Expression of AMPA-type glutamate receptors in HEK cells and

cerebellar granule neurons impairs CXCL2-mediated chemotaxis

Cristina Limatolaa,*, Sabrina Di Bartolomeoa, Flavia Trettela,b, Clotilde Lauroa,Maria T. Ciottic, Delio Mercantic, Loriana Castellanid, Fabrizio Eusebia,b

aDipartimento di Fisiologia Umana e Farmacologia, Universita di Roma «La Sapienza», Piazzale Aldo Moro 5, I00185 Rome, ItalybDipartimento di Scienze Internistiche, San Raffaele alla Pisana, Tosinvest-Sanita, Via della Pisana 235, I-00163 Rome, Italy

c Istituto Neurobiologia CNR Viale Marx, Rome, ItalydDipartimento Scienza e Societa Universita di Cassino, and INFM sez. B, Universita di Roma «Tor Vergata», Rome, Italy

Received 17 July 2002; received in revised form 23 October 2002; accepted 23 October 2002

Abstract

We find that cerebellar granule neurons (CGN) obtained from newborn rats (p3) migrate in response to both CXC chemokine ligand-2

(CXCL2) and -12 (CXCL12), while CGN from p7 rats are unresponsive to CXCL2. The expression of a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionate (AMPA)-type glutamate receptor 1 (GluR1) greatly impairs the chemotaxis induced by CXCL2 in CXCR2-expressing

HEK cells. By immunoprecipitation, we show that CXCR2 is associated with AMPA receptors (AMPARs) in p7 CGN, and with GluR1 co-

expressed in HEK cells. Taken together, these results suggest that the association between CXCR2 and AMPARs results in the inhibition of

CXCL2-dependent chemotaxis, and may represent a molecular mechanism underlying the modulation of nerve cell migration.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Chemokines; Glutamate; Chemotaxis; Rat cerebellar neurons

1. Introduction

Most central nerve cells express several types of both

ionotropic glutamate (Glu) and CXC chemokine receptors

(Bigge, 1999; Asensio and Campbell, 1999). AMPA-type

Glu receptors (AMPARs) are ligand-gated receptors that

mediate the fast synaptic transmission and play a key role

in synaptic plasticity (Mack et al., 2001; Carroll et al.,

2001); are composed of the Glu receptor subunits 1–4

(GluR1–4); and their subunit composition and stoichiom-

etry affect channel properties (for reviews, see Swanson et

al., 1997; Dingledine et al., 1999). The CXC receptor 2

(CXCR2) is a G-protein-coupled receptor that exerts a

chemotactic activity on neutrophils and lymphocytes and

mediates leukocyte infiltration upon neurological diseases

(Glabinski and Ransohoff, 1999; Horuk, 2001). In the

CNS, the CXCR2-activating chemokines, the growth-

related gene product a (CXCL1), the growth-related gene

product h (CXCL2) and interleukin-8 (CXCL8), all mod-

ulate the cerebellar synaptic transmission, suppressing LTD

induction and increasing both the frequency of spontane-

ous events and the amplitude of the evoked post-synaptic

currents (Giovannelli et al., 1998; Ragozzino et al., 1998).

Neuromodulatory effects of CXCL8 have been also

described in hippocampal (Meucci et al., 1998), and

cholinergic septal cultured neurons (Puma et al., 2001).

CXCR2 plays a role in CNS cell survival (Araujo and

Cotman, 1993; Robinson et al., 1998; Saas et al., 1999;

Bruno et al., 2000; Wu et al., 2000), and both CXCL2 and

CXCL8 exert a neurotrophic activity on cerebellar granule

neurons (CGN), through the functional expression of

AMPARs (Limatola et al., 2000, 2002). Furthermore,

chemokines maintain their own chemotactic activity

toward CNS cells: astrocytes and developing nerve cells

migrate in response to many different chemokines (Hes-

selgesser et al., 1997; Dorf et al., 2000), while CXC

chemokine receptor 4 (CXCR4) plays determinant roles

0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0165 -5728 (02 )00401 -0

Abbreviations: CXCR, CXC chemokine receptor; CXCL, CXC

chemokine ligand; Glu, glutamate; AMPAR, a-amino-3-hydroxy-5-meth-

yl-4-isoxazolepropionate receptor; CGN, cerebellar granule neurons; GFP,

green fluorescence protein; hMCD, h-methyl cyclodestrin.

* Corresponding author. Tel.: +39-6-49910434; fax: +39-6-49910851.

E-mail address: cristina.limatola@uniroma1.it (C. Limatola).

www.elsevier.com/locate/jneuroim

Journal of Neuroimmunology 134 (2003) 61–71

in cerebellar and hippocampal neuron migration during

development (Ma et al., 1998; Zou et al., 1998; Klein et

al., 2001; Lu et al., 2002). For all these reasons, we found

it of interest to study the chemotactic activity of CXCL2

on CGN during development, when AMPARs are differ-

entially expressed, and to analyze the effects of GluR1 on

CXCL2-mediated chemotaxis of HEK cells stably express-

ing CXCR2.

In general, chemokine receptors undergo internalization

upon agonist binding (Pelchen-Matthews et al., 1999); both

receptor internalization and membrane reinsertion to the cell

surface seem to be crucial events for modulating the chemo-

kine-induced cell migration. Specifically, agonist-mediated

CXCR2 internalization is regulated by a C-terminal LLKIL

motif that drives receptor association with the adaptor

protein AP2 (Fan et al., 2001a), while a number of different

proteins physically interact with CXCR2, regulating recep-

tor signaling and internalization (Fan et al., 2001b, 2002). In

addition, CXCR2 phosphorylation as well as the presence of

intact actin filaments may be required for receptor internal-

ization and recycling (Ben-Baruch et al., 1997; Grimm et

al., 1998; Zaslaver et al., 2001). Similarly, the growing

family of AMPARs-interacting proteins regulates membrane

expression, synaptic localization and trafficking of AMPARs

(Martin et al., 1998; Srivastava et al., 1998; Xia et al., 1999;

Wyszynski et al., 1999, 2002). Another concept in the field

of receptor signaling is the ability of some receptors to

integrate external signals for a coordinate cellular response

through their direct association with other receptors. Exam-

ples of these associations enabling receptor cross-talk are

the physical coupling between the neurotransmitter recep-

tors GABAA and dopamine D5 (Liu et al., 2000), and the

hetero-oligomerization of dopamine and somatostatin

receptors (Rocheville et al., 2000). Furthermore, the het-

ero- or the homodimerization of chemokine receptors

yields distinct unique effects on receptor signaling and

biological activity (Mellado et al., 2001). In this paper, we

report the functional coupling between CXCR2 and

AMPARs influencing cell migration, likely induced by

the association of CXCR2 and AMPARs in a multi-protein

complex.

2. Materials and methods

2.1. Materials

Polyclonal antibodies (Abs) to CXCR2 (K19, C19),

CXCR4 (C20), GFP (FL), and monoclonal Abs (mAbs) to

CXCR1 (B1) and to CXCR2 (E2) were purchased from

Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal

Abs to GluR1–4 were from Chemicon Int. (Temecula, CA);

mAb to human h3 (CD61) was obtained from Immunotech

(Marseille, France); recombinant rat and human CXCL2,

human CXCL12, human CXCL8 and anti-rat CXCL2 were

from Peprotech (London, UK); transwell cell culture inserts

were from Becton Dickinson Labware (Franklin Lakes, NJ);

AMPA and CNQX were from Tocris (Bristol, UK). 125I-

CXCL8 was from Amersham Biosciences Italia (Milan,

Italy). Other drugs and chemicals were purchased from

Sigma Aldrich Italia (Milan, Italy). All culture media were

purchased from Life Technology Italia (Milan, Italy).

2.2. Generation of GluR1 and CXCR2 constructs

C- (L638) and N-terminal (M537) GluR1 deleted

mutants and C-terminal CXCR2 deleted mutants (G354,

F321, A315) were generated by PCR and cloned into the

expression vector pCEP4 (Invitrogen, Groningen, The Neth-

erlands). Wt GluR1 and I826, L638, M537 deleted con-

structs, generated by PCR, were also cloned into pEGFP-C3

expression vector in order to obtain N-terminal green

fluorescence protein (GFP) tagged receptors. GluR1 pCEP4

constructs were generated using 5V-CCTGAAGCTTA-

TGCAGCACATT-3V and 5V-ATTAGGATCCTTACAG-

GAAGGAGGCGGC-3V primers for the L638 mutant,

5V-CAGGAAGCTTATGTATGAGATTTGGATG-3Vand 5V-ATTAGGATCCTTACAATCCCGTGGC-3Vprimers for the

M537mutant.CXCR2pCEP4constructsweregeneratedusing

5V-CGGCGGTACCATGGAAGATTTTAAC-3Vand 5V-TAATCTCGAGTTACCCTGAAGAAGAG-3Vprimers for

G354;5V-CGGCGGTACCATGGAAGATTTTAAC-3Vand5V-TAATCTCGAGTTAAAACTTCTGGCCAATG-3Vfor F321,5V-GATCAAGCTTATGGAAGATTTTAAC-3Vand 5V-GATCCTCGAGTTAGGCGTAGATGAG-3Vfor A315. FortheGFPconstructs,weused the following specific primers: 5V-ATTAGGATCCTTACAATCCCGTGGC-3Vfor wt, 5V-AATAGGATCCTTAGATTAAGGCAAC-3Vfor I826, 5V-AATAGGATCCTTACAGGAAGGCGGC-3Vfor L638, andthe universal primer 5V-CCTGAAGCTTATGCAGCACATT-3V. For theM537 construct, primers 5V-GAGGAAGCTTATG-TATGAGATTTGGAT-3Vand 5V-ATTAGGATCCTTACA-ATCCCGTGGC-3Vwere used. All constructs were checked

by DNA sequencing; their expression in HEK cells was

analyzed by Western blot and immunofluorescence.

2.3. Cell transfection

Parental HEK 293 cells (HEK cells) and HEK cells

stably expressing (a) the rat subunit GluR1-flip (HEK-

GluR1, kindly provided by Dr. Piotr Bregestovski, Institut

Pasteur, Paris, France), (b) the human CXCR2 or (c) the

human CXCR1 (HEK-CXCR2 and HEK-CXCR1, kindly

provided by Dr. Massimo Locati, University of Brescia,

Italy) were plated on polylysine-coated 35-mm dishes

(150,000 cells/dish) and transfected 24 h later using a

calcium-phosphate procedure. Routinely, cells were used

for experiments 48 h after transfection. cDNAs encoding for

human CXCR2, CXCR1 (kindly provided by Dr. Massimo

Locati) or human GluR1 (from ATCC; Manassas, VA) were

subcloned in the expression vector pCEP4 and used for cell

transfection.

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–7162

2.4. Cultures of cerebellar granule neurons (CGN)

Primary cultures of CGN were obtained from 8-day-old

(p8) Wistar rats (Charles River, Como, Italy) as reported

(Limatola et al., 2002) and cultured in BME, supplemented

with 10% heat-inactivated foetal bovine serum and 25 mM

KCl. Cells were plated onto polylysine-coated Petri dishes,

or multiwells according to experimental requirements at a

density of 2.5� 105 cells/cm2. To prevent glial cell prolif-

eration, 20 h after plating, cultures were treated with

cytosine-h-D-arabinofuranoside at a final concentration of

10 AM. These cultures were used 7–8 days after plating,

when the cell population comprises 95% CGN and 5% of

other cell types including astrocytes.

2.5. Immunoprecipitation

Both CGN and transfected HEK cells, when necessary,

were serum starved for 1–2 h and then stimulated with

vehicle, AMPA (100 AM), CXCL2 (125 nM) or CNQX (50

AM) for 5 min or with tunicamycin (5 Ag/ml) for 16 h. Cells

were washed once with phosphate-buffered saline (PBS),

and lysed in a buffer containing 50 mM Tris–HCl pH 8, 20

mM EDTA, 1% Nonidet P-40, 10 Ag/ml leupeptin, 10 Ag/ml

aprotinin, 10 mM NaF, 1 mM phenylmethylsulfonyl fluo-

ride, for 15 min on ice. Alternatively, cells were lysed in this

same buffer with 0.1% SDS or with a buffer containing the

following: 30 mM Tris–HCl pH 7.5, 150 mM NaCl, 1%

Triton, 10 Ag/ml leupeptin, 10 Ag/ml aprotinin, 10 mM NaF,

1 mM phenylmethylsulfonyl fluoride. Cell lysates, after

centrifugation at 15,000� g for 10 min at 4 jC, were pre-

cleared with pre-immune rabbit IgG or normal mouse serum

and then incubated for 16 h with polyclonal anti-CXCR2

Abs (4 Ag/ml) or with the same amount of polyclonal rabbit

IgG for CGN and with a mAb anti-CXCR2 or anti-GFP

(both 8 Ag/ml) for HEK cells. The immunocomplexes were

separated on SDS polyacrylamide gel electrophoresis and

analyzed by Western blotting with Abs specific for GluR1,

GluR2/3, GluR4 or CXCR2. For experiments with h-methyl

cyclodestrin (hMCD), immunoprecipitated material was

further incubated 1 h, shaking at 37 jC, with 5 mM hMCD.

2.6. Immunofluorescence

Transfected HEK cells were analyzed for protein local-

ization by immunofluorescence. Cells were fixed for 10 min

with 4% paraformaldehyde in PBS at room temperature and

permeabilized with ethanol/acetic acid (20:1 v/v) for 10 min

at � 20 jC. Incubation with primary Abs was carried out

for 1 h at room temperature or overnight at 4 jC. Afterextensive washing with PBS, cells were incubated with the

corresponding secondary Abs (FITC- and TRITC-conju-

gated goat anti-rabbit and anti-mouse Abs). Confocal anal-

ysis was carried out with a Leica (Heidelberg, Germany)

TCS 4D system, equipped with 40� 1.00–0.5 and 100�1.3–0.6 oil immersion lenses. Images of double-labeled

samples were recorded with simultaneous excitation and

emission of both dyes to ensure their alignment. To correct

for possible cross-talk resulting from overlapping excitation

and emission of the dyes, recorded images were corrected,

when necessary, with the Multi Colour analysis package

software from Leica and compared to images recorded with

single dye excitation and detection.

2.7. Chemotaxis assay

CXCL2-induced chemotaxis was investigated in GluR1-

or mock- (pCEP4) transfected HEK-CXCR2 cells. Fourty-

eight hours after transfection, cells were trypsinized, washed

twice in RPMI with 1% BSA and 25 mM HEPES, pH 7.4,

and plated (500,000/well) on collagen-pre-coated 12-mm

transwells (12-Am pore size filters) in this same medium.

Cerebellar neurons were obtained from rats of different ages,

p3 and p7, as described above, and immediately plated in

DMEM and HAM’S 12 (3:1), supplemented with insulin,

putrescin, transferrin, Na-selenite and progesterone (Bottes-

tein et al., 1979), onto polylysine-treated 12-mm transwells

(3-Am pore size filters, 500,000 cells/well). When necessary,

cells were preincubated 5 min with AMPA (100 AM) or

CNQX (50 AM), or 1 h with pertussis toxin (PTX, 1 Ag/ml)

that were also present in the upper chamber during the assay.

The lower chambers contained vehicle (water), CXCL2 (60

nM), CXCL8 (60 nM) or CXCL12 (previously named SDF-

1a 60 nM). In some experiments, anti-rat CXCL2 (20 Ag/ml)

was present in the lower chamber, together with CXCL2.

After 2 h of incubation at 37 jC, cells were treated with 10%trichloroacetic acid on ice for 5 min. Cells adhering to the

upper side of the filter were scraped off, while cells on the

lower side were stained with a solution containing 50%

isopropanol, 1% formic acid and 0.5% (w/v) brilliant blue R

250. Stained cells were visually counted in more than 20

fields with either a 20� (HEK cells) or a 63� objective

(CGN). Chemotactic index is obtained by the ratio between

chemokine-treated vs. untreated cells, for each type of trans-

fection (HEK cells) or cerebellar neurons.

2.8. Saturation binding experiments

HEK and HEK-GluR1 cells were transiently transfected

with cDNA encoding for CXCR2 and, 24 h after trans-

fection, enzymatically removed from the dish and plated on

polylysine-pre-coated 24-multiwell dishes (80,000 cells/

well). After an additional 24 h, cells were washed with,

and then incubated in BSA medium (50 mM HEPES, pH

7.2, containing 0.5% BSA, 5 mM MgCl2 and 1 mM CaCl2)

in the presence of different doses of 125I-CXCL8 for 2 h on

ice. Cells were then washed two times with 25 mM HEPES,

pH 7.2, containing 0.5 M NaCl, and lysed with 0.1 M

NaOH for 10 min at room temperature. Lysates were

counted in a g-counter and part of them was used for protein

quantification with a bicinchoninic acid-based method

(Pierce, Rockford, IL).

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–71 63

2.9. Whole-cell patch-clamp recordings

When necessary, whole-cell currents were recorded in

HEK cells with experimental procedures detailed before

(Lax et al., 2002).

3. Results

3.1. CXCL2-induced CGN migration is age-dependent

It has been shown that cerebellar neurons express

CXCR2 (Horuk et al., 1997; Giovannelli et al., 1998), while

the expression of AMPARs is developmentally regulated,

being correlated with the timing of CGN migration from the

external to the inner granular layer (Martin et al., 1998;

Altman, 1972a,b; Smith et al., 1999, 2000). We investigated

the ability of CGN to migrate upon CXCL2 stimulation

throughout neuronal maturation, when the AMPARs expres-

sion increases. The chemotactic action of CXCL2 was

studied in CGN obtained from rats at different ages, p3 or

p7, corresponding to before and at the beginning of granule

neurons migration, respectively (Altman, 1972a,b). It was

found (Table 1) that CXCL2 induces a chemotactic activity

in neurons from p3 rats, and that this activity is reduced in

CGN derived from animals at p7, at a time when the

functional expression of AMPARs is detectable (Smith et

al., 2000). Comparable results were obtained with another

CXCR2-activating chemokine, CXCL8, both at p3 and p7

(data not shown). This age-dependent differential stimula-

tion of nerve cell migration by CXCL2 was rather specific,

since migration of p3 vs. p7 CGN remained stable upon

CXCR4 stimulation with CXCL12. The reduced migration

of p7 CGN upon CXCR2 stimulation was not caused by a

reduction of CXCR2 expression over age, since immuno-

logical analysis of extracts derived from these cells clearly

showed a progressive increase of both GluR1 and CXCR2

accumulation (see below). To investigate whether CXCR2

acts independently of AMPARs activity, the effect of the

competitive AMPARs blocker CNQX was tested in the

chemotaxis assays. Data reported in Table 1 indicate that

CNQX treatment was unable to affect p3 CGN migration

upon CXCL2 or CXCL12 treatment while it increased the

chemotactic index of p7 CGN only upon CXCL2 stimula-

tion. To address whether, upon CNQX treatment, CGN

response is still CXCL2-specific, in some experiments (both

p3 and p7) anti-CXCL2 Ab (20 Ag/ml) was included

together with CXCL2 in the lower chamber. Alternatively,

for the same reasons, CNQX-treated neurons were preincu-

bated for 1 h with PTX (1 Ag/ml). In both cases, the

chemotactic activities of CXCL2 were completely abolished

(data not shown), indicating that the effects observed are

CXCL2-specific and depend on the activation of receptor

coupled to PTX-sensitive G proteins. In addition to p3 and

p7, older rats have been tested both for GluR1/CXCR2

expression and chemotaxis, to investigate what happens

when CGN migration in the inner layer is at the maximum.

Neurons obtained from p11/p12 rats migrated similarly to

those from p7 rats in response to both chemokines (CXCL2:

1.1F 0.5; CXCL12: 3.6F 0.3) and the expression levels of

CXCR2 and GluR1 maintained the trend toward an increase

(data not shown). We have also tried to test neurons

obtained from older rats, namely p30, but the number of

viable cells obtained under our experimental conditions was

negligible, hampering to test their chemotactic response to

chemokines.

3.2. GluR1 co-expression with CXCR2 inhibits CXCL2-

induced HEK cell migration

We investigated whether CXCR2 and AMPAR co-

expression could influence the chemotactic activity of

CXCR2 in HEK cells. Experiments were performed in

GluR1-transfected HEK-CXCR2 cells stimulated with

CXCL2 vs. mock-transfected HEK-CXCR2 cells. It was

found that the co-expression of GluR1 with CXCR2 impairs

the CXCL2-induced chemotactic activity of HEK cells

(Table 2). This inhibitory effect was not influenced by

AMPA, but was blocked by CNQX (Table 2). CNQX was

able to block AMPARs at the used concentrations as tested

by whole-cell patch-clamp recordings of AMPA-induced

currents from HEK-GluR1 cells (data not shown). To

determine the GluR1 regions potentially involved in the

inhibition of cell migration, a series of drastically truncated

Table 1

Chemotactic indexes of chemokine-stimulated CGN

Rat

age

CXCL2 CXCL2+

CNQX

CXCL12 CXCL12+

CNQX

p3 2.0F 0.1 (6)* 1.8F 0.1 (3)* 2.2F 0.4 (6)* 1.8F 0.1(3)*

p7 1.1F 0.1 (8) 3.5F 0.7 (5)* 2.2F 0.5 (5)* 1.8F 0.1 (3)*

Numbers in brackets = n of experiments. The mean n of cells for field in p3

untreated, unstimulated samples (63� objective; 20–30 fields analyzed)

was 35.7F 9 (this number was not altered by CNQX pretreatment or by rat

age). Asterisk indicates paired t-test value (between chemokine-untreated

and -treated cells for each group of experimental conditions and rat age)

< 0.02.

Table 2

Chemotactic indexes of CXCL2-stimulated HEK-CXCR2 cells

Transfection + treatment Chemotactic index

Mock 2.0F 0.1 (9)**

GluR1 1.0F 0.15 (8)

GluR1 +AMPA 1.0F 0.05 (4)

GluR1 +CNQX 2.6F 0.5 (4)**

GluR1-L638 2.8F 0.4 (4)**

GluR1-M537 1.35F 0.08 (4)

Numbers in brackets same as in Table 1; ** and * indicate paired t-test

values (between the chemokine-untreated and -treated cells for each group

of transfection and cell treatment), respectively, < 0.007 and < 0.02. The

mean n of cells for field (20� objective; 20–30 fields analyzed) in mock-

transfected HEK-CXCR2 cells was 38.3F 0.4 (not altered throughout the

various experimental conditions).

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–7164

GluR1 were generated and transfected to HEK-CXCR2

cells, as described (see Section 2). Immunohistochemical

(not shown) and Western blotting analysis (see below) and

the AMPA-elicited whole-cell currents supported the ex-

pression of the truncated receptors. Among the deletions

tested, L638, lacking the whole intracellular C-terminal

domain, the M4 transmembrane segment and the entire

nearby extracellular region, was unable to inhibit CXCL2-

mediated chemotaxis of HEK-CXCR2 cells; while M537,

lacking the whole N-terminal domain, still retained its

inhibitory potential (Table 2). Considered together, these

results indicate that the C-terminal domain of GluR1 may be

involved in the inhibition of CXCL2-induced chemotaxis.

As a control for specificity, HEK-CXCR1 cells were trans-

fected with either pCEP (mock) or GluR1 and analyzed as

above for CXCL8 (12 nM)-induced chemotaxis; the chemo-

tactic indexes obtained were, respectively, 2.2F 0.3 and

2.9F 0.4, indicating a specific inhibition of CXCR2-medi-

ated chemotaxis by GluR1.

3.3. Expression of GluR1 does not influence the CXCR2

binding affinity

To analyze whether the inhibition of cell migration could

be explained by a change of CXCR2 affinity for the agonist

as a consequence of its co-expression with GluR1, satura-

tion-binding assays were performed with [125I] CXCL8 and

CXCR2-transfected HEK vs. CXCR2-transfected HEK-

GluR1 cells. It was found that the co-expression of CXCR2

with GluR1 does not significantly alter the Kd for CXCL8

being respectively 1.4F 0.2 nM in CXCR2-transfected

HEK cells and 1.61F 0.3 nM in CXCR2-transfected

HEK-GluR1 cells (nonlinear regression, not shown). These

results indicated that the co-expression of CXCR2 with

GluR1 does not modify the agonist binding affinity at

CXCR2, at difference with the Glu affinity at GluR1 (Lax

et al., 2002).

3.4. CXCR2 and AMPAR subunits co-immunoprecipitate in

both CGN and HEK cells

To determine whether the receptor association could

mediate the inhibition of cell migration, lysates of cultured

CGN obtained from p7 rats were subjected to immunopre-

cipitation with CXCR2-specific Abs and the immunocom-

plexes were analyzed by Western blot. We found the

presence of GluR1 and GluR2/3 in the complexes, while

GluR4 was not detected (Fig. 1A). When cellular lysates

were immunoprecipitated with pre-immune rabbit IgG, no

association was detected (not shown). Some minor GluR1

immunoreactivity was also detected when neuronal lysates

were immunoprecipitated with aCXCR4 (Fig. 1B), although

this association apparently does not abrogate CXCL12-

mediated chemotaxis (see above). The neuronal expres-

sion of this chemokine receptor was supported by Western

blot analysis (Fig. 1D). The association of CXCR2 with

AMPARs was independent of receptor stimulation, as

observed upon granule cells treatment with the respective

receptor agonists, CXCL2 and AMPA (Fig. 1C); and similar

results were obtained with GluR1 (not shown). Interestingly,

in p3 CGN, we failed to co-immunoprecipitate CXCR2 with

GluR1 (Fig. 2A) or with GluR2–4 subunits (not shown),

although both receptors were present in the extracts (Fig.

Fig. 1. CXCR2 and AMPAR subunits co-immunoprecipitate in rat CGN.

Immunoblot analysis of CGN protein extracts immunoprecipitated with

Abs specific for CXCR2 (K19, A, C), or CXCR4 (C20, B), and of CGN

total lysates. The blots were probed with Abs specific for GluR1 (A, B),

GluR2/3 (A, C) and GluR4 (A), as indicated. In C, cells were pretreated

with CXCL2 (60 nM), AMPA (100 AM) or both for 5 min before

immunoprecipitation. CGN lysates were analyzed for chemokine receptor

expression in D; arrows indicate the putative chemokine receptors, as

indicated. The apparent molecular weights for these proteins were the

following: CXCR2, 40 kDa; CXCR4, 40 kDa.

Fig. 2. GluR1 and CXCR2 co-immunoprecipitation in rat cerebellar

neurons is age-dependent. (A) Lysates from CGN at p3 and p7 (about

6� 106 cells/point) were immunoprecipitated with Ab anti-CXCR2 and the

immunocomplexes analyzed for GluR1 (indicated by arrow). Total cellular

lysate from p7 (lysate) was run as control for GluR1 position. Note the lack

of the GluR1 in the immunoprecipitates at p3. (B) Cerebellar neurons,

obtained from p3 and p7 rats as described in Section 2, were analyzed for

CXCR2 and GluR1 expression by Western blot; equal protein loading was

verified analyzing actin content. Three different experiments gave

comparable results. Note the increased accumulation of both receptors

over age, while actin content remained constant.

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–71 65

2B). Since both CXCR2 and GluR1 levels increase in p7 vs.

p3 CGN (Fig. 2B), it may also be possible that the lack of

co-immunoprecipitation at p3 is due to the low expression

level of these receptors.

To investigate further the association of CXCR2 with

AMPAR subunits, HEK-GluR1 cells were transiently trans-

fected with an expression vector encoding for CXCR2.

Immunocomplexes, obtained by subjecting cell lysates to

immunoprecipitation with a CXCR2 mAb, displayed spe-

cific GluR1 immunoreactivity, again revealing the presence

of multi-protein complexes (Fig. 3A). The same results

were obtained when cells were lysed and proteins immu-

noprecipitated with buffers containing different detergents

(0.1% SDS or 1% Triton X-100) or NaCl (150 mM, see

Section 2; data not shown). To address the specificity of

CXCR2 and GluR1 association, HEK-GluR1 cells were

transiently transfected with a vector encoding for the

chemokine CXC receptor 1 (CXCR1), and cell lysates

immunoprecipitated using a specific CXCR1 mAb (B1).

Western blot analysis of these immunocomplexes did not

reveal any immunoreactivity for GluR1 (Fig. 3B), indicat-

ing that GluR1 was specifically associated to CXCR2.

Furthermore, to investigate whether receptor association

was dependent on the activation of the receptors, co-

immunoprecipitation was performed on GluR1 transfected

HEK-CXCR2 cells pretreated with either AMPA (100 AM)

together with CXCL2 (120 nM), or CNQX (50 AM).

Results reported in Fig. 3C indicate that comparable

amounts of GluR1 were co-immunoprecipitated with

CXCR2 independent of either stimulation or blockage of

the receptors.

Membrane domains with specific lipid composition (lipid

rafts) seem to be involved in receptor clustering and local-

ization of signaling complexes even in nerve cells (Masser-

ini et al., 1999; Suzuki et al., 2001; Becher et al., 2001). We

investigated whether the co-localization of the two receptors

in insoluble, cholesterol-rich membrane microdomains

could be responsible of this association. With this aim, the

immunoprecipitated complexes were treated with h-MCD

(5 mM) at 37 jC for 15 min. By analyzing GluR1 immu-

Fig. 3. CXCR2 and GluR1 co-immunoprecipitate in HEK cells. (A) HEK and HEK-GluR1 cells were either mock (pCEP)-transfected (� ) or transfected (+)

with CXCR2. Protein extracts were immunoprecipitated with mAb specific for CXCR2 (E2). Corresponding Western blot was probed with Ab specific for

GluR1. Total lysate was used as a control for GluR1 identification, as indicated. (B) HEK-GluR1 cells were transfected with CXCR1 (+), CXCR2 (+), or pCEP

(� ), and treated as described in A. (C) HEK-GluR1 cells were transfected with CXCR2 (+) or pCEP (� ) and treated for 5 min with AMPA (100 AM) and

CXCL2 (60 nM) or CNQX (50 AM), and treated as described in A. (D) HEK-GluR1 cells were transfected with CXCR2 (+) or pCEP (� ) and treated as

described in A, except that the immunoprecipitated material was treated for 30 min at 37 jC with hMCD (5 mM). (E) HEK-GluR1 cells were transfected with

CXCR2 (+) or pCEP (� ), incubated for 16 h with tunicamycin (5 Ag/ml, E) and treated as described in A (top panel). Bottom panel indicates the relative

positions of the deglycosylated and glycosylated GluR1 proteins (indicated by arrows).

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–7166

noreactivity (Fig. 3D), receptor association was again found.

The same results were obtained when cells were pretreated

with h-MCD for 1 h before the immunoprecipitation (not

shown).

N-glycosylation has been shown to be involved in

mediating receptor subunit interaction for many different

receptors (Li et al., 1998; Ramanathan and Hall, 1999;

Fernandes et al., 2001). We investigated whether receptor

de-glycosylation with tunicamycin (2.5 Ag/Al, 48 h) could

prevent CXCR2-GluR1 association. Fig. 3E (top) shows

that tunicamycin treatment does not abolish protein asso-

ciation, and that the GluR1 co-immunoprecipitating with

CXCR2 is completely deglycosylated. Total cell lysates

from tunicamycin-treated cells contained two GluR1-immu-

noreactive bands (Fig. 3E, bottom), likely indicative of total

and partial deglycosylated forms of GluR1. Conversely,

CXCR2 was completely deglycosylated by this treatment

(not shown). Given that CXCR2 surface expression in

neutrophils is dependent on N-glycosylation (Ludwig et

al., 2000), these findings indicate that the GluR1 and

CXCR2 interaction may take place also intracellularly.

3.5. Truncated receptors co-immunoprecipitate with wild

type (wt) receptors

Progressive deletions of portions of both CXCR2 and

GluR1, generated to identify the determinants of this puta-

tive physical interaction, are schematically depicted in Fig.

4. CXCR2 deletion mutants were generated at the C-

terminal region: CXCR2-G354 lacks the last 6 aa, including

the potential RGS12 PDZ-binding domain (Snow et al.,

1998); CXCR2-F321 lacks all but the last 6 aa of the C-

terminal region, while CXCR2-A315 lacks the whole C-

terminal region. Fig. 5A shows the expression of wt and

deleted CXCR2 constructs in HEK cells: the main immu-

noreactive bands detected are two proteins of about 38 and

35 kDa, plus an additional protein of about 45 kDa that

disappeared upon tunicamycin treatment (not shown). The

same pattern has been described for human neutrophils and

transfected HEK cells (Ludwig et al., 2000). In CXCR2

mutants, a progressive shift towards lower molecular

weights is detectable for the two smaller proteins (Fig.

5A). Fig. 5B indicates that when HEK-GluR1 cells were

transiently transfected with the mutated CXCR2 constructs,

GluR1 immunoreactivity was always associated with immu-

noprecipitated CXCR2 (Ab E2). Deleted constructs were

also generated as GFP-fusion proteins for GluR1 (Fig. 4).

GluR1-I826 lacks the whole C-terminal region, while

GluR1-I638 also lacks the M4 domain and the extracellular

loop; GluR1-M537 lacks the big (537 aa) N-terminal

domain. Fig. 5C demonstrates the expression of GFP-GluR1

proteins in HEK cells, with the reduction in molecular

weight upon receptor truncation. When HEK cells co-

expressing CXCR2 and GluR1-GFP constructs were immu-

noprecipitated with anti-GFP Ab (FL), the immunoprecipi-

tated materials also contained CXCR2 immunoreactivity,

again indicating that both N-terminal and C-terminal trun-

cated GFP-GluR1 receptors co-immunoprecipitate with

CXCR2 (Fig. 5D).

3.6. CXCR2 and GluR1 co-localize in HEK cells

HEK-GluR1 cells were transiently transfected with

CXCR2 and studied by immunofluorescence and confocal

analysis for GluR1 and CXCR2 localization (Fig. 6A).

Staining for GluR1 (Fig. 6A, left upper panel) was

detected in the majority of the cells both on the plasma

membrane and on the cytosolic compartments (likely

indicative of receptor being synthesized). The localization

of CXCR2 (Fig. 6A, right upper panel) in transfected cells

matched that of GluR1, as highlighted by merging images

showing in yellow extensive areas of co-localization (Fig.

6A, lower panel). These findings are consistent with an

association of CXCR2 together with AMPARs. As a

Fig. 4. Schematic structures of GluR1 and CXCR2 constructs generated.

Left panel indicates the GluR1 expressed as GFP fusion proteins (indicated

by the circle); the wt GluR1 is the full-length protein that contains 906 aa;

I826 and L638 are C-terminally truncated GluR1: the letters indicate the last

aa present in the sequence and the numbers refer to the total number of aa.

M537 is the N-terminus deleted GluR1 that only contains the last 537 aa.

Wt CXCR2 (360 aa) and the three C-terminus deleted constructs G354

(lacking 6 aa), F321 (lacking 39 aa) and A315 (lacking 45 aa) are

represented on the right panel.

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–71 67

control, a similar double-staining experiment was per-

formed using an irrelevant Ab; Fig. 6B shows that when

GluR1 staining was compared with that obtained with

another ubiquitous protein, like h3 (CD61, right upper

panel), no similar co-localization was obtained (lower

panel).

4. Discussion

In this study we show that the co-expression of AMPARs

and their assembly into a multi-protein complex with

CXCR2 negatively modulate CXCL2-mediated CGN

migration. These findings represent the first report of the

Fig. 5. CXCR2 and GluR1 deletion mutants co-immunoprecipitate in HEK cells. (A) Western blot analysis of HEK cells transiently transfected with pCEP

(mock), wt CXCR2, or the indicated deletion mutants of CXCR2, and analyzed with mAb E2. Positions of molecular weight standards are indicated on the left.

(B) The same CXCR2 mutants as in A were transiently expressed in HEK-GluR1 cells, immunoprecipitated with mAb E2 and blotted for GluR1 reactivity

(Ab1504), as indicated by arrow. Note that the different intensity of immunoprecipitated GluR1 was not statistically significant. (C) HEK cells were transiently

transfected with CXCR2 and the indicated GFP-GluR1 constructs and cell lysates were tested for GFP-GluR1 expression by anti-GFPAb (FL). Note that the wt

GFP-GluR1 protein has a molecular weight of about 125 kDa. Molecular weight markers are indicated on the right. (D) Parallel transfected cultures as in C

were immunoprecipitated with anti-GFP Ab (FL) and analyzed for CXCR2 immunoreactivity (Ab C19). Arrows indicate the position of CXCR2 proteins.

Fig. 6. GluR1 and CXCR2 co-localize in HEK-GluR1 cells transiently expressing CXCR2. Cells, processed for immunofluorescence 48 h after transfection,

were double-labelled with polyclonal Abs to GluR1 (revealed with FITC-conjugated anti-rabbit Abs, left upper panels of A and B) and mAb to CXCR2 or h3(revealed with TRITC-conjugated anti-mouse Abs, right upper panels, respectively, of A and B) and visualized by confocal microscopy. Lower panels show

merging of the images of the two couples of receptors, clearly highlighting regions of co-localization, in yellow, only for A. Scale bars, 10 Am.

C. Limatola et al. / Journal of Neuroimmunology 134 (2003) 61–7168

involvement of AMPARs in CGN migration. The migration

of CGN from p10 mice has been elsewhere reported to be

independent of AMPAR activity, but profoundly influenced

by NMDA receptors (Komuro and Rakic, 1993). The

involvement of AMPARs on CGN migration is suggested

by the absence of migration in p7 neurons that express high

levels of AMPARs, and that instead migrate upon CNQX

treatment. This involvement seems to be rather specific for

chemotaxis induced by CXCR2-activating chemokines,

because CXCR4-mediated chemotaxis is not altered in p3

vs. p7 neurons and is not affected by CNQX. The lack of

effect on CXCR4 indicates that its chemoattractant activity

is not modulated by its interaction with AMPARs, because

either this interaction involves only a minority of CXCR4

expressed or does not impair the CXCR4 activity. The

observation that a functional interaction between CXCR2

and GluR1 receptors can be reproduced in HEK cells by

their co-expression, reinforces the data obtained in cerebel-

lar neurons, even considering the differences between the

two cellular systems and their AMPAR subunit expression;

and suggests a direct coupling between the two receptors.

This phenomenon could play a role in the developing

cerebellum where CGN begin to express AMPARs at the

time of their migration from the external to the inner granule

layer and fully express functional AMPARs at the end of

their migration (Martin et al., 1998; Altman, 1972a,b; Smith

et al., 1999, 2000). We speculate that AMPARs could

modulate chemokine-dependent CGN migration through

their progressive association with CXCR2 during cerebellar

development. A role played by CXCR2 in neuronal process

trafficking has been recently suggested on the basis of its

differential expression during mouse brain development

(Luan et al., 2001), and our findings are compatible with

a functional role for CXCR2 in the early phases of postnatal

cerebellar development. However, CXCR2-deficient mice

(Cacalano et al., 1994) have an apparently normal cerebellar

structure. Considering the functional redundancy described

so far for most chemokine receptors (Horuk, 2001), it is

likely that other chemokine receptors could compensate the

CXCR2 biological function. Alternatively, CXCR2 � /�mice manifest neurological defects under stress conditions

(Luan et al., 2001), being highly vulnerable to environ-

mental stresses (Devalaraja et al., 2000).

We also report the association between CXCR2 and

AMPARs, with molecular links worthy to be ascertained.

The association of AMPAR subunits with PDZ (PSD-95/

Dlg/ZO-1) domain-containing proteins (Srivastava et al.,

1998; Xia et al., 1999; Wyszynski et al., 1999; Dong et

al., 1997; Leonard et al., 1998), and the molecular inter-

actions of CXCR2 with the regulator of G-protein-signalling

protein 12 (RGS12) (Snow et al., 1998), with the protein

phosphatase 2A, and with the Hsc/Hsp70-interacting protein

(Fan et al., 2001b, 2002), involve direct binding to the

intracellular C-terminal receptor domains. We hypothesize

both the receptor direct association and the interposition

between receptor domains of accessory proteins; the asso-

ciation of CXCR2 with GluR1 does not rely uniquely on C-

terminal domains, since we failed to dissociate them upon

generation of mutated receptors lacking the whole C-termi-

nal regions. This result may indicate that either these

domains are not involved or the association requires more

than one single domain.

Interestingly, the GluR1-L638 deletion mutant allows full

migratory capacity of HEK-CXCR2 cells, indicating that

mutated GluR1 receptor looses its inhibitory activity even if

it maintains its association with CXCR2 and its functional

properties (Lax et al., 2002). This observation, together with

the fact that the competitive AMPAR antagonist CNQX is

unable to interfere with CXCR2/GluR1 association, but

restores CXCL2-mediated chemotaxis both in HEK cells

and in p7 CGN, indicate that the mutual interaction between

these two receptors is an event necessary but not sufficient

to play a functional role. The same conclusion is supported

by the experiments on both p3 and p7 CGN: p7 neurons fail

to migrate in response to CXCL2, while the receptors co-

immunoprecipitation is detectable; p3 neurons actively

respond to CXCL2 treatment, while the co-immunoprecipi-

tation is not observed.

Previously we described that the functional expression of

AMPARs in cultured CGN is necessary, but not sufficient,

for the neurotrophic activity of CXCR2 (Limatola et al.,

2000, 2002). Results herein reported confirm that AMPARs

alter the functional properties of CXCR2 and give evidence

of the presence of their association. Taken together, these

data suggest that the modulation of AMPAR expression

during CNS development could drive the functional proper-

ties of chemokine receptors in neurons, and represent a

novel mechanism of cross-talk between the immune and the

central nervous systems.

Acknowledgements

We thank Drs. Adit Ben-Baruch, Francesca Grassi and

Andres Morales for critical reading of the manuscript. This

work was supported by Telethon (grant n. E0912 to F.E.)

and by Ministero dell’Universita e della Ricerca Scientifica

e Tecnologica (to F.E.). We thank Dr. Piotr Bregestovski for

providing HEK-GluR1 cells and Dr. Massimo Locati for

providing HEK-CXCR2, HEK-CXCR1 cells and cDNAs

encoding for CXCR2 and CXCR1.

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