Critical residues for ligand binding in blade 2 of the propeller domain of the integrin alphaIIb subunit.
Ligand binding to integrin alphaIIbbeta3 is a key event of thrombus formation. The propeller domain of the alphaIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the alphaV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the alphaIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the alphaIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the alphaV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to alphaIIbbeta3 induced by different types of integrin activation including activation of alphaIIbbeta3 by beta3 cytoplasmic mutation. CHO cells stably expressing recombinant alphaIibbeta3 bearing Q111A, H112A or N114A mutation did not exhibit alphaIibbeta3-mediated adhesion to fibrinogen. According to the crystal structure of alphaVbeta3, the alphaV residue corresponding to alphaIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the alphaIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.
Tamura
Tatsushiro
T
Department of Internal Medicine, Ehime University School of Medicine Shigenobu, Ehime 791-0295, Japan.
Hato
Takaaki
T
Yamanouchi
Jun
J
Fujita
Shigeru
S
D000409:Alanine / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D016023:Integrins / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; D040201:Platelet Membrane Glycoprotein IIb; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D000409:Alanine