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: cristina.limatola@uniroma1.it (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.