Expression and function of calcium binding domain chimeras of the integrins alpha(IIb) and alpha(5). J Biol Chem 2000 Mar 03;275(9):6680-8
Date
02/29/2000Pubmed ID
10692478DOI
10.1074/jbc.275.9.6680Scopus ID
2-s2.0-0034051173 (requires institutional sign-in at Scopus site) 4 CitationsAbstract
To further identify amino acid domains involved in the ligand binding specificity of alpha(IIb)beta(3), chimeras of the conserved calcium binding domains of alpha(IIb) and the alpha subunit of the fibronectin receptor alpha(5)beta(1) were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of alpha(IIb) with those of alpha(5), or deleted all four calcium binding domains were synthesized but not expressed on the cell surface. Additional chimeras exchanged subsets or all of the variant amino acids in the second calcium binding domain, a region implicated in ligand binding. Cell surface expression of each second calcium binding domain mutant complexed with beta(3) was observed. Each second calcium binding domain mutant was able to 1) bind to immobilized fibrinogen, 2) form fibrinogen-dependent aggregates after treatment with dithiothreitol, and 3) bind the activation-dependent antibody PAC1 after LIBS 6 treatment. Soluble fibrinogen binding studies suggested that there were only small changes in either the K(d) or B(max) of any mutant. We conclude that chimeras of alpha(IIb) containing the second calcium binding domain sequences of alpha(5) are capable of complexing with beta(3), that the complexes are expressed on the cell surface, and that mutant complexes are capable of binding both immobilized and soluble fibrinogen, suggesting that the second calcium binding domain does not determine ligand binding specificity.
Author List
Gidwitz S, Lyman S, White GC 2ndAuthor
Gilbert C. White MD Professor in the Medicine department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Antibodies, Monoclonal
Binding Sites
COS Cells
Calcium
Cell Aggregation
Dimerization
Fibrinogen
Flow Cytometry
Molecular Sequence Data
Mutation
Platelet Glycoprotein GPIIb-IIIa Complex
Protein Binding
Receptors, Fibronectin
Recombinant Fusion Proteins
Transfection