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Vol. 12, Issue 10, 3214-3225, October 2001
9 Subunit
Requires the Adaptor Protein Paxillin to Inhibit Cell Spreading but
Promotes Cell Migration in a Paxillin-independent Manner




and
*Lung Biology Center, Department of Medicine, University of
California, San Francisco, San Francisco, California 94110;
Department of Vascular Biology, Scripps Research
Institute, La Jolla, California 92037; §Department of
Internal Medicine, Department of Laboratory Medicine, National
Hiroshima Hospital, 513 Jike, Saijoh, Higashi-Hiroshima, 739; and
Cancer Biology Program, Division of Hematology-Oncology,
Department of Medicine, Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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The integrin
9 subunit forms a single heterodimer,
9
1. The
9 subunit is most closely related to the
4 subunit,
and like
4 integrins,
9
1 plays an important role in
leukocyte migration. The
4 cytoplasmic domain preferentially
enhances cell migration and inhibits cell spreading, effects that
depend on interaction with the adaptor protein, paxillin. To determine
whether the
9 cytoplasmic domain has similar effects, a series of
chimeric and deleted
9 constructs were expressed in Chinese hamster
ovary cells and tested for their effects on migration and spreading on
an
9
1-specific ligand. Like
4, the
9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also
specifically bound the
9 cytoplasmic domain and to a similar level
as
4. In paxillin
/
cells,
9 failed
to inhibit cell spreading as expected but surprisingly still enhanced
cell migration. Further, mutations that abolished the
9-paxillin
interaction prevented
9 from inhibiting cell spreading but had no
effect on
9-dependent cell migration. These findings suggest that
the mechanisms by which the cytoplasmic domains of integrin
subunits enhance migration and inhibit cell spreading are distinct and
that the
9 and
4 cytoplasmic domains, despite sequence and
functional similarities, enhance cell migration by different
intracellular signaling pathways.
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INTRODUCTION |
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Integrins are a family of
transmembrane receptors composed of at least 25 different 
heterodimers that mediate both cell-substrate and cell-cell adhesion
(Hynes, 1992
). Among their other functions, integrins play a
central role in cell migration. Integrin-dependent migration is
important in many biologic processes including embryonic development,
wound healing, inflammation, and tumor metastasis.
Cell migration is a complex and poorly understood process that involves
both actin reorganization and integrin-dependent focal adhesion
remodeling. For cells to migrate on a substrate, they must adhere and
de-adhere in a coordinated manner and generate tensile force in the
direction of migration. The force required to promote migration is
generated by the actin cytoskeleton and integrin-dependent
protein complexes that anchor actin to specific sites on the cell
membrane (Lauffenburger and Horwitz, 1996
). The actin reorganization
required to promote cell polarization and directional migration is
regulated by specific intracellular signaling pathways (Lauffenburger
and Horwitz, 1996
; Horwitz and Parsons, 1999
). Although most members of
the integrin family are capable of mediating cell migration
(Lauffenburger and Horwitz, 1996
), experiments utilizing chimeric or
truncated integrins indicate that the cytoplasmic domain of the
4 subunit preferentially enhances cell migration and inhibits cell
spreading compared with other
subunits of the
1 subclass of
integrins (Chan et al., 1992
; Kassner et
al., 1995
). In addition, unlike most integrins,
4
1 is relatively excluded from mature focal adhesion complexes (Kassner et al., 1995
). These findings support the hypothesis that
4-containing integrins, which are widely expressed on
leukocytes, play a specialized role in promoting rapid cell migration.
Further, these findings support a model that links inhibition of cell
spreading (i.e., cell rounding) to enhanced migration. Recently, the
integrin
9
1, an integrin that is structurally
most closely related to
4
1, was shown to promote transendothelial
neutrophil migration through its interactions with vascular cell
adhesion molecule-1 on activated endothelial cells (Taooka et
al., 1999
). The cytoplasmic domain of the
9 subunit shares 52%
homology with the
4 subunit but is divergent from all other
integrin-cytoplasmic domains. We therefore questioned whether
the
9 cytoplasmic domain would also specifically enhance cell
migration and inhibit cell spreading.
Recently,
4
1-dependent cell migration was shown to be dependent
on the specific interaction of the
4 cytoplasmic domain with the
adapter protein paxillin (Liu et al., 1999
). Paxillin participates in several intracellular signaling pathways that influence
cell migration (Clark and Brugge, 1995
; Turner, 2000
). Site-directed
mutagenesis of a tyrosine (Y) residue to an alanine (A) residue at
position 991 (Y991A) in the
4 cytoplasmic domain that inhibited the
4-paxillin interaction also inhibited
4
1-dependent migration
and cell shape changes (Liu et al., 1999
; Liu and Ginsberg, 2000
). In the current study, we utilized a series of chimeric and
truncated versions of the
9 subunit to determine whether the
9
cytoplasmic domain preferentially enhances migration and inhibits cell
spreading on an
9
1-specific ligand and what role, if any,
paxillin plays in these processes.
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MATERIALS AND METHODS |
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Reagents and Antibodies
The
9
1-specific ligand used in this study was a
recombinant form of the third fibronectin type III repeat of chicken
tenascin-C (Prieto et al., 1993
) containing alanine (A)
substitutions for both glycine (G) and aspartate (D) residues within
the arginine (R)-G-D site (TNfn3RAA; Yokosaki et al., 1998
).
The cDNA for TNfn3RAA was obtained from Anita Prieto and Kathryn
Crossin (Scripps Research Institute, La Jolla, CA) and was prepared in
Escherichia coli as previously described (Prieto et
al., 1993
). The mouse mAb, Y9A2, increased against human
9
1,
was prepared as previously described (Wang et al., 1996
).
The following monoclonal antibodies were purchased commercially:
monoclonal antibodies against paxillin (clone 349, BD-Biosciences-Transduction Laboratories, Lexington, KY) and against
hemagglutinin (HA)-tag (12CA5, American Type Culture Collection
[ATCC], Rockville, MD).
Generation of
9 Constructs
The previously described pBlueScript (BS)-SK
9 cDNA plasmid
was used as the template to generate all
9 constructs (Yokosaki et al., 1994
). To generate the
9 chimeras containing the
cytoplasmic domains of
2,
5, and
4, a mutation in the
9
sequence was generated at amino acid position 972 in the transmembrane
domain near the start of cytoplasmic domain that changed a leucine
residue to a valine residue and created an SpeI restriction
site. The mutation was created by polymerase chain reaction (PCR) with
the use of a 5' forward primer that was upstream of an EcoRI
site in the extracellular domain, 5'-tttcctttcatgaggtca-3' and a 3'
reverse primer that created the SpeI restriction site for
subcloning into the multiple-cloning site of pBS-SK
9, 5'-ttct
ta ctag tacggccagcagcaggaagat-3'. The
nucleotides in bold type represent the sites of mutagenesis. The PCR
product was digested with EcoRI and SpeI,
purified, and subcloned into pBS-SK
9. The
2 and
5 cDNA used in
the PCR reactions to generate the cytoplasmic domains was from a
teratocarinoma-2 cell line (ATCC). To generate the
9
2 chimera, a
5' forward primer that was specific for the cytoplasmic domain of
2
and contained an SpeI restriction site, 5'-ggcttactagtctggaagctcggcttcttc-3', and a 3' reverse primer specific
for the cytoplasmic domain of
2 that contained a NotI restriction site for subcloning into the multiple-cloning site of
pBS-SK
9, 5'-atcttgcggccgcaagaaatccatgcacgcaaa-3', were used. The PCR
product was digested with SpeI and NotI,
purified, and subcloned into pBS-SK
9. To generate the
9
5
chimera, a 5' forward primer that was specific for the cytoplasmic
domain of
5 and contained an SpeI restriction site,
5'-ttcttactagtctggaaacttggattcttcaaacgc-3', and a 3' reverse primer
specific for the cytoplasmic domain of
5 that contained an
XbaI restriction site for subcloning into the
multiple-cloning site of pBS-SK
9,
5'-atctttctagagtggggggactggttcttca-3', were used. To generate the
9
4 chimera, the
4 expression plasmid, pc
4DM8 (generously
provided by Dr. David Erle), was used as a template, and a 5' forward
primer that was specific for the cytoplasmic domain of
4 and
contained an SpeI site,
5'-gctccactagtctggaaggctggcttcttt-3', and a 3' reverse primer specific
for the cytoplasmic domain of
4 that contained a NotI
site,
5'-ctgctgctgctggcggccgcggtaccttattaatcatcatt-gcttttac-3', were used. To generate the
9 cytoplasmic deletion mutants (
9DMs), the pBS-SK
9 was used as the template in PCR reactions that used the
5' forward primer, 5'-tttcctttcatgaggtca-3', for all of the
9DMs
(Figure 1), and the 3' reverse primers
specific for the
9 cytoplasmic domain that contained a
NotI restriction site, 5'-ttcttgcggccgcttacatcttccagagcagcacggc-3',
5'-ttcttgcggccgcttatcggcgaaagaagcccatctt-3', 5'-ctgctgctgctggcggccgctctagattattattctttgtacc-ttcggcg-3',
5'-ctgctgctgctggcggccgctctagattattacttctcagcttcgataat-3', 5'-ctgctgctgctggcggccgctctagattattattcattctctttccggtt-3',
5'-ctgctgctgctggcggccgctctagattattactggacccagtcccaact-3', for
9DM1-
9DM6, respectively. All reverse primers were designed to
introduce two translation stop codons in tandem at the end of the
coding sequence before the restriction sites, and all constructs were
confirmed by nucleotide sequencing. The
9 site-directed mutants
containing a tryptophan (W) to A substitution at either position 999 (W999A) or 1001 (W1001A) in the
9 subunit were generated from
pBS-SK
9 with the use of a QuikChange Site-Directed
Mutagenesis kit (Stratagene, La Jolla, CA) according to the
manufacturer's protocol. The 5' forward and 3' reverse primers used to
generate
9(W999A) were 5'-ggaaagagaatgaagacagt
gcggactgggtccagaaaaacc-3' and 5'-ggtttttctggac
ccagtccgcactgtcttcattctctttcc-3', and the primers used to
generate
9(W1001A) were 5'-ggaaagagaatgaagacagttgggac gcggtccagaaaaacc-3' and 5'-ggtttttctggaccgcgtc
ccaactgtcttcattctctttcc-3'. The nucleotides in bold type
represent the sites of mutagenesis. All constructs were confirmed by
nucleotide sequencing. All
9 constructs were subcloned into the
previously described full-length
9 expression plasmid pcDNAIneo
9
(Yokosaki et al., 1994
) after excision of the pBS-SK
9
constructs with HindIII and NotI and subcloning
into pcDNAIneo
9. For subcloning into pBABEpuro, the
9 constructs
were excised from pBS-SK
9.
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Generation of Stable Cell Lines
The CHO cells lines were generated by calcium phosphate
precipitation with vectors made in pcDNAIneo (Invitrogen, San Diego, CA.) and were maintained in Dulbecco's minimal essential medium (DMEM)
supplemented with 10% fetal calf serum (FCS) and the neomycin analogue
G418 (1 mg/ml; Life Technologies, Rockville, MD). Transfected cells
were analyzed for expression of
9
1 integrins by flow
cytometry with the anti-
9
1 antibody, Y9A2. Mouse embryonic
fibroblasts (MEF; from ATCC) and MEF
paxillin
/
cell lines were infected with
the use of
9 constructs in the retroviral vector pBABEpuro
(Morgenstern and Land, 1990
). Retroviruses were generated by calcium
phosphate-mediated transfection into the Phoenix-E
replication-incompetent ecotropic virus packaging cell line (Kinoshita
et al., 1998
; Swift et al., 1999
).
Specifically, 8 µg of plasmid DNA were added to 70% confluent
Phoenix-E cells growing in 60-mm tissue culture dishes at 37°C in 3 ml of 10% FCS DMEM for 16 h. The medium was removed, 3 ml of
fresh 10% FCS DMEM were added, and the cells were cultured for 16 h. Virus-containing supernatants were harvested and filtered through a
0.22-µm filter and then added to 50% confluent cultures in the
presence of 5 µg/ml polybrene and cultured for 18-20 h. The
virus-containing medium was removed and the cells were cultured in 10%
FCS DMEM supplemented with 10 µg/ml puromycin (Sigma, St. Louis, MO).
MEF and paxillin
/
MEF cells expressing
the
9
1 constructs were identified by flow cytometry with the
anti-
9
1 antibody, Y9A2. Fluorescence-activated cell sorting was
performed to isolate heterogeneous populations of cells expressing high
levels of
9
1 integrins on their cell surfaces (Yokosaki
et al., 1998
). All cell lines continuously expressed high
surface levels of
9
1 as determined by flow cytometry with Y9A2.
Flow Cytometry
Cultured cells were harvested by trypsinization and rinsed with phosphate-buffered saline (PBS). Nonspecific binding was blocked with normal goat serum at 4°C for 10 min. Cells were then incubated with primary antibody for 20 min at 4°C, followed by a secondary goat anti-mouse antibody conjugated with phycoerythrin (Chemicon, Temecula, CA). Between incubations cells were washed twice with PBS. The stained cells were resuspended in 100 µl of PBS, and fluorescence was quantified on 5000 cells with a FACScan (Becton Dickinson, Rutherford, NJ) flow cytometer.
Cell Adhesion Assays
The wells of nontissue culture 96-well microtiter plates (Nunc, Naperville, IL) were coated by incubation with 100 µl of TNfn3RAA for 1 h at 37°C. After incubation, wells were washed with PBS and then blocked with 1% bovine serum albumin (BSA) in DMEM at 37°C for 30 min. Control wells were filled with 1% BSA in DMEM. The cells were detached with the use of 2.5 ml of trypsin solution (Sigma), followed by 2.5 ml of trypsin-neutralizing solution (Sigma), washed once in DMEM, and resuspended in DMEM at 5 × 105 cells/ml. The cells were incubated with or without 50 µg/ml Y9A2 for 20 min at 4°C before plating. Plates were centrifuged (top side up) at 10 × g for 5 min before starting the incubation for 1 h at 37°C in humidified 5% CO2. Nonadherent cells were then removed by centrifugation (top side down) at 48 × g for 5 min. Attached cells were fixed and stained in 40 µl of a 1% formaldehyde, 0.5% crystal violet, 20% methanol solution for 30 min, after which the wells were washed three times with PBS. The relative number of cells in each well was evaluated after solubilization in 40 µl of 2% Triton X-100 by measuring the absorbance at 595 nm in a microplate reader (Bio-Rad, San Francisco, CA). All determinations were carried out in triplicate, and the data represent the means ± SEM for a minimum of three experiments.
Cell Migration Assays
For chemotactic migration assays, 24-well Transwell plates
(Costar, Cambridge, MA) were used. The lower side of the Transwell filters (6.5-mm diameter, pore size 8.0 µm) were coated with TNfn3RAA dissolved in 250 µl of DMEM for 60 min at 37°C. After incubation with TNfn3RAA, filters were washed by adding 100 µl of PBS to the top
well and 500 µl of PBS to the bottom well. After washing twice,
filters were blocked with 1% BSA in DMEM for 30 min and again washed
once in PBS. Cells were detached as described above and resuspended in
DMEM at 5 × 105 cells/ml. Migration and
adhesion assays were performed at the same time, and the cells from the
same dishes were used for both assays. Cells were incubated for 20 min
on ice with or without the anti-
9
1 antibody, Y9A2 (50 µg/ml),
and then 100 µl were loaded (50,000 cells/chamber) in each chamber.
Each chamber was inserted into a well containing 600 µl of DMEM
supplemented with 1% FCS to serve as a chemoattractant and incubated
at 37°C in humidified 5% CO2 for 2 h for
the CHO cells or 3 h for the MEF cells. Medium was then aspirated
and the filters washed once with PBS. Cells on the bottom of the
filters were fixed for 20 min in 500 µl of DifQuik fixative (Fisher,
Springfield, NJ), and the nonmigrated cells on the top of the filter
were gently removed with a Q-tip. Filters were allowed to completely
dry, stained by DifQuik, washed in running distilled
H20 and allowed to destain in distilled
H2O for 1 h. Filters were air-dried (
3 h),
removed from the chamber with a scalpel, and mounted onto glass slides with the use of a Permamount/xylene solution, and the migrated cells
were counted. Migrated cells were counted under a 25× objective with
the use of a gridded eyepiece (reticule). Ten high-powered fields (HPF)
per slide were counted, the average was taken, and the number of
migrated cells was expressed as migrated cells per 10 HPF. The data
represent means ± SEM from a minimum of three experiments
Cell-spreading Assays
Glass coverslips (12-mm circle, Fisher) sterilized in 100% ethyl alcohol were placed into the wells of 24-well plates, washed twice with 500 µl of PBS, and coated with TNfn3RAA in 400 µl of serum-free DMEM for 60 min at 37°C. After incubation with TNfn3RAA, coverslips were washed twice with 500 µl of PBS and then blocked with 1% BSA in DMEM for 30 min at 37°C. Cells were detached (as described above) and resuspended in DMEM at 5 × 105 cells/ml with 100 µl (50,000 cells), loaded onto coated coverslips, and incubated for either 3 h (MEF cells) or 6 h (CHO cells) at 37°C in humidified 5% CO2. Medium was then removed, and the cells were washed in PBS once and fixed in 2% (freshly made) paraformaldehyde for 20 min at room temperature. Coverslips were mounted and analyzed under a 25× objective with the use of a reticule. Ten HPF per slide were observed, and the number of spread cells and the number of total cells were counted. Time-course experiments were first performed to determine the time required to display the greatest difference in cell spreading for each cell type studied. The difference in the rate of cell spreading for a given time was expressed as the number of spread cells/number of total cells in 10 HPF × 100. Data represent the mean percentages of spread cells ± SEM for a minimum of three experiments.
Coimmunoprecipitation and Western Blot Analysis
CHO cell lines expressing different chimeric
9
1
integrins were surface labeled with
sulfo-N-hydroxysuccinimide-biotin (Pierce, Rockford, IL)
according to the manufacturer's protocol. Cells were then lysed on ice
for 30 min in an immunoprecipitation buffer: 20 mM Tris-HCl (pH 7.4),
150 mM NaCl, 10 mM EDTA, 10 mM benzamidine HCl, 0.02% sodium azide,
1% Triton X-100, 0.05% Tween 20, 2 mM phenylmethylsulfonyl fluoride,
5 µg/ml aprotinin, and 5 µg/ml leupeptin, as previously described
(Liu et al., 1999
). Briefly, cell lysates were clarified by
centrifuging at 16,000 × g for 20 min at 4°C and
then incubated with protein G-Sepharose coated with the anti-
9
1
antibody, Y9A2, or an irrelevant mouse immunoglobulin (Ig) G at 4°C
overnight. The beads were washed with the same buffer four times, and
precipitated polypeptides were extracted with SDS sample buffer.
Precipitated cell surface biotin-labeled polypeptides were separated by
SDS-PAGE under nonreducing conditions and detected with streptavidin
peroxidase followed by ECL (Amersham Pharmacia Biotech, Piscataway,
NJ). In parallel, lysates of unmodified cells were precipitated with
the anti-
9
1 antibody, Y9A2, and coimmunoprecipitated paxillin was
detected with biotin-labeled anti-paxillin antibodies (BD-Biosciences-Transduction Laboratories) as previously described (Liu
and Ginsberg, 2000
).
Integrin Cytoplasmic Domain Model Proteins and Affinity Chromatography
The design and recombinant production of cytoplasmic domain
model proteins was performed as previously described (Liu et
al., 1999
; Liu and Ginsberg, 2000
). Briefly, PCR was used to
generate a HindIII-BamHI fragment for each
wild-type or mutant integrin cytoplasmic domain, and this
fragment was subcloned into the modified pET15b vector as previously
described (Liu and Ginsberg, 2000
). Recombinant proteins were expressed
in BL21 (DE3) pLysS cells (Novagen, Madison, WI), isolated by
Ni2+-charged resins, and further purified to
>90% homogeneity with the use of a reverse phase
C18 high-performance liquid chromatography column
(Vydac, Hesperia, CA). Masses of all proteins were assessed by
electrospray ionization mass spectrometry on an API-III quadrupole spectrometer (Sciex, Toronto, Canada) and varied by <0.1% from the
predicted masses. Recombinant integrin cytoplasmic tails were bound to Ni2+-charged His-Bind resins (Novagen)
and used for affinity chromatography as previously described (Liu and
Ginsberg, 2000
). Briefly, 1 mg of each recombinant integrin
tail dissolved in 5 ml of 20 mM 1,4-piperazinediethanesulfonic acid, 50 mM NaCl, pH 6.8 (PN buffer), plus 1 ml of 100 mM sodium acetate (pH
3.5) was bound to 100 µl of Ni2+-charged
His-Bind resins (Novagen) at 4°C overnight. Resins were then washed
with PN buffer twice and stored in an equal volume of PN buffer plus
0.1% sodium azide. The expression and isolation of recombinant human
paxillin was performed as previously described (Liu et al.,
1999
; Liu and Ginsberg, 2000
). Aliquots of recombinant HA-tagged
glutathione S-transferase (GST)-paxillin were mixed with 300 µl of buffer A plus 20 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 0.1% Triton X-100, 3 mM
MgCl2, and 1 mg/ml BSA, added to integrin
tail-loaded resins, and incubated at room temperature with rotation for
2 h (Liu et al., 1999
; Liu and Ginsberg, 2000
). Resins
were washed three times with the same buffer, and bound proteins were
extracted with SDS sample buffer, separated on SDS-PAGE, and detected
with antibody specific for HA-tag.
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RESULTS |
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The
9 Cytoplasmic Domain Specifically Enhances Cell Migration
To determine whether the cytoplasmic domain of the
9 subunit
specifically enhances cell migration, chimeric
subunits composed of
the extracellular and transmembrane domain of the
9 subunit fused to
the cytoplasmic domains of
4,
2, or
5 were constructed (Figure
1A). A comparison of the cytoplasmic sequences of
9,
4,
2, and
5 reveals that all 4
subunits contain a similar membrane-proximal
region but that otherwise only the
9 and
4 sequences are similar.
Overall, the
9 cytoplasmic domain is 52% homologous to the
4
cytoplasmic domain.
The full-length
9 subunit, the
9
4,
9
2, and
9
5
chimeras, and vector alone were stably expressed in CHO cells that do not express endogenous
9 and were examined for their ability to
promote migration on the
9
1-specific ligand, TNfn3RAA. To determine whether the
9 constructs were expressed on the cell surface as
9
1 integrins and to sort cells expressing high
levels of the
9
1 integrins, flow cytometry was performed
with the anti-
9
1 antibody, Y9A2 (Figure
2A). All stably transfected cells used in
subsequent adhesion and migration assays expressed similar high surface
levels of
9
1 integrins. Cell adhesion assays performed on
TNfn3RAA (Figure 2, B and D) demonstrated that each chimera supports
equivalent cell adhesion at both 3 µg/ml (Figure 2B) and 10 µg/ml
(Figure 2D), and in each case adhesion was blocked by the anti-
9
1
antibody, Y9A2. To determine whether the
9 cytoplasmic domain
preferentially enhances migration compared with the
2 and
5
cytoplasmic domains, as had been previously demonstrated for the
4
cytoplasmic domain, chemotactic migration assays were performed on
filters coated with either 3 µg/ml (Figure 2C) or 10 µg/ml (Figure
2E) of TNfn3RAA. As expected, wild-type
9 and each of the chimeras
tested supported greater migration on TNfn3RAA than that seen in
mock-transfected cells. However, both the
9 and the
4 cytoplasmic
domains caused similar enhancement of cell migration compared with the
cytoplasmic domains of
2 and
5. Migration of all
9-expressing
cells was also inhibited by the anti-
9
1 antibody, Y9A2,
demonstrating that the enhanced migration was specific to the
9
1
integrins. These results indicate that the
9 cytoplasmic
domain preferentially enhances cell migration and to the same level as
the
4 cytoplasmic domain.
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The Membrane-proximal 17 Amino Acids of the
9
Cytoplasmic Domain Are Sufficient to Mediate Enhanced Cell Migration
To identify the cytoplasmic sequences critical for mediating
9-dependent enhancement of cell migration, a series of
9DMs (Figure 1B) was stably expressed in CHO cells and examined for their
ability to mediate
9
1-dependent adhesion and migration. All of
the
9DMs were stably expressed on the cell surface at similarly high
levels (Figure 3A). The cells expressing
9DM3-
9DM6 all bound to TNfn3RAA-coated plates at similar levels
and to the same level as cells expressing full-length
9 (Figure 3, B
and D). The most severe truncations,
9DM1 and
9DM2, impaired
9
1-mediated adhesion, especially to 10 µg/ml TNfn3RAA. As
expected from their lack of adhesion,
9DM1 and
9DM2 were unable
to mediate
9
1-dependent migration to the same level as the
full-length
9 subunit (Figure 3, C and E). In contrast, the
9DM4-
9DM6 all mediated migration comparable to full-length
9.
However,
9DM3 was unable to mediate
9
1-dependent migration,
although it mediated adhesion to TNfn3RAA to the same level as the
9DM4-
9DM6. These results indicate that
9-mediated enhancement
of cell migration requires the 17 amino acids retained in the
9DM4
construct and is particularly sensitive to the loss of amino acids
within the sequence IIEAEK that is present in
9DM4 but absent in
9DM3.
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The
9 Cytoplasmic Domain Inhibits Cell Spreading
The cytoplasmic domain of
4 has previously been demonstrated to
inhibit cell spreading compared with other
subunits of the
1
subclass of integrins (Kassner et al., 1995
). To
determine whether the
9 cytoplasmic domain could also inhibit cell
spreading, spreading assays were performed with cells expressing each
of the constructs described above on coverslips coated with 10 µg/ml TNfn3AA (Figure 4). After 6 h, the
greatest difference in cell spreading was evident with cells expressing
the full-length
9
1 and the
9
4 chimera being less spread
(~10% spread) than cells expressing either the
9
2 or
9
5
chimeras (~35% spread). Similarly, cells expressing the
9DM3,
which mediates adhesion but not migration on TNfn3RAA, were spread to
the same level as cells expressing the
9
2 and
9
5 chimeras
that do not mediate enhanced migration. The
9DM4-
9DM6 that
mediate enhanced migration also inhibited cell spreading to similar
levels as that of
9
1- and
9
4
1-expressing cells. These
results indicate that the membrane-proximal 17 amino acids of the
9
cytoplasmic domain are sufficient to mediate both enhanced migration
and impaired spreading and that the
9 and
4 cytoplasmic domains
share both functional properties.
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The
9 Cytoplasmic Domain Associates with the Focal Adhesion
Adapter Protein, Paxillin
Recently, the
4 cytoplasmic domain was demonstrated to
associate with and directly bind to the adapter protein, paxillin, and
this interaction was shown to be critical for
4-dependent enhanced
migration and impaired cell spreading (Liu et al., 1999
). Because the
9 cytoplasmic domain is highly homologous to the
4
cytoplasmic domain (52% homology), we predicted that the
9 cytoplasmic domain would also associate with paxillin. Indeed, bacterially expressed GST-paxillin directly and specifically bound to
the recombinant
9 cytoplasmic domain immobilized on a
Ni2+ resin-charged column but not the
IIb
cytoplasmic domain in vitro (Figure 5A).
To determine whether
9 binds paxillin with a similar affinity as
4, recombinant protein-binding assays were again performed. Both the
9 and
4 cytoplasmic domains specifically bound paxillin in a
concentration-dependent manner with very similar binding affinities,
suggesting that the strength of the
9-paxillin and
4-paxillin
interactions is quite similar (Figure 5B). To determine whether
paxillin associates with
9 in vivo and to the same level as
4,
cell lysates from CHO cells expressing
9
1,
9
4
1, and
9
2
1 were immunoprecipitated with the anti-
9
1 antibody,
Y9A2. The precipitates were resolved with the use of SDS-PAGE and
immunoblotted with an anti-paxillin antibody. Paxillin was
coimmunoprecipitated with full-length
9 and the
9
4 chimera to
similar levels but not with the
9
2 chimera (Figure 5C). These combined results demonstrate that the
9 cytoplasmic domain, like the
4 cytoplasmic domain, directly interacts with paxillin both in vitro
and in vivo.
|
The Binding of Paxillin to
9DM4 and
9DM5 Correlates with
Enhanced Migration and Inhibition of Cell Spreading
To determine whether paxillin binds to the
9DM3-
9DM5,
recombinant protein-binding assays were performed as described above. Both
9DM4 and
9DM5 bound paxillin to similar levels and to the same level as the
9 cytoplasmic domain (Figure
6). However, the
IIb cytoplasmic
domain, as expected, and
9DM3 did not bind paxillin. The inability
of
9DM3 to bind paxillin and promote enhanced migration and inhibit
cell spreading suggests that an
9-paxillin interaction may be
required for
9
1-dependent migration and inhibition of cell
spreading. The fact that both
9DM4 and
9DM5 bind paxillin and
support enhanced migration and inhibition of cell spreading further
suggests that
9, like
4, may require paxillin to mediate enhanced
migration and inhibit cell spreading.
|
The
9 Cytoplasmic Domain Mediates
9
1-dependent Migration
in Paxillin Null Cells
To determine whether paxillin is required for
9-mediated
enhanced migration, MEF cells null for paxillin
(paxillin
/
) were infected with
retroviruses encoding
9,
9
4,
9
5, or vector alone and
examined for their ability to promote migration on TNfn3RAA. As a
control, wild-type MEF cells that contain endogenous paxillin were
similarly infected. Neither the wild-type MEF nor the
paxillin
/
MEF cells express endogenous
9. As in CHO cells, all constructs were surface expressed at similar
high levels (Figure 7A), and all
supported adhesion to TNfn3RAA (Figure 7, B and D). In the wild-type
MEF cells,
9 and
9
4 mediated enhanced migration compared with
9
5 on both 3 µg/ml (Figure 7C) and 10 µg/ml (Figure 7E) TNfn3AA, as expected. In the paxillin
/
MEF cells, however, the
9 cytoplasmic domain mediated enhanced migration (Figure 7, C and E). At both 3 µg/ml (Figure 7C) and 10 µg/ml (Figure 7E) TNfn3RAA, the
9
1-expressing
paxillin
/
MEF cells demonstrated
enhanced migration, whereas the
9
4-expressing paxillin
/
MEF cells did not. Migration
mediated by wild-type
9 and each
9 chimeric integrin was
inhibited by the anti-
9
1 antibody, Y9A2, in both the wild-type
MEF and paxillin
/
MEF cells.
Surprisingly, these findings suggest that paxillin is not required for
9-dependent enhancement of cell migration, as is the case for
4.
|
Paxillin Is Required for
9
1-dependent Inhibition of Cell
Spreading
To determine whether paxillin is required for
9-mediated
inhibition of cell spreading, MEF and
paxillin
/
MEF cells stably infected
with
9,
9
4,
9
5, or vector alone were analyzed in
cell-spreading assays on 10 µg/ml TNfn3AA (Figure 8). After 3 h, the greatest
difference in cell spreading was evident. The wild-type MEF cells
expressing
9
5 were approximately twice as spread as cells
expressing either
9 or
9
4, as expected. However, the
paxillin
/
MEF cells expressing either
9 or
9
4 were at least as well spread as cells expressing
9
5. Thus, paxillin appears to be critical for both
9- and
4-dependent inhibition of cell spreading.
|
Point Mutations in the
9 Cytoplasmic Domain That Abolish
Paxillin Binding Do Not Inhibit Cell Migration
Point mutations in the
4 cytoplasmic domain have previously
been shown to both inhibit paxillin binding and abolish
4-mediated enhancement of cell migration and inhibition of cell spreading (Liu
et al., 1999
; Liu and Ginsberg, 2000
). To confirm our
results that an
9-paxillin interaction is not required for
9-dependent enhancement of cell migration, two point mutations were
made in the
9 cytoplasmic domain,
9(W999A) and
9(W1001A),
based on mutations in the
4 cytoplasmic domain shown to inhibit the
4-paxillin interaction (Figure 9A).
Although, as shown in Figure 6, the region of the
9 cytoplasmic
domain containing these residues is not required for paxillin binding,
recombinant protein-binding studies indicated that the
9(W999A) and
9(W1001A) mutations abolished the
9-paxillin interaction in vitro
and the interaction of
9 with the paxillin family member, Hic-5
(Young, Taooka, Liu, Askins, Yokosaki, Thomas, and Sheppard,
unpublished results). Thus, these mutations were used as additional
tools to evaluate the in vivo significance of the
9-paxillin
interaction. To evaluate the effects of the
9(W999A) and
9(W1001A) mutations in vivo,
9(W999A) and
9(W1001A)
were stably expressed in CHO cells. Both mutants were similarly
expressed at the same high surface levels as wild-type
9 (Figure
9B). Immunoprecipitation studies performed with the use of the
anti-
9
1 antibody, Y9A2, demonstrate that both the W999A and
W1001A point mutations in
9 dramatically inhibit
coimmunoprecipitation of paxillin with
9 (Figure 9C). In functional
assays, cells expressing either
9(W999A) or
9(W1001A) mediated
adhesion (Figure 9D) and migration (Figure 9E) to TNfn3AA (10 µg/ml),
as well as cells expressing the wild-type
9
1 integrin.
These results confirm our earlier findings and indicate that an
9-paxillin interaction is not required for
9
1-dependent
enhancement of cell migration.
|
9
1-dependent Inhibition of Cell Spreading Is Dependent on an
9-Paxillin Interaction
To confirm that an
9-paxillin interaction is required for
9
1-dependent inhibition of cell spreading, the
9 mutants,
9(W999A) and
9(W1001A), that do not bind paxillin described above
were analyzed in a cell-spreading assay on 10 µg/ml TNfn3AA (Figure 10). After 6 h, the greatest
difference in cell spreading was evident. Cells expressing wild-type
9 were less spread (~12%) than cells expressing the
9
5
chimera (~30%), as expected. However, cells expressing the
9
mutants,
9(W999A) and
9(W1001A), were even more spread (~50%)
than cells expressing the
9
5 chimera, indicating that an
9-paxillin interaction is required for
9 to inhibit cell
spreading. These results confirm our earlier findings and suggest that
an
9-paxillin interaction is required for
9
1-dependent inhibition of cell spreading. In addition, these combined results (Figures 7-10) suggest that
9 may use different intracellular
signaling pathways to promote enhanced migration and inhibition of cell spreading.
|
| |
DISCUSSION |
|---|
|
|
|---|
In this study, we demonstrate that the
9 cytoplasmic domain
specifically promotes cell migration and inhibits cell spreading. In
these experiments, all performed on a ligand specific for the
9
1
extracellular domain, TNfn3RAA, the
9 cytoplasmic domain preferentially enhanced cell migration and inhibited cell spreading compared with the cytoplasmic domains of
2 or
5. These results were nearly identical to those reported previously for the
4 cytoplasmic domain utilizing similarly constructed chimeric
integrin subunits containing the extracellular domain and
transmembrane domains of either the
2 or
4 subunit (Chan et
al., 1992
; Kassner et al., 1995
). As expected, based on
these earlier studies, the
4 cytoplasmic domain also enhanced
migration and inhibited spreading in our study. These results, together
with recent studies demonstrating that
9
1 and
4
integrins are both critical to transendothelial leukocyte
migration (Issekutz et al., 1996
; Gao and Issekutz, 1997
;
Taooka et al., 1999
), establish
9 and
4
integrins as members of a functional subfamily of
integrins. This classification is further supported by the
sequence similarity between
9 (Palmer et al., 1993
) and
4 (Takada et al., 1989
) and by several recent reports of
overlapping ligand-binding specificity for the
9 and
4
integrins (Taooka et al., 1999
; Eto et
al., 2000
; Takahashi et al., 2000
)
Critical
9 cytoplasmic sequences required for promotion of
9
1-dependent migration and inhibition of cell spreading were localized to a region encompassing the last six amino acids in the
9DM4 (Figures 3 and 4). The
9DM4, which contains 17 of the 33 amino acids of the
9 cytoplasmic domain (Figure 1B), was able to
mediate
9
1-dependent migration to the same extent as the full-length
9 subunit or the
9
4 chimera. In addition,
9 DM4 was able to inhibit cell spreading compared with cells expressing either the
2 or the
5 cytoplasmic domains (Figure 4). The
deletion mutant,
9DM3, which was unable to promote enhanced
9
1-dependent migration, did not inhibit cell spreading onTNfn3RAA
with cells expressing the
9DM3 being as well spread as cell
expressing the
9
2 and
9
5 chimeras (Figure 4). The
4
cytoplasmic domain has previously been shown to directly bind to the
adaptor protein, paxillin, and this
4-paxillin interaction has been
reported to be critical to
4
1-dependent enhancement of cell
migration and inhibition of cell spreading (Liu et al.,
1999
). Based on the high degree of sequence similarity between the
9
and
4 cytoplasmic domains, we were not surprised that the
9
cytoplasmic domain also associates with paxillin (Figures 5, 6, and 9).
In addition, the
9 cytoplasmic domain specifically and directly
bound paxillin (Figure 5 and 6) and to the same level as the
4
cytoplasmic domain (Figure 5). However, the site(s) of interaction
between each cytoplasmic domain and paxillin appears to differ. Whereas
the paxillin binding site in
4 has been localized to a region close
to the carboxy terminus (Liu and Ginsberg, 2000
), the analogous region
can be deleted in
9 without eliminating binding, demonstrating the
existence of a more membrane-proximal paxillin-binding site in
9.
The elimination of paxillin binding by point mutations in the carboxyl
terminal region of
9 (i.e., residues 999 and 1001) suggests that
either these mutations change the conformation of the more proximal
binding site or the
9 cytoplasmic domain contains more than one such binding site.
As for the
4 cytoplasmic domain, the
9-paxillin interaction
appears to be required for
9-mediated inhibition of cell spreading. This conclusion is supported by the fact that the
9 cytoplasmic domain did not inhibit cell spreading in the
paxillin
/
MEF cells (Figure 8) and by
the fact that two point mutations in the
9 cytoplasmic domain that
abolish association with paxillin (W999A and W1001A) also abolished
9-dependent inhibition of cell spreading (Figure 10). Surprisingly,
the
9-paxillin interaction does not appear to be required for the
9-dependent enhancement of cell migration. The
9 cytoplasmic
domain was able to promote enhanced migration in the
paxillin
/
MEF cells to a similar level
as that seen in wild-type MEF cells (Figure 7). In addition, the same
mutations in
9 (W999A and W1001A) that abolished the
9-paxillin
interaction had no effect on
9-dependent enhanced migration (Figure
9). These results indicate that an
9-paxillin interaction is not
required for
9
1-dependent enhanced migration. Although it is
possible that other known or as yet to be identified paxillin family
members could be required for
9-dependent enhanced migration, the
9(W999A) and
9(W1001A) mutants that inhibited the
9-paxillin
interaction also inhibited the interaction of
9 with Hic-5, the only
other paxillin family member known to be expressed in our cell lines.
The fact that
9 mediates enhanced migration in a
paxillin-independent manner and inhibits cell spreading in a
paxillin-dependent manner suggests that
9 may use different
intracellular signaling pathways to regulate cell migration and cell
spreading. The intracellular signaling pathway(s) activated by
9 and
4 that promote enhanced migration and inhibit cell spreading remains
to be determined. Our finding that the first 17 membrane-proximal amino
acids of the
9 cytoplasmic domain are sufficient to mediate these
effects and our identification of several mutants that do or do not
support enhanced migration and inhibit spreading provide important
tools for mapping the intracellular signaling pathway(s) responsible for
9
1-dependent enhancement of cell migration and inhibition of
cell spreading.
It is important to note that the
9- and
4-containing
integrins are clearly not unique in their ability to support
cell migration. In fact, extensive previous studies suggest that all
members of the integrin family can support substrate-specific
migration to varying degrees (Lauffenburger and Horwitz, 1996
). This
conclusion is further supported by the findings in previous studies
utilizing integrin
subunit chimeras (Chan et
al., 1992
; Kassner et al., 1995
; Liu et al.,
1999
) and in the current study showing that the chimeras containing the
structurally dissimilar
2 or
5 cytoplasmic domains still
supported levels of migration greater than that seen in mock
transfectants. However, the unique contributions of the
9 and
4
cytoplasmic domain are that they enhance the rate of cell migration.
One explanation for this effect would be that these cytoplasmic domains
simply alter the confirmation of the extracellular domains and reduce
avidity of ligand binding. Such an explanation is unlikely, however,
because enhanced migration was seen over a range of ligand-coating
concentrations and all of the chimeras and most of the deletion mutants
examined mediated comparable degrees of cell adhesion. Further, the two
deletion mutants that inhibited cell adhesion both supported decreased, not increased migration.
In summary, the findings in this paper contribute to a body of evidence
defining the
9 and
4 integrins as members of a unique integrin subclass that preferentially promotes enhanced cell
migration and inhibits cell spreading. Most leukocytes, cells that
require rapid migration to exit the vasculature and traffic to
extravascular sites of inflammation and immune response, express either
the
9 integrin,
4 integrins, or both. The
9
and
4 cytoplasmic domains each contain specific amino acid sequences
that enhance cell migration and inhibit cell spreading. Both
cytoplasmic domains associate with the adaptor protein, paxillin, and
this association is critical for the ability of the
9 and
4
subunits to inhibit cell spreading. However, whereas paxillin binding
is critical for
4-mediated enhancement of cell migration,
paxillin-independent pathways are sufficient for
9-mediated enhanced
migration. These combined findings suggest that, despite their
structural and functional similarities, the
9 and
4 cytoplasmic
domains can activate different intracellular signaling pathways to
regulate enhanced cell migration.
| |
ACKNOWLEDGMENTS |
|---|
We thank Mark Ginsberg for his many helpful suggestions. This work was supported by National Institutes of Health grants HL 47412, HL53949, and HL56385 (to D.S.) and CA 75621, by a Leukemia and Lymphoma Society Scholar Award (to S.M.T.), by training grant GM 20839 (to B.A.Y.), and by a Scientist Development Grant from the American Heart Association (to S.L.).
| |
FOOTNOTES |
|---|
These authors contributed equally to the work.
¶ Corresponding author. E-mail address:deans{at}itsa.ucsf.edu.
| |
ABBREVIATIONS |
|---|
Abbreviations used:
9DM,
9 cytoplasmic deletion mutant;
ATCC, American Type Culture Collection;
BSA, bovine serum albumin;
CHO, Chinese hamster ovary;
FCS, fetal calf serum;
GST, HA, hemagglutinin;
HPF, high-powered fields;
Ig, immunoglobulin;
MEF, mouse embryonic
fibroblasts;
PBS, phosphate-buffered saline;
PCR, polymerase chain
reaction;
TNfn3RAA, an RAA for RGD mutant fragment of the third
fibronectin type III repeat of tenascin-C.
| |
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