Expression and Function of Calcium Binding Domain Chimeras of the Integrins alpha sub(IIb) and alpha sub(5)

• Published in: The Journal of biological chemistry 2000-03, Vol.275 (9), p.6680-6688
• Main Authors: Gidwitz, S, Lyman, S, White II, GC
• Format: Article
• Language: English
• Subjects: fibronectin receptors
• ISSN: 0021-9258
• DOI: 10.1074/jbc.275.9.6680

To further identify amino acid domains involved in the ligand binding specificity of alpha sub(IIb) beta sub(3), chimeras of the conserved calcium binding domains of alpha sub(IIb) and the alpha subunit of the fibronectin receptor alpha sub(5) beta sub(1) were constructed. Chimeras that replaced all four calcium binding domains, replaced all but the second calcium binding domain of alpha sub(IIb) with those of alpha sub(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 sub(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 sub(d) or B sub(max) of any mutant. We conclude that chimeras of alpha sub(IIb) containing the second calcium binding domain sequences of alpha sub(5) are capable of complexing with beta sub(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.