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of June 18, 2017.
Cutting Edge: Association of the Motor
Protein Nonmuscle Myosin Heavy Chain-IIA
with the C Terminus of the Chemokine
Receptor CXCR4 in T Lymphocytes
Mercedes Rey, Miguel Vicente-Manzanares, Fernando
Viedma, María Yáñez-Mó, Ana Urzainqui, Olga Barreiro,
Jesús Vázquez and Francisco Sánchez-Madrid
References
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2002; 169:5410-5414; ;
doi: 10.4049/jimmunol.169.10.5410
http://www.jimmunol.org/content/169/10/5410
The Journal of Immunology
●
Cutting Edge: Association of the Motor Protein
Nonmuscle Myosin Heavy Chain-IIA with the C
Terminus of the Chemokine Receptor CXCR4 in
T Lymphocytes1
Mercedes Rey,* Miguel Vicente-Manzanares,* Fernando Viedma,*
Marı́a Yáñez-Mó,* Ana Urzainqui,* Olga Barreiro,* Jesús Vázquez,†
and Francisco Sánchez-Madrid2*
myosins are heterohexamers composed of a pair of H chains and two
pairs of L chains. Each H chain contains a globular region at the N
terminus that catalyzes ATP hydrolysis and binds to actin, and an
␣-helical C-terminal tail region, responsible for the formation of an
extended parallel-coiled coil and the assembly of bipolar myosin filaments (7, 8). The two isoforms of nonmuscle myosin H chain
(NMMHC)3-II show distinct patterns of intracellular localization and
biological properties (9 –11). However, the specific functional roles of
these NMMHC-II isoforms at a cellular level are not well known.
The aim of this study was to determine the cytoskeletal molecules that interact with the C-termini of chemokine receptors. Using biochemical approaches, we found an association between the
motor protein NMMHC-IIA and the C-termini of CXCR4 and
CCR5. This interaction also occurred in intact cells, and the association was also found for the myosin L chain (MLC). Finally, the
colocalization of NMMHC-IIA, CXCR4, MLC, and filamentous
actin (F-actin) at the leading edge of polarized migrating T lymphocytes suggests that these receptor-motor protein complexes
have a key role in cell chemotaxis.
Materials and Methods
C
hemokines have a very important role in inflammation
and the generation of the immune response. These cytokines bind to G protein-coupled receptors, triggering different signaling cascades, including activation of Gi proteins,
phosphatidylinositol 3-kinase, Janus kinase/STAT proteins, the
mitogen-activated protein kinase pathway, and the Rho-p160
ROCK axis (1–5). However, the intracellular signals regulating
lymphocyte polarization and chemotaxis are still largely unknown.
Nonmuscle myosin II is a motor protein present in all cell types;
there are at least two distinct isoforms, IIA and IIB, which are encoded by genes located in different chromosomes (6). Nonmuscle
*Servicio de Inmunologı́a, Hospital de la Princesa, Universidad Autónoma de Madrid,
and †Centro de Biologı́a Molecular Severo Ochoa, Facultad de Ciencias, Universidad
Autónoma de Madrid, Madrid, Spain
Received for publication July 12, 2002. Accepted for publication September 20, 2002.
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.
1
This work was supported by Grants BMC02-00563 and European Community
QLRT-1999-010-36 (to F.S.-M.).
2
Address correspondence and reprint requests to Dr. Francisco Sánchez-Madrid, Servicio de Inmunologı́a, Hospital de la Princesa, c/Diego de León, 62, E-28006 Madrid,
Spain. E-mail address: [email protected]
Copyright © 2002 by The American Association of Immunologists, Inc.
●
Cells, cell lines, Abs, and reagents
Peer ␥␦ CD3⫹ and Jurkat human T cell lines were grown in RPMI 1640
(Flow Laboratories, Irvine, U.K.) with 10% FCS. PBLs were obtained as
described (2).
The affinity-purified polyclonal anti-NMMHC-IIA was a kind gift of
Drs. R. S. Adelstein and Q. Wei (National Institutes of Health, Bethesda,
MD). Rabbit polyclonal Ab against CD69 and mAb TP1/24 anti-ICAM-3
have been described elsewhere (12, 13). Rabbit polyclonal Ab anti-CXCR4
and mAb MY-21 anti-MLC were from Sigma-Aldrich (St. Louis, MO).
Recombinant human stromal-derived factor-1␣ (SDF-1␣) was purchased
from R&D Systems (Minneapolis, MN).
GST preparation and pull-down assays
GST fusion proteins were prepared by PCR amplification of the C-termini
of the chemokine receptors CXCR4 and CCR5, and subcloning of these
PCR products into EcoRI and XhoI sites in PGEX-4T-2 vector (Amersham
Pharmacia Biotech, Uppsala, Sweden). Primers used were: forward primer,
5⬘-CCGGGAATTGCCAAATTTAAAACC-3⬘, reverse primer, 5⬘-CCGCTC
GAGTTAGCTGGAGTGAAA-3⬘ (for CXCR4); and forward primer, 5⬘CCGGGAATTGAGAAGTTCAGAAAC-3⬘, reverse primer, 5⬘-CCGCTC
GAGTCACAAGCCCACAGA-3⬘ (for CCR5). The ICAM-3 cytoplasmic
region GST-fusion protein, and the GST production method have been previously described (14).
3
Abbreviations used in this paper: NMMHC, nonmuscle myosin H chain; SDF-1␣,
stromal-derived factor-1␣; MLC, myosin L chain; F-actin, filamentous actin; cyt,
cytoplasmic; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight.
0022-1767/02/$02.00
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The binding of chemokines to their receptors guides lymphocyte
migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by
the actomyosin complex of the cell has not been elucidated. Using
biochemical approaches and mass spectrometry analysis, we
found an association between the C-termini of CXCR4 and CCR5
and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As
expected, myosin L chain was also associated with CXCR4.
Confocal microscopy analysis showed that CXCR4 and motor
protein nonmuscle myosin H chain-IIA colocalize at the leading
edge of migrating T lymphocytes, together with filamentous actin
and myosin L chain. These results provide the first evidence of a
biochemical association between chemokine receptors and motor
proteins, a mechanosignaling mechanism that may have a key
role in lymphocyte migration. The Journal of Immunology,
2002, 169: 5410 –5414.
The Journal of Immunology
5411
For pull-down assays, 108 cells were lysed in lysis buffer containing
TBS, 1% Nonidet P-40 (Boehringer Mannheim, Germany), and COMPLETE protease inhibitor mixture (Roche, Mannheim, Germany), and centrifuged at 15,000 ⫻ g for 15 min at 4°C. The supernatant was incubated
twice with GST for 2 h, and then overnight with GST, GST-ICAM-3cytoplasmic (cyt), GST-CXCR4cyt, or GST-CCR5cyt, respectively. Then,
glutathione-coupled Sepharose beads were washed twice with lysis buffer,
once with lysis buffer plus 0.1% SDS, and once with lysis buffer plus 0.5
M NaCl, and resuspended in Laemmli buffer. Samples were separated in
SDS-PAGE gels and processed to mass spectrometry techniques, or transferred to a nitrocellulose membrane for immunoblot analysis with an Ab
against NMMHC-IIA, respectively.
“In gel” digestion of proteins, peptide extraction, and mass
spectrometry analysis
A total of 10% SDS-PAGE gels were stained with Gelcode Blue Stain
Reagent (Pierce, Rockford, IL), and the bands of interest excised and subjected to in situ digestion with trypsin as described (15). A small aliquot
(0.5 ␮l) of the extract was taken up and analyzed by matrix-assisted laser
desorption ionization-time of flight (MALDI-TOF) mass spectrometry, as
described (16).
Immunoprecipitation and Western blot
Normal lymphocytes (2 ⫻ 107) were stimulated with 10 nM SDF-1␣ for
the indicated times under continuous shaking before being washed twice in
cold PBS. Then, cells were lysed in lysis buffer (TBS, 1% Nonidet P-40,
1 mM Cl2 Mg) for 30 min at 4°C and centrifuged (15,000 ⫻ g for 15 min).
Protein extracts precleared by incubation with protein A-Sepharose were
immunoprecipitated with the anti-CXCR4 or anti-CD69 polyclonal Abs
conjugated to protein A-Sepharose (5 ␮g Ab per sample, overnight at 4°C).
Sepharose pellets were washed twice with lysis buffer and three times with
50 mM Tris-HCl, pH 7 (15000 ⫻ g for 1 min at 4°C) and resuspended in
Laemmli buffer.
Samples were separated in a SDS-6% PAGE, transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA), and incubated with Abs to
NMMHC II-A, CXCR4, or MLC at 4°C overnight. Then, membranes were
incubated with a peroxidase-conjugated secondary Ab (Pierce) at room
temperature for 1 h, and proteins were visualized using a SuperSignal West
Pico Luminol/Enhancer solution (Pierce).
Immunofluorescence studies
PBLs (2 ⫻ 106) in 500 ␮l complete medium were allowed to adhere at
37°C, 30 min to coverslips coated with 50 ␮g/ml human fibronectin (Sigma-Aldrich, St. Louis, MO), and then fixed in 2% formaldehyde in PBS for
10 min at room temperature. CXCR4 was visualized with a biotinylated
anti-CXCR4 mAb (BD PharMingen, San Diego, CA) plus a biotinylated
anti-mouse Ab (Amersham Pharmacia Biotech and Molecular Probes, Eugene, OR) and Rhodamine X-labeled streptavidin (Molecular Probes). For
NMMHC-IIA, MLC, and actin staining, cells were permeabilized by incubation for 10 min at room temperature with FACS lysing solution (BD
Biosciences, San Jose, CA) and then appropriate primary and secondary
Abs or Alexa 568-phalloidin (Molecular Probes) were used. Images were
acquired with a Leica TCS-SP (Leica Microsystems, Heidelberg, Germany) confocal microscope.
Results and Discussion
To identify intracellular ligands of CXCR4 and CCR5, GST fusion
proteins containing the C-termini of the chemokine receptors
CXCR4 and CCR5 were generated (Fig. 1A). Pull-down assays
with cell lysates of Peer T lymphocytes (Fig. 1B) revealed the
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FIGURE 1. Identification of NMMHC-IIA as a protein associated to the C termini of CXCR4 and CCR5 by
mass spectrometry analysis. A, GST-fusion proteins including the C termini of chemokine receptors CXCR4
and CCR5 and the adhesion molecule ICAM-3. B, GSTfusion proteins incubated with Peer T cell lysates as
indicated. Specific bands of 230 kDa (spots X and Y)
and 40 kDa (spots A and B) appear in the precipitates
corresponding to chemokine receptors, but not in those
of GST alone or GST-ICAM-3. C and D, Identification
of the 230-kDa bands by mass spectrometry. C,
MALDI-TOF mass-map of peptides obtained after in
gel digestion with trypsin. Peptides whose mass corresponds to a tryptic peptide of NMMHC-IIA are labeled
with a Œ. D, Sequencing of one of the peptides from
NMMHC-IIA by nanospray-ion trap tandem mass espectrometry. The fragment MS/MS spectrum from the
ion specie at m/z 578.0 (M ⫹ 2H⬘), which corresponds
to the peptide at m/z 1155.0 (M ⫹ H⬘) in the MALDITOF spectrum is shown. In the area labeled x5, the scale
was expanded five times relative to the y-axis. The assigned peptide sequence is indicated, detailing the observed backbone fragment ions (34).
5412
CUTTING EDGE: MYOSIN IIA ASSOCIATES WITH CHEMOKINE RECEPTORS
they are supposed to guide F-actin polymerization and contractility. Therefore, we have assessed the subcellular localization of
NMMHC-IIA, CXCR4, MLC, and the actin cytoskeleton in migrating polarized lymphocytes using confocal microscopy. We
found a clear-cut colocalization pattern of NMMHCIIA and Factin (Fig. 3A), and also with CXCR4 (Fig. 3B) at the leading edge
of migrating T lymphocytes. Likewise, NMMHC-IIA and MLC
also colocalized at the advancing front of migrating T lymphocytes
(Fig. 3C). We used ICAM-3 staining as a control of the trailing
edge (uropod) of the cell (12), and we found that NMMHC-IIA
was localized at the opposite pole from that stained by ICAM-3
(Fig. 3D and sideview).
NMMHC-IIA is one of the two different isoforms of the H chain
of type II myosin in nonmuscular cells. Although these two isoforms were described long ago (18), their specific functional roles
remain controversial and poorly understood. It has been postulated
that both proteins are functionally redundant, although with different efficiency given their different features, e.g., their velocity of
movement into the cell (11). Nevertheless, their distinct subcellular localization and different enzymatic kinetics suggest specific
functions for each isoform (19, 20). Interestingly, the localization
of NMMHC-IIA in polarized migrating T cells differs from that
of the isoform IIB, located at the uropod base (12). The latter
isoform has been implicated in cerebellar and heart development
(21–23), and in other processes like growth cone motility (24). In
contrast, the isoform IIA has been implicated in the control of cell
FIGURE 2. Western blot analysis of CXCR4-NMMHC-IIA association. Peer T cell lysates (A) or PBL lysates (B) were pulled-down with GST and
GST-CXCR4Ct and analyzed by Western blot. SDS-PAGE gels (10%) were used in all cases. Coomassie blue staining is shown. C, Jurkat T cells lysed,
immunoprecipitated with Abs against CXCR4 and CD69, and revealed for NMMHC-IIA (a), MLC (b), and CXCR4 (c) by Western blot. D, Cells pulsed
for the different times with 10 nM SDF-1␣. The association between CXCR4 and NMMHC-IIA was assessed as in C.
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presence of protein bands of 40 (spots A and B) and 230 kDa
(spots X and Y) that were specifically associated to CXCR4 and
CCR5. These polypeptides were absent in GST and GST-ICAM3-C terminus precipitates used as controls (Fig. 1B). To identify
the proteins corresponding to the spots X and Y (230 kDa), the
bands were excised, digested with trypsin, and the peptides were
analyzed by mass spectrometry (Fig. 1C). The 230-kDa band was
unequivocally identified as NMMHC-IIA by both peptide mass
fingerprinting (Fig. 1C) and MS/MS analysis of some of the peptides (Fig. 1D). Similarly, bands A and B were identified as actin,
both by fingerprinting and MS/MS analysis and also by Western
blot (data not shown).
To confirm the identity of the 230-kDa protein, Western blot
analysis with an Ab against NMMHC-IIA was performed in Peer
T cells (Fig. 2A) and PBL (Fig. 2B) lysates pulled-down with GSTCXCR4-Ct. This protein was specifically recognized by the antiNMMHC-IIA Ab. Furthermore, immunoprecipitation assays confirmed the association between the endogenous motor protein and
chemokine receptor in T lymphocytes (Fig. 2Ca). Likewise, MLC
also coimmunoprecipitated with the CXCR4-NMMHC-IIA complex (Fig. 2Cb). These associations seemed to be constitutive,
since addition of SDF-1␣, the ligand of CXCR4, did not apparently modify the association of CXCR4 with NMMHC-IIA at any
time points studied (Fig. 2D).
The chemokine receptor CXCR4 and MLC have been shown to
localize at the leading edge of motile lymphocytes (5, 17), where
The Journal of Immunology
5413
motor protein participates in the redistribution of adhesion receptors
toward the immunological synapse (31). Furthermore, previous studies in the amoeba Dictyostelium discoideum have suggested that the H
chain of myosin II is involved in processes such as the tuning of
pseudopod formation and chemotaxis, as a result of the fine regulation
of other processes (32, 33). Finally, our results demonstrating a novel
association between a chemotactic receptor and myosin point to a key
functional role of this mechanotransducing complex in the directional
migration of immune cells.
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In this work, we show an association between the C terminus of
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edge of T lymphocytes. These data provide for the first time evidence of a connection between chemokine signaling machinery
and the contractile forces generated by the cell actomyosin system.
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The involvement of myosin II in cell movement has been assessed previously in different works (28 –30). In addition, this
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FIGURE 3. Colocalization of NMMHC-IIA, CXCR4, MLC, and F-actin at the leading edge of migrating T lymphocytes. Confocal microscopy
images of SDF-1␣-treated PBLs for inducing polarization are shown. Cells
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Alexa 568-phalloidin (A), CXCR4 (B), MLC (C), and ICAM-3 (D). Colocalization histograms are shown at the right side for each case. A
three-dimensional reconstruction is also shown for D.
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