CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase
Activation and Segregation into Membrane Domains Is
Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells1
Yi Le, Marek Honczarenko, Aleksandra M. Glodek, Daniel K. Ho, and Leslie E. Silberstein2
CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the
mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation
of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After
CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is mem-
brane cholesterol dependent and impaired by metabolic inhibitors of Gi, Src family, and the GTPase-activating protein, regulator
of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature
B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion
are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overex-
pression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains
plays an important role in CXCL12/CXCR4-induced cellular responses. The Journal of Immunology, 2005, 174: 2582–2590.
veloping B-lineage cells, bone marrow stromal cells also secrete a
number of factors that are indispensable in B cell development (1).
One of these molecules is the chemokine CXCL12, also known as
stromal cell-derived factor-1. Targeted disruption of the CXCL12
gene is lethal in mice and is accompanied by defects in B cell
lymphopoiesis (2). Our recent studies have shown that although
developing bone marrow B cells maintain high levels of CXCR4
surface expression, their responsiveness to CXCL12 diminishes
with B cell maturation (3, 4). Notably, CXCL12 triggers increased
chemotactic and adhesive responses in progenitor B cells com-
pared with mature B cells in bone marrow. Moreover, the in-
creased responsiveness correlates with prolonged activation of fo-
cal adhesion kinase (FAK),3observed only in progenitor B cells.
However, the mechanisms that regulate CXCL12-induced cellular
responses and the CXCL12/CXCR4-FAK pathway during B cell
development are not clear. We hypothesized that these CXCL12/
CXCR4-mediated responses might be dependent on raft-associated
membrane domains, because these membrane regions play a cen-
tral role in regulating B cell Ag, e.g. immune receptor, responses
(5). Because raft domains are not readily visible by light micros-
cell development is dependent on the nonlymphoid stro-
mal cells found in the bone marrow microenvironment.
In addition to making specific adhesion contacts with de-
copy and are heterogeneous with respect to cholesterol and gly-
cosphingolipid content, a combination of approaches is used to
localize receptors, including CXCR4, and signaling proteins in or
near raft-associated membrane domains, e.g. lipid rafts (6–9). Be-
sides lipid rafts, we hypothesized that intracellular proteins known
as regulators of G protein-signaling (RGS) also might play a role
in the modulation of CXCL12/CXCR4-induced responses in de-
veloping B cells. RGS has been demonstrated to modulate G pro-
tein responses by accelerating the GTPase activity of Gi? proteins
(10). Overexpression of RGS1, RGS3, and RGS13 in transfected
lymphoid cells has an inhibitory effect on CXCL12-mediated che-
motaxis (10–13). Moreover, germinal center B lymphocytes dis-
tinctly express high levels of RGS1 and RGS13, correlating with
their refractoriness to CXCL12-induced chemotaxis (12, 13). The
latter suggests that RGS proteins might developmentally modulate
Gisignaling responses. In the present study we demonstrate that
lipid rafts and RGS1 play key roles in CXCL12/CXCR4-FAK sig-
naling and in CXCL12-induced cellular responses of progenitor B
cells. Moreover, we find that these roles are interrelated, suggest-
ing that CXCL12-induced FAK activation and its recruitment to
lipid rafts are critical parameters of CXCL12-induced chemotactic
and adhesive responses.
Materials and Methods
mAbs against phosphotyrosine (4G10) were provided by Dr. T. Roberts
(Dana-Farber Cancer Institute, Boston, MA). Antisera against FAK,
p130Cas, and Lyn were purchased from Santa Cruz Biotechnology. Anti-
sera against PI3K(p85) were obtained from Upstate Biotechnology. Abs
against MAPK and phospho-MAPK were obtained from New England
Biolabs. HRP-conjugated goat anti-mouse and goat anti-rabbit secondary
Abs were purchased from Caltag Laboratories and Bio-Rad, respectively.
Cholera toxin conjugated to HRP (CTX-HRP) was purchased from Sigma-
FAK (PY397) Abs (BioSource International), Alexa 568-conjugated goat
anti-rabbit IgG, Alexa 488-conjugated goat anti-mouse IgM, and Alexa
488- or Alexa 555-conjugated cholera toxin subunit B (Molecular Probes).
Joint Program in Transfusion Medicine, Children’s Hospital Boston, Harvard Medical
School, Boston, MA 02115
Received for publication July 20, 2004. Accepted for publication December 17, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant POI84800.
2Address correspondence and reprint requests to Dr. Leslie E. Silberstein, Depart-
ment of Pathology, Joint Program in Transfusion Medicine, Harvard Medical School,
Children’s Hospital Boston, Bader 4, 300 Longwood Avenue, Boston, MA 02115.
E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: FAK, focal adhesion kinase; CTX-HRP, cholera
toxin conjugated to HRP; DRM, detergent-resistant membrane; GPCR, G protein-
coupled receptor; MCD, methyl-?-cyclodextrin; PP2, 4-amino-5-(4-chlorophenyl)-7-
(t-butyl)pyrazolo(3,4-d)pyrimidine; PTX, pertussis toxin; RGS, regulator of G protein
signaling; GAP, GTPase-activating protein.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
Cell culture and isolation
Human REH pro-B cells (American Type Culture Collection) were main-
tained in RPMI 1640 supplemented with 10% FBS, 1% penicillin and
streptomycin, and 2 mM glutamine (all from Invitrogen Life Technolo-
gies). Heparinized bone marrow was obtained by iliac crest aspiration from
healthy adult volunteers in accordance with guidelines approved by the
institutional review committees of the Dana-Farber Cancer Institute. Pe-
ripheral blood cells were isolated from the buffy coats of donated whole
blood. Mononuclear cells were isolated by Ficoll-Hypaque (Amersham
Biosciences) gradient centrifugation (density, 1.077 g/ml). Pelleted cells
were collected and washed three times in PBS. Bone marrow cells were
stained and sorted into two populations, designated early lineage and late
lineage B cells, at ?98.5% purity. The early lineage B cell population
included both pro-B and pre-B cell subsets (CD19?, ??/??), whereas the
late lineage B cell population included immature and mature B cells
(CD19?, ??/??) (3, 14). Peripheral B cells were stained and sorted with
allophycocyanin-labeled anti-CD19 (Caltag Laboratories) and were re-
garded as mature B cells. Isolated B cells were stored at 37°C in StemSpan
H2000 serum-free medium (StemCell Technologies) for 16 h before
Gene mutation and transfection
The human RGS1 cDNA, a gift from Dr. J. Kehrl (National Institutes of
Health, Bethesda, MD), was cloned into the GFP-C1 vector (Clontech Lab-
oratories). An alanine substitution at Cys105of RGS1 was generated by
QuikChange site-directed mutagenesis following the manufacturer’s pro-
tocols (BD Clontech). Vectors were amplified in Escherichia coli strain
JM109 and then sequenced for verification. The GFP-RGS1, GFP-
RGS1(C105A), and GFP vectors were linearized by digestion with SalI
(New England Biolabs) and transfected into REH cells by electroporation.
Neomycin-resistant clones were analyzed for GFP expression by FACS.
For analyses, a minimum of 10 positive clones from each stably transfected
cell line were pooled and sorted by GFP expression on a MoFlo cytometer
(DakoCytomation) to establish comparable expression levels (15).
Cholesterol extraction and use of metabolic inhibitors
Cultured cells were resuspended in RPMI 1640 medium lacking FBS, then
stimulated with 100 nM recombinant human CXCL12 (R&D Systems) at
37°C for the indicated times before being stopped by addition of ice-cold
PBS. Pretreatment of cells with inhibitors was performed by incubating
cells in RPMI 1640 supplemented with 100 ng/ml pertussis toxin (PTX;
Invitrogen Life Technologies), 10 ?M 4-amino-5-(4-chlorophenyl)-7-
(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2), or 100 nM wortmannin (all from
Calbiochem) for 30 min at 37°C. To deplete cholesterol from the lipid rafts,
cells were incubated with 10 mM methyl-?-cyclodextrin (MCD; Sigma-
Aldrich) for 30 min at 37°C. After washing with RPMI 1640 medium to
remove pretreatment chemicals, the cells were stimulated with CXCL12 or
10 ng/ml PMA (Sigma-Aldrich). To replenish cholesterol, MCD-treated
cells were incubated in RPMI 1640 medium containing 2 ?g/ml free cho-
lesterol (Sigma-Aldrich) for 30 min at 37°C. Cell viability was not affected
by any of these treatments, as determined by trypan blue exclusion.
After CXCL12 stimulation, cells were washed with ice-cold PBS and lysed
for 30 min at 4°C in buffer A (150 mM NaCl, 50 mM Tris-HCl (pH 7.6),
1 mM PMSF, 10 ?g/ml aprotinin, 10 ?g/ml leupeptin, 1 mM sodium
orthovanadate, and 10 mM sodium fluoride) containing 1% Triton X-100.
Cell lysates were clarified by centrifugation at 13,000 ? g for 10 min at
4°C, incubated with 2 ?g of anti-FAK or anti-p130CasAbs for 1 h, then
incubated with 20 ?l of protein A-Sepharose 4B beads (Amersham Bio-
sciences) for 2 h at 4°C. Immunoprecipitates were washed three times with
wash buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.6), and 0.1% Triton
X-100) to remove nonspecifically bound proteins. Bound proteins were
eluted by boiling in SDS-PAGE loading buffer, then analyzed by Western
blotting. The relative intensities of bands detected by ECL Western Blot-
ting Detection Reagents (Amersham Biosciences) were quantified by den-
sitometry using ImageQuant software (Molecular Dynamics).
Lipid raft isolation and immunoblotting
Lipid rafts were isolated by fractionation on sucrose gradients as previ-
ously described (16). Briefly, REH cells were stimulated by treatment with
100 mM CXCL12 at 37°C for 10 min and stopped by addition of ice-cold
PBS. Cells were lysed in 1 ml of buffer A containing 0.2% Triton X-100.
After 20-min incubation at 4°C, the cell lysates were homogenized with 10
strokes in a Dounce homogenizer (Wheaton) and adjusted to 40% sucrose
by addition of an equal volume of ice-cold 80% (w/v) sucrose prepared in
buffer A. The mixture was transferred to an SW55 ultracentrifuge tube
(Beckman Coulter) and overlayered successively with 2 ml of 30% and 1
ml of 5% (w/v) sucrose, also prepared in buffer A. After centrifugation at
39,000 rpm in a Sorvall AH-650 rotor at 4°C for 16 h, 0.4-ml fractions
were collected from the top of the sucrose gradient designated from 1 (top)
to 12 (bottom). Equal aliquots of each fraction were loaded on SDS-PAGE
and run at constant voltage. In some experiments, fractions 2–4 were
pooled to concentrate the detergent-resistant membrane (DRM) fractions,
and fractions 10–12 were pooled to represent soluble fractions. Proteins
were transferred to nitrocellulose membranes (Bio-Rad) for immunoblot-
ting, and bound HRP-conjugated secondary Abs were detected by ECL.
For GM1 detection, aliquots of each fraction were spotted onto nitrocel-
lulose membranes and blotted with CTX-HRP.
For surface staining of GM1, stimulated or unstimulated cells were washed
with PBS containing 2% BSA, labeled with Alexa-conjugated CTX (10
?g/ml) for 30 min at 4°C, washed with three times PBS, and fixed for 30
min in 1% paraformaldehyde in PBS at 4°C. For intracellular staining of
phospho-FAK(PY397), paraformaldehyde-fixed cells were permeabilized
with 0.1% Triton X-100 in PBS for 1 min at 4°C, washed with PBS con-
taining 2% BSA, and incubated with rabbit anti-FAK(PY397) Ab (1/100
dilution) or rabbit IgG isotype at 4°C for 30 min. Cells were subsequently
washed and incubated with Alexa-conjugated secondary Ab (1/500 dilu-
tion) at 4°C for 1 h. Cells were washed and mounted onto glass slides, and
data were collected from a Zeiss LSM510 confocal laser scanning micro-
scope and analyzed by LSM510 software.
Chemotaxis and cell adhesion assays
Chemotaxis assays were performed using previously described methods
(3). Migratory cells were collected and counted by timed acquisition (60
s/sample) on a FACSCalibur flow cytometer (BD Biosciences). Cell ad-
hesion assays were performed as previously described (4). The number of
bound cells was determined using a CyQuant Cell Proliferation Assay kit
(Molecular Probes). Briefly, cells were stained with CyQuant DNA dye,
and the percentage of bound cells was calculated using a microtiter plate
fluorometer (Dynex Technologies). In some cases 100 ng/ml PMA was
added to the cells instead of CXCL12 before the 30-min incubation in
VCAM-coated wells. For Mn2?-induced adhesion, cells were washed with
2 mM EDTA in a modified adhesion medium (HBSS containing HEPES,
but lacking Ca2?and Mg2?). Cells were subsequently incubated with 2
mM MnCl2and CaCl2in the modified adhesion medium.
Total RNA was purified using TRIzol reagent following the manufacturer’s
protocol (Invitrogen Life Technologies). Purified RNA from early lineage,
late bone marrow, and peripheral B cells was diluted to 1 ?g/?l and treated
with RNase-free DNase I (Invitrogen Life Technologies). RT-PCR was
performed on 1 ng of DNase I-treated RNA using an iCycler real-time
detection system (Bio-Rad) and the SYBR Green RT-PCR kit (Qiagen).
The following primer pairs (Invitrogen Life Technologies) were used to
amplify the RGS1, RGS3, and GAPDH cDNAs, respectively: 5?-AGAAG
GAATGTGCCAGTATG-3? and 5?-TCTGCGCCTGGATAACTTTCA-3?,
5?-GTGAGGAGAATCTGGAGTT-3? and 5?-CCATCTTGGACTGTGA
CTT-3?, and 5?-CAGAAGACTGTGGATGG-3? and 5?-GCTTCACCAC
CTTCTTG-3?. The real-time thermocycler conditions used to amplify
these cDNAs included 30-min incubation at 50°C, followed by 15 min at
95°C and 50 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 30 s. Each
sample was assayed in triplicate, and the number of copies of the gene of
interest in each sample was extrapolated from a corresponding standard
curve and normalized by the amount of GAPDH cDNA amplified from the
same RNA sample. Primer specificity was confirmed by electrophoretic
analysis of the RT-PCR products and by use of template- and reverse-
CXCL12 stimulation recruits FAK and PI3K to lipid raft
One of the principal properties of lipid rafts is their ability to in-
clude or exclude certain signaling molecules. Because FAK and
PI3K are thought to play important roles in chemotaxis and adhe-
sion responses, we investigated their localization with respect to
2583The Journal of Immunology
raft-associated membrane domains in B cells after CXCL12 stim-
ulation. The Lyn kinase served as a raft marker and was predom-
inantly localized in DRM sucrose gradient fractions 2, 3, and 4
(Fig. 1A, bottom panels) (17). FAK was not localized in the DRM
fractions in unstimulated cells, but was recruited to rafts after stim-
ulation with CXCL12 (Fig. 1A, top panels). Because PI3K signal-
ing is associated with CXCL12-induced tyrosine FAK phosphor-
ylation (18), we reprobed the same membrane with anti-PI3K(p85)
Ab and found that substantial amounts of PI3K(p85) were trans-
located to raft fractions in CXCL12-stimulated cells (Fig. 1A, cen-
ter panels). Thus, both FAK and PI3K are recruited into DRM
domains after CXCL12 stimulation.
We also examined whether lipid rafts are important components
of the FAK and PI3K signal transduction pathways by using a
raft-disrupting agent, MCD, which disrupts rafts by cholesterol
extraction from the plasma membrane. FAK, PI3K(p85), and Lyn
were no longer present in lipid raft fractions after CXCL12 stim-
ulation in MCD-treated REH cells (Fig. 1B, left panels). However,
replenishment of MCD-treated cells with cholesterol allowed par-
tial reassociation of FAK, PI3K(p85), and Lyn with lipid rafts after
CXCL12 stimulation (Fig. 1B, right panels). These results sug-
gested that FAK and PI3K(p85) are recruited to cholesterol-en-
riched membrane domains. We were concerned that MCD treat-
ment could induce conformational changes in CXCR4. To this
end, we stained untreated and 10 mM MCD-treated REH cells with
four different commercially available monoclonal anti-CXCR4
Abs (R&D Systems) and found no difference in immunofluores-
cence staining. Moreover, binding of biotinylated CXCL12 to un-
treated vs MCD-treated cells was unchanged. These data (not
shown) thus argue that MCD treatment of REH pro-B cells did not
induce conformational changes in CXCR4.
Recruitment of FAK into lipid rafts is dependent on the Gi
protein and Src family signaling pathways
To further characterize the CXCR4-FAK pathway, we used vari-
ous kinase inhibitors to determine whether any of these might af-
fect recruitment of FAK into lipid rafts. The inhibitors PTX, PP2,
and wortmannin block the Giprotein, Src family, and PI3K signal
transduction pathways, respectively. After CXCL12 stimulation,
FAK was detected in the pooled lipid raft fractions (Fig. 1C, lane
2). In contrast, no FAK localized to the lipid rafts in the absence of
CXCL12 stimulation (Fig. 1C, lane 1). Inhibition of Giprotein sig-
naling by PTX significantly reduced CXCL12-induced recruitment of
FAK into lipid rafts (Fig. 1C, lane 3), indicating that association of
FAK with lipid rafts is dependent on Giprotein signaling.
Pretreatment of cells with PP2, a strong inhibitor of Src family
proteins, also abolished CXCL12-induced recruitment of FAK to
lipid rafts (Fig. 1C, lane 4). In contrast, pretreatment of cells with
wortmannin, an inhibitor of PI3K, did not significantly alter FAK
recruitment (Fig. 1C, lane 2 vs lane 5). Interestingly, only wort-
mannin, not PTX or PP2, blocked the CXCL12-induced recruit-
ment of PI3K(p85) into lipid rafts (Fig. 1C, middle panel), sug-
gesting that recruitment of PI3K(p85) is only dependent on its
kinase activation, not on the Giprotein and Src family pathways.
Lyn, whose recruitment is not affected by these inhibitors, served
as a loading control (Fig. 1C, lower panel).
CXCL12 stimulation induces tyrosine phosphorylation of FAK,
which colocalizes to raft-associated membrane domains
The results presented in Fig. 1 demonstrate that CXCL12 stimu-
lation induces the translocation of FAK into the lipid rafts in a
signaling-dependent manner. We next investigated whether lipid
rafts play a role in FAK phosphorylation. To this end, REH pro-B
cells were treated with or without MCD, then lysed and immuno-
precipitated with anti-FAK Abs, followed by Western blot with an
anti-phosphotyrosine Ab 4G10 to examine total FAK phosphory-
lation. CXCL12 stimulation induced strong tyrosine phosphoryla-
tion of FAK (5.9-fold increase) in REH cells (Fig. 2A, lane 2).
However, tyrosine phosphorylation of FAK was significantly re-
duced in MCD-treated cells (Fig. 2A, lane 3) and was partially
restored after replenishment of MCD-treated cells with cholesterol
(Fig. 2A, lane 4). No change in CXCL12-induced FAK phosphor-
ylation was observed in REH cells that were treated only with
cholesterol (Fig. 2A, lane 5). We also found that PMA induced
tyrosine phosphorylation of FAK in REH cells (Fig. 2A, lane 7).
FAK phosphorylation was abolished in MCD-treated cells (lane 8)
and was restored after replenishment of MCD-treated cells with
cholesterol (lane 9). The blots were subsequently reprobed with
anti-FAK Abs to demonstrate that equivalent amounts of FAK
were loaded onto each lane.
CXCL12 stimulation. A, REH cells were stimulated by treatment with 100
nM CXCL12 at 37°C for 10 min. Cell lysates were prepared and fraction-
ated on sucrose gradients as described in Materials and Methods. Fractions
were resolved by SDS-PAGE, analyzed by Western blot (WB) using anti-
FAK Abs, and reprobed (RP) with anti-PI3K or anti-Lyn Abs. The arrows
indicate the fraction numbers loaded onto each lane. DRM fractions 2, 3,
and 4 are indicated by the short bars. Data shown are from one of four
independent experiments. B, REH cells were pretreated with MCD to dis-
rupt lipid rafts before stimulation with CXCL12. To replenish cholesterol
(MCD?Cho), MCD-treated cells were incubated for 30 min at 37°C in
medium containing 2 ?g/ml free cholesterol, washed, then stimulated with
CXCL12. Cell lysates were prepared, fractionated, and analyzed as de-
scribed above. C, Effects of PTX, PP2, and wortmannin on the CXCL12-
induced recruitment of FAK to lipid rafts. REH cells were incubated with
100 ng/ml PTX, 10 ?M PP2, or 100 nM wortmannin before stimulation
with CXCL12. After sucrose gradient centrifugation, fractions 2, 3, and 4
were pooled and analyzed by Western blotting as described above. The
numbers indicate the relative fold intensities of FAK and PI3K(p85) pro-
tein in the lipid rafts, determined by densitometry. Representative data
from one of three experiments are shown.
Recruitment of FAK and PI3K into lipid rafts after
2584 REGULATION OF CXCL12 SIGNALING BY RGS1 IN LIPID RAFTS
We subsequently used sucrose gradient centrifugation and con-
focal microscopy to examine the cellular localization of phosphor-
ylated FAK by using an Ab specific for the FAK autophosphory-
lation site on tyrosine 397 (PY397). In Fig. 2B, we show that,
similar to total FAK, phosphorylated FAK cosegregates with Lyn
and GM1 to the DRM fractions. For confocal microscopic analysis
(Fig. 2C), REH cells were stained for phosphorylated FAK and for
the lipid raft component GM1, which, like other raft markers, may
not always be associated with cholesterol; they may be resistant to
MCD treatment (8). As reported for other lymphoblastoid cells (6),
we noted that GM1 membrane domains were detectable by fluo-
rescence microscopy in unstimulated REH pro-B cells. In contrast,
primary PBL required stimulation with CXCL12 to allow GM1
visualization (data not shown). In unstimulated REH cells, phos-
pho-FAK(PY397) was not detectable (Fig. 2C). After CXCL12
stimulation (3 min), we noted significant phospho-FAK(PY397)
staining distributed primarily near the cell membrane. The yellow
margin observed after merging the two images indicates that a
significant portion of phospho-FAK(PY397) colocalizes with lipid
raft marker GM1 after CXCL12 stimulation (Fig. 2C). After
CXCL12 stimulation for 10 min, we observed a clustering of lipid
rafts and colocalization of phospho-FAK(PY397) with GM1.
Thus, these fluorescence microscopy studies are in agreement with
our biochemical studies illustrating cosegregation of FAK(PY397)
to DRM fractions after CXCL12 stimulation (Fig. 2B).
Mutation of Cys105reduces the association of RGS1 with
The cellular localization of RGS proteins is diverse. Certain RGS
proteins are tightly membrane bound and behave as hydrophobic
molecules, whereas others are soluble and are found in the cyto-
plasm (19–22). Another subset of RGS proteins displays interme-
diate behavior and is found in both membrane fractions and the
cytoplasm (19). The mechanism by which RGS proteins associate
with lipid membranes is not clear. As with other membrane-asso-
ciated signaling proteins, it has been proposed that palmitoylation
of cysteine residues helps anchor the RGS proteins to lipid bilay-
ers. Moreover, palmitoylation of conserved cysteine residues in the
RGS domain modulates the GTPase-activating protein (GAP) ac-
tivities of RGS4, RGS10, and RGS16 (19, 23, 24). In RGS1, the
conserved cysteine residue in the RGS domain is positioned at
residue 105 (Cys105; Fig. 3A). To determine whether this con-
served cysteine is important for the function and/or membrane lo-
calization of RGS1, we generated a mutant form of RGS1 in which
Cys105was substituted with alanine, RGS1(C105A). We trans-
fected GFP-RGS1 and GFP-RGS1(C105A) expression vectors as
well as a GFP control into REH cells. A minimum of 10 GFP-
positive clones were pooled and sorted for similar levels of GFP
expression. Three representative pooled cell lines containing 95–
97% GFP-positive cells are shown in Fig. 3B. We subsequently
examined the association of RGS1 and lipid rafts by sucrose gra-
dient fractionation, followed by Western blot analysis with anti-GFP
Abs. The majority of GFP-RGS1 fusion protein was detectable in the
DRM fractions, whereas there was very little detected in the soluble
fractions. In contrast, the mutant GFP-RGS1(C105A) protein exhib-
ited significantly reduced association with lipid rafts, and relatively
more mutant protein was present in the soluble fractions (Fig. 3C). In
Fig. 3D, we compared the relative amounts of GFP-RGS1 vs GFP-
RGS1(C105A) in the pooled DRM and pooled soluble fractions. In
this experiment the pooled DRM fractions of each of the transfectants
were run on one gel, and the same was done for the pooled soluble
fractions. In the DRM fractions, with and without CXCL12 stimula-
tion, the relative amount of GFP-RGS1(C105A) was one-third that of
GFP-RGS1. By contrast, the pooled soluble fractions showed almost
twice as much GFP-RGS1(C105A) compared with GFP-RGS1.
Disruption of lipid rafts impairs both CXCL12- and PMA-induced tyrosine
phosphorylation of FAK. Untreated, MCD-treated, or cholesterol-replen-
ished REH cells were stimulated with CXCL12 or PMA. Cell lysates were
immunoprecipitated with anti-FAK Ab, analyzed by Western blot using
anti-phosphotyrosine Ab 4G10 (upper panel), and reprobed with anti-FAK
Ab after stripping (lower panel). The numbers indicate the relative fold in-
crease in the phosphoprotein bands relative to the unstimulated control (set at
1.0). Data shown are from one of three representative experiments. B,
CXCL12-induced phosphor-FAK distributed in both DRM and soluble frac-
tions. REH cells were stimulated by treatment with 100 nM CXCL12 at 37°C
for 10 min. Cell lysates were prepared and fractionated on sucrose gradients as
described in Materials and Methods. Fractions were resolved by SDS-PAGE,
analyzed by Western blot (WB) using anti-phospho-FAK(PY397) Ab, and
reprobed (RP) with an anti-Lyn Ab. GM1 was detected with CTX-HRP. The
arrows indicate the fraction numbers that were loaded onto each lane. DRM
fractions 2, 3, and 4 are indicated by the short bars. C, Colocalization of
FAK(PY397) with the lipid raft marker GM1. REH cells were nonstimulated
or stimulated with 100 nM stromal cell-derived factor-1 for 3 or 10 min at
37°C. Cells were then labeled with Alexa 488-conjugated CTX. Subsequently,
the cells were fixed, permeabilized, and stained with anti-FAK(PY397) Ab,
followed by Alexa 568-conjugated secondary Ab. Cells were then mounted on
glass slides and analyzed by confocal microscopy. Graphs show the fluores-
shown in the merged images (3 and 10 min).
Role of lipid rafts in CXCL12-induced FAK activation. A,
2585The Journal of Immunology
Moreover, CXCL12 stimulation did not alter the distribution of GFP-
RGS1 or the GFP-RGS1(C105A) mutant protein in the sucrose gra-
dient fractions (Fig. 3D).
The cellular localization of GFP-RGS1 and GFP-RGS1(C105)
was similar when investigated by confocal microscopy (Fig. 3E).
GM1 staining is indicated in red, whereas GFP-RGS1 and GFP-
RGS1(C105) staining are indicated in green in Fig. 3. The yellow
margin observed after merging the images indicates that the ma-
jority of GFP-RGS1 colocalizes with GM1. In contrast, mutant
GFP-RGS1(C105A) was localized to a large extent in the cyto-
plasm (Fig. 3E, middle panel). As expected, the GFP control did
not colocalize with GM1 (Fig. 3E, bottom panel). Taken together,
these findings from biochemical and fluorescence microscopy
studies suggested that Cys105is important in mediating the asso-
ciation of RGS1 with lipid rafts.
Overexpression of RGS1 inhibits CXCL12-induced recruitment
of FAK and p130Casinto lipid rafts
We next determined whether RGS1 could influence the recruit-
ment of FAK to lipid rafts after CXCL12 stimulation. After
CXCL12 stimulation, the recruitment of FAK and its downstream
signaling molecule p130Casto lipid rafts was impaired in GFP-
RGS1-transfected cells compared with cells transfected with the
GFP control (Fig. 4A, top panel). In contrast, recruitment of FAK
and p130Casto raft domains was retained in cells expressing GFP-
RGS1(C105A) (Fig. 4A). Taken together, these findings suggested
that the ability of RGS1 to inhibit the recruitment of FAK and
P130Cas was linked to the residence of RGS1 in or near raft-
associated membrane domains. However, we cannot exclude the
possibility that mutation of RGS1 Cys105to alanine could influ-
ence GAP activity independently of RGS1 cellular location.
Interestingly, the recruitment of PI3K was not affected by the
expression of RGS1 (Fig. 4A, third panel). The membrane was
reprobed with anti-Lyn Abs to control for DRM loading (Fig. 4A,
fourth panel). Moreover, levels of FAK, p130Cas, and PI3K were
unchanged in the soluble fractions of CXCL12-stimulated cell
lines transfected with GFP, GFP-RGS1, or GFP-RGS1(C105A)
(Fig. 4A, right panels). Densitometric histograms of relative band
intensities from Western blot experiments of DRM fractions are
illustrated in Fig. 4B.
RGS1 down-regulates CXCL12-induced tyrosine phosphorylation
of FAK and p130Cas
As previously reported for primary pro-B cells (4), CXCL12
quickly induced sustained tyrosine phosphorylation of both FAK
and p130Casin GFP-transfected REH pro-B cells (Fig. 4, C and D),
lasting up to 30 min. Overexpressed GFP-RGS1 markedly im-
paired the initiation and duration of FAK phosphorylation,
whereas GFP-RGS1(C105A) did not have a striking effect on FAK
phosphorylation. Interestingly, GFP-RGS1 similarly affected the
initiation and duration of p130CAS phosphorylation, whereas
GFP-RGS1(C105) had minimal or no effect. Thus, the effects of
RGS1 on FAK and p130Casphosphorylation are also correlated
with the localization of RGS1 in or near raft membrane domains.
CXCL12-induced MAPK activation is dependent on lipid rafts
and is modulated by RGS1
The MAPK pathway is activated by CXCL12 and plays an im-
portant role in cell growth and survival (25). In Fig. 5, A and B, we
found that MCD-treated cells significantly reduced CXCL12-in-
duced ERK and p38 phosphorylation. We also investigated the
kinetics of ERK and p38 activation in GFP-, GFP-RGS1-, and
GFP-RGS1(C105A)-transfected REH cells. In GFP-RGS1-trans-
fected REH cells, CXCL12 stimulation resulted in a significant
form of RGS1 in REH cells. A, Sequence alignment of the ?4 region
(indicated by the box) of the RGS domains of human RGS1, RGS2, RGS3,
and RGS4. The arrow indicates the cysteine residue that was mutated to
alanine in RGS1 for transfection assays. B, Similar levels of gene expres-
sion in transfected REH cells. GFP, GFP-RGS1, or GFP-RGS1(C105A)
expression vectors were transfected into REH cells and sorted by GFP
expression using a MoFlo cytometer as described in Materials and Methods.
The number in each histogram indicates the percentage of GFP-positive cells
in each population. C, The RGS1(C105A) mutant alters the localization of
RGS1 in lipid rafts (determined by sucrose gradient fractionation). Transfected
fractionated on sucrose gradients, and lipid rafts were isolated as described in
Materials and Methods. The association of each GFP fusion protein with lipid
rafts fractions was determined by Western blotting with anti-GFP Abs. Mem-
branes were reprobed with anti-Lyn Abs. D, Comparison of GFP-RGS1 vs
GFP(C105) in DRM and soluble fractions. Cells were either nonstimulated or
stimulated with CXCL12. Cells were prepared and fractionated, and DRM and
soluble fractions were pooled. E, The RGS1(C105A) mutant alters the local-
ization of RGS1 in lipid rafts (determined by confocal microscopy). Trans-
fected cells were fixed on a coverslip as described in Materials and Methods.
After blocking, cells were incubated with Alexa 555-conjugated CTX, then
analyzed by confocal microscopy. Cells in each image field are representative
of at least 100 cells of each transfected cell line.
Overexpression of human RGS1 and the C105A mutant
2586REGULATION OF CXCL12 SIGNALING BY RGS1 IN LIPID RAFTS
reduction in the level of activation of both ERK and p38 compared
with that in GFP-transfected cells (Fig. 5, C and D). The duration
of MAPK activation in GFP-RGS1-transfected cells was also
shorter than that observed in GFP-transfected cells. In contrast,
overexpression of GFP-RGS1(C105A) restored the activation of
both ERK and p38 and extended the duration of MAPK activation
compared with cells expressing GFP-RGS1.
Increased expression of RGS1 during B cell development
RGS proteins deactivate G protein signaling pathways by acceler-
ating the GTPase activity of the G protein ? subunit (26). Because
CXCL12-induced responses are down-regulated during B cell mat-
uration, we examined the expression of RGS1 during B cell de-
velopment. We isolated CD34?CD19?early (pro- and pre-) and
late (immature and mature) B cells from bone marrow. We also
isolated CD19?B cells from peripheral blood. Because insufficient
primary cells could be isolated from bone marrow to detect RGS
protein expression by immunoblot analysis, we performed quan-
titative RT-PCR. RGS1 expression, which was normalized to the
housekeeping gene GAPDH, was low in early lineage B cells, and
was dramatically increased in late B cells (Fig. 6A). Normalized
RGS1 was most highly expressed in peripheral B cells (?60- to
70-fold more than in early bone marrow B cells; Fig. 6B), indi-
cating that increased RGS1 levels correlate with decreased
CXCL12-mediated adhesion to VCAM-1 of mature B cells. In
contrast, there was no difference in the expression of GAPDH-
normalized RGS3 in any of the three populations of B cells we
tested. For comparison, we performed quantitative RT-PCR on the
transfected REH pro-B cells. Both GFP-RGS1 and GFP-RGS1
(C105A) expressed comparable levels of RGS1 as peripheral
blood B cells (Fig. 6, A and C).
CXCL12-induced chemotaxis and adhesion to VCAM-1 are impaired
by membrane cholesterol depletion and overexpression of RGS1
MCD-treated cells had impaired B cell chemotaxis and adhesion to
VCAM-1 after CXCL12 stimulation (Fig. 7, A and B), and replen-
ishing MCD-treated cells with cholesterol restored chemotaxis and
cell adhesion to VCAM-1 in response to CXCL12. Cells treated
only with cholesterol exhibited no difference in adhesion to
VCAM-1 compared with untreated cells (data not shown). In the
presence of EDTA or EGTA, the divalent cation Mn2?can acti-
vate cell adhesion by binding to integrin ectodomains, e.g.,
through outside-in signaling (27). To confirm that MCD itself does
not affect the binding capacity of integrins, we investigated the
effect of Mn2?on cell adhesion to VCAM-1. There was no sta-
tistical difference in Mn2?-induced adhesion between MCD-
treated and untreated cells, indicating that MCD does not alter the
binding capacity of integrins (Fig. 7C). Only background levels of
cell adhesion were observed after treatment of cells with medium.
CXCL12-mediated chemotaxis and adhesion were also impaired
by overexpression of GFP-RGS1 (Fig. 7, D and E). Furthermore,
this reduction was abrogated in GFP-RGS1(C105A)-transfected
cells, which showed equivalent CXCL12-mediated chemotaxis
and adhesion as GFP-transfected cells. Transfection alone did not
alter the adhesion of REH cells, because stimulation of cells with
PMA (which triggers adhesion through G protein-coupled receptor
hesion in all transfected cell lines (Fig. 7F). Interestingly, CXCL12-
induced adhesion was less sensitive to RGS1 action than chemotaxis.
A similar observation was previously noted in experiments using a
murine cell line stimulated with the synthetic peptide FMLP (28). The
importance of RGS proteins in the desensitization of CXCR4 signal-
ing in rgs1?/?mice was discussed in a recent report (29).
stimulated with CXCL12 and fractionated on sucrose gradients as described in Materials and Methods and Fig. 4. Samples were resolved by SDS-PAGE
and analyzed by Western blot (WB) using anti-FAK Abs. Lanes containing pooled DRM fractions 2, 3, and 4 (DRM) or soluble cell components from
fractions 10, 11, and 12 (soluble) are marked. Membranes were subsequently stripped and reprobed (RP) with anti-p130Cas, anti-PI3K(p85), or anti-Lyn
Abs, as indicated. B, Densitometric histograms of relative band intensities from Western blot experiments. The relative fold increases in FAK protein levels
in lipid rafts are shown as the mean ? SD from three experiments. C and D, RGS1 attenuates CXCL12-induced FAK and p130Casactivity. Transfected
cells were stimulated with CXCL12 for the indicated times. Cell lysates were immunoprecipitated (IP) with Abs against FAK (C) or p130Cas(D) as
described in Materials and Methods. Immunoprecipitates were resolved by 6% SDS-PAGE and analyzed by Western blotting (WB) with anti-
phosphotyrosine mAb 4G10 (left panels). Membranes were then stripped and reprobed (RP) with anti-FAK (C) or anti-p130Cas(D) Abs to confirm equal
protein loading (right panels). The numbers indicate relative fold increases in tyrosine phosphorylation, as determined by densitometric analysis of Western
blots. Representative results from one of three independent experiments are shown.
RGS1 inhibits the recruitment of FAK and p130Casto lipid rafts. A, GFP-, GFP-RGS1-, or GFP-RGS1(C105A)-transfected cells were
2587The Journal of Immunology
In the present study we have explored the importance of lipid raft
membrane domains and RGS proteins in the CXCR4-FAK signaling
pathway. We present the novel finding that FAK is recruited to lipid
rafts upon CXCL12 stimulation in REH pro-B cells (Fig. 1A). Addi-
tionally, disruption of lipid rafts by MCD inhibits both FAK recruit-
ment and FAK activation in response to CXCL12 stimulation (Figs.
1B and 2A). We also found that phosphorylated FAK colocalizes with
lipid rafts after CXCL12 stimulation (Figs. 1B and 2C). These obser-
vations suggest a significant role for lipid rafts as a platform for sig-
naling molecules to facilitate CXCL12/CXCR4 signal transduction.
In general, there are two mechanisms by which signaling mol-
ecules can associate with lipid rafts (30). Molecules such as Lyn,
Lck, Fyn, LAT, and the CXCR4-associated signaling units, the G
proteins, may constitutively associate with membrane rafts (31).
Other molecules, such as Zap-70, PKC, PLC?, or Vav, inducibly
associate with lipid rafts in response to receptor cross-linking or
other stimuli. Our results show that FAK inducibly associates with
lipid rafts upon CXCL12 stimulation and that this translocation is
dependent on Giand Src family protein-mediated signaling (Fig.
1). The precise mechanism by which FAK translocation occurs is
not clear. Based on our findings, however, we postulate the fol-
lowing sequence of signaling events. As previously suggested by
other groups, CXCR4-mediated signaling occurs in or near lipid
rafts (6, 32, 33). The initial signaling event involves the association
of the CXCR4 receptor with Gisignaling units, which, in turn,
leads to the activation of several effector pathways (18, 34). We
hypothesize that the activation of Src family proteins, e.g., Lyn,
which constitutively reside in lipid rafts, is a proximal event in
CXCR4 signaling and occurs upstream of FAK (35). The latter is
in agreement with our observation that inhibition of Src proteins
by PP2 abrogates FAK recruitment to lipid rafts after CXCL12
stimulation (Fig. 1C). The phosphorylation of FAK is sensitive to
membrane cholesterol depletion (Fig. 2A) and thus may require the
close association of FAK and activated Src proteins in lipid rafts.
Alternatively, however, after CXCL12 stimulation, FAK may first
be phosphorylated in nonraft domains and subsequently recruited
to the lipid raft signaling complex. To explore this latter possibil-
ity, we stimulated REH pro-B cells while they were in suspension
for 3 min with CXCL12 or with the phorbol ester PMA, which,
unlike CXCL12/CXCR4, does not require lipid rafts for the initi-
ation of signaling. In both instances we found that FAK phosphor-
ylation was inhibited by cholesterol depletion (Fig. 2A), suggesting
that FAK phosphorylation, induced by either CXCL12 or PMA, is
dependent on intact lipid raft domains. Thus, FAK is probably first
recruited to lipid raft domains and subsequently phosphorylated. It
is important to note that the REH pro-B cells were stimulated in
rafts and is modulated by RGS1. REH cells (2 ? 106) with (A) or without
(B) MCD treatment, or transfected cells (C and D) were stimulated with
100 nM CXCL12 for the indicated times. Cells lysates were prepared as
described in Materials and Methods. Samples were analyzed by Western
blotting (WB) with anti-phospho-ERK1/2 (A and C) or with anti-phospho-
p38 Abs (B and D; New England Biolabs). The membranes were then
stripped and reprobed (RP) with anti-ERK1/2 or anti-p38 Abs (New England
Biolabs) to control for protein loading. The numbers represent the relative fold
changes in phosphoprotein intensity as determined by densitometry. Repre-
sentative results from one of three independent experiments are shown.
CXCL12-induced MAPK activation is dependent on lipid
measured by quantitative RT-PCR. RGS1 and RGS3 expressions were de-
termined by RT-PCR of mRNA extracted from bone marrow and periph-
eral B cells as described in Materials and Methods. GAPDH-normalized
mRNA expression levels are indicated as log units (A) or as the fold dif-
ference (B) among B cell populations relative to mRNA expression levels
from early bone marrow B cells. The developmental state of each B cell
population is indicated. Early BM B cells are combined populations of
pro-B and pre-B cells from bone marrow (?50% of total bone marrow B
cells). Late BM B cells are combined populations of immature and mature
B cells from bone marrow. Peripheral B cells are mature B cells isolated
from circulating blood. Representative results from one of three independent
normalized RGS1 mRNA expression levels are indicated in log units.
Increased RGS1 expression during B cell development
2588REGULATION OF CXCL12 SIGNALING BY RGS1 IN LIPID RAFTS
suspension for only 3 min. Under these conditions, integrin en-
gagement is considered to be minimal and thus represent inside-
out, e.g., PMA-FAK or CXCL12/CXCR4-FAK, signaling. In con-
trast, in adherent cells, where integrins are activated through
outside-in signaling, FAK phosphorylation appears independent of
cholesterol-enriched raft domains, suggesting that FAK is not re-
cruited to lipid raft membrane domains (36).
Subsequently, we explored the role of RGS molecules in
CXCL12–FAK signaling and CXCL12-induced cellular re-
sponses. Using REH pro-B cells expressing wild-type (GFP-
RGS1) or mutant RGS1 (C105) proteins, our studies indicate that
GFP-RGS1 protein constitutively associates with raft membrane
domains, suggesting that RGS1 is not translocated to raft mem-
brane domains after ligand engagement of GPCRs. Furthermore,
the association with lipid rafts is affected by mutation of the con-
served cysteine residue at position 105 (Fig. 3, C and E). Palmi-
toylation of additional cysteine residues in the RGS1 protein is
needed to direct RGS1 to the plasma membrane microdomains,
because the alanine substitution at Cys105did not completely abol-
ish the association of mutant RGS1(C105A) with lipid rafts (Fig.
3C). Moreover, RGS1 function, as measured by its ability to im-
pair CXCL12-mediated lipid raft recruitment and activation of
FAK and p130Cas, correlates with RGS1 segregation to lipid raft
domains (Fig. 4). However, we cannot exclude the possibility that
the C105A mutation alters RGS1 GAP activity regardless of its
localization in lipid rafts. Western blot analyses conducted at var-
ious time intervals of CXCL12 stimulation suggest that RGS ac-
tion inhibits the initiation and duration of FAK and p130Cassig-
naling, whereas this effect is attenuated for RGS1(C105A) (Fig. 4,
C and D). Thus, the presence of FAK in lipid rafts correlates with
prolonged activation of FAK and its downstream signaling mole-
cule p130Cas. This finding is intriguing in view of recent data pro-
posing that FAK signaling prevents internalization of raft mem-
brane domains (36–38). Taken together, the current data argue that
both the initiation of CXR4-mediated signaling as well as desen-
sitization by RGS1 occur in or near lipid raft membrane domains.
Interestingly, the recruitment of PI3K(p85) to lipid rafts is not
affected by RGS1 (Fig. 4A) or PTX (Fig. 1C), suggesting that
activation of the p85/110 isoform of PI3K may be due to binding
of an adapter protein to the CXCR4 receptor and is Gi-protein-
independent (39). The CXCL12-induced activation of PI3K (p85)
appears distinct from other major CXCR4-mediated signaling
pathways, including CXCL12-mediated phosphorylation of FAK
(Fig. 1) and MAPK (Fig. 5), both of which depend on proximal
coupling of the CXCR4 receptor to heterotrimeric G proteins and
are desensitized by RGS1 (Figs. 4 and 5). Moreover, FAK recruit-
ment to lipid rafts is not affected by the PI3K inhibitor, wortman-
nin (Fig. 1C), further supporting the idea that CXCR-FAK and
CXCR4-PI3K pathways may be unlinked.
Based on current data and previous studies (4), we propose the
following model to explain the roles of the CXCL12/CXCR4 axis
and RGS1 in B cell development. In the bone marrow, CXCL12,
which is secreted by stromal cells, binds and activates CXCR4 on
Chemotaxis assays were performed as described in A. The data shown are
the mean ? SD from six separate experiments. E, Transfected cells were
subjected to an adhesion assay as described in B. The data shown are the
mean ? SD from five separate experiments. F, Transfected cells were
stimulated with PMA, then incubated in VCAM-1-coated wells for 30 min.
After washing, adherent cells were counted. These data are the mean ? SD
from four separate experiments. Statistically significant data relative to
results from GFP-RGS1-transfected cells were calculated using Student’s t
test: ??, p ? 0.001.
decrease pro-B cell migration and adhesion to VCAM-1. A, Untreated,
MCD-treated, and cholesterol-replenished REH cells were seeded in the
upper chamber of CXCL12-coated Transwells and incubated for 1 h to
measure chemotaxis. Migratory cells were harvested and quantified by
FACS analysis. The extent of cell migration was calculated as a percentage
of total cell input. The data shown are the mean ? SD from six independent
experiments. Statistically significant results relative to MCD-treated cells
were calculated using Student’s t test: ?, p ? 0.005; and ??, p ? 0.001. B,
Untreated, MCD-treated, and cholesterol-replenished REH cells were sub-
jected to a cell adhesion assay as described in Materials and Methods. REH
cells were placed on VCAM-1-coated wells for 28 min and stimulated with
1.0 ?M CXCL12 for 2 min. After washing, adherent cells were counted.
The data shown are the mean ? SD from four or five separate experiments.
Statistically relevant results relative to the MCD-treated cells are indicated
as described above. C, Untreated or MCD-treated cells were incubated with
medium or Mn?and then assayed for cell adhesion as described in B. Data
are the mean ? SD from four independent experiments. D, Transfected
cells were seeded in the upper chamber of CXCL12-coated Transwells.
Disruption of lipid rafts and/or overexpression of RGS1
2589 The Journal of Immunology
progenitor B cells. Activated CXCR4 receptors form clusters in Download full-text
lipid raft domains (6) and subsequently trigger signaling mole-
cules, e.g., Gi protein, Src family proteins, and FAK. Progenitor B
cell surface integrins, such as VLA-4, are activated via inside-out
signaling, bind to VCAM-1, and form close contacts with stromal
cells. This process retains B cells in the bone marrow microenvi-
ronment (40) and promotes their growth and differentiation. Dur-
ing B cell differentiation, however, CXCL12-induced signaling
and cellular responses are down-regulated (4), which may be at-
tributed in part to RGS1 action. In this regard, in primary B cells
from bone marrow and peripheral blood, RGS1 expression is low
in progenitor (pro- and pre-) B cells and highest in mature B cells
(Fig. 6). Moreover, overexpression of RGS1 in progenitor pro-B
cells (which have little endogenous RGS1) impairs CXCL12-in-
duced FAK activation, chemotaxis, and adhesion to VCAM-1 (Fig.
7). The relatively increased RGS1 expression in mature B cells
correlates with decreased CXCL12-induced adhesion to VCAM-1
(4), a process that might contribute to the release of mature B cells
from the bone marrow into the peripheral circulation. Besides the
action of RGS proteins, cells potentially use other mechanisms to
desensitize signaling via GPCRs, including G protein degradation,
GPCR sequestration, and phosphatases targeting phosphorylated
signaling proteins (41–43). Collectively, these findings argue that
in the bone marrow, RGS1 may developmentally regulate
CXCL12-mediated responses, which play a critical role in progen-
itor B cell positioning and maturation.
We gratefully acknowledge the technical assistance of Harry Leung and
Tao Lu. We thank Dr. James J. Campbell for his helpful discussions.
The authors have no financial conflict of interest.
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2590REGULATION OF CXCL12 SIGNALING BY RGS1 IN LIPID RAFTS