Department of Medicine, and Department of Microbiology and Immunology, University of California, Los Angeles, California 90095
The cytoplasmic domains of integrins are essential for cell adhesion. We report identification of a
novel protein, ICAP-1 (integrin cytoplasmic domain-
associated protein-1), which binds to the 1 integrin cytoplasmic domain. The interaction between ICAP-1
and
1 integrins is highly specific, as demonstrated by
the lack of interaction between ICAP-1 and the cytoplasmic domains of other
integrins, and requires a
conserved and functionally important NPXY sequence
motif found in the COOH-terminal region of the
1 integrin cytoplasmic domain. Mutational studies reveal that Asn and Tyr of the NPXY motif and a Val residue
located NH2-terminal to this motif are critical for the
ICAP-1 binding. Two isoforms of ICAP-1, a 200-amino
acid protein (ICAP-1
) and a shorter 150-amino acid
protein (ICAP-1
), derived from alternatively spliced
mRNA, are expressed in most cells. ICAP-1
is a phosphoprotein and the extent of its phosphorylation is regulated by the cell-matrix interaction. First, an enhancement of ICAP-1
phosphorylation is observed when
cells were plated on fibronectin-coated but not on nonspecific poly-L-lysine-coated surface. Second, the expression of a constitutively activated RhoA protein that
disrupts the cell-matrix interaction results in dephosphorylation of ICAP-1
. The regulation of ICAP-1
phosphorylation by the cell-matrix interaction suggests an important role of ICAP-1 during integrin-dependent
cell adhesion.
INTEGRINS comprise a family of heterodimeric cell adhesion receptors responsible for attachment of cells to
the extracellular matrix or to specific cell surface
counterreceptors (Hynes, 1992 Although the cytoplasmic domains of integrins lack any
known enzymatic activity or sequence motif involved in
protein-protein interaction, studies have shown that the
short cytoplasmic tails of How the integrin A second unresolved issue is whether the adhesive function and cytoskeletal interaction of different integrins are
regulated by a common mechanism or by similar but distinct processes. Despite the remarkable similarities in the
amino acid sequences of different integrin In the present study, we report a novel polypeptide
named ICAP-1 Cell Lines and Antibodies
293, HeLa, Jurkat, K562, and Cos-7 cells were obtained from American
Type Culture Collection (Rockville, MD). SaOS, Rat-1, NIH3T3, 2f-TGH
(human fibroblast cells), and UTA-6 (Englert et al., 1995 Cells expressing constitutively activated RhoA were generated using
UTA-6 (Englert et al., 1995 An anti- Polyclonal rabbit antisera against the Yeast Genetic Screening
The yeast genetic screening for the isolation of proteins interacting with
the cytoplasmic domain of Northern Blot Analysis and cDNA Cloning
The cDNA insert from Clone E16-1 was used as the probe to screen a
multiple tissue mRNA blot (Clontech, Palo Alto, CA). A full-length
cDNA was isolated from a HeLa cell cDNA library (a gift from K. Shuai,
UCLA) using Clone E16-1 as the probe. An expressed sequence tag
(EST) clone corresponding to ICAP-1 In Vitro Interaction Assay
For in vitro GST "pull-down" experiments, the COOH-terminal cytoplasmic domains of Eukaryotic Expression Plasmids
The coding sequences of human The full-length ICAP-1 Eukaryotic Expression and In Vivo Interaction Assay
5-10 µg of plasmid DNA was transfected into 293T cells by using a
Ca2PO4 precipitation method (Ausubel et al., 1994 In Vitro Phosphatase Assay
50 µg NP-40 detergent lysate in 40 µl of 25 mM Tris-HCl, pH 8.0, 50 mM
NaCl, and 10 mM MgCl2, was incubated at 30°C for 30 min, either in the
presence or absence of phosphatase inhibitors (1 mM NaVO3 and 0.5 µM
calyculin A). The reaction was terminated by adding SDS sample buffer
and boiling for 5 min. 15 µg of lysates were analyzed on Western blot using anti-ICAP1 antibody.
Adhesion Assay
Cell adhesion was carried out using six-well tissue culture plates. Each 35-mm well was coated with 1 ml of fibronectin (20 µg/ml) or poly-L-lysine
(PLK) (50 µg/ml) in TBS (25 mM Tris-HCl, pH 8.0, 150 mM NaCl) overnight at 37°C. The coated wells were subsequently blocked with 1% BSA
(Faction V; GIBCO BRL, Gaithersburg, MD) in TBS before the addition
of cells. Adhesion was carried out using 5 × 105 cells per well at 37°C for
15-30 min and bound cells were lysed in 0.2% SDS in TE (25 mM Tris-HCl, pH 8.0, 1 mM EDTA).
Cloning of ICAP-1
A genetic screening based on the protein-protein interaction in yeast (Gyuris et al., 1993 Using the COOH-terminal 21 aa of integrin Table I.
ICAP1: Integrin Cytoplasmic Domain Interaction in Yeast Genetic Screen
). Each subunit consists of a
large extracellular domain that participates in the ligand
recognition, a transmembrane region, and a short cytoplasmic domain. In adherent cells, the ligand binding induces recruitment of integrins to the focal adhesion
plaques or focal contacts, where actin cytoskeletons converge onto the site of cell-extracellular matrix contact (for
review see Burridge and Chrzanowska-Wodnicka, 1996
).
Studies have shown that the integrin-dependent cell adhesion can be regulated either by direct affinity modulation of integrins (Bennett and Vilaire, 1979
; Altieri and Edgington, 1988
; Faull et al., 1993
; Stewart et al., 1996
) or by
clustering of integrins, which requires cytoskeletal rearrangement (Hermanoswki-Vosatka et al., 1988; Haverstick et al., 1992
; van Kooyk et al., 1994
; Stewart et al.,
1996
). Either through recruitment of regulatory proteins
such as adaptor protein Shc or focal adhesion kinase (FAK)1 to the focal contacts or by inducing reorganization
of actin cytoskeleton, integrins function as transmembrane
receptors for extracellular signals and participate in the activation of cytoplasmic signaling cascade (for review see
Schwartz et al., 1995
). The dependence of cell proliferation, prevention of apoptosis, and cell differentiation on
the cell-matrix interaction mediated by integrins illustrates the importance of this adhesion-dependent cell signaling.
or
subunits are important for
the regulation of integrin affinity and cytoskeletal interaction (Sastry and Horwitz, 1993
; Schwartz et al., 1995
). The
cytoplasmic domains of different
subunits are similar in
size and sequence. Mutational analysis of the cytoplasmic
domain of integrin
1 has identified three regions that are
important for the recruitment of integrins to the focal contacts (Marcantonio et al., 1990
; Reszka et al., 1992
). The
first region, located in the membrane-proximal region, is rich in charged residues and predicted to form an
-helical
structure. The second and third region consist of short sequences Asn-Pro-X-Tyr (NPXY). The NPXY motif was initially recognized as a sequence motif required for receptor-mediated endocytosis (Chen et al., 1990
) and represents a
unique structural motif capable of generating a reverse
turn in solution (Bansal and Gierasch, 1991). The two tandem NPXY motifs of the integrins are situated in the
membrane-distal region that is known to undergo alternative splicing (Languino and Ruoslahti, 1992
; Zhidkova et
al., 1995
). Naturally occurring splicing variants of the
1 integrin lacking the NPXY motifs do not localize to the focal
contacts (Balzac et al., 1993
). The first NPXY motif (membrane-proximal), in addition to playing a role in integrin-
cytoskeleton interaction (Reszka et al., 1992
; Ylanne et al.,
1995
), is also involved in affinity regulation of integrins
(O'Toole et al., 1995
) and integrin-dependent endocytic processes (Van Nhieu et al., 1996
). Mutational studies
have shown that the second NPXY motif (membrane-distal), like the first NPXY motif, is important for the focal
contact localization of
1 integrins (Reszka et al., 1992
)
and cell adhesion by the
2 integrins (Hibbs et al., 1991
;
Peter and O'Toole, 1995
).
subunit cytoplasmic domain participates in the regulation of cell-matrix interaction has not
been resolved. The initial molecular models for the adhesion-dependent recruitment of integrins to the focal contacts were based on the observation that talin (Horwitz et
al., 1986
) and
-actinin (Otey et al., 1990
) bind to the
1 integrins. As both of these proteins can bind actin, either directly as in
-actinin or through interaction with vinculin
as in talin, the proposed function of talin and
-actinin in
linking integrins to the cytoskeletal structures remains an
attractive model. Other proteins that have been shown to
bind
1 integrins include FAK (Schaller et al., 1995
), paxillin (Schaller et al., 1995
), and a Ser/Thr kinase (ILK-1)
(Hannigan et al., 1996
). Of these proteins, FAK and
ILK-1, because of their ability to affect cell adhesion and
cell spreading, represent potential regulators of the integrin-matrix interaction.
subunit cytoplasmic domains, each
subunit displays distinct differences in its ability to localize to the focal contacts (Wayner
et al., 1991
; LaFlamme et al., 1994
), to induce tyrosine phosphorylation of cytoplasmic proteins upon surface
clustering (Freedman et al., 1993
), and to participate in
gene induction (Yurochko et al., 1992
). In particular, a direct comparison of
1 and
5 cytoplasmic domains using a
chimeric
1-
5 construct where the cytoplasmic domain of
1 was replaced with that of
5 has demonstrated that the
5 cytoplasmic domain, unlike the
1 counterpart, does not
efficiently direct integrins to the focal contact or promote
cell proliferation (Pasqualini and Hemler, 1994
). Recently
identified
3-endonexin (Shattil et al., 1995
) and cytohesin-1 (Kolanus et al., 1996
), which display restricted binding to the
3 and
2 cytoplasmic domain, respectively,
suggest the presence of proteins that can discriminate the
subtle differences in the amino acid sequence of different
subunits. These
subunit cytoplasmic domain-specific
binding proteins may allow specific regulation of individual
subunits.
(integrin cytoplasmic domain-associated
protein-1) that binds to the
1 integrin cytoplasmic domain. The interaction, which can be demonstrated both in
vitro and in vivo, is specific for the
1 integrins and requires the Asn and Tyr residues of the membrane-distal
NPXY motif. The ability of ICAP-1
to interact only with
the
1 cytoplasmic domain is attributed to an additional requirement of Val residue NH2-terminal to the NPXY.
The functional role of ICAP-1 in cell adhesion is suggested
by the observation that ICAP-1
is a phosphoprotein and
that the degree of phosphorylation is regulated by integrin-dependent, cell-matrix interaction.
Materials and Methods
) were gifts
from C. Sawyers (University of California, Los Angeles, CA [UCLA]),
K. Shuai (UCLA), and D. Haber (Massachusetts General Hospital, Boston, MA).
), a derivative of U2OS cells (osteosarcoma
cell line) expressing tetracycline-repressible transactivator (Gossen and
Bujard, 1992
). An NH2-terminal FLAG epitope-tagged RhoA(Q63L)
(Coso et al., 1995
) was cloned into pTPH-1 (Gossen and Bujard, 1992
).
The expression construct was introduced into UTA-6 cells using a
Ca2PO4-precipitation method (Ausubel et al., 1994
) and hygromycin-
resistant clones were isolated in the presence of 1 µg/ml tetracycline.
1 integrin mAb producing hybridoma cell line, TS2/16, was
generously provided by M. Hemler (Dana Farber Cancer Institute, Boston, MA). A mouse hybridoma cell line TS1/18 producing anti-
1 integrin
mAb were obtained from ATCC. Hybridoma cell lines were cultured in
DME + 10% CPSR-3 (Sigma Chemical Co., St. Louis, MO). mAb from
tissue culture supernatant was purified on protein A-Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ).
subunit of LFA-1 (
L
2) were
prepared by immunizing rabbits with bacterially expressed glutathione-S-transferase (GST) fusion protein containing the entire 58-amino acid
(aa) cytoplasmic domain (aa 1,088-1,145). For the generation of rabbit antisera against ICAP-1, the entire ICAP-1
coding sequences were cloned
in pET16b (Novagen Inc., Madison, WI) and expressed as a histidine-tagged protein in Escherichia coli DE21 (Studier and Moffatt, 1986
). His-tagged ICAP-1
was purified on nickel charged column (Novagen Inc.)
under denaturing conditions following the manufacturer's recommendations and used as the immunogen.
1 integrin was carried out essentially as described previously (Gyuris et al., 1993
). COOH-terminal 21 aa (GENPIYKSAVTTVVNPXYEGK) of the
1 subunit was cloned in frame into
LexA coding sequence to generate a "bait" plasmid pNlex-
1cyto. The resulting Lex-
1cyto fusion protein was able to bind LexA operator in yeast,
but displayed no basal transcriptional activity. A yeast expression library
was generated from oligo dT-primed cDNA from JY cell (human B cell
line) mRNA. The cDNA was cloned unidirectionally into the EcoRI/
XhoI sites of a yeast expression vector pJG4-5. This cDNA library that
had the complexity of >106 was amplified once and used to transform a
yeast strain EGY48 harboring pNlex-
1cyto and JK103 lacZ reporter plasmid. Approximately 2 × 106 independent yeast transformants were
pooled and subjected to selection as described. Eight positive clones obtained all had identical cDNA insert. Plasmid DNA from one isolate
(Clone E16-1) was rescued using E. coli KC8 (Gyuris et al., 1993
) and amplified for further analysis. All bait constructs containing various integrin cytoplasmic domains or
1 integrin cytoplasmic domain mutants were
tested for proper expression in a "suppression assay" in yeast using a reporter construct JK101 (Gyuris et al., 1993
).
-galactosidase activity measurement was carried out as described previously (Ausubel, 1994).
was obtained from the IMAGE
Consortium (these sequence data are available EMBL/GenBank/DDBJ under accession number T69975). The presence of cDNA corresponding to ICAP-1
was independently confirmed by PCR (40 cycles: 94°C for 45 s;
55°C for 45 s; 72°C for 30 s) using primers A3 (5
-CCCAGCAAGATGGAAAGTTGCC-3
) and B2 (5
-GATCAGCATTTTACACAATCCA-3
) flanking the deleted sequences, which generated a 459- (ICAP-1
) or a
shorter 308-bp fragment (ICAP-1
).
1 subunit (see above),
2 subunit (DNPLFKSATTTVMNPKFAES), and
L (KVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLASGQEAGDPGCLKPLHEKDSESGGGD) were individually ex-pressed in bacteria as GST fusion proteins. The EcoRI/XhoI insert of
Clone E16-1 encoding ICAP-1
(aa 54-200) was cloned into the EcoRI/
XhoI site of an in vitro transcription vector pCITE-3a (Novagen, Inc.).
The resulting pCITE-ICAP1
plasmid was used as the template in
cotranscription/translation reaction using T7 RNA polymerase and rabbit
reticulocyte lysate (Promega Corp., Madison, WI) to generate [35S]methionine-labeled polypeptides. An equal amount of labeled polypeptides
was added to ~2 µg of GST fusion proteins bound on glutathione-Sepharose beads (Pharmacia LKB Biotechnology Inc.) and incubated
overnight at 4°C in NET (25 mM Tris-HCl, pH 7.6, 100 mM NaCl, 3 mM
EDTA) containing 1 mM DTT, 1% BSA, and 0.1% Triton X-100. Beads
were washed twice in the binding buffer and twice in 0.05% Triton X-100
in NET. Bound proteins were eluted by boiling in SDS sample buffer and
analyzed by SDS gel electrophoresis.
L integrin (CD11a) and human
2
(CD18) from the expression vectors previously described (Hibbs et al.,
1991
) were cloned into the XbaI site of the eukaryotic expression vector
pcDNA3 (Invitrogen Inc., Madison, WI). To construct a hybrid
2.1 subunit, two PCR-generated fragments corresponding to the amino acids
634-749 of
2 and amino acids 778-798 of
1 were ligated together using a
XcmI site that was introduced during the PCR. This fragment was cloned
into the BstBI and NotI site of pcDNA3/
2 to generate pcDNA/
2.1. The
sequence of the cytoplasmic domain in this hybrid
2.1 subunit consists
of NH3-KALIHLSDLREYRRFEKEKLKSQWNGENPIYKSAVTTVV- NPXYEGK-COOH.
cDNA was cloned into the EcoRV site of
pcDNA3 to generate pcDNA/ICAP-1
. To generate the full-length ICAP-1
coding sequence, the 5
half of the above PCR fragment and the
3
half of a second PCR fragment amplified from T69975 clone (see
above) were ligated in frame using a unique HpaII restriction site. The ligation product was cloned into the EcoRV site of pcDNA3 to produce
pcDNA/ICAP-1
. The GST-tagged ICAP-1 constructs were generated
using eukaryotic expression vector pEBG. The coding sequence of the
partial ICAP-1
(aa 54-200) was derived from Clone E16-1.
). 48 h after transfection, cells were lysed in TBSM (25 mM Tris-HCl, pH 7.6, 150 mM NaCl,
and 2 mM MgCl2) containing 0.5% NP-40, leupeptin, aprotinin, and
PMSF. Detergent insoluble materials were removed by centrifugation at
12,000 g for 15 min. 500 µg of cleared lysates were mixed with an equal
volume of TBSM to reduce the final detergent concentration to 0.25%
NP-40 and incubated with glutathione-Sepharose beads for 3 h at 4°C.
Beads were washed with TBSM + 0.25% NP-40 once, TBSM + 0.1% NP-40 twice, and TBSM alone twice. Coprecipitation of
1 integrins with
bound GST-ICAP1 was determined on a Western analysis using mAb
TS2/16 (anti-
1 integrin).
Results
) was used to identify
polypeptides that interact with the
1 integrin cytoplasmic
domain. Of the 47 aa that comprise the entire
1 integrin
cytoplasmic domain, the COOH-terminal 21 aa were
cloned in frame into the DNA-binding domain of bacterial
protein LexA. Mutational studies and analysis of naturally
occurring splicing variants lacking this 21-aa region have
shown that this region is involved in the localization of
1
integrins to the focal contacts and proper adhesive function of integrins (Balzac et al., 1993
; Meredith et al., 1995
).
1 as the
bait, a ~1.1-kb partial cDNA was isolated in a yeast two-hybrid screening of a human B cell cDNA library. This
partial cDNA insert was used to obtain a full-length
cDNA encoding a polypeptide of 200 aa. In a yeast two-hybrid assay, both the original partial cDNA (aa 154-200)
and the full-length cDNA exhibited comparable interaction with the
1 integrin cytoplasmic domain but not with
unrelated baits (Table I). In addition, the partial or full-length cDNA clones interacted with baits containing either the
1 integrin COOH-terminal 21 aa or a longer 47 aa that comprise the complete cytoplasmic domain. The
interaction was specific towards only the
1 integrin as neither the partial nor the full-length cDNA clone interacted
with the
2,
3, or
5 integrin cytoplasmic domain. The isolated clone hereon will be referred as ICAP-1
.
The deduced amino acid sequence of ICAP-1 was unrevealing except for the preponderance (especially in the
NH2-terminal region) of Ser and Thr, several of which represented potential phosphorylation sites (Fig. 1 A). In particular, Ser20, Ser46, and Ser197 represent potential phosphorylation sites by protein kinase C (Woodgett et al.,
1986
), and Ser10 is present in sequence context favorable
for phosphorylation by cyclic nucleotide dependent protein kinases (Glass et al., 1986
). The initiation codon ATG
in the sequence was preceded by an in frame termination
codon and was present in correct sequence context for
translational initiation (Kozak, 1992
). Although clones corresponding to the ICAP-1
cDNA were represented in
the National Center for Biotechnology Information (NCBI)
EST database, analyses of the NCBI Non-Redundant database using the BLAST Enhanced Alignment Utility algorithm failed to identify any significant similarities to
known proteins.
In Northern analyses, ICAP-1 transcripts of 0.9 and 1.3 kb in size were detected in mRNA isolated from several
tissues (Fig. 1 B). The
1 integrins are ubiquitously expressed and ICAP-1, as a
1 integrin-binding protein, is
expected to have a similar broad expression in various tissues and cell lines. The differences in the size of two transcripts probably reflect the usage of two different polyadenylation sites observed during cDNA sequence analysis.
In Western analyses using polyclonal anti-ICAP1 antibodies, two distinct polypeptides of 20 and 16 kd were detected (Fig. 1 C, lane 1). In hypotonic cell lysis, ICAP-1
fractionates in the cytosolic fraction suggesting ICAP-1
is
a cytoplasmic protein (data not shown). Expression of
ICAP-1
cDNA in 293T cells produced the exact 20-kD
polypeptides (Fig. 1 C, lane 2). The shorter 16-kD species
likely represent ICAP-1
, an alternatively spliced variant of ICAP-1
lacking internal 50 aa (Fig. 1 D). A cDNA
corresponding to ICAP-1
mRNA was recognized during
the database search. The presence of ICAP-1
cDNA in
several human cDNA libraries was subsequently confirmed by PCR analyses (data not shown). The expression
of ICAP-1
cDNA in 293T cells, as expected, produced
the exact 16-kD polypeptide that comigrated with the endogenous ICAP-1
polypeptides (Fig. 1 C, lane 3). Interestingly, ICAP-1
did not interact with the integrin
1 cytoplasmic domain in a yeast two-hybrid assay (data not
shown).
1 Integrin Cytoplasmic Domain Restricted Binding
of ICAP-1
To verify the specific interaction between ICAP-1 and
the
1 integrin cytoplasmic domain seen in the yeast genetic screening, in vitro-translated ICAP-1
was incubated with various integrin cytoplasmic domains expressed in bacteria as GST fusion proteins (Fig. 2 A).
After incubation, GST fusion proteins were isolated on
glutathione-Sepharose beads and the bound ICAP-1
polypeptides were analyzed by a SDS gel electrophoresis.
As in a yeast two-hybrid assay, ICAP-1
was bound only
to the GST-
1 fusion protein and not to the GST-
2 or
GST-
L fusion proteins.
Reciprocal GST pull-down experiments were used to
demonstrate the interaction between ICAP-1 and endogenous
1 integrins in vivo. GST epitope-tagged ICAP-1
was transiently expressed in 293T cells. Glutathione-
Sepharose beads were used to purified GST-ICAP1
and
copurification of endogenous
1 integrins was tested on a
Western analysis using anti-
1 antibody. Confirming the
results of the yeast two-hybrid assays and in vitro binding
studies,
1 integrins copurified with GST-ICAP1
, but not
with unrelated GST fusion proteins or GST alone (Fig. 2 B).
Finally, to demonstrate that ICAP-1 is a specific
1 integrin binding protein in vivo, GST epitope-tagged ICAP-1
was coexpressed in 293T cells with the integrin
2 subunit
or a chimeric
2 subunit (
2.1) constructed by replacing the
COOH-terminal 20 aa region of the
2 subunit with the
corresponding sequences of the
1 subunit. The expression
of
2 integrins is limited to cells of hematopoietic origin
and 293T cells completely lack expression of
2 integrins (Liu, J., and D. Chang, unpublished observation). To ensure a proper cell surface expression, the
L subunit capable of forming stable heterodimer with the
2 subunit was
also expressed. A comparable surface expression of
L
2 and
L
2.1 was seen on FACS® analysis (data not shown). After precipitation of GST-ICAP1
on glutathione-Sepharose
beads, copurification of
2 integrin was indirectly tested
in Western analysis using anti-
L antibody. In GST pull-down experiments, as predicted from the yeast two-hybrid assays and in vitro binding assays, only
L
2.1 and not
L
2
copurified with ICAP-1
(Fig. 2 C). These findings demonstrate the ability of ICAP-1
to interact with integrin
heterodimer in a
1 cytoplasmic domain-dependent
fashion.
Conserved NPXY Motif of Integrin 1 Cytoplasmic
Domain is Required for the ICAP-1
Binding
ICAP-1 interacts with the
1 integrins through the
COOH-terminal 21 aa region. Naturally occurring, alternatively spliced variants of
1 integrins, which lack this region or mutant
1 integrins containing amino acid substitutions at either of the two conserved NPXY motifs do not
localize to the focal contacts (Reszka et al., 1992
; Balzac et
al., 1993
). Furthermore, mutations in the analogous region
of
2 or
3 integrins affect the leukocyte integrin
L
2
(LFA-1)-dependent cell adhesion to intercellular cell adhesion molecules-1 or the affinity regulation of the platelet integrin
IIb
3 (Hibbs et al., 1991
; O'Toole et al., 1995
; Peter and O'Toole, 1995
).
The exact ICAP-1 binding site within the
1 integrin
cytoplasmic domain was further delineated by testing the
interaction between ICAP-1
and truncated
1 integrin
cytoplasmic domain (Table II). NH2-terminal deletions up
to 8 aa did not affect the interaction with ICAP-1
. A 3-aa
deletion in the COOH terminus, however, completely abolished the interaction. Therefore, a minimum binding
site for ICAP-1
on the
1 integrin cytoplasmic must consist of this 13-aa region that included one of the two
NPXY motifs. The NPXY motif is required for the interaction as amino acid substitutions at the conserved Asn to
Glu or Ala abolished the binding. Similarly, a substitution
of Tyr to Ala disrupted the ICAP-1
binding whereas a
more conservative replacement of Tyr to Phe had no effect.
Table II.
ICAP1: |
In yeast interaction and in vitro binding assays,
ICAP-1 failed to interact with the
2 integrin cytoplasmic
domain, which is closely related to the
1 integrin cytoplasmic domain in sequence (Table III). The molecular basis
of this highly discriminatory binding specificity of ICAP-1
was investigated by progressively mutating the
2 integrin
cytoplasmic domain. There are two conserved and four
nonconserved amino acid differences between the
1 and
2 integrins within the minimum 13 aa ICAP-1
binding
region. A replacement of three consecutive nonconserved
residues found in the COOH terminus of
2 integrin from
Ala-Glu-Ser to Glu-Gly-Lys found in the
1 subunit did
not allow the ICAP-1
binding. Interestingly, a single
amino acid replacement at the
11 position from Thr to Val allowed the mutant
2 integrin to interact with ICAP-1
, demonstrating that in addition to the NPXY motif, Val
at the
11 position is essential for the interaction between
1 integrins and ICAP-1
.
Table III.
ICAP1: |
ICAP-1 Is a Phosphoprotein
In addition to the 20- and 16-kD polypeptides corresponding to ICAP-1 and ICAP-1
, heterogeneous bands migrating slower than the 20-kD band were detected in
Western analysis of 293T cell lysates using polyclonal anti-ICAP1 antisera (data not shown). Because the amino acid
composition of ICAP-1 is rich in Ser and Thr, the possibility that this mobility difference is due to protein phosphorylation was raised. When total detergent lysates of several different cell lines were surveyed by Western analysis,
there was a significant variation in the relative amount of
slow migrating species (Fig. 3 A). In particular, two osteosarcoma cell lines, SaOS (Fig. 3 A, lane 2) and UTA-6
(Fig. 3 A, lane 3), displayed high abundance of the slow
migrating species. To directly confirm that the slow migrating species represented phosphorylated forms of
ICAP-1
, UTA-6 cell lysates incubated at 30°C to activate
endogenous phosphatases (Fig. 3 B). The slow migrating
species disappeared with a concomitant increase in the 20 kD ICAP-1
upon 30°C incubation (Fig. 3 B, lane 3). The
addition of phosphatase inhibitors, sodium vanadate, and
calyculin A, completely prevented the conversion of the
slow migrating species to the 20 kD ICAP-1
(Fig. 3 B, lanes 2 and 4), confirming the mobility difference is due to
protein phosphorylation. Interestingly, during in vitro
phosphatase experiment, we failed to detect any mobility
shift in ICAP-1
, suggesting that either ICAP-1
is not
phosphorylated or the mobility of ICAP-1
does not
change significantly by protein phosphorylation.
Regulation of ICAP-1 Phosphorylation by
Integrin-dependent Cell-Matrix Interaction
Many focal contact proteins, FAK, paxillin, and tensin being a few representative ones, have been shown to undergo protein phosphorylation during cell attachment
(Clark and Brugge, 1995). The possibility that the phosphorylation of ICAP-1
may be regulated in a similar
manner during cell attachment was directly addressed by
binding UTA-6 cells to either PLK or fibronectin (FN)-
coated surface (Fig. 4). Western analyses of cell lysates revealed an enhanced phosphorylation of ICAP-1
when
cells adhere to the FN-coated surface. The maximum enhancement occurred within the minimum 30 min required
for sufficient number of cells to adhere and undergo cell
spreading (Fig. 4, lanes 2 and 4). A longer incubation did not further enhance ICAP-1
phosphorylation (data not
shown). This enhancement was specific for cell attachment
to FN-coated surface, which requires
1 integrins. On
PLK-coated surface, cells adhere very efficiently, but fail
to initiate subsequent cell spreading. Under these conditions, the phosphorylation of ICAP-1
remained at a reduced level (Fig. 4, lanes 1 and 3).
During integrin-dependent cell adhesion, the rearrangement of cytoskeletal actin stress fibers and assembly of focal adhesion plaques are regulated by Rho-family GTPases
(Hall, 1994; Burridge and Chrzanowska-Wodnicka, 1996
).
In UTA-6 derivative cell lines expressing constitutively
activated RhoA(Q63L) under the control of tetracycline-
inducible system, the induction of RhoA(Q63L) expression results in the formation of dense stress fibers and increase in the number of focal adhesion plaques (Wong, C.,
and D. Chang, manuscript in preparation). These cells display a significantly delayed and incomplete cell spreading
when plated on FN-coated surface (Fig. 5 A). To test
whether the alteration in cell-matrix interaction induced
by the expression of RhoA(Q63L) had any effect on the
phosphorylation status of ICAP-1
, a Western analysis
was carried out using the lysates from RhoA(Q63L) expressing cells. The lysates prepared from cells grown in the
presence of tetracycline, which represses the expression of
RhoA(Q63L), were used as controls. Fig. 5 B shows that
the expression of constitutively activated RhoA(Q63L) reduces the extent of ICAP-1
phosphorylation in two independently derived cell lines. Thus, taken together, these findings suggest that phosphorylation of ICAP-1
is regulated during integrin-dependent cell adhesion and spreading.
Integrins, through binding to both extracellular matrix
proteins and cytoskeletal structures, provide a direct linkage between the extracellular environment and the cell interior. The cytoplasmic domains of integrins have been implicated in the integrin affinity regulation and localization
of integrins to the focal contacts. Understanding how the
short cytoplasmic tails of integrins affect the functions of
integrins requires characterization of cellular proteins that
bind, either directly or indirectly, to the integrin cytoplasmic domains. To this end, we have used a yeast two-hybrid screen to identify proteins that directly bind to the 1 integrin cytoplasmic domain. In particular, we have focused
on the COOH-terminal 21-aa region of the
1 integrin,
which is required for the localization of integrins to the focal contacts. In this study, we report identification and
characterization of ICAP-1
, a novel 200-aa protein that
specifically interacts with the
1 integrin cytoplasmic domain through a conserved and functionally important
NPXY sequence motif. In addition, ICAP-1
undergoes
protein phosphorylation that is subject to regulation, suggesting that ICAP-1
plays an important role during integrin-dependent cell adhesion.
The broad tissue distribution of ICAP-1 mRNA and
the detection of ICAP-1
protein in cell lines of various
tissue origins indicate ICAP-1
, like
1 integrins, is ubiquitously expressed. In contrast, the expression of ICAP-1
,
an alternatively spliced variant of ICAP-1
that does not
bind to the
1 integrin cytoplasmic domain, was more variable in cell lines we have tested. In particular, the ICAP-1
expression was low or absent in three cell lines, SaOS
(human osteosarcoma line), TPH-1 (human monocytic
line), and Cos-7 (monkey kidney line). The differences in
the ability to interact with the
1 cytoplasmic domain and
in the expression level provide a mechanism for regulation
of ICAP-1
function by ICAP-1
. It is noteworthy that
3-endonexin likewise has an alternatively spliced variant
that does not interact with the
3 integrin cytoplasmic domain (Shattil et al., 1995
).
The amino acid sequence of ICAP-1 is unique and displays no similarities to any known proteins. Several proteins, including known focal contact proteins,
-actinin,
paxillin, talin, and FAK, as well as recently identified potential regulatory proteins such as
3-endonexin, ILK-1,
and cytohesin-1 have been shown to bind integrins
through the
subunit cytoplasmic domain (for review see
Sastry and Horwitz, 1993
; Shattil et al., 1995
; Hannigan et al., 1996
; Kolanus et al., 1996
). In contrary to
-actinin,
FAK, and ILK-1, which interact with more than one type
of
subunit, the interaction of ICAP-1
is restricted to the
1 subunit only. This property of ICAP-1
is similar to two
recently identified proteins,
3-endonexin and cytohesin-1,
which bind specifically to integrins
3 and
2, respectively
(Shattil et al., 1995
; Kolanus et al., 1996
). As these proteins
are not related in sequence to each other or to ICAP-1
, it
remains to be seen how these unrelated proteins are coupled to the
integrin cytoplasmic domains, which are related and functionally interchangeable in some cases.
The results from our mutagenesis studies indicate that
the COOH-terminal 13-aa region of the 1 subunit, which
includes a conserved NPXY motif, is sufficient to bind
ICAP-1
. Several studies have shown that this region, especially the NPXY motif, is important for the recruitment
of
1 integrins to the focal contacts (Marcantonio et al.,
1990
; Reszka et al., 1992
; Peter and O'Toole, 1995
; Ylanne et al., 1995
) and the colocalization of talin, FAK, and actin with
1 integrins (Lewis and Schwartz, 1995
). Our finding
that Asn to Ala (or Glu), or Tyr to Ala substitution within
the NPXY motif completely abolished the interaction between ICAP-1
and integrins, while a more conservative
replacement of Tyr with Phe had no effect, demonstrates a
remarkable similarity between the sequence requirement
for the binding of ICAP-1
to integrins and localization of
1 integrins to the focal contacts and suggests that ICAP-1
may play a role in the recruitment of
1 integrins to the
focal contacts. Alternatively, the COOH-terminal 13-aa region may be involved in initiating signal transduction
events required to trigger cell spreading and ICAP-1
may
participate in the initiation of this signaling event. As
2,
3, and
5 integrin cytoplasmic domains (which do not
bind ICAP-1
) can also direct integrins to the focal contacts (LaFlamme et al., 1994
; Peter and O'Toole, 1995
),
there must be parallel mechanisms for recruiting various
integrins to the focal contacts.
The COOH-terminal regions of different integrins are
somewhat diverse in amino acid sequence, which may explain in part the observed specificity of ICAP-1
interaction with the
1 integrin cytoplasmic domain. The basis of
this restricted specificity, however, was attributed to a single Val residue NH2-terminal to the conserved NPXY motif. In
2 or
3 subunits, the corresponding position is occupied by Thr (Sastry and Horwitz, 1993
). Confirming the
importance of Val at this position, the replacement of the Thr with a Val in the
2 subunit cytoplasmic tail allowed it
to bind ICAP-1
. The
3 integrin cytoplasmic domain, in
addition, has a less conserved NITY in place of the NPXY
motif, which may also account for the lack of interaction
with ICAP-1
. A recent study on the amino acid sequence
requirement for the interaction between
3-endonexin and
3 integrin cytoplasmic domain indicated that the NITY
motif is critical for the binding specificity (Eigenthaler et
al., 1997
). Altogether these findings suggest that the
COOH-terminal regions of different
integrins may constitute specific binding sites for different cytoplasmic proteins. In addition, the existence of integrin
subunit specific binding proteins indicates that the functions of
individual subunits can be differentially regulated.
The presence of ICAP-1 immunoreacting species,
which migrated slower than the expected molecular
weight of 20 kD predicted from the ICAP-1
reading
frame suggested that ICAP-1
undergoes posttranslational modification. The conversion of slower migrating species to the expected 20-kD species during 30°C incubation, which activates the endogenous phosphatases present
in nonionic detergent lysates, and the observation that this
conversion can be effectively blocked by the addition of
known phosphatase inhibitors demonstrate that ICAP-1
is a phosphoprotein.
The most intriguing property of ICAP-1 is that its
phosphorylation is regulated during cell adhesion and by
RhoA protein. We suspect that the effect on ICAP-1
phosphorylation seen during cell adhesion on FN-coated
surface and in cells expressing constitutively activated
RhoA(Q63L) are related events, reflective of the cytoskeletal rearrangement that occurs during cell adhesion. It is
well known that cells, soon after making an initial contact
on FN-coated surface, undergo cell spreading that involves
the formation of focal adhesion plaques and actin stress fibers (for review see Hall, 1994
). Both these events are
known to be regulated by RhoA (Ridley and Hall, 1992
;
Nobes and Hall, 1995
). Furthermore, both matrix assembly and cell spreading are integrin-dependent processes and require integrin cytoplasmic domain (for review see
Burridge and Chrzanowska-Wodnicka, 1996
; LaFlamme
et al., 1994
). On a nonspecific PLK-coated surface, cells
make initial contact but fail to promote matrix assembly
and initiate spreading. Cells expressing RhoA(Q61L) also
display a delayed and incomplete spreading, presumably
due to the interference from dense stress fibers. According
to this scenario, the diminished ICAP-1
phosphorylation
observed during cell adhesion on PLK-coated surface and
in RhoA(Q63L) expressing cells is a result of inefficient
cell spreading. Our findings, however, do not rule out the
possibility that phosphorylation of ICAP-1
may be a direct consequence of the interaction between integrins and
fibronectin. Regardless of the exact mechanism underlying the control of ICAP-1
phosphorylation, our data clearly
demonstrate that the phosphorylation of ICAP-1
is regulated during the cell-matrix interaction.
It remains to be seen whether the enhancement in
ICAP-1 phosphorylation during cell attachment involves
Ser/Thr phosphorylation or Tyr phosphorylation. Although Ser/Thr phosphorylation is suspected based on the
amino acid composition of ICAP-1
(21% Ser/Thr) and the presence of potential protein kinase C and protein kinase A phosphorylation sites, the exact amino acid residues that are phosphorylated are not known. We have not
been able to demonstrate the immunoreactivity of
ICAP-1
with PY20 anti-phosphotyrosine antibody (data
not shown).
In summary, we present initial characterization of
ICAP-1, a novel
1 integrin cytoplasmic domain binding
protein. Two observations, (a) the binding of ICAP-1
to
a conserved region of
1 integrin cytoplasmic domain that
previously has been shown to be important for the adhesive function and focal contact localization of integrins, and (b) the extent of ICAP-1
phosphorylation is regulated during cell-matrix interaction, suggest that ICAP-1
plays a role during integrin-dependent cell adhesion.
Integrin cytoplasmic domains likely contain overlapping
binding sites for both structural and regulatory proteins
that coordinate cell adhesion and subsequent cytoskeletal
rearrangement. The identification of ICAP-1
together with the characterization of the sequences on
1 integrin
cytoplasmic domain required for the ICAP-1
binding
should facilitate future studies on how specific integrin-
dependent cellular events are regulated.
Received for publication 23 April 1997 and in revised form 3 July 1997.
Please address all correspondence to David D. Chang, UCLA School of Medicine, Division of Heme-Onc, Factor 11-934, 10833 Le Conte Avenue, Los Angeles, CA 90095. Tel.: (310) 825-9759. Fax: (310) 825-6192. e-mail: dchang{at}medicine.medsch.ucla.eduWe are grateful to R. Brent (Massachusetts General Hospital, Boston, MA) for providing the yeast strains and plasmids for the yeast genetic screening, to J. Gutkind (National Institutes of Health, Bethesda, MD), for providing mAbs, cell lines, and DNA constructs. We thank T. Kim for technical assistance and K. Shuai for helpful comments on the manuscript.
This work was supported by the grants from Searle Scholars Program/ The Chicago Community Trust, James S. McDonnell Foundation, and Jonnson Comprehensive Cancer Center of UCLA.
aa, amino acid; FAK, focal adhesion molecule; FN, fibronectin; GST, glutathione-S-transferase; ICAP-1, integrin cytoplasmic domain-associated protein-1; PLK, poly-L-lysine.
1. |
Altieri, D.C., and
T.S. Edgington.
1988.
The saturable high affinity association
of factor X to ADP-stimulated monocytes defines a novel function of the
Mac-1 receptor.
J. Biol. Chem.
263:
7007-7015
|
2. | Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1994. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York. |
3. |
Balzac, F.,
A.M. Belkin,
V.E. Koteliansky,
Y.V. Balabanov,
F. Altruda,
L. Silengo, and
G. Tarone.
1993.
Expression and functional analysis of a cytoplasmic domain variant of the ![]() |
4. | Bansal, A., and L.M. Geirasch. 1991. The NPXY internalization signal of the LDL receptor adopts a reverse-turn conformation. Cell. 67: 1195-1201 |
5. | Bennett, J.S., and G. Vilaire. 1979. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J. Clin. Invest. 64: 1393-1401 |
6. | Burridge, K., and M. Chrzanowska-Wodnicka. 1996. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12: 463-519 . |
7. |
Chen, W.-J.,
J.L. Goldstein, and
M.S. Brown.
1990.
NPXY, a sequence often
found in cytoplasmic tails, is required for coated pit-mediated internalization
of the low density lipoprotein receptor.
J. Biol. Chem.
265:
3116-3123
|
8. | Clark, E.A., and J.S. Brugge. 1995. Integrins and signal transduction pathways: the road taken. Science (Wash. DC). 268: 233-239 |
9. | Coso, O.A., M. Chiariello, J.C. Yu, H. Teramoto, P. Crespo, N. Xu, T. Miki, and J.S. Gutkind. 1995. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 81: 1137-1146 |
10. |
Eigenthaler, M.,
L. Hofferer,
S. J. Shattil, and
M. H. Ginsberg.
1997.
A conserved sequence motif in the integrin ![]() ![]() |
11. | Englert, C., X. Hou, S. Maheswaran, P. Bennett, C. Ngwu, G.G. Re, A.J. Garvin, M.R. Rosner, and D.A. Haber. 1995. WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. EMBO (Eur. Mol. Biol. Organ.) J. 14: 4662-4675 [Abstract]. |
12. |
Faull, R.J.,
N.L. Kovach,
J.M. Harlan, and
M.H. Ginsberg.
1993.
Affinity modulation of integrin ![]() ![]() |
13. |
Freedman, A.S.,
K. Rhynhart,
Y. Nojima,
J. Svahn,
L. Eliseo,
C.D. Benjamin,
C. Morimoto, and
E. Vivier.
1993.
Stimulation of protein tyrosine phosphorylation in human B cells after ligation of the ![]() |
14. |
Glass, D.B.,
M.R. el-Maghrabi, and
S.J. Pilkis.
1986.
Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases.
J. Biol. Chem.
261:
2987-2993
|
15. | Gossen, M., and H. Bujard. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA. 89: 5547-5551 [Abstract]. |
16. | Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell. 75: 791-803 |
17. | Hall, A.. 1994. Small GTP-binding proteins and the regulation of the actin cytoskeleton. Annu. Rev. Cell Biol. 10: 31-54 . |
18. |
Hannigan, G.E.,
C. Leung-Hagesteijn,
L. Fitz-Gibbon,
M.G. Coppolino,
G. Radeva,
J. Filmus,
J.C. Bell, and
S. Dedhar.
1996.
Regulation of cell adhesion and anchorage-dependent growth by a new ![]() |
19. | Haverstick, D.M., H. Sakai, and L.S. Gray. 1992. Lymphocyte adhesion can be regulated by cytoskeleton-associated, PMA-induced capping of surface receptors. Am. J. Physiol. 262: 916-926 . |
20. |
Hermanowski-Vosatka, A.,
P.A. Detmers,
O. Gotze,
S.C. Silverstein, and
S.D. Wright.
1988.
Clustering of ligand on the surface of a particle enhances adhesion to receptor-bearing cells.
J. Biol. Chem.
263:
17822-17827
|
21. |
Hibbs, M.L.,
S. Jakes,
S.A. Stacker,
R.W. Wallace, and
T.A. Springer.
1991.
The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1![]() |
22. |
Horwitz, A.,
K. Duggan,
C. Buck,
M. C. Beckerle, and
K. Burridge.
1986.
Interaction of plasma membrane fibronectin receptor with talin![]() |
23. | Hynes, R.O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25 |
24. | Kolanus, W., W. Nagel, B. Schiller, L. Zeitlmann, S. Godar, H. Stockinger, and B. Seed. 1996. Alpha L beta 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell. 86: 233-242 |
25. | Kozak, M.. 1992. Regulation of translation in eukaryotic systems. Annu. Rev. Cell Biol. 8: 197-225 . |
26. | LaFlamme, S.E., L.A. Thomas, S.S. Yamada, and K.M. Yamada. 1994. Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126: 1287-1298 [Abstract]. |
27. |
Languino, L.R., and
E. Ruoslahti.
1992.
An alternative form of the integrin beta
1 subunit with a variant cytoplasmic domain.
J. Biol. Chem.
267:
7116-7120
|
28. | Lewis, J.M., and M.A. Schwartz. 1995. Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants. Mol. Biol. Cell. 6: 151-160 [Abstract]. |
29. |
Marcantonio, E.E.,
J.-L Guan,
J.E. Trevithick, and
R.O. Hynes.
1990.
Mapping
of the functional determinants of the integrin ![]() |
30. |
Meredith, J. Jr,
Y. Takada,
M. Fornaro,
L.R. Languino, and
M.A. Schwartz.
1995.
Inhibition of cell cycle progression by the alternatively spliced integrin
![]() |
31. | Nobes, C.D., and A. Hall. 1995. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 81: 53-62 |
32. |
Otey, C.A.,
F.M. Pavalko, and
K. Burridge.
1990.
An interaction between ![]() ![]() |
33. |
O'Toole, T. E.,
J. Ylanne, and
B. M. Culley.
1995.
Regulation of integrin affinity states through an NPXY motif in the beta subunit cytoplasmic domain.
J.
Biol. Chem.
270:
8553-8558
|
34. |
Pasqualini, R., and
M. E. Hemler.
1994.
Contrasting roles for integrin ![]() ![]() |
35. |
Peter, K., and
T.E. O'Toole.
1995.
Modulation of cell adhesion by changes in ![]() ![]() |
36. |
Reszka, A. A.,
Y. Hayashi, and
A. F. Horwitz.
1992.
Identification of amino
acid sequences in the integrin ![]() |
37. | Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 70: 389-399 |
38. | Sastry, S.K., and A.F. Horwitz. 1993. Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr. Opin. Cell Biol. 5: 819-831 |
39. |
Schaller, M.D.,
C.A. Otey,
J.D. Hildebrand, and
J.T. Parsons.
1995.
Focal adhesion kinase and paxillin bind to peptides mimicking ![]() |
40. | Schwartz, M.A., M.D. Schaller, and M.H. Ginsberg. 1995. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11: 549-599 . |
41. |
Shattil, S.J.,
T. O'Toole,
M. Eigenthaler,
V. Thon,
M. Williams,
B.M. Babior, and
M.H. Ginsberg.
1995.
![]() ![]() |
42. | Stewart, M.P., C. Cabanas, and N. Hogg. 1996. T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1. J. Immunol. 156: 1810-1817 [Abstract]. |
43. | Studier, F.W., and B.A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130 |
44. | van Kooyk, Y., P. Weder, K. Heije, and C.G. Figdor. 1994. Extracellular Ca2+ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion. J. Cell Biol. 124: 1061-1070 [Abstract]. |
45. |
Van Nhieu, G.T.,
E.S. Krukonis,
A.A. Reszka,
A.F. Horwitz, and
R.R. Isberg.
1996.
Mutations in the cytoplasmic domain of the integrin ![]() |
46. |
Wayner, E.A.,
R.A. Orlando, and
D.A. Cheresh.
1991.
Integrin, ![]() ![]() ![]() ![]() |
47. | Woodgett, J.R., K.L. Gould, and T. Hunter. 1986. Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements. Eur. J. Biochem. 161: 177-184 [Abstract]. |
48. |
Ylanne, J.,
J. Huuskonen,
T.E. O'Toole,
M.H. Ginsberg,
I. Virtanen, and
C.G. Gahmberg.
1995.
Mutation of the cytoplasmic domain of the integrin beta 3 subunit. Differential effects on cell spreading, recruitment to adhesion
plaques, endocytosis, and phagocytosis.
J. Biol. Chem.
270:
9550-9557
|
49. | Yurochko, A.D., D.Y. Liu, D. Eierman, and S. Haskill. 1992. Integrins as a primary signal transduction molecule regulating monocyte immediate-early gene induction. Proc. Natl. Acad. Sci. USA. 89: 9034-9038 [Abstract]. |
50. |
Zhidkova, N.I.,
A.M. Belkin, and
R. Mayne.
1995.
Novel isoform of ![]() |