Identification of a novel calcium-binding protein that interacts with the integrin α(IIb) cytoplasmic domain

Abstract

The mechanism by which platelets regulate the function of integrin α(IIb)β3 (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(IIb)β3 indicates a specific interaction between CIB and intact α(IIb)β3. These results suggest that CIB is a candidate regulatory molecule for integrin α(IIb)β3.