COMMUNICATION:
Identification of a Novel Calcium-binding Protein That Interacts with the Integrin alpha IIb Cytoplasmic Domain*

(Received for publication, September 20, 1996, and in revised form, December 3, 1996)

Ulhas P. Naik Dagger §, Pankaj M. Patel Dagger and Leslie V. Parise Dagger

From the Dagger  Department of Pharmacology, Center for Thrombosis and Hemostasis, and  Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The mechanism by which platelets regulate the function of integrin alpha IIbbeta 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 alpha 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 alpha IIb, since it does not interact with the alpha v, alpha 2, alpha 5, beta 1, or beta 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 alpha IIbbeta 3 indicates a specific interaction between CIB and intact alpha IIbbeta 3. These results suggest that CIB is a candidate regulatory molecule for integrin alpha IIbbeta 3.


INTRODUCTION

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 alpha IIbbeta 3, the platelet fibrinogen receptor, is specific to the megakaryocytic lineage. In unstimulated platelets the majority of alpha IIbbeta 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 alpha IIbbeta 3 to an active, high affinity state, capable of binding soluble ligands. This activation process is termed inside-out signaling (2).

The role of the alpha IIb and beta 3 cytoplasmic domains in inside-out signaling has been indicated using mutants of alpha IIbbeta 3 transfected into Chinese hamster ovary cells (3). When wild type alpha IIbbeta 3 is expressed in Chinese hamster ovary cells, it is unable to bind soluble fibrinogen. However, when an alpha IIbbeta 3 construct lacking the alpha 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 alpha IIb cytoplasmic domain makes the integrin constitutively active. These data suggest that the alpha IIb cytoplasmic domain helps to maintain a default low affinity state of alpha IIbbeta 3. Interestingly, a naturally occurring point mutation, Ser-752 right-arrow Pro in the beta 3 cytoplasmic domain, disrupts inside-out signaling (5), indicating that the beta 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 alpha IIb and beta 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 beta 3-endonexin, which interacts with the cytoplasmic domain of the beta 3 integrin.

Fibrinogen binding to alpha IIbbeta 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 beta  cytoplasmic domain is required for integrin localization in focal contacts, while the alpha  cytoplasmic domain is required for selective association of alpha IIbbeta 3 with focal contacts (3). The alpha IIb and/or beta 3 cytoplasmic domains have been shown to interact with talin (7), Shc, and Grb2, proteins potentially involved in these processes (8).

We have identified a novel Ca2+-binding protein that interacts specifically with the integrin alpha IIb subunit cytoplasmic domain. This protein is expressed in platelets and binds to heterodimeric integrin alpha IIbbeta 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 alpha IIbbeta 3.


EXPERIMENTAL PROCEDURES

Construction of Vectors

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' end of the Gal4-(1-147) DNA binding domain.

Two-hybrid Library Screening

Human cDNA libraries derived from fetal liver in pGAD10 (Clontech) were screened in the yeast two-hybrid system (9, 10), for alpha IIb cytoplasmic domain binding proteins, as described by the manufacturer.

Expression of GST-fusion Protein

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.

45Ca2+ Binding

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).

RT-PCR

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'-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 alpha IIb.

Antibodies

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).

In Vitro Binding Assay

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 alpha IIbbeta 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 alpha IIbbeta 3 and recombinant CIB were determined using anti-mouse and anti-rabbit secondary antibody conjugated with alkaline phosphatase, respectively.


RESULTS

Isolation and Sequence Analysis of CIB cDNA

Using the alpha IIb cytoplasmic domain (alpha 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 alpha IIb-cyt insert, or (c) a pGBT9 hybrid with the unrelated protein, lamin C (Fig. 1). Only transformants that received the alpha IIb-cyt hybrid and clone 8 were positive for beta -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 alpha IIb, alpha v, alpha 2, alpha 5, beta 1, or beta 3, and beta -galactosidase activity was quantified. The product of clone 8 failed to interact with the cytoplasmic domains of integrins alpha v, alpha 2, alpha 5, beta 1, and beta 3, indicating a relatively specific interaction with the alpha IIb cytoplasmic domain (Fig. 1).


Fig. 1. Specificity of interaction of the clone 8 protein with the alpha 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 beta -galactosidase activity in both filter and liquid assays.
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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' 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.
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Calcium Binding to Recombinant CIB

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).


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.
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Expression of CIB in Human Platelets

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, alpha 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 alpha 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, alpha 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.
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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 of CIB to Heterodimeric Integrin alpha IIbbeta 3

In vitro binding assays were used to determine whether CIB interacts with the intact heterodimeric alpha IIbbeta 3 integrin. In these experiments, purified recombinant CIB was immobilized to microtiter wells, wells were blocked with BSA, and alpha IIbbeta 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 alpha IIbbeta 3 than did immobilized BSA in the absence of CIB (Fig. 5A). Similar results were obtained in a reverse experiment in which heterodimeric integrin alpha IIbbeta 3 from platelet lysate was captured on immobilized mAb 10E5 (Fig. 5B). Significantly more recombinant CIB bound to wells containing antibody-captured integrin alpha IIbbeta 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 alpha IIbbeta 3.


Fig. 5. In vitro binding of CIB to integrin alpha IIbbeta 3. Binding assays were performed as described under "Experimental Procedures." A, binding of integrin alpha IIbbeta 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 alpha IIbbeta 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.
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DISCUSSION

In the present study, we attempted to identify molecules that might interact with cytoplasmic domains of alpha IIbbeta 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 alpha 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 alpha IIbbeta 3.

Because CIB appears to interact only with alpha IIb and not with other integrin alpha  subunits (alpha v, alpha 2, and alpha 5) in the yeast two-hybrid system, it is unlikely that CIB binds to the membrane-proximal GFFKR region of the alpha IIb cytoplasmic domain, since this sequence is common to all integrin alpha  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 alpha IIb cytoplasmic domain may include the highly acidic portion distal to the membrane.

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 alpha 5beta 1 in vitro has also been shown to be regulated by calcineurin (18).

It was further demonstrated that Ca2+ and calcineurin regulate recycling of integrin alpha vbeta 3 to the leading edge of migrating neutrophils (17, 19). The alpha IIbbeta 3 integrin also undergoes recycling, in a process that down-regulates platelet aggregation (20). This event appears to be independent of the beta 3 cytoplasmic domain (21) and probably dependent on the alpha 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.

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 alpha IIbbeta 3 correlates under some conditions with phosphorylation of the beta 3 subunit of alpha IIbbeta 3 although the extent of beta 3 phosphorylation is unclear (25, 26). In addition, a protein kinase that interacts with the beta 1 and beta 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 alpha IIb, identify other molecules that bind to CIB, and delineate functional details of this novel Ca2+-binding protein.


FOOTNOTES

*   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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U82226[GenBank].


§   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.

Acknowledgments

We thank R. L. Juliano, University of North Carolina at Chapel Hill, for providing alpha 5 and beta 1 cDNA constructs, Pam Conley, COR Therapeutics, South San Francisco, CA, for alpha IIb, alpha v, and beta 3 constructs, Martin Hemler, Dana Farber Institute, Harvard Medical School, for alpha 2 constructs, and Christy Turbeville for help in purifying recombinant CIB.


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