Expression and function of calcium binding domain chimeras of the integrins alpha(IIb) and alpha(5).
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.
Gidwitz
S
S
Center for Thrombosis and Hemostasis, University of North Carolina, Chapel Hill, North Carolina 27599, USA. gidwitz@med.unc.edu
Lyman
S
S
White
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002449:Cell Aggregation; D019281:Dimerization; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D008969:Molecular Sequence Data; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017469:Receptors, Fibronectin / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen; D002118:Calcium