The Center for Cell Biology and Cancer Research, Albany Medical College,
Albany, NY 12208, USA
*
Author for correspondence (e-mail:
laflams{at}mail.amc.edu
)
Accepted April 23, 2001
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SUMMARY |
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Key words: Integrin ß cytoplasmic domains, Tyrosine phosphorylation, Cell attachment, Cell spreading
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INTRODUCTION |
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Integrin ß1 and ß3 cytoplasmic domains share a significant degree
of amino acid homology, and several proteins such as talin, -actinin,
FAK, paxillin, filamin and the integrin-linked kinase (ILK) can bind to both
the ß1 and ß3 tails (Dedhar and Hannigan,
1996
; Hemler,
1998
; Liu et al.,
2000
). One or more of these
proteins may interact with ß cytoplasmic domains to regulate integrin
function. Interestingly, endogenous ß1 integrin conformation and function
in cell attachment and cell spreading can be inhibited by both tac-ß1 and
tac-ß3 chimeras, suggesting that these processes require at least one
protein that can bind to both the ß1 and the ß3 cytoplasmic domains
(LaFlamme et al., 1994
;
Mastrangelo et al., 1999b
;
Berrier et al., 2000
). Proteins
that specifically interact with either the ß1 or ß3 cytoplasmic
domain have also been identified; integrin cytoplasmic domain-associated
protein-1 (ICAP-1) and ß3-endonexin have been shown to bind specifically
to the ß1 and ß3 cytoplasmic domains, respectively (Chang et al.,
1997
; Zhang et al.,
1999
; Shattil et al.,
1995
). Thus, it is possible
that the ß1 and ß3 cytoplasmic domains may regulate similar aspects
of integrin function by interacting with proteins that can associate with both
ß1 and ß3 tails, as well as proteins that associate specifically
with only the ß1 or ß3 cytoplasmic domain.
Because integrin ß cytoplasmic domains are small (50 amino acids)
and are capable of binding to a number of different proteins, it is likely
that these proteins have overlapping binding sites. For this reason, we
generated a series of substitution mutants to ask whether distinct amino acid
residues are involved in the ability of the ß cytoplasmic domain to
regulate specific aspects of integrin function. Our goal was to identify
mutations that can inhibit some aspects of ß cytoplasmic domain function
and not others, so that in future studies these mutations could be used to
identify the protein interactions pertinent to individual aspects of integrin
function. Our experimental approach was to express wild-type and mutant ß
cytoplasmic domains in the context of tac chimeras and to compare the ability
of these chimeras to activate tyrosine phosphorylation and to inhibit ß1
integrin conformation and function in cell attachment and spreading. With this
approach, we demonstrate that the ß1 and ß3 cytoplasmic domains are
sufficient to activate the tyrosine phosphorylation of p130CAS and paxillin in
addition to FAK. We also report three categories of ß cytoplasmic domain
mutants: (1) mutants that behaved like wild-type tac-ß1 and tac-ß3
in terms of their ability to activate tyrosine phosphorylation, to regulate
ß1 integrin conformation and to inhibit endogenous integrin ß1
function in cell attachment and spreading; (2) mutants that inhibited
tac-ß1 or tac-ß3 function in all of these assays; and (3) mutants
that inhibited tac-ß1 or tac-ß3 function in only a subset of assays.
For example, we identified mutations in the WDT motif that inhibited the
ability of tac-ß1 to regulate ß1 integrin conformation and function
in cell spreading, but had little effect on cell attachment. We also
identified mutations in the membrane-proximal region of the ß3
cytoplasmic domain and the C-terminal region of the ß1 cytoplasmic domain
that greatly diminished the ability of tac-ß1 and tac-ß3 to inhibit
cell spreading, but had little effect in other assays. Interestingly, these
latter mutations were previously shown by other laboratories to inhibit the
interaction of FAK, paxillin and ICAP-1 with ß cytoplasmic domains
(Schaller et al., 1995
; Chang
et al., 1997
). Future studies
will examine the role of these proteins in mediating ß cytoplasmic domain
function in cell spreading.
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MATERIALS AND METHODS |
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Plasmids and ß cytoplasmic domain mutants
The construction of plasmids encoding chimeric receptors containing the
extracellular and transmembrane domains of the tac subunit of IL-2 receptor
connected to the wild-type ß1, ß3, ß3B, and ß4 cytoplasmic
domains has been described previously (LaFlamme et al.,
1992; LaFlamme et al.,
1994
; Homan et al.,
1998
). The construction of the
tac chimeras containing the ß3 cytoplasmic domain mutants has also been
described (Tahiliani et al.,
1997
). Tac chimeras containing
mutations in the ß1 cytoplasmic domain were constructed as outlined
below. Site-directed mutagenesis was performed by PCR using wild-type
tac-ß1 chimera as template DNA, except where indicated. The following
mutants were constructed by one-stage PCR: ß1-758, 760 (*/A)
using the forward primer
5'-CTCATCTGGAAGCTTTTAATGATAATTGCTGACGCAAGGGAGTTTGCTAAA-3', and the
reverse primer 5'-TTACCTTAGAGCTTTAAATC-3' (SH17); ß1-783,795
(Y/F) using the forward primer 5'-CCATGGAGACGTCCA-3' (SP3) and the
reverse primer
5'-GAGTCACTCGAGTCATTTTCCCTCAAACTTCGGATTGACCACAGTTGTTACGGCACTCTTAAAAATAGGATTTTCACC-3';
ß1-787-789 (*/A), 793 (P/I), using SP3 as the forward primer
and 5'-GAGTCACTCGAGGTCATTTTCCCTCATACTTGATATTGACCACAGCTGC-3' as the
reverse primer, and the ß1-787-789 (*/A) mutant (described
below) as the template. The following mutants were constructed by two-stage
PCR using internal overlapping primers, together with the outside primers SP3
and SH17. The internal overlapping primer pairs were as follows: for
ß1-783 (Y/A), 5'-GGTGAAAATCCTATTGCTAAGAGTGCCGTAACA-3' and
5'-TGTTACGGCACTCTTAGCAATAGGATTTTCACC-3'; for ß1-763,765
(*/A), 5'-CATGACAGAAGGGAGGCTGCTGCATTTGAAAAGGAGAAAATG-3'
and 5'-TTTCTCCTTTTCAAATGCAGCAGCCTCCCTTCTGTCATG-3'; for
ß1-775,776 (*/A),
5'-AAAATGAATGCCAAAGCGGCCACGGGTGAAAATCCT-3' and
5'-AGGATTTTCACCCGTGGCCGCTTTGGCATTCATTTTCTC-3'; for ß1-775,777
(*/A), 5'-AAAATGAATGCCAAAGCGGACGCGGGTGAAAATCCTATT-3'
and 5'-AATAGGATTTTCACCCGCGTCCGCTTTGGCATTCATTTTCTC-3'; for
ß1-787-789 (*/A),
5'-ATTTATAAGAGTGCCGCTGCAGCTGTGGTCAATCCGAAG-3' and
5'-CTTCGGATTGACCACAGCTGCAGCGGCACTCTTATAAATAGG-3'; for ß1-787
(*/A), 5'-ATTGACCACAGTTGTTGCGGCACTCTTATAAAT-3' and
5'-ATTTATAAGAGTGCCGCAACAACTGTGGTCAAT-3'. For ß1-787
(*/A), the outside primers were 5'-GCAGTGGCCGGCTGTG-3'
and SH17. For all constructs, the resulting PCR products were digested with
the restriction enzymes HindIII and XhoI and ligated in
place of the HindIII/XhoI fragment of the control receptor.
Plasmids were sequenced to confirm the integrity of the newly constructed
mutants.
Analysis of phosphotyrosine signaling
Purified 7G7B6 mouse mAb was used to coat magnetic beads conjugated with
goat anti-mouse IgG (Polysciences). To activate signaling, chimeric receptors
were clustered on the cell surface by incubating transfected cells with the
antibody-coated magnetic beads as previously described (Akiyama et al.,
1994; Tahiliani et al.,
1997
; Mastrangelo et al.,
1999a
). Positively expressing
cells were magnetically sorted and then lysed on ice for 15 minutes in mRIPA
containing 50 mM Tris (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM
NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM
sodium vandate and 1 mM sodium fluoride. Protein concentrations were
determined using a MicroBCA assay (Pierce). Lysates were either analyzed
directly by western blotting or used for immunoprecipitations. Paxillin and
p130CAS were immunoprecipitated using monoclonal antibodies (mAbs) to paxillin
(clone 349, Transduction Laboratories) or to p130CAS (clone 21, Transduction
Laboratories). Cell lysates or immunoprecipitates were separated by 10%
SDS-PAGE and analyzed on western blots with the mAb 4G10 to phosphotyrosine
(Upstate Biotechnology) and visualized by enhanced chemiluminescence
(Amersham). Blots were reprobed for p130CAS or paxillin as previously
described (Tahiliani et al.,
1997
). To quantitatively
compare the ability of the different tac-ß tail mutants to trigger
tyrosine phosphorylation, the tyrosine phosphorylation signal, including
signals in the MW range of FAK and p130CAS, was normalized to the amount of
protein present as assayed by reprobing the blot for p130CAS or paxillin using
a PSDI scanner from Molecular Dynamics and ImageQuant software.
The surface expression of the different tac chimeras was analyzed and compared by flow cytometry using mAbs specific for the tac subunit of the IL-2 receptor. Similar surface expression of the various mutant tac chimeras and the wild-type tac-ß1 and tac-ß3 chimera was observed by flow cytometry in several independent experiments. However, for signaling experiments, transfected cells were also routinely examined for the expression of the tac chimeras by immunofluorescence microscopy using a FITC-conjugated mAb to the tac subunit of the IL-2 receptor (Accurate Chemical and Scientific Corporation and Becton Dickinson) to ensure similar expression levels of the different tac chimeras in individual experiments.
Analysis of 9EG7 epitope expression and cell attachment
The ability of tac chimeras containing integrin ß cytoplasmic domains
to inhibit the expression of the 9EG7 epitope was analyzed by two-color flow
cytometry as previously described (Mastrangelo et al.,
1999a; Mastrangelo et al.,
1999b
). Briefly, human
fibroblasts transiently transfected with the various tac chimeras were
harvested and then allowed to recover at 37°C for 15 minutes in serum-free
DMEM. Each sample was stained with rat mAb, 9EG7 (Pharmingen) and then with a
fluorescein-conjugated mouse anti-rat (Pharmingen) and a phycoerythrin
(PE)-conjugated mouse mAb specific for the tac subunit of the IL-2 receptor
(Pharmingen). The levels of expression of the 9EG7 epitope and the various tac
chimeras were analyzed on individual cells using a FACScan flow cytometer
(Becton Dickinson). To normalize 9EG7 expression to total ß1 integrin
expression, an aliquot of each sample was doubly stained with mAb K20 to the
ß1 subunit and a mAb to the tac subunit of the IL-2 receptor (Mastrangelo
et al., 1999a
). The ratio of
the fluorescence of 9EG7 to K20 was used to calculate the relative expression
of the 9EG7 epitope ((mean fluorescence intensity of 9EG7/mean fluorescence
intensity of K20) x100). However, it is important to note that the
expression of the K20 epitope on normal fibroblasts was not significantly
changed by the expression of the various tac-ß tail constructs (data not
shown). The inhibition of expression of the 9EG7 epitope was then calculated
by comparing the relative expression of the 9EG7 epitope on tac-ß tail
expressing cells to its expression on cells expressing the control
receptor.
The ability of tac-ß1 chimeras to inhibit cell attachment was analyzed
as previously described (Mastrangelo et al.,
1999a; Mastrangelo et al.,
1999b
). Human fibroblasts were
transiently transfected with the various tac chimeras, harvested and allowed
to recover as described above, and then 6x105 cells from each
transfection were allowed to attach for 30 minutes at 37°C in serum-free
DMEM to a well of a six-well tissue culture plate previously coated with human
fibronectin (10 µg/ml). The plates were then rotated on an orbital shaker
for 30 seconds at 150 rpm and the medium containing the unattached cells was
removed. Each well was gently washed twice with 0.8 ml of PBS, which was added
to the unattached samples. The attached cells were then removed with
trypsin/EDTA. The expression level of the various tac chimeras was then
analyzed on the unattached cells, the attached cells and a sample of the
starting population of cells by flow cytometry using a mAb specific for the
tac subunit. To quantitatively recover and analyze unattached cells,
3x105 untransfected fibroblasts were added to each sample
containing the unattached cells. The expression of the tac chimeras on the
attached and unattached samples was then compared to their expression on the
starting population.
Analysis of cell spreading
Transiently transfected fibroblasts were harvested and allowed to recover
for 20 minutes at 37°C. Approximately 2x105 cells in 2
mls of serum-free medium were plated onto glass coverslips coated with 10
µg/ml fibronectin placed in six-well tissue culture dishes. The cells were
allowed to spread for 60 minutes at 37°C. Spreading assays using A5 and
ETC12 CHO cell lines were performed similarly; however, cells were allowed to
recover for 60 minutes and then A5 cells were plated on 15 µg/ml fibrinogen
for 30 minutes and ETC12 cells were plated for 2 hours on 10 µg/ml
fibronectin. The cells were then fixed and stained with a FITC-conjugated mAb
to the tac subunit (Becton Dickinson). Microscopy was performed using an
Olympus Provos microscope equipped with phase contrast and epifluorescence and
attached to a SPOT, low light camera interfaced with a PC computer. Cell area
and fluorescence intensity were measured using Image Pro-Plus software (Media
Cybernetics). Round cells that had not begun to spread were found to have cell
areas of less than 600 µm2 (Berrier et al.,
2000).
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RESULTS |
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Mutations in some conserved amino acid motifs of the integrin ß1
and ß3 cytoplasmic domains inhibit their ability to activate tyrosine
phosphorylation
We targeted mutations to motifs conserved in the ß1 and ß3
cytoplasmic domains (Fig. 2),
and then compared the ability of these mutant ß tails to trigger tyrosine
phosphorylation. The ß1-758,760 (*/A) and ß1-763,765
(*/A) mutants targeted either the histidine (H) and arginine (R) or
the phenylalanine (F) and lysine (K) residues, respectively, in the conserved
membrane-proximal HDR(RorK)EFAK motif. We found
that the ß1-758,760 (*/A) mutant triggered tyrosine
phosphorylation to similar levels as the wild-type ß1 cytoplasmic domain
(Fig. 3A). Although there was
variability between experiments, on average the ß1-763,765
(*/A) mutant triggered slightly reduced levels of tyrosine
phosphorylation compared to wild-type ß1
(Fig. 3D). The ß1-775,776
(*/A) and the ß1-775,777 (*/A) mutants with alanine
substitutions in the conserved WDT motif consistently showed a reduced ability
to activate tyrosine phosphorylation, with only FAK showing significant
phosphorylation in cell lysates (Fig.
3A). Changing the membrane-proximal NPXY motif to NPXA in the
ß1-783 (Y/A) mutant abrogated the ability of the ß1 cytoplasmic
domain to activate tyrosine phosphorylation. The level of tyrosine
phosphorylation observed was comparable to that in lysates from cells
expressing the control receptor (Fig.
3A). By contrast, the substitution of phenylalanine for the
tyrosine residues in both the membrane-proximal and the membranedistal NPXY
motifs in the ß1-783,795 (Y/F) mutant consistently resulted in only
slightly reduced tyrosine phosphorylation
(Fig. 3B). Thus, tyrosine
phosphorylation of the ß1 cytoplasmic domain itself is not required to
activate these signaling events. Additionally, the ß1-787-789
(*/A) mutant containing AAA in place of the VTT motif consistently
resulted in a loss of tyrosine phosphorylation, similar to the ß1-783
(Y/A) mutant (Fig. 3A). Thus,
it seems that both a membrane-proximal NPXY motif and VTT motif are necessary
for the ß1 cytoplasmic domain to activate tyrosine phosphorylation.
|
|
The results for the ß3 mutants were similar to those for the ß1 mutants. The ß3-723,726 (*/A) mutant, which targets the aspartic acid (D) and glutamic acid (E) residues in the conserved membrane-proximal HDR(RorK)EFAK motif, signaled as efficiently as the wild-type ß3 cytoplasmic domain (Fig. 3E). Thus, the membrane-proximal region does not appear to be required for either the ß1 or the ß3 cytoplasmic domain to activate tyrosine phosphorylation. An alanine substitution for the tyrosine in the membrane-proximal NPXY motif in the ß3-747 (Y/A) mutant abolished the ability of the ß3 cytoplasmic tail to signal (Fig. 3E), whereas the more conservative substitution of phenylalanine for tyrosine in the ß3-747 (Y/F) mutant did not inhibit tyrosine phosphorylation (Fig. 3E). These results are similar to those obtained for the analogous ß1 mutants (Figs 2, 3). Interestingly, the ß3-751-753 (*/A) mutant containing AAA for the TST motif consistently triggered tyrosine phosphorylation, although at reduced levels compared to the wild-type ß3 tail (Fig. 3E). This is different from the analogous ß1-787-789 (*/A) mutant, which lost the ability to trigger tyrosine phosphorylation. Thus, the TST motif in the ß3 tail plays a more modulatory role, whereas the analogous VTT motif in the ß1 tail appears to be required for the activation of tyrosine phosphorylation.
A side-by-side comparison of the signaling phenotypes of analogous ß1-787-789 (*/A) and ß3-751-753 (*/A) mutants confirmed that the ß1-787-789 (*/A) mutant was unable to activate tyrosine phosphorylation, whereas the ß3-751-753 (*/A) mutant triggered the tyrosine phosphorylation, albeit not as efficiently as the wild-type ß cytoplasmic domains (Fig. 4). The differences were not time-dependent as this same pattern of tyrosine phosphorylation was observed following either 40 minutes or 90 minutes of clustering (Fig. 4A). Additionally, the surface expression of the ß1-787-789 (*/A) mutant was similar to that of the ß3-751-753 (*/A) mutant and the levels of cell-surface expression of these mutants were comparable to the levels of expression of the tac chimeras containing wild-type ß cytoplasmic domains (Fig. 4B). Immunoprecipitation of p130CAS (Fig. 4C) and paxillin (Fig. 4D) also demonstrated that their phosphorylation was triggered by the ß3-751-753 (*/A) mutant, but not the ß1-787-789 (*/A) mutant (Fig. 4C,D).
The ß1-787-789 (*/A), 793 (P/I) mutant does not
trigger tyrosine phosphorylation, whereas the ß1-787 (V/A) mutant
does
The VTT motif in the ß1 cytoplasmic domain is flanked by two NPXY
motifs (NPIY and NPKY), whereas the TST motif in the ß3 cytoplasmic
domain is flanked by a NPXY motif and a NXXY motif (NPLY and NITY). Therefore,
the conformation of the ß1 and ß3 tails may differ in the regions of
the VTT and TST motifs, and this difference may be responsible for the
different phenotypes of the ß3-751-753 (*/A) and
ß1-787-789 (*/A) mutants. To test this hypothesis, we
constructed the ß1-787-789 (*/A), 793(P/I) mutant, which
contains a substitution of an isoleucine for the proline in the NPKY motif of
ß1. However, we found that the ß1-787-789 (*/A), 793
(P/I) mutant did not trigger tyrosine phosphorylation
(Fig. 3C), suggesting that the
difference between these ß1 and ß3 mutants is not simply the
presence of a proline residue in the membrane-distal NPXY motif of the ß1
tail.
In addition, as the VTT motif in the ß1 cytoplasmic domain overlaps a
region previously shown to be important in the binding of ICAP-1 to the
ß1 tail (Chang et al.,
1997), we tested whether a more
conservative mutation, previously shown to inhibit the interaction of ICAP-1
with the ß1 tail (Chang et al.,
1997
), would inhibit signaling
initiated by clustering the ß1 cytoplasmic domain. We constructed the
ß1-787(V/A) mutant, which contains an alanine substitution for valine
787. However, we found that the ß1-787 (V/A) mutant was able to activate
tyrosine phosphorylation (Fig.
3C) to levels only slightly reduced compared to wild-type
tac-ß1. Although this result suggests that ICAP-1 binding to the ß1
tail is not required to activate tyrosine phosphorylation, we have not yet
tested whether this mutation affects the binding of ICAP-1 to tac-ß1.
Comparison of the ability of various ß1 cytoplasmic domain
mutants to regulate ß1 integrin conformation and function in cell
attachment
Previous studies had demonstrated that high levels of tac-ß1 or
tac-ß3 can inhibit specific ß1 integrin conformations, as well as
ß1 function in cell attachment (Mastrangelo et al.,
1999b). To determine whether
distinct regions of the integrin ß1 tail regulate these processes, we
compared the ability of wild-type and mutant tac-ß1 chimeras to regulate
ß1 integrin conformation and to inhibit ß1 function in cell
attachment. To monitor the conformation of the ß1 subunit, we assayed the
expression of the 9EG7 epitope (Lenter et al.,
1993
). Previous studies
indicated that the expression of the 9EG7 epitope on the ß1 subunit can
be regulated by divalent cations (Bazzoni et al.,
1998
) and ligand binding and
depends on the amino acid sequence of the integrin ß cytoplasmic domain
(Belkin et al., 1997
; Sakai et
al., 1998
; Wennerberg et al.,
1998
). In addition, the
expression of the 9EG7 epitope correlates with
5ß1 function in
ligand binding and in some instances with integrin function in cell attachment
(Mastrangelo et al., 1999b
;
Belkin et al., 1997
; Bazzoni et
al., 1998
; Sakai et al.,
1998
; Wennerberg et al.,
1998
).
When we compared the expression of the 9EG7 epitope in cells transiently expressing either the wild-type or mutant tac-ß1 chimeras by two-color flow cytometry, we found that tac-ß1 chimeras containing either the ß1-758,760 (*/A), the ß1-763,765 (*/A), the ß1-787 (V/A), or the ß1-783, 795 (Y/F) mutant inhibited the expression of the 9EG7 epitope similar to wild-type tac-ß1 (Fig. 5A). By contrast, tac-ß1 chimeras containing the either the ß1-775,776 (*/A), the ß1-775,777 (*/A), the ß1-783 (Y/A), ß1-787-789 (*/A) or the ß1-787-789 (*/A), 793 (P/I) mutant showed diminished abilities to inhibit the 9EG7 expression (Fig. 5A). These results suggest that the NPXY, WDT and VTT motifs in the ß1 tail are important for protein interactions with ß cytoplasmic domains that regulate ß1 integrin conformation. Interestingly, the same mutations affected the ability of the ß cytoplasmic domain to activate tyrosine phosphorylation; however, mutations in the WDT motif had an intermediate effect on the activation of tyrosine phosphorylation (Fig. 2).
|
To compare the ability of wild-type and mutant tac-ß1 chimeras to
inhibit cell attachment on fibronectin, normal human fibroblasts transiently
expressing the various tac-ß1 chimeras were allowed to adhere to
fibronectin for 30 minutes at 37°C. The levels of expression of the
chimeric receptors on the starting, attached and unattached populations of
cells were compared by flow cytometry. Inhibition of cell attachment results
in an increase in the mean fluorescence intensity of chimeric receptor
expression in the unattached population of cells. We found that tac-ß1
chimeras containing either the ß1-758,760 (*/A),
ß1-763,765 (*/A), ß1-775, 776 (*/A),
ß1-783,795 (Y/F) or the ß1-787 (V/A) mutant inhibited cell
attachment similar to wild-type tac-ß1
(Fig. 5B). The tac-ß1
chimeras containing the ß1-775, 777 (*/A) mutant also
inhibited cell attachment, but not as dramatically. The tac-ß1 chimeras
containing either the ß1-783 (Y/A), ß1-787-789 (*/A), or
the ß1-787-789 (*/A), 793 (P/I) mutant did not inhibit cell
attachment to fibronectin. These results suggest that the NPXY and the VTT
motifs of the ß1 tail are required for the regulation of ß1 integrin
conformation and for ß1 cytoplasmic domain function in cell attachment
(Fig. 2). The analogous motifs
in the ß3 tail had similar phenotypes in these assays (Mastrangelo et
al., 1999b; see summary in
Fig. 2). Interestingly,
mutations in the WDT motif of the ß1 tail that inhibited the ability of
tac-ß1 to regulate ß1 integrin conformation had little effect on the
ability of tac-ß1 to inhibit cell attachment. This was especially true of
the ß1-775,776 (*/A) mutant. These results suggest that
distinct protein interactions are involved in the ability of the ß
cytoplasmic domain to regulate ß1 integrin conformation and ß1
function in cell attachment.
Comparison of the ability of the various ß1 and ß3
cytoplasmic domain mutants to regulate cell spreading
Previous studies had indicated that moderate levels of expression of either
tac-ß1 or tac-ß3 allow cell attachment, but inhibit cell spreading
(LaFlamme et al., 1994;
Mastrangelo et al., 1999b
;
Berrier et al., 2000
). To
compare the ability of the wild-type and mutant tac-ß1 and tac-ß3
chimeras to inhibit cell spreading, we measured the extent of cell spreading
as a function of the expression level of the chimeric receptor (Berrier et
al., 2000
). For these
experiments, normal fibroblasts transiently expressing tac-ß tail
chimeras were plated onto fibronectin-coated glass coverslips and allowed to
spread for one hour. Adherent cells were stained for tac expression. The
fluorescence intensity and area of positively transfected cells were
determined as described in Materials and Methods. We found that moderate
levels of expression of wild-type tac-ß1 inhibited cell spreading
(Fig. 6) as previously reported
(LaFlamme et al., 1994
;
Berrier et al., 2000
). When we
compared the ability of various tac-ß1 mutants to inhibit cell spreading,
we found that the ß1-758,760 (*/A) and the ß1-763,765
(*/A) mutants with mutations in the membrane-proximal motif
inhibited cell spreading similar to wild-type tac-ß1
(Fig. 6A). The ß1-783,795
(Y/F) mutant also inhibited cell spreading, but was not as potent an inhibitor
as the wild-type tac-ß1 (Fig.
6A). By contrast, tac-ß1 chimeras containing either the
ß1-775,776 (*/A), ß1-775, 777 (*/A), or
ß1-783 (Y/A) mutant were each poor inhibitors of cell spreading
(Fig. 6A). Thus, alanine
substitutions in the WDT motif or the membrane-proximal NPXY motif of the
ß1 tail abrogated the ability of tac-ß1 to regulate cell
spreading.
|
In a second series of experiments, we compared the ability of tac-ß3 mutants and additional tac-ß1 mutants to inhibit cell spreading on fibronectin. We found that wild-type tac-ß1 and tac-ß3 inhibited cell spreading as expected (Fig. 6B); however, tac-ß1 consistently inhibited cell spreading better than tac-ß3. Tac-ß1 and tac-ß3 chimeras containing either the ß1-787 (V/A), ß1-787-789 (*/A), ß1-787-789 (*/A), 793(P/I), ß3-723,726 (*/A), ß3-751-753 (*/A) or the ß3-747 (Y/A) mutant failed to inhibit cell spreading on fibronectin. However, it is important to note that the spread cells expressing the ß1-787 (V/A) mutant tended to have smaller areas than cells expressing the control receptors. These results suggest that three regions of the ß cytoplasmic domain are important for regulating cell spreading: the membrane-proximal region in the ß3 tail, the NPXY motif in the ß1 and ß3 tails and the analogous VTT and TST motifs in the ß1 and ß3.
Tac-ß1 chimeras containing the ß1-787 (V/A) mutant fail to
inhibit cell spreading mediated by either ß1 or ß3 integrins
The ß1-787 (V/A) mutant and the ß3-723,726 (*/A)
mutant are of particular interest because they behaved similar to wild-type
tac-ß tail chimeras in their ability to trigger tyrosine phosphorylation,
regulate ß1 integrin conformation and inhibit ß1 function in cell
attachment (Fig. 2), suggesting
that these mutations inhibit protein interactions that specifically regulate
cell spreading. Because the ß1-787 (V/A) mutation was previously shown to
inhibit the interaction of ß1-tail specific binding protein ICAP-1 (Chang
et al., 1997), we were
interested in whether the diminished ability of tac-ß1 containing this
mutation to inhibit cell spreading was specific for spreading mediated by
ß1 integrins. To test this hypothesis, we used two CHO cell lines that
stably express either wild-type integrin
IIbß3 (A5 cells) or
IIbß3 containing a ß3 cytoplasmic domain truncation,
IIbß3
727 (ETC12 cells). A5 cells adhere and spread on
fibrinogen via
IIbß3 and ETC12 cells spread on fibronectin via
their endogenous
5ß1 integrin (Ylanne et al.,
1993
). Therefore, we compared
the ability of tac-ß1 containing the ß1-787 (V/A) mutant to inhibit
cell spreading of A5 cells on fibrinogen and ETC12 cells on fibronectin. We
found that the ß1-787 (V/A) mutant failed to inhibit cell spreading both
on fibrinogen and on fibronectin (Fig.
6C). This result suggests that either the binding of ICAP-1 to the
ß1 tail is required to nucleate protein interactions, which are common to
both ß1 and ß3 and are required for cell spreading, or that the
ß1-787 (V/A) mutation inhibits the binding of a protein other than ICAP-1
that binds to both ß1 and ß3 tails to regulate cell spreading.
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DISCUSSION |
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Interestingly, the two mutations that specifically abrogated the ability of
tac-ß1 and tac-ß3 to inhibit cell spreading were previously
demonstrated to inhibit known protein interactions with ß cytoplasmic
domains. The ß3-723, 726 (*/A) mutation had previously been
shown to inhibit the in vitro interaction of FAK and paxillin with peptides
modeled after the ß cytoplasmic domain (Schaller et al.,
1995). Thus, the binding of
FAK or paxillin to ß tails may be important for cell spreading. However,
Rack1 and talin have also been shown to bind to the conserved
membrane-proximal domain of the ß1 and ß3 tails (Liliental and
Chang, 1998
; Patil et al.,
1999
). These mutations may
also inhibit the binding of these proteins with ß tails.
The ß1-787 (V/A) mutation had previously been shown to inhibit the
interaction of ICAP-1 with the ß1 cytoplasmic domain (Chang et al.,
1997). Because ICAP-1 is
ubiquitously expressed (Chang et al.,
1997
; Zhang and Hemler,
1999
) and can easily be
detected in lysates from normal human fibroblasts (A.L.B., unpublished),
tac-ß1 may inhibit cell spreading by titrating ICAP-1 from endogenous
ß1 integrins. However, chimeric receptors containing the ß1-787
(V/A) mutant also had a diminished ability to inhibit cell spreading on
fibrinogen mediated by recombinant
IIbß3. Therefore, although one
could argue that the binding of ICAP-1 is required for the ß1 tail to
bind other proteins that normally also bind the ß3 tail to regulate cell
spreading, it is also possible that the ß1-787 (V/A) mutation is
affecting a different protein interaction required for cell spreading. For
example, valine 787 is in a region of the ß1 tail that is important for
ILK association (Dedhar and Hannigan,
1996
) and for the binding of
filamin to ß tails in vitro (Loo et al.,
1998
). Thus, there are several
protein interactions that may be important in mediating ß cytoplasmic
domain function in cell spreading that could be disrupted by these mutations.
More studies are required to identify the relevant ones.
Alanine substitutions of the tryptophan and aspartic acid residues in the
WDT motif of the ß1 tail inhibited the ability of tac-ß1 to regulate
integrin conformation and function in cell spreading. Interestingly, the WDT
motif and the membrane-proximal NPXY motif overlap a putative binding site for
talin (Horwitz et al., 1986;
Pfaff et al., 1998
; Kaapa et
al., 1999
). Expression of
recombinant forms of the head domain of talin can activate
IIbß3
(Calderwood et al., 1999
),
suggesting that the interaction of the head domain of talin with integrin
ß cytoplasmic domains may function in integrin activation. A similar
interaction with the ß1 cytoplasmic domain may regulate ß1 integrin
conformation and 9EG7 expression. Additionally, other studies have implicated
the binding of talin to the ß1 tail in regulating integrin function in
cell spreading (Kaapa et al.,
1999
). Our data indicating
that mutations in the WDT motif inhibit the ability of the ß tail to
regulate integrin conformation and function in cell spreading is consistent
with ß tail-talin interactions having a role in regulating these
processes. However, it is not known whether mutations in the WDT motif inhibit
talin binding. In addition, other studies have suggested that the talin head
domain does not bind to this region of the ß tail, but interacts with the
membrane-proximal region instead (Patil et al.,
1999
). More studies are needed
to understand how talin interacts with the ß tail and the functional
significance of the interaction.
As reported previously and in the current study, mutation of the
membrane-proximal NPXY motif to NPXA appears to completely inhibit the
function of ß1 and ß3 cytoplasmic domains (Reszka et al.,
1992; O'Toole et al.,
1995
; Ylanne et al.,
1995
; Tahiliani et al.,
1997
; Schaffner-Reckinger et
al., 1998
). Because integrin
ß cytoplasmic domains containing NPXA lose their abilities to bind to
talin and filamin in vitro (Pfaff et al.,
1998
), it is possible that one
or both of these protein interactions with ß cytoplasmic domains is
required for all aspects integrin function requiring integrin ß
cytoplasmic domains. However, it is also possible that this mutation changes
the overall conformation of integrin ß cytoplasmic domains, thus
nonspecifically inhibiting multiple protein interactions and integrin function
in multiple assays. It seems likely that structural information on ß
cytoplasmic domains will be necessary to understand how mutations in this NPXY
motif affect specific protein interactions.
In addition, we have found that alanine substitutions in the VTT motif of
the ß1, the ß1-787-789 (*/A), or the TST motif of
ß3, the ß3-751-753 (*/A) mutant, inhibited the ability of
chimeric receptors containing these ß tails to regulate endogenous
ß1 integrin conformation and function in cell attachment and cell
spreading (also see Mastrangelo et al.,
1999b). Additionally, the
ß1-787-789 (*/A) mutant also abrogated the ability of
tac-ß1 to activate tyrosine phosphorylation; however, tac-ß3 with
the analogous mutation was able to signal, albeit to lower levels than
tac-ß3 containing a wild-type tail. Consistent with our findings, alanine
substitutions at the threonine residues in the VTT motif of the ß1 tail
in the context of heterodimeric integrins inhibited the expression of the 9EG7
epitope on these integrins, as well as their function in cell attachment
(Wennerberg et al., 1998
).
Interestingly, these mutant ß1 integrins were still able to trigger FAK
phosphorylation. This difference in phenotype could be due to the additional
alanine substitution for valine in the VTT motif of our mutant.
Interestingly, the substitution of phenylalanine for tyrosines in the
ß1 and ß3 tails did not have profound effects in our studies,
although the ability of tac-ß1 containing the ß1-783,795 (Y/F)
mutant to inhibit cell spreading was somewhat diminished. However, Wennerberg
and colleagues demonstrated that changing tyrosine residues to phenylalanine
in the ß1 cytoplasmic domain dramatically inhibited the ability of
ß1 integrins to trigger FAK phosphorylation and paxillin phosphorylation
and to mediate cell spreading (Wennerberg et al.,
2000). These differences could
reflect differences in cell type: the use of GD25 derivatives (Wennerberg et
al., 2000
) and normal human
fibroblasts (present study). We have previously found differences in the
behavior of these two cell types in our assays. For example, treating GD25
cells re-expressing the mouse ß1 subunit with Mn+2 does not increase 9EG7
expression as in human fibroblasts and treating these same GD25 cells with
sodium vanadate does not inhibit the expression of 9EG7 or inhibit integrin
function in cell attachment as in human fibroblasts (A.M.M., unpublished).
However, these differences may also reflect differences in chimeric receptors
and heterodimeric integrins.
Other laboratories have also identified mutations that result in the
inhibition of some integrin-mediated processes and not others. A substitution
mutant in the putative -actinin binding region of recombinant
IIbß3 has been identified that was able to trigger FAK
phosphorylation, but was unable to retract a fibrin clot (Lyman et al.,
1997
). A different
substitution mutant in the same region of the ß3 tail was identified that
inhibited the ability of recombinant
IIbß3 to be recruited to
pre-established focal adhesions and to be internalized, but that still
functioned in cell spreading and focal adhesion formation (Ylanne et al.,
1995
). In addition, a deletion
of the membrane-proximal HDRRE motif inhibited the ability of a tac-ß1
chimera to be recruited to focal adhesions and to activate tyrosine
phosphorylation, but not to trigger cell detachment in response to LPA (David
et al., 1999
). In the present
study, we identified two mutations that specifically affect the ability of
tac-ß1 and tac-ß3 to function as dominant negative inhibitors of
cell spreading. Clearly, evidence is accumulating to support the notion that
distinct protein interactions with integrin ß cytoplasmic domains are
required to regulate different aspects of integrin function. The task for the
future is to identify the proteins involved and to determine how these
interactions with the ß cytoplasmic domain are regulated.
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ACKNOWLEDGMENTS |
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