Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain.
The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (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 alphaIIb 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 alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 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 ( approximately 25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3 indicates a specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3.
Naik
U P
UP
Department of Pharmacology, Center for Thrombosis and Hemostasis, The University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Patel
P M
PM
Parise
L V
LV
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D017421:Sequence Analysis
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D019039:Platelet Glycoprotein GPIIb-IIIa Complex