Abstract
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.
Highlights
Integrins function as adhesion receptors that mediate cellextracellular matrix, cell-cell, and cell-soluble ligand interactions [1]
Expression of Mutant Constructs—To determine if the various ␣IIb calcium binding domain mutants were expressed on the cell surface, Chinese hamster ovary K1 (CHO) cells co-transfected with the mutants and wild type 3 were analyzed by flow cytometry using Tab, a murine monoclonal antibody that binds ␣IIb
Cell with the same dissociation constant, Kd, as reported for AP2 binding to platelets [38]. More extended chimeras such as IIb␣5, a chimera in which the entire calcium binding domain of ␣IIb was replaced with the corresponding sequence of ␣5, and D2, a chimera in which the first, third, and fourth calcium binding domains of ␣IIb were replaced by the corresponding sequences of ␣5, were not recognized by flow cytometric analysis with any anti-␣IIb or complex-specific antibody tested, indicating lack of expression on the cell surface
Summary
Integrins function as adhesion receptors that mediate cellextracellular matrix, cell-cell, and cell-soluble ligand interactions [1]. The results indicate that these mutants are expressed on the cell surface, complex with 3 but not 1, and function in a manner indistinguishable from wild type ␣IIb3, indicating that the second calcium binding domain of ␣IIb3 does not determine ligand binding specificity.
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