually replaced with
Ser or Leu residues. Results indicate that no Cys sub-
stitution in CXCR2 is sufficient per se to abolish dimer
formation, in line with the observation that other kinds of
molecular interactions, involving TM3 region, are impli-
cated [16]. On the other hand, we report that Cys mutations
did not severely impair CXCR2 surface expression, with
only minor effects on receptor trafficking observed for
Cys39 and Cys286, whose mutation produced small but
significant reduction of surface receptors. In addition, we
report that both wt- and all the Cys-mutated CXCR2 were
also expressed at the cell surface as dimers, and that
biotinylated dimers were converted to monomers under
reducing conditions. These results indicate that both
monomeric and dimeric CXCR2 are expressed on cell
surface. Interestingly, three extracellular (C119L, C196L,
C286S) and the seventh TM domain (C308L) Cys-mutated
CXCR2 could not bind the agonist CXCL8, indicating
structural changes interfering with agonist recognition. A
Fig. 9. Effects of Cys mutations on receptor internalization. wt and Cys mutants CXCR2 were transiently transfected in HEK cells and analyzed by confocal-
microscopy after incubation with or without CXCL8 (1 h, 600 nM). Staining with anti-CXCR2 antibody (E2) shows membrane surface expression of receptors
in all non-stimulated cells (right panels). After CXCL8 stimulation, membrane staining is lost for wt, C39S, C139L, C166S, C230S, C263S mutants, while it is
still present in C119L, C196L, C286S, and C308L transfected cells. Scale bar, 10 Am.
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–75 73
similar effect of extracellular Cys substitution on agonist
binding was shown for vasopressin, A opioid, and chemo-
kine CCR6 receptors [24,30,32].
In addition, we report that all the extracellular and TM
Cys mutants result greatly deficient in chemotactic activity
and in intracellular signaling, analyzed as ERK1/2 phos-
phorylation and Ca2+ transients, with the unique exception
of C166S CXCR2 mutant, which fully retains its signaling
properties. The functional effects induced by this mutation
are particularly intriguing, because this behavior is reminis-
cent of what is already described for CXCR2 when it is
physically associated with the AMPA-type glutamate
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–7574
receptor [16], suggesting comparable conformational effects
behind the same functional alterations. We demonstrated
that the different phenotypes described for some agonist-
binding mutants, like C166S, which maintains its signaling
properties while losing the chemotactic activity, were not
due to major differences in receptor internalization with
wtCXCR2. These results indicate that substitution of
Cys166 residue in CXCR2 determines conformational
changes that specifically impair chemotactic function,
without altering receptor coupling to intracellular signaling
pathways (at least the ERK1/2 and Ca2+ pathways) and
receptor internalization upon agonist binding.
The impairment of chemotaxis and intracellular signaling
found in the Cys mutants generated indicates that each Cys
substitution, either in the helices of TM segments or in the
extracellular regions, determines structural changes, all
incompatible with receptor activity. Similar effects on
receptor function upon Cys substitution have been reported
for CCR6 [24], V2 vasopressin receptor [30], mGluR1–5
[20,29], Ca2+ receptor [21], D2 dopamine receptor [31], and
A opioid receptor [32], indicating a common structural role
for Cys in GPCR function. From all these data, we conclude
that the Cys residues in CXCR2 are critical for correct
protein folding, agonist recognition, and signaling; the
presence of Cys residues could also be necessary per se to
determine receptor structure. To our knowledge, this is the
first demonstration that Cys residues in CXCR2 are
necessary for its functional activity.
Acknowledgments
The authors thank Dr. Antonella Sucapane for help with
fluorescence measurements. This work was supported by
grants from MIUR to FE. MC is a fellow of PhD in
Neuroscience of the University of Rome La Sapienza.
References
[1] M. Baggiolini, B. Dewald, B. Moser, Human chemokines: an update,
Annu. Rev. Immunol. 15 (1997) 675–705.
[2] A. Ben-Baruch, M. Grimm, K. Bengali, G.A. Evans, O. Chertov, J.M.
Wang, O.M. Howard, N. Mukaida, K. Matsushima, J.J. Oppenheim,
The differential ability of IL-8 and neutrophil-activating peptide-2 to
induce attenuation of chemotaxis is mediated by their divergent
capabilities to phosphorylate CXCR2 (IL-8 receptor B), J. Immunol.
158 (1997) 5927–5933.
[3] A. Ben-Baruch, K. Bengali, K. Tani, L. Xu, J.J. Oppenheim, J.M.
Wang, IL-8 and NAP-2 differ in their capacities to bind and
chemoattract 293 cells transfected with either IL-8 receptor type A
or type B, Cytokine 9 (1997) 37–45.
[4] R. Feniger-Barish, M. Ran, A. Zaslaver, A. Ben-Baruch, Differ-
ential modes of regulation of CXC chemokine-induced internal-
ization and recycling of human CXCR1 and CXCR2, Cytokine 11
(1999) 996–1009.
[5] O. Meucci, A. Fatatis, A.A. Simen, R.J. Miller, Expression of
CX3CR1 chemokine receptors on neurons and their role in neuronal
survival, Proc. Natl. Acad. Sci. U. S. A. 97 (1998) 8075–8080.
[6] D. Ragozzino, A. Giovannelli, A.M. Mileo, C. Limatola, A. Santoni,
F. Eusebi, Modulation of the neurotransmitter release in rat cerebellar
neurons by GRO beta, NeuroReport 9 (1998) 3601–3606.
[7] A. Giovannelli, C. Limatola, D. Ragozzino, A.M. Mileo, M.T. Ciotti,
D. Mercanti, A. Santoni, F. Eusebi, CXC chemokines interleukin-8
(IL-8) and growth-related gene product alpha (GRO alpha) modulate
Purkinje neuron activity in mouse cerebellum, J. Neuroimmunol. 92
(1998) 122–132.
[8] C. Puma, M. Danik, R. Quirion, F. Ramon, S. Williams, The
chemokine interleukin-8 acutely reduces Ca2+ currents in identified
cholinergic septal neurons expressing CXCR1 and CXCR2 receptor
mRNAs, J. Neurochem. 78 (2001) 960–971.
[9] P. Lax, C. Limatola, S. Fucile, F. Trettel, S. Di Bartolomeo, M. Renzi,
D. Ragozzino, F. Eusebi, Chemokine receptor CXCR2 regulates the
functional properties of AMPA-type glutamate receptor GluR1 in
HEK293 cells, J. Neuroimmunol. 129 (2002) 66–73.
[10] H. Xiong, J. Boyle, M. Winkelbauer, S. Gorantla, J. Zheng, A.
Ghorpade, Y. Persidsky, K.A. Carlson, H.E. Gendelman, Inhibition of
long-term potentiation by interleukin-8: implications for human
immunodeficiency virus-1-associated dementia, J. Neurosci. Res. 71
(2003) 600–607.
[11] G.H. Fan, W. Yang, J. Sai, A. Richmond, Phosphorylation-independ-
ent association of CXCR2 with the protein phosphatase 2A core
enzyme, J. Biol. Chem. 276 (2001) 16960–16968.
[12] G.H. Fan, W. Yang, X.J. Wang, Q. Qian, A. Richmond, Identification
of a motif in the carboxyl terminus of CXCR2 that is involved in
adaptin 2 binding and receptor internalization, Biochemistry 40 (2001)
791–800.
[13] G.H. Fan, W. Yang, J. Sai, A. Richmond, Hsc/Hsp70 interacting
protein (hip) associates with CXCR2 and regulates the receptor
signaling and trafficking, J. Biol. Chem. 277 (2002) 6590–6597.
[14] R.M. Richardson, R.J. Marjoram, L.S. Barak, R. Snyderman, Role
of the cytoplasmic tails of CXCR1 and CXCR2 in mediating
leukocyte migration, activation, and regulation, J. Immunol. 170
(2003) 2904–2911.
[15] C. Limatola, S. Di Bartolomeo, F. Trettel, C. Lauro, M.T. Ciotti, D.
Mercanti, L. Castellani, F. Eusebi, Expression of AMPA-type
glutamate receptors in HEK cells and cerebellar granule neurons
impairs CXCL2-mediated chemotaxis, J. Neuroimmunol. 134 (2003)
61–71.
[16] F. Trettel, S. Di Bartolomeo, C. Lauro, M. Catalano, M.T. Ciotti, C.
Limatola, Ligand-independent CXCR2 dimerization, J. Biol. Chem.
278 (2003) 40980–40988.
[17] S.S. Karnik, C. Gogonea, S. Patil, Y. Saad, T. Takezako, Activation of
G-protein-coupled receptors: a common molecular mechanism,
Trends Endocrinol. Metab. 14 (2003) 431–437.
[18] S. Cvejic, L.A. Devi, Dimerization of the delta opioid receptor:
implication for a role in receptor internalization, J. Biol. Chem. 272
(1997) 26959–26964.
[19] B.A. Jordan, L.A. Devi, G-protein-coupled receptor heterodimeriza-
tion modulates receptor function, Nature 399 (1999) 697–700.
[20] K. Ray, B.C. Hauschild, Cys-140 is critical for metabotropic
glutamate receptor-1 dimerization, J. Biol. Chem. 275 (2000)
34245–34251.
[21] G.F. Fan, K. Ray, X.M. Zhao, P.K. Goldsmith, A.M. Spiegel,
Mutational analysis of the cysteines in the extracellular domain of the
human Ca2+ receptor: effects on cell surface expression, dimerization
and signal transduction, FEBS Lett. 436 (1998) 353–356.
[22] Z. Zhang, S. Sun, S.J. Quinn, E.M. Brown, M. Bai, The extracellular
calcium-sensing receptor dimerizes through multiple types of inter-
molecular interactions, J. Biol. Chem. 276 (2001) 5316–5322.
[23] S. Venkatesan, A. Petrovic, M. Locati, Y.O. Kim, D. Weissman, P.M.
Murphy, A membrane-proximal basic domain and cysteine cluster in
the C-terminal tail of CCR5 constitute a bipartite motif critical for cell
surface expression, J. Biol. Chem. 276 (2001) 40133–40145.
[24] L.S. Ai, F. Liao, Mutating the four extracellular cysteines in the
chemokine receptor CCR6 reveals their differing roles in receptor
C. Limatola et al. / Experimental Cell Research 307 (2005) 65–75 75
trafficking, ligand binding, and signaling, Biochemistry 41 (2002)
8332–8341.
[25] M. Bai, Dimerization of G-protein-coupled receptors: roles in signal
transduction, Cell Signalling 16 (2004) 175–186.
[26] S. Terrillon, M. Bouvier, Roles of G-protein-coupled receptor
dimerization, EMBO Rep. 5 (2004) 30–34.
[27] G. Milligan, D. Ramsay, G. Pascal, J.J. Carrillo, GPCR dimerisation,
Life Sci. 74 (2003) 181–188.
[28] S.P. Lee, B.F. O’Dowd, S.R. George, Homo- and hetero-oligomeriza-
tion of G protein-coupled receptors, Life Sci. 74 (2003) 173–180.
[29] C. Romano, J.K. Miller, K. Hyrc, S. Dikranian, S. Mennerick, Y.
Takeuchi, M.P. Goldberg, K.L. O’Malley, Covalent and noncovalent
interactions mediate metabotropic glutamate receptor mGlu5 dimeri-
zation, Mol. Pharmacol. 59 (2001) 46–53.
[30] R. Schqlein, K. Zuhlke, A. Oksche, R. Hermosilla, J. Furkert, W.
Rosenthal, The role of conserved extracellular cysteine residues in
vasopressin V2 receptor function and properties of two naturally
occurring mutant receptors with additional extracellular cysteine
residues, FEBS Lett. 466 (2000) 101–106.
[31] S.P. Lee, B.F. O’Dowd, R.D. Rajaram, T. Nguyen, S.R. George, D2
dopamine receptor homodimerization is mediated by multiple sites of
interaction, including an intermolecular interaction involving trans-
membrane domain 4, Biochemistry 42 (2003) 11023–11031.
[32] P. Zhang, P.S. Johnson, C. Zollner, W. Wang, Z. Wang, A.E. Montes,
B.K. Seidleck, C.J. Blaschak, C.K. Surratt, Mutation of human Aopioid receptor extracellular bdisulfide cysteineQ residues alters ligandbinding but does not prevent receptor targeting to the cell plasma
membrane, Mol. Brain Res. 72 (1999) 195–204.
Top Related