Identification of a Novel Calcium-binding Protein That Interacts with the Integrin αIIb Cytoplasmic Domain

doi:10.1074/jbc.272.8.4651

Identification of a Novel Calcium-binding Protein That Interacts with the Integrin α_IIb Cytoplasmic Domain*

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

^§ 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.

Abstract

The mechanism by which platelets regulate the function of integrin α_IIbβ₃ (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 α_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 α_IIb, since it does not interact with the α_v, α₂, α₅, β₁, or β₃ 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 α_IIbβ₃ indicates a specific interaction between CIB and intact α_IIbβ₃. These results suggest that CIB is a candidate regulatory molecule for integrin α_IIbβ₃.

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 GenBank™/EMBL Data Bank with accession number(s) U82226[GenBank].
↵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.
- Received September 20, 1996.
- Revision received December 3, 1996.