Mutations in and near the second calcium-binding domain of integrin alphaIIb affect the structure and function of integrin alphaIIbbeta3.
Calcium-binding domains in the alpha-subunit of integrins contain a central loop structure. To examine the importance of the loop structure, a series of alphaIIb mutants containing changes to the calcium-liganding amino acids have been constructed. Significantly, none of the mutant alphaIIbbeta3 complexes was detected on the surface of transfected cells, but mutant pro-alphaIIb was detected in cell lysates in complex with beta3. To study the importance of the regions flanking the second calcium-binding domain for ligand-binding and ligand-binding specificity, three alphaIIb/alpha5 chimaeras containing alpha5 sequences flanking or flanking and including the second calcium-binding domain were constructed. The chimaera containing both alpha5-flanking regions was not expressed on the cell surface, but FR1 and FR2, substituting either the first or second flanking region, were expressed. FR1beta3-transfected cells lost the ability to adhere to fibrinogen and to support aggregation and had minimal fibrinogen-binding ability. The heterodimer complex was less stable than the wild-type. FR2beta3-transfected cells adhered to fibrinogen and bound soluble fibrinogen with higher affinity when compared with wild-type. In addition, the heterodimer complex was more stable than wild-type. These results indicate that the conformation of the second calcium-binding domain is critical for maturation of the alphaIIbbeta3 complex and expression on the cell surface and that the surrounding sequences are critical for alphaIIbbeta3 function.
Gidwitz
Susan
S
Division of Hematology and Oncology, Department of Medicine, Center for Thrombosis and Hemostasis, University of North Carolina, Chapel Hill, NC 27599-7035, USA. gidwitz@med.unc.edu
Temple
Brenda
B
White
Gilbert C
GC
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D008024:Ligands; D008958:Models, Molecular; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism*; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D040201:Platelet Membrane Glycoprotein IIb; D011993:Recombinant Fusion Proteins; D002118:Calcium