From the
The cytoplasmic domain of the
The
effects of these and other mutations on additional functions of
Integrins are transmembrane glycoproteins that mediate cell
adhesion and migration by functionally linking extracellular structures
to the cytoskeleton (see Refs. 1-5). Integrins can induce
intracellular signals, which are needed for cytoskeletal rearrangements
(see Ref. 6), for maintenance of anchorage-dependent cell growth (see
Refs. 1 and 7) and for prevention of apoptosis
(8, 9) .
The signals require integrin clustering, which may generate signaling
complexes of cytoskeletal proteins and regulatory proteins such as
tyrosine kinases (see Refs. 10 and 11). In several adherent cells,
adhesion plaques or focal contacts may represent integrin-induced
signaling complexes
(11) .
The cytoplasmic domains of
integrin
In the present work, we have used
recombinant
From the
final PCR products, a MluI- XhoI fragment or an
AflII- XhoI fragment (restriction enzymes were from
New England Biolabs, Beverly, MA) was purified from an agarose gel by
using the Geneclean II kit (Bio 101, La Jolla, CA) and cloned into the
original vector. The PCR-derived parts of the mutant clones were
verified by sequencing (T7 sequencing kit, Pharmacia Biotech).
Supercoiled plasmid DNA was purified using CsCl (Pharmacia) gradient
centrifugation
(27) .
For cell adhesion experiments,
fibrinogen and fibronectin were purified from human plasma
(28, 29) . Contaminating fibronectin was removed from
the fibrinogen preparations by gelatin-Sepharose affinity
chromatography. Round glass coverslips (13 mm in diameter,
Menzel-Gläzer, Germany) or cell culture plastics (Falcon) were
treated with 30 µg/ml of fibrinogen or 10 µg/ml of fibronectin
in phosphate-buffered saline (PBS: 0.14
M NaCl 10 m
M sodium phosphate, pH 7.4) overnight at +4 °C, followed by
treatment with 1% bovine serum albumin (Sigma) in PBS for 1 h at 22
°C, and washed twice with PBS before the cells were seeded in
serum-free medium. For phase contrast photographs or counting of spread
cells
(30) , the specimens were fixed with 2% paraformaldehyde
in PBS for 10 min on ice. The proportions of spread cells to adherent
cells were determined from triplicate samples by counting more than 150
cells/sample in a double-blind manner. For immunostaining, cells were
fixed with 2% paraformaldehyde, containing 0.5% Triton X-100 (BDH Ltd.,
Poole, UK) in PBS for 10 min on ice.
The ability of the
The major findings of this study are as follows.
1) By
using COOH-terminal deletions we could determine that the sequence up
to Ile
2) The Y747A substitution abolished
3) Residues Phe
4) The
cytoplasmic domain of
5) The cytoplasmic domain of
The results are summarized in
. Fig. 9 summarizes our current thinking about the
functional roles of the different parts of the cytoplasmic domain of
the
The ability of our
We also mutated Ser
The mutation
Y747A disrupted all of the functions dependent on the COOH-terminal
part of the
As far as no physical data are
available on the three-dimensional structure of the cytoplasmic domains
of integrin
We found that point mutations at
Phe
Integrins are known to participate in
phagocytosis or in internalization of particles coated with ligands.
The major complement iC3b receptor in leukocytes is
In contrast to
integrin-mediated internalization of ligand-coated particles, the
constitutive intake of
The
present identification of important residues required for
integrin-mediated cell spreading should be helpful in resolving the
detailed mechanisms of integrin function and transmission of
integrin-induced signals. Our results imply that factors that are
absolutely required for integrin-mediated cell spreading bind to the
The data in the table
have been presented in Figs. 2-8. ND, not determined.
We thank Aili Grundström and Pipsa Kaipainen for
technical help, Dr. Matti Korhonen for helpful discussions, and Dr.
Keith Burridge (Chappel Hill, NC) for providing anti-talin antiserum.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit of the
integrin is required for cell
spreading on fibrinogen. Here we report that deletion of six amino
acids from the COOH terminus of the
(I
TYRGT) totally abolished cell spreading and
formation of adhesion plaques, whereas retaining Ile
partially preserved these functions. We further found that
substitution of Tyr
with Ala also abolished
-mediated cell spreading.
were also studied. Progressive
truncations of
, in which stop codons were inserted at
amino acid positions 759-756, caused partial defects in the
recruitment of
to preestablished
adhesion plaques and a gradual decrease in the ability of
to mediate internalization of
fibrinogen-coated particles. The Tyr
Ala
substitution mutant was almost totally inactive in both of these
assays. Point mutations at Tyr
, and at a conserved area
close to the transmembrane domain of
, decreased
integrin recruitment to preestablished adhesion plaques but allowed
-mediated formation of these
structures and partial cell spreading. Deletion of the cytoplasmic
domain of
did not affect the constitutive endocytosis
of
.
-subunits are highly conserved in all integrins that are
linked to adhesion plaques (see Ref. 12). The cytoplasmic domains of
and
mediate interactions with the
cytoskeletal proteins talin and
-actinin, which are concentrated
in adhesion plaques
(13, 14, 15) . Two kinds of
observations point out the critical role of the cytoplasmic domains of
integrin
-subunits in integrin function: their removal inhibits
integrin-mediated cell adhesion
(16) and cell spreading
(17) , and their autonomous overexpression causes cell rounding
by uncoupling integrin-induced signals from cell adhesion
(18, 19) .
integrins expressed in
CHO
(
)
cells to study which areas of the
cytoplasmic domain of the
subunit are needed for cell
spreading and other functions of
.
This system allows us to study
mutants with minimal
interference by endogenously expressed integrins. It has been
previously shown that no prior activation events are needed for
-mediated cell adhesion to
fibrinogen-coated surfaces
(20) , although the binding of
to soluble fibrinogen requires
activation
(21) . We show that an area of
containing Tyr
and Ile
is essential
for
-mediated cell spreading. On
the other hand, mutations at amino acid positions 727-733 or 759
caused defects in the recruitment of
to preestablished adhesion plaques and in
-mediated internalization of
fibrinogen-coated particles but still allowed the formation of adhesion
plaques and cell spreading. We also show that neither the cytoplasmic
domain of the integrin
subunit nor the NPLY
sequence motif are needed for constitutive endocytosis of
.
Plasmid Constructs and Mutations
The pCDM8
(Invitrogen, San Diego, CA) derived plasmids containing human
or
integrin cDNAs or their
mutants
(
996),
(
728), and
(S752P) have been described previously
(17, 22, 23, 24, 25, 26) .
In some experiments, a pCDM8 construct, CN2b, was used containing
and neomycin resistance genes
(22) .
Mutations were introduced into the
plasmid by
amplifying by PCR a segment of the insert with a common 5` primer
(5`GGCTGGCTGGGATCCCAGTGTGAG3`) and a 3` primer containing the
appropriate mutation. The mutant primers were as follows:
747,
5`-ATATCTCGAGTCATGGGTTGTTGGCTGTGTCCC-3`;
752,
5`-ATATCTCGAGTCACGTGGCCTCTTTATACAGTGG-3`;
755,
5`-ATATCTCGAGTCAG-AAGGTAGACGTGGCCTC-3`;
756,
5`-ATATCTCGAGTCAGGTGAAGGTAGACGTGGCC-3`;
757,
5`-ATATCTCGAGTCAATTGGTGAAGGTAGACG3`;
758,
5`-ATATCTCGAGTCAGATATTGGTGAAGGTAGACG-3`;
759,
5`-ATATCTCGAGTCACGTGATATTGGTGAAGGT-AG-3`; S752A,
5`-ATATCTCGAGTTAAGTGCCCCGGTACGTGATATTGGTGAAGGTAGCCGTGGCCTC-3`; and
Y759A, 5`-ATATCTCGAGTTAAGTGCCCCGGGCCGTGATATTGG-3`. All of these primers
contained a XhoI restriction site at their 3` end. Internal
point mutations were generated by a two-step PCR reaction, where the
common 5` primer and a mutant 3` primer were first used (mutant primers
were (F727A/F730L/E733A): 5`-GCTCTGGCGCGTACTTCCTCAAGTTTAGCGGCTTCTT-3`
and Y747A, 5`-GGCCTCTTTAGCCAGTGGGTTGTTGGC-3`). The PCR product was then
purified and used as a 5` primer together with a 3` primer containing a
XhoI site (5`-ATATCTCGAGGACATTCTCCCAACCTACCC-3`). The
oligonucleotides were made with a PCR-mate 391 DNA synthesizer (Applied
Biosystems, Foster City, CA). All PCR reactions were done by using the
Dynazymethermostable DNA polymerase (Finnzymes, Espoo, Finland) and
a Perkin-Elmer/Cetus DNA thermal cycler (Perkin-Elmer).
Cell Culture and Transfections
Chinese hamster
ovary cells (CHO-K1, ATCC CCL 61) were from the American Type Culture
Collection (Rockville, MD). They were maintained in Dulbecco's
modified Eagle's medium (Sigma) supplemented with 10% fetal
bovine serum (Biological Industries, Kibbutz Beth Haemek, Israel), 100
units/ml penicillin, 0.1 mg/ml streptomycin (Biological Industries),
and nonessential amino acids (Life Technologies, Inc.). The expression
vectors were transiently introduced into the cells by using the
LipofectAMINE reagent (Life Technologies, Inc.). Briefly, 2
10
cells were seeded on a 100-mm diameter cell culture dish
(Falcon, Becton Dickinson UK, Plymouth, United Kingdom) 24 h before
transfection. Two µg of
and
plasmids were incubated in 200 µl of nonsupplemented medium
with 20 µl of LipofectAMINE for 10 min; the cells were washed twice
with nonsupplemented medium, and the DNA-LipofectAMINE mixture was
added to the cells in 3.8 ml of nonsupplemented medium. After
incubation for 6 h, the medium was changed to 10 ml of normal cell
culture medium, which was further changed 24 h after transfection. The
cells were analyzed 48 h after the start of transfection. For stable
transfections, either the LipofectAMINE method or a calcium-phosphate
precipitation method was used
(27) . Stable cell lines
expressing the transfected integrins were selected in 0.75 mg/ml
Geneticin(G418)-disulfate (Sigma), and further cloned until cell lines
of similar expression levels were established. The stable cell lines
expressing
(
728) and
(S752P) have been characterized
earlier
(25, 26) .
Antibodies, Flow Cytometry, and Microscopy
The
antibodies used in the study were as follows: monoclonal
anti-PL98DF6
(31) , monoclonal
anti-
PL90BB10
(31) , monoclonal
anti-fibrinogen (clone D1G10VL2, Immunotech, Marseille, France),
polyclonal anti-fibronectin receptor (Telios Pharmaceuticals, La Jolla,
CA), polyclonal anti-talin
(32) , and polyclonal anti-vinculin
(rabbit 901, 33). For flow cytometry and immunofluorescence microscopy,
diluted polyclonal antisera or 10 µg/ml purified IgG of the
monoclonal antibodies were used, followed by appropriate secondary
antibodies (fluorescein isothiocyanate-coupled rabbit anti-mouse
immunoglobulin (Dakopatts, Glostrup, Denmark), tetramethylrhodamine
isothiocyanate-coupled goat anti-mouse IgG (Jackson Immunoresearch
Laboratories, West Grove, PA), or fluorescein isothiocyanate-coupled
goat anti-rabbit IgG (Cappel, Organon Teknika, West Chester, PA)). For
the immunofluorescence stainings, controls were routinely made that
showed negligible nonspecific staining with the primary and secondary
antibodies. Flow cytometry was done with a FACScaninstrument (Becton
Dickinson Immunocytometry Systems, San Jose, CA). A Leiz Aristoplan
fluorescence microscope (Leica Mikroskopie und Systeme, Wetzlar,
Germany) was used with a plan Apochromat 63
oil immersion
objective, and photographs were taken using Kodak Tmax 400 film
(Eastman Kodak Co.).
mutations
to mediate recruitment of
(
996)
to adhesion plaques
(17) was scored, after transient
transfections, by counting the proportion of
(
996)
-expressing spread cells
in which adhesion plaque-like immunolocalization could be revealed with
the anti-
monoclonal antibody. For this purpose, the
cells were allowed to adhere on fibronectin for 2 h. Triplicate coded
samples were counted, and the results of one representative experiment,
out of at least three, are shown.
Endocytosis
Endocytosis of integrins was measured
by a method modified from Bretscher
(34) . Briefly, 2
10
cells were cultured overnight on 35-mm diameter tissue
culture dishes, washed twice with Dulbecco's modified PBS (Life
Technologies, Inc.), and labeled for 1 h on ice with 1 ml of 0.2 mg/ml
NHS-SS-biotin (Pierce) in Dulbecco's modified PBS. Labeled cells
were washed twice and incubated in serum-free culture medium containing
10 m
M HEPES (Sigma), either at 37 °C or on ice, for 15 min
followed by washing with Dulbecco's modified PBS and three
reductions of 5 min on ice with a solution containing 50 m
M 2-mercaptoethanesulfonic acid (Sigma), 10 m
M NaCl, 1
m
M EDTA, 50 m
M Tris, 0.2% bovine serum albumin, pH
8.6. The cells were lysed with 200 µl of 200 m
M n-octyl-
-
D-glycopyranoside (Sigma), 1
m
M phenylmethylsulfonyl fluoride (Sigma) in Dulbecco's
modified PBS at +4 °C and centrifuged at 12,000
g, for 5 min. After this, an equal volume of 100 m
M Tris, 150 m
M NaCl, 1 m
M CaCl
, 1%
Triton X-100, 0.1% SDS (BDH Ltd), 0.1% Nonidet P-40 (BDH Ltd), pH 7.4,
was added. Preabsorption was done for 30 min at 4 °C by the
addition of 25 µl of a 50% suspension of protein-G-Sepharose 4 fast
flow (Pharmacia) and 1 µl of normal mouse serum to the lysate. The
Sepharose was discarded, and 25 µl of protein-G-Sepharose,
equilibrated with 2 µg of PL98DF6 IgG, was added to the lysate.
After continuous rotation for 2 h at +4 °C, the supernatant
was discarded, the Sepharose was washed 4 times, and bound proteins
were eluted with 70 µl of electrophoresis sample buffer
(35) without reducing agents. A 6.5% acrylamide slab gel was run
and the proteins were transferred to Immobilon membrane (Millipore,
Bedford, MA) using 25 m
M Tris, 192 m
M glycine, 20%
methanol, pH 8.3
(36) . Biotin-labeled proteins were visualized
using 2 µg/ml peroxidase-coupled streptavidin (Pierce) and a
chemiluminescence Western blotting kit (Boehringer Mannheim).
Internalization of Fibrinogen-coated
Beads
Polystyrene beads (5 10
(4.5 µm),
Polysciences, Inc, Warrington, PA) were coated for 20 h at 22 °C
with 100 µg/ml of fibrinogen in 250 µl of 0.25
M Na
B
O
, pH 9.5, followed by
incubation for 20 h at 4 °C with 0.1% bovine serum albumin in PBS.
3
10
cells were seeded in 400 µl of serum-free
media on fibronectin-coated coverslips on 24-well cell culture plates
for 2 h, and 5
10
fibrinogen-coated beads were
added to the wells. After further incubation for 2 h, the cells were
washed with PBS, fixed with 4% paraformaldehyde in PBS, and stained
simultaneously with PL98DF6 and monoclonal anti-fibrinogen followed by
the fluorescein isothiocyanate-labeled secondary antibody. The
proportion of cell bound nonfluorescent beads was determined using a
fluorescence microscope. The samples were coded so that the examiners
could not identify them. The results are shown as mean values from six
different fields containing about 100 beads.
Cell Spreading
To study which residues of the
cytoplasmic domain of are needed for
-induced cell spreading, we
constructed a series of point mutations and COOH-terminal deletions.
The mutations used in this study are listed in . All of the
mutants were expressed in CHO cells together with the
subunit, which is not expressed at the CHO cell surface without
transfected
(17, 23) . Cells
transiently transfected with
and
mutants were allowed to adhere to fibrinogen. All of the
constructs were expressed at similar levels, but only some
transfectants were able to promote cell spreading on fibrinogen
(Fig. 1). Deletions at which the stop codon was inserted at or
before Ile
as well as the point mutations Y747A and S752P
were not able to mediate cell spreading. Few partially spread cells
were detected among these cells. The deletions
758 and
759 as
well as the point mutations F727A/F730L/E733V, S752A, and Y759A were
able to mediate cell spreading on fibrinogen. Double immunofluorescence
studies by using anti-talin (Fig. 2, a, c,
e, and g) or anti-vinkulin (not shown) and
anti-
(Fig. 2, b, d,
f, and h) revealed that the wild-type
(Fig. 2, a and
b) and
expressed together with the
deletion mutations up to the
758 (Fig. 2, g and
h) were able to form and localize to adhesion plaques in cells
cultured on fibrinogen. However, in cells expressing the
757
(Fig. 2, e and f) or further deletion
(
747; Fig. 2, c and d), no such structures
were found. Similarly, cells transiently expressing the point mutations
Y747A (Fig. 3, b and c) or S752P (Fig. 3 d)
did not reveal any adhesion-plaque like structures when cultured on
fibrinogen. Even the few partially spread cells expressing these
mutations (Y747A; Fig. 3 c) lacked adhesion plaques.
However, those point mutations that were able to mediate clear cell
spreading, were also found in adhesion plaques (Fig. 3,
a, e, and f).
Figure 1:
Ability of cells transiently
transfected with and
or its
mutants to spread on fibrinogen. In each case, the percentage of
-expressing cells, determined by
flow cytometry analysis using anti-
, is indicated.
Phase contrast photomicrographs are shown from samples allowed to
adhere on fibrinogen for 2 h. Note that the microscope was focused on
the level of the substratum so that mostly only the adherent cells are
seen. There were no or very few spread cells transfected with
and the
757 deletions of
or
with the mutations Y747A or S752P.
Figure 2:
Double immunofluorescence micrograph of
CHO cells transiently transfected with and some
COOH-terminal deletion mutants of
. Cells were allowed
to adhere on fibrinogen for 2 h and stained with anti-talin
( a, c, e, and g) and
anti-
( b, d, f, and
h). Cells were transfected with wild-type
( a and b), or
with the
mutants
747 ( c and d),
757 ( e and f), or
758
( g and h). Bar, 10
µm.
Figure 3:
Immunofluorescence micrographs with
transiently transfected cells expressing with the
mutants F727A/F730L/E733V ( a), Y747A ( b and c), S752P ( d), S752A ( e), or Y759A
( f). All of the samples were stained with
anti-
. Note clear reactivity at adhesion plaques in
a, e, and f, whereas in b,
c, and d no adhesion plaques were detected either in
round or partially spread cells. Bar, 10
µm.
For more detailed studies
of cell spreading, cell lines stably expressing the various mutants with
were established. When analyzed
by flow cytometry, the expression levels of the different
mutants were similar, only the
S752P- and
728-bearing cells had somewhat higher expression levels
of
than the others (Fig. 4).
By using the stable cell lines, we could verify the results obtained
with the transiently transfected cells, indicating that deletion
mutants up to the
757 and the point mutant Y747A were not able to
mediate cell spreading. The mutants
759,
758,
F727A/F729L/E733V, S752P, and Y759A were not as effective in mediating
cell spreading as wild-type
(Fig. 5). The stable cell
line expressing the S752P mutant behaved slightly differently than
transient transfectants with the same mutation. This was apparently due
to the relatively high expression level of the transfected integrin.
The S752P cell line was able to form small but well organized adhesion
plaques on fibrinogen
(26) . In control experiments, all of the
cell lines spread equally well on fibronectin (not shown). Together,
the experiments using transiently transfected cells and stably
transfected cell lines indicated that a difference of one amino acid
(Ile
) in COOH-terminal deletion mutants or the single
Y747A substitution of
determined whether adhesion
plaques were formed and the cells spread or not. On the other hand, the
point mutation F727A/F729L/E733V and point and deletion mutations
around Tyr
caused small defects in cell spreading, but
still allowed the formation of adhesion plaques.
Figure 4:
Flow
cytometry analysis of stable cell lines expressing
with wild-type
or the
mutants
728,
747,
756,
757,
758,
759,
F727A/F730L/E733V, Y747A, S752A, S752P, or Y759A. In each case,
fluorescence histograms with negative control antibody ( dashed line) or anti-
( solid line) with fluorescein isothiocyanate-labeled secondary
antibody are shown.
Recruitment to Adhesion Plaques
As there may be
differences in the ability of the mutants to mediate de novo formation of adhesion plaques, and to be recruited to
preestablished adhesion plaques, we studied the recruitment by
utilizing the ability of truncated (
996) to
allow ligand-independent movement of
to adhesion plaques
(17) . In this assay, wild-type
or the mutants are expressed transiently in CHO cells
together with the
(
996) cells are cultured on
fibronectin-coated surfaces, fixed, and stained for immunofluorescence
with an anti-
monoclonal antibody. The percentage of
spread positive cells having an adhesion plaque-like localization of
(
996)
was calculated. Fig. 6
shows that all of the mutants except S752A and Y759A were significantly
defective in recruitment to adhesion plaques. A repeatable, but less
significant defect was also detected in the case of the S752A and Y759A
mutants. No correlation was found between the level of transient
expression of the mutants and their ability to be recruited to adhesion
plaques. This suggests that we had managed to mutate residues that are
involved in integrin-cytoskeleton interactions at adhesion plaques.
Constitutive Endocytosis
The tyrosine residues at
positions 747 and 759 of are located in putative
internalization signal sequences
(37, 38) . Because some
integrins are known to be constitutively endocytosed and recycled
(34, 39) , mutations deleting or altering these signals
could also alter the endocytotic cycle of integrins. To study this,
cell lines expressing wild-type
or
with the extensive COOH-terminal deletion of
(
728) were surface-labeled with NHS-SS-biotin at
0 °C, incubated either at 0 or 37 °C for 15 min, and exposed to
a membrane-impermeable reducing agent. Immunoprecipitation followed by
blotting with streptavidin-coupled peroxidase showed that in both cell
lines 10-20% of
became
protected from reduction at 37 °C (Fig. 7 A),
indicating that constitutive endocytosis of
took place in CHO cells,
regardless of the presence or absence of the
cytoplasmic domain. The Y747A (Fig. 7 B) and Y759A
mutants (not shown) were also found to be endocytosed similar to
wild-type
. Thus, neither the
cytoplasmic domain of
nor the sequences
N
PKY
or N
ITY
are needed for the constitutive endocytosis of
in CHO cells.
Figure 7:
Measurement of integrin endocytosis.
Panel A, comparison of wild-type
and
(
728); panel B, comparison of wild-type
and
(Y747A). Sample
1, surface labeling with NHS-SS-biotin without reduction.
Indicated amounts, given as percentage of duplicate samples, were
pipetted on the gel to facilitate quantitation; sample 2,
surface labeling, 15-min incubation at 0 °C, and reduction with
2-mercaptoethanesulfonic acid; sample 3, surface labeling,
15-min incubation at 37 °C, and reduction with
2-mercaptoethanesulfonic acid. Under each lane, corresponding
results of densitometric scanning of the
subunit band
is shown ( OD, optical density, arbitrary units). In
A, comparison of the sample 3 bands to the standards
of sample 1 indicated that 17% of the wild-type and 13% of the
728 mutant were detected (reduction controls in samples 2 subtracted). In B, samples 3 constituted 12% of
the wild-type and 18% of the Y747A mutant.
Phagocytosis of Ligand-coated Particles
Certain
integrins participate in phagocytosis
(40, 41) or in
the internalization of particles coated with their ligands
(42, 43) . To study the effects of the cytoplasmic domain mutations in
-mediated particle internalization,
fibrinogen-coated polystyrene beads were allowed to settle on the
cells, and internalized beads were counted by using immunolabeling
techniques. CHO cells expressing wild-type
, or the mutants S752A and Y759A
internalized 60-80% of the beads in 2 h, whereas a significantly
smaller intake was detected in cells expressing other mutations (Fig.
8). Control experiments showed that the beads bound poorly to parental
CHO cells, and that less than 1% of the bound beads were taken into
untransfected CHO cells under these conditions. The results show that
the
subunit cytoplasmic domain is necessary for
-mediated internalization of
fibrinogen-coated particles, and that the same mutations that cause
defects in recruitment to adhesion plaques also cause defects in
particle internalization.
of
was necessary for
integrin-induced cell spreading and formation of adhesion plaques.
-mediated cell
spreading.
-Glu
and
Tyr
define areas involved in integrin-adhesion plaque
interactions so that mutations at these positions slow down cell
spreading, but still allow formation of adhesion plaques.
is not needed for the
constitutive endocytosis of
in CHO
cells.
is needed for
-mediated internalization of
ligand-coated particles, and the same mutations that affect
recruitment to adhesion plaques
also affect internalization.
integrin subunit.
mutations to be recruited to preestablished adhesion
plaques largely correlated to what has been previously found by using
corresponding mutation in the
integrin subunit
(44) . However, functional differences of COOH-terminal deletion
mutations of
showed that the cytoplasmic domain up to
Ile
is specifically needed for cell spreading. The
mutant, in which Ile
was the first of the deleted amino
acids, was unable to mediate
-dependent formation of adhesion
plaques and cell spreading, but retained partial activity in recruiting
the integrin to preestablished adhesion plaques and in mediating
internalization of ligand-coated particles. Although Ile
of
is not conserved in other integrins, the
sequences around this position are homologous
(V
TTVVNPKY
in
,
T
TTVMNPKF
in
, and
T
STFTNITY
in
).
Which residues of this stretch are critical for cell spreading has not
been studied in the case of other integrins. In point mutation studies
of
and
, this conserved stretch has,
however, been implicated for cytoskeletal interactions
(44) and
integrin activation
(45) . Comparison of the different studies
is difficult, due to different experimental conditions. It is, however,
possible that the area around Ile
of
could be part of a binding site for some signal-transducing
proteins, which are necessary for the formation of adhesion plaques.
Such signal-transducing proteins
(7) may be scarce at adhesion
plaques, and, thus, the addition or removal of the binding site would
not increase integrin recruitment to preestablished structures.
Actually, COOH-terminal deletions ending at amino acid positions
755-758 were all similarly active in the recruitment. Mutations
at Pro
in
, which corresponds to
Ile
of
, have no effect of recruitment
either
(44) . In addition to amino acid homology,
interchangeable functions of the cytoplasmic domains of some integrin
-subunits
(46) favor the hypothesis of a common signaling
function for this area.
, which is
located at the conserved stretch discussed above. A serine to proline
substitution at this position has been found in a patient with
Glanzmann thrombasthenia
(47) . This mutation makes
incapable to be activated by
intracellular signals
(26) and decreases its capacity to
mediate cell spreading (this study and Ref. 26). Here we showed that a
serine to alanine mutation at this position had only minimal effects on
the recruitment of
to adhesion
plaques and had no detectable effects on
-mediated cell spreading.
Previously, substitution of the homologous sequence in
, T
TT, with AAA, has been shown to cause
a major decrease in leukocyte adhesion to ICAM-1, whereas Thr
Ala mutation in
had only a a minor
effect
(45) . It is possible that this area is involved in both
integrin activation and in integrin-cytoskeletal interactions in
and
integrins.
cytoplasmic domain. The same defects were
observed in the COOH-terminal deletion mutants where a stop-codon was
inserted before or at position 755. Thus, the simplest explanation for
the effects of the Y747A mutation is that it disrupts the folding of
the COOH terminus of
. Sequences such as
N
PIY
of
make a
-turn
in several proteins
(37) , and a tyrosine to alanine
substitution in this motif may disrupt this turn. A similar explanation
has been suggested in the case of the corresponding mutation in
(12) .
subunits, we must also consider other than
conformational hypotheses for the functional effects of the Y747A
mutation. There is evidence that talin-integrin interaction is
inhibited by phosphorylation of the corresponding tyrosine in the
subunit
(13, 48) . Tyrosine to
phenylalanine substitution has, however, no effect to the recruitment
of the
subunit to adhesion plaques
(44) . A
synthetic peptide from this area inhibits integrin-talin interaction
(13) . If this is the case also in
, our
results imply that the cytoskeletal interactions mediated by the region
around Tyr
would be absolutely required for
integrin-mediated formation of adhesion plaques. This is further
supported by findings that cytochalasin D inhibits integrin-mediated
signals
(49) .
-Glu
and Tyr
and
COOH-terminal deletions ending at Ile
or T
slowed down
-mediated cell
spreading on fibrinogen but were able to support formation of adhesion
plaques. Furthermore, we found that these mutants were defective in
integrin recruitment to preestablished adhesion plaques. Thus, these
mutations abrogated some, but not all,
-cytoskeletal
interactions. They could still perform functions that need sequences
around Tyr
or before and at Ile
(that is,
the organization of adhesion plaques). This suggests that the mutations
abrogate binding sites for some proteins at adhesion plaques. The area
defined by Phe
-Glu
may be an
interaction site of
-actinin, because peptides from the
corresponding region of
have been found to bind to
this protein
(15) .
(CD11b/CD18) (see Refs. 2 and 40).
This integrin binds to complement-coated particles and cells, and
mediates their phagocytosis. Some pathogens also directly use integrins
for entering the cells
(43, 50) ( e.g. enteropathogenic Yersinia strains, which bind to
integrins through their surface protein invasin
(42) ). It has been proposed that for effective invasion of
bacteria, the bacterial protein-integrin interaction must be of
sufficiently high affinity and that the host cells must be able to
undertake extensive reorganization of the cytoskeleton induced by
ligand binding to integrins
(51) . Our results support this by
showing that similar determinants of the cytoplasmic domain of the
integrins are needed for internalization of ligand-coated particles and
for integrin recruitment to adhesion plaques.
was not
found to be dependent on the cytoplasmic domain of
.
Several integrins are internalized via the endocytotic pathway and are
rapidly recycled back to the cell surface
(34, 39) .
Different integrins seem to have different abilities to take part in
the endocytotic cycle. For example,
,
, and
(CD11b/CD18) were found to circulate in all cells studied,
whereas
,
, and
(CD11a/CD18) did not circulate in any cells
(39) .
Typically, circulating and noncirculating integrins have circulating
rates of 1-2% and less than 0.1% of surface protein/min,
respectively
(39) . The size of the intracellular pool is
8-20% for circulating integrins but less than 1.3% for
noncirculating integrins
(39) . We showed here that 10-20%
of cell surface-labeled
was
internalized in 15 min in spite of the large COOH-terminal deletion of
or the mutations Y747A and Y759A. These values
clearly fall in the range expected for circulating integrins.
758 deletion mutant of
, but do not bind either
to the
757 deletion mutant or the Y747A substitution mutant. It is
possible that binding of these putative factors to integrins represent
early, still unidentified steps in the post-ligand binding events of
integrin function.
Table:
Mutations used in this study
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.