(Received for publication, September 20, 1996, and in revised form, December 3, 1996)
From the The mechanism by which platelets regulate the
function of integrin Integrin-mediated adherence of cells to extracellular matrix is
important for a variety of physiological processes, such as hemostasis,
angiogenesis, and cell differentiation (1). Integrin The role of the Fibrinogen binding to We have identified a novel Ca2+-binding protein that
interacts specifically with the integrin The cytoplasmic domains of integrin
subunits were amplified using polymerase chain reaction
(PCR)1 from corresponding plasmid
constructs and directionally cloned into pGBT9, a yeast expression
vector (Clontech, Palo Alto, CA). DNA sequencing (UNC-CH Automated DNA
Sequencing Facility) indicated an in-frame fusion of each cytoplasmic
domain to the 3 Human cDNA libraries derived
from fetal liver in pGAD10 (Clontech) were screened in the yeast
two-hybrid system (9, 10), for The CIB cDNA insert
was subcloned into pGEX 2T (Pharmacia Biotech Inc.). The in-frame
fusion was confirmed by DNA sequencing. The GST-fusion protein was
purified by glutathione-Sepharose (Pharmacia) affinity chromatography
following elution with 10 mM reduced glutathione. Recombinant CIB was released from GST by cleavage of the GST fusion protein while on the column with 10 units of thrombin as described by
the manufacturer.
Proteins were
separated by SDS-PAGE and electrophoretically transferred to a PVDF
membrane (0.2-µ pore size). Blots were overlaid with
45Ca2+ (1 µCi/ml) for 10 min as described by
Maruyama et al. (11) with or without excess unlabeled
CaCl2 (10 mM).
Total RNA was isolated using TRIZOL reagent (Life
Technologies, Inc.) from washed human platelets made free of leukocytes and other cells. The RNA was reverse-transcribed using the 1st Strand
cDNA Synthesis Kit (Boehringer Mannheim) and random primers. PCR
amplification of specific fragments was performed for 30 cycles using
specific sense (5 Polyclonal antibodies were raised against an
antigenic synthetic peptide derived from the amino acid sequence
(KQEILLAHRRFCELLPQEQR) of CIB. The antisera were analyzed by
enzyme-linked immunosorbent assay and Western blotting to determine
specificity and titer for recombinant CIB. Monoclonal antibodies
against CIB were developed by immunizing mice with GST-CIB and
screening the culture supernatants with thrombin-cleaved CIB (North
Carolina State University Hybridoma Facility).
Microtiter wells were coated with 10 µg/ml recombinant CIB, monoclonal antibody 10E5 (provided by Dr.
Barry Coller, Mt. Sinai Medical Center, New York), or BSA. After
blocking with 1% BSA, 5-fold diluted platelet extract in 1% Triton
X-100 containing 2 mM Ca2+ was added to each well
and incubated for 1 h. Wells were washed four times with PBST. Wells
containing immobilized CIB and BSA were incubated with
Using
the
Sequence analysis of this clone indicated an 855-bp insert and an ATG
sequence (bp 47-49) in a region making it the likely translation start
site (12). The open reading frame of 573 bp encodes a polypeptide of
191 amino acids with a predicted mass of 21.7 kDa (Fig.
2A). A consensus sequence for a
polyadenylation recognition site (AATAAA) is located 20 bp before the
3
To determine whether
Ca2+ specifically bound to CIB, we performed
45Ca2+ blot overlay assays. Purified bovine
calmodulin was used as a positive control. Recombinant CIB, which was
purified free from GST as described under "Experimental
Procedures," had a mass of ~25 kDa. 45Ca2+
bound to CIB and calmodulin; the binding was abolished upon inclusion of 10 mM unlabeled Ca2+, indicating a specific
interaction (Fig. 3).
To address whether CIB
is expressed in human platelets and other hematopoietic cells or cell
lines, we isolated total RNA from platelets, leukocytes, HEL cells, and
K-562 cells. RT-PCR was performed using CIB-specific sense and
antisense primers designed to amplify a 0.5-kb segment. As a control
for the quality of platelet RNA,
We also performed Western blot analysis using two different monoclonal
anti-CIB antibodies in an attempt to detect CIB in platelets, HEL, and
K-562 cells. Both mAbs detected an ~25-kDa band in platelet but not
in HEL or K-562 cell extracts (Fig. 4B) despite the
detection of CIB mRNA in HEL cells. The slightly higher than
predicted molecular mass observed on Western blots may be due to
post-translational modification of CIB.
In vitro binding
assays were used to determine whether CIB interacts with the intact
heterodimeric
In the present study, we attempted to identify molecules that
might interact with cytoplasmic domains of
Because CIB appears to interact only with It is well documented that a rise in intracellular Ca2+ is
a prerequisite for platelet activation by many agonists. How
intracellular calcium regulates the function of integrins is not
clearly understood. One possibility is that an increase in
intracellular Ca2+ may regulate integrin function through
CIB or a CIB-like protein. In fact, it has been shown that neutrophil
migration on vitronectin is regulated by calcineurin; migration is
decreased by inhibitors of calcineurin or intracellular
Ca2+ chelators, suggesting that increases in
[Ca2+]i promote neutrophil migration and reduce
integrin-mediated cell adhesion to vitronectin (17). Interaction of
fibronectin and integrin It was further demonstrated that Ca2+ and calcineurin
regulate recycling of integrin Both serine/threonine and tyrosine phosphorylation and
dephosphorylation events appear to play key roles in the regulation of
integrin function (24). Recently, it has been shown that exposure of
ligand binding sites on The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U82226[GenBank]. We thank R. L. Juliano, University of North
Carolina at Chapel Hill, for providing
Department of Pharmacology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
IIb
3 (or
GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may
involve the binding of proteins or other factors to integrin
cytoplasmic domains. To identify candidate cytoplasmic domain binding
proteins, we screened a human fetal liver cDNA library in the yeast
two-hybrid system, using the
IIb cytoplasmic domain as
"bait," and isolated a novel 855-base pair clone. The open reading
frame encodes a novel 191-amino acid polypeptide (termed CIB for
calcium- and integrin-binding protein) that appears to be specific for
the cytoplasmic domain of
IIb, since it does not
interact with the
v,
2,
5,
1, or
3 integrin cytoplasmic domains in
the yeast two-hybrid system. This protein has sequence homology to two
known Ca2+-binding regulatory proteins, calcineurin B (58%
similarity) and calmodulin (56% similarity), and has two EF-hand
motifs corresponding to the two C-terminal Ca2+ binding
domains of these proteins. Moreover, recombinant CIB specifically binds
45Ca2+ in blot overlay assays. Using reverse
transcriptase-polymerase chain reaction and Western blot analysis, we
detected CIB mRNA and protein (~25 kDa), respectively, in human
platelets. An enzyme-linked immunosorbent assay performed using either
immobilized recombinant CIB or monoclonal antibody-captured
IIb
3 indicates a specific interaction
between CIB and intact
IIb
3. These
results suggest that CIB is a candidate regulatory molecule for
integrin
IIb
3.
IIb
3, the platelet fibrinogen receptor,
is specific to the megakaryocytic lineage. In unstimulated platelets
the majority of
IIb
3 is in an inactive
conformation or low affinity state and is unable to bind soluble
ligands. Platelet agonists through their respective receptors transduce
a cascade of intracellular signals ultimately converting
IIb
3 to an active, high affinity state,
capable of binding soluble ligands. This activation process is termed
inside-out signaling (2).
IIb and
3 cytoplasmic
domains in inside-out signaling has been indicated using mutants of
IIb
3 transfected into Chinese hamster
ovary cells (3). When wild type
IIb
3 is
expressed in Chinese hamster ovary cells, it is unable to bind soluble
fibrinogen. However, when an
IIb
3
construct lacking the
IIb cytoplasmic domain is
expressed, the integrin becomes constitutively active and binds soluble
fibrinogen with high affinity (4). Moreover, deletion of the conserved
GFFKR motif of the
IIb cytoplasmic domain makes the
integrin constitutively active. These data suggest that the
IIb cytoplasmic domain helps to maintain a default low
affinity state of
IIb
3. Interestingly, a
naturally occurring point mutation, Ser-752
Pro in the
3 cytoplasmic domain, disrupts inside-out signaling (5),
indicating that the
3 cytoplasmic domain is involved in
inducing or maintaining the high affinity state. Thus, inside-out
signaling probably requires both integrin cytoplasmic domains. It seems
likely that during platelet activation, the conformations of the
IIb and
3 cytoplasmic domains are altered
as a result of an interaction with cytoplasmic factors. It is therefore
of interest to identify candidate cytoplasmic factors and determine
their roles in integrin affinity modulation. In this regard, Shattil
et al. (6), using the yeast two-hybrid system, recently
identified a novel protein termed
3-endonexin, which
interacts with the cytoplasmic domain of the
3
integrin.
IIb
3 causes
receptor clustering, which induces outside-in signaling, leading to
cytoskeletal rearrangements and localization of the integrin in focal
contacts. The mechanism of integrin-mediated signaling and localization
is unclear but is believed to involve the interaction of integrin
cytoplasmic domains with specific proteins. The
cytoplasmic domain
is required for integrin localization in focal contacts, while the
cytoplasmic domain is required for selective association of
IIb
3 with focal contacts (3). The
IIb and/or
3 cytoplasmic domains have
been shown to interact with talin (7), Shc, and Grb2, proteins
potentially involved in these processes (8).
IIb subunit
cytoplasmic domain. This protein is expressed in platelets and binds to
heterodimeric integrin
IIb
3. It has
significant homology to two well known Ca2+-binding
proteins, calmodulin and calcineurin B, which are involved in
regulating the activity of a variety of proteins. Thus, our studies
identify a novel candidate intracellular regulatory molecule for
platelet integrin
IIb
3.
Construction of Vectors
end of the Gal4-(1-147) DNA binding domain.
IIb cytoplasmic domain
binding proteins, as described by the manufacturer.
-CGAGTTGGCGGAGCTGT-3
) and antisense
(5
-AGGATGTTGTCGATGAG-3
) primers to amplify a 0.5-kb fragment of CIB,
and sense (5
-GGCATTCAGTCGCTGTCA-3
) and antisense
(5
-CTCGTTGGCTGCGTCCA-3
) primers to amplify a 1.05-kb fragment of
IIb.
IIb
3 complex-specific mAb 10E5, whereas wells
containing immobilized mAb 10E5 were incubated with recombinant CIB (10 µg/ml) followed by anti-CIB antiserum. The amount of bound
IIb
3 and recombinant CIB were determined using
anti-mouse and anti-rabbit secondary antibody conjugated with alkaline
phosphatase, respectively.
Isolation and Sequence Analysis of CIB cDNA
IIb cytoplasmic domain (
IIb-cyt) as
bait, we screened a human fetal liver cDNA library constructed in
plasmid pGAD10 encoding the GAL4 activation domain. A human fetal liver library was of special interest because the fetal liver produces platelets, making it a likely source of relevant platelet-related cDNA. Out of 2 × 106 total clones screened, we
isolated a single clone (clone 8). In an experiment designed to
eliminate false positives, plasmid DNA from clone 8 was isolated,
purified, and retransformed into the yeast SFY526 strain alone or with
one of the following: (a) pGBT9, (b) a pGBT9
hybrid with an
IIb-cyt insert, or (c) a pGBT9 hybrid with the unrelated protein, lamin C (Fig. 1).
Only transformants that received the
IIb-cyt hybrid and
clone 8 were positive for
-galactosidase activity, indicating a true
positive interaction. The clone 8 cDNA was also transformed into
the yeast SFY526 strain along with plasmid pGBT9 encoding cytoplasmic
domains of integrins
IIb,
v,
2,
5,
1, or
3, and
-galactosidase activity was quantified. The
product of clone 8 failed to interact with the cytoplasmic domains of
integrins
v,
2,
5,
1, and
3, indicating a relatively
specific interaction with the
IIb cytoplasmic domain (Fig. 1).
Fig. 1.
Specificity of interaction of the clone 8 protein with the IIb cytoplasmic domain. Clone 8 plasmid DNA was co-transformed in yeast SFY526 along with empty vector,
plasmid encoding the unrelated protein lamin C, or plasmids encoding
various integrin cytoplasmic domains as indicated, fused with the Gal4
DNA binding domain. The co-transformants were assayed for
-galactosidase activity in both filter and liquid assays.
[View Larger Version of this Image (54K GIF file)]
end. A search of GenBankTM using the cDNA sequence
revealed no significant homology to any published genes. Kyte-Doolittle
hydropathy analysis (13) of the deduced amino acid sequence predicts
that the protein is highly hydrophilic. CIB has a large number of
acidic amino acid residues with a predicted isoelectric point of 4.48 and net charge of
13. Sequence analysis of CIB using "Prosite"
revealed an N-terminal myristoylation site, two consensus sites for
phosphorylation by protein kinase C, and five consensus sites for
phosphorylation by casein kinase II (Fig. 2A). A search of
protein data banks indicated that the deduced amino acid sequence is
novel but shares homology with calcineurin B, the regulatory subunit of
protein phosphatase 2B (28% identity, 58% similarity) and calmodulin
(27% identity, 55% similarity) (Fig. 2B). Further sequence
analysis of CIB indicated the presence of two EF-hand motifs that
correspond to the two adjacent C-terminal Ca2+ binding
domains in calcineurin B and calmodulin. The identity between EF-hand
motif I of CIB and EF-hand 3 of calcineurin B is 39% and that of
EF-hand II of CIB and EF-hand 4 of calcineurin B is 35% (Fig.
2B).
Fig. 2.
Sequence analysis of clone 8. A,
cDNA sequence and deduced amino acid sequence of clone 8. A
consensus sequence for a polyadenylation recognition site (AATAAA) is
underlined. The two EF-hand motifs are boxed.
Putative sites of phosphorylation by protein kinase C are shown by
shaded circles and by casein kinase II are shown by
unshaded circles. A putative myristoylation site is
indicated by a square. B, amino acid sequence alignment of
CIB with calcineurin B (CALB) and calmodulin
(CAM). Identical residues are shaded. The two
EF-hand motifs of CIB are indicated by solid lines, whereas
the EF-hand motifs of CALB and CAM are indicated by broken
lines.
[View Larger Version of this Image (39K GIF file)]
Fig. 3.
Blot overlay assay of
45Ca2+ binding to CIB. Purified calmodulin
or CIB (2 µg each) was separated by SDS-PAGE and transferred to PVDF
membranes, and the membranes were overlaid with
45Ca2+ (1 µCi/ml) for 10 min in the absence
(A) and in the presence of 10 mM unlabeled
CaCl2 (B). Lane 1, molecular size
markers; lane 2, purified bovine calmodulin; lane
3, recombinant CIB.
[View Larger Version of this Image (60K GIF file)]
IIb-specific primers,
designed to amplify a 1.05-kb segment, were used. With CIB-specific
primers, we observed the amplification of a 0.5-kb band from RNA
derived from platelets and HEL cells (Fig.
4A) indicating the presence of CIB-specific
mRNA. A slightly smaller sized band was observed from leukocytes
and K-562 cells, the identity of which is not yet known. In contrast,
none of these bands were amplified in a breast cancer cell line (T47D).
Amplification of a fragment of the expected size (~1 kb) from
platelet RNA with
IIb primers served as a positive
control and demonstrates the integrity of the platelet RNA.
Fig. 4.
Expression of CIB in human platelets.
A, RT-PCR amplification of RNA. Total RNA was isolated and
cDNA was synthesized as described under "Experimental
Procedures." Aliquots of the cDNA synthesis reaction mixture of
various cellular RNAs were amplified for 30 cycles. Lanes 1 and 7, platelets; lane 2, leukocytes; lane
3, HEL cells; lane 4, T47D cells (a transformed breast
epithelial cell line); lane 5, K-562 cells. In lane
6, clone 8 cDNA was used as template (a positive control).
Lanes 1-6, CIB specific primers, 500 bp apart, were used;
whereas in lane 7, IIb-specific primers, 1050 bp apart, were used. B, Western blot analysis of CIB.
Proteins (200 µg) from cells lysed in Laemmli buffer were separated
using SDS-PAGE, transferred to PVDF membranes, and blotted with mAb UN2
and mAb UN7, both raised against recombinant CIB.
[View Larger Version of this Image (48K GIF file)]
IIb
3
IIb
3 integrin. In these experiments, purified recombinant CIB was immobilized to microtiter wells, wells were blocked with BSA, and
IIb
3 from platelet extract was allowed to
bind. The amount of integrin bound was quantified using mAb 10E5, an
integrin complex specific antibody. Immobilized CIB bound significantly
higher levels of
IIb
3 than did
immobilized BSA in the absence of CIB (Fig.
5A). Similar results were obtained in a
reverse experiment in which heterodimeric integrin
IIb
3 from platelet lysate was captured on
immobilized mAb 10E5 (Fig. 5B). Significantly more
recombinant CIB bound to wells containing antibody-captured integrin
IIb
3 than to similarly treated wells lacking mAb 10E5, as detected with anti-CIB antiserum. Moreover, CIB
did not bind to immobilized mAb 10E5 alone (data not shown). These
results demonstrate an in vitro interaction of CIB with integrin
IIb
3.
Fig. 5.
In vitro binding of CIB to integrin
IIb
3. Binding assays were performed
as described under "Experimental Procedures." A, binding
of integrin
IIb
3 to immobilized BSA or
CIB; B, binding of CIB to wells lacking mAb 10E5 (BSA-coated
wells) and treated with platelet lysate or to wells with immobilized
10E5 and treated with platelet lysate to capture integrin
IIb
3. As controls, CIB-coated wells
incubated with mAb 10E5 and mAb 10E5-coated wells incubated with CIB
followed by anti-CIB antiserum gave background level readings. Each
value is the mean of triplicates and is representative of two separate
experiments.
[View Larger Version of this Image (10K GIF file)]
IIb
3, using the yeast two-hybrid system
(9, 10). We isolated a novel full-length human cDNA that encodes a
polypeptide with a predicted molecular mass of 21.7 kDa, which binds to
the integrin
IIb cytoplasmic domain and is expressed in
platelets. The structural properties of CIB indicate that it is a
hydrophilic calcium-binding protein, most similar to calcineurin B and
calmodulin. Calcineurin B is a small regulatory subunit (19 kDa) of
phosphoprotein phosphatase 2B or calcineurin, the only known protein
phosphatase that is regulated by Ca2+ and calmodulin (14).
Calcineurin is a heterodimer that also consists of a large catalytic
subunit, calcineurin A. The A subunit binds calmodulin, whereas the B
subunit binds four atoms of Ca2+ (14) and is a member of
the "EF-hand" family of Ca2+-binding proteins.
Calmodulin is a well described Ca2+-dependent
regulatory molecule for several different enzymes. Unlike calcineurin B
and calmodulin, CIB has two rather than four EF-hand motifs.
Nonetheless, the similarity between CIB and these regulatory molecules
raises the question as to whether CIB also serves as a regulatory
subunit for an unknown enzyme(s) as well as for integrin
IIb
3.
IIb and not
with other integrin
subunits (
v,
2,
and
5) in the yeast two-hybrid system, it is unlikely
that CIB binds to the membrane-proximal GFFKR region of the
IIb cytoplasmic domain, since this sequence is common to
all integrin
subunits. This is in contrast to calreticulin, a
Ca2+-binding protein that binds to GFFKR (15) and
co-purifies with several integrins (16). This suggests that the CIB
binding site on the
IIb cytoplasmic domain may include
the highly acidic portion distal to the membrane.
5
1 in
vitro has also been shown to be regulated by calcineurin (18).
v
3 to the
leading edge of migrating neutrophils (17, 19). The
IIb
3 integrin also undergoes recycling,
in a process that down-regulates platelet aggregation (20). This event
appears to be independent of the
3 cytoplasmic domain
(21) and probably dependent on the
IIb cytoplasmic
domain. Thus, it is possible that calcineurin or a calcineurin-like
regulatory molecule such as CIB contributes to recycling. In this
regard, Barroso et al. (22) have reported the cloning of a
novel 22-kDa calcium-binding protein (p22) that is also highly
homologous to calcineurin B and required for constitutive membrane
traffic. Another protein called CHP, which is 99% identical to p22, is involved in regulating the Na+/H+ antiporter by
interacting with a specific domain of the cytoplasmic tail (23). Both
of these proteins have two distinct C-terminal EF-hand motifs, like
CIB. However, their amino acid sequences are only 53% homologous (29%
identical) to CIB, but do suggest the existence of a family of small
regulatory Ca2+-binding proteins with two EF-hand
motifs.
IIb
3 correlates
under some conditions with phosphorylation of the
3
subunit of
IIb
3 although the extent of
3 phosphorylation is unclear (25, 26). In addition, a
protein kinase that interacts with the
1 and
3 cytoplasmic domains has been identified (27). It is
therefore possible that a protein phosphatase may be directly involved
in integrin regulation. Since CIB shows structural similarity to
calcineurin B, it is conceivable that CIB may also be a subunit of a
multisubunit protein phosphatase. Thus, it will be of interest to
determine specific sites that mediate CIB binding to
IIb, identify other molecules that bind to CIB, and
delineate functional details of this novel Ca2+-binding
protein.
*
This work was supported by National Institutes of Health
Grant 1-P01-HL45100 (to L. V. P.). Additional support was provided by
the University of North Carolina Research Council Award (to U. P. N.)
and the Medical Faculty Research Award (to U. P. N.). The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
University of North Carolina, CB# 7365, Chapel Hill, NC 27599. Tel.:
919-962-1058; Fax: 919-966-5640.
1
The abbreviations used are: PCR, polymerase
chain reaction; RT-PCR, reverse transcriptase-PCR; CIB, calcium- and
integrin-binding protein; GST, glutathione S-transferase;
HEL, human erythroleukemia; mAb, monoclonal antibody; PBST,
phosphate-buffered saline containing 0.1% Tween 20; PAGE,
polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride;
bp, base pair(s); kb, kilobase(s); BSA, bovine serum albumin.
5 and
1 cDNA constructs, Pam Conley, COR Therapeutics,
South San Francisco, CA, for
IIb,
v, and
3 constructs, Martin Hemler, Dana Farber Institute,
Harvard Medical School, for
2 constructs, and Christy
Turbeville for help in purifying recombinant CIB.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.