Effects of limiting extension at the alphaIIb genu on ligand binding to integrin alphaIIbbeta3.
Structural data of integrin alphaIIbbeta3 have been interpreted as supporting a model in which: 1) the receptor exists primarily in a "bent," low affinity conformation on unactivated platelets and 2) activation induces an extended, high affinity conformation prior to, or following, ligand binding. Previous studies found that "clasping" the alphaIIb head domain to the beta3 tail decreased fibrinogen binding. To study the role of alphaIIb extension about the genu, we introduced a disulfide "clamp" between the alphaIIb thigh and calf-1 domains. Clamped alphaIIbbeta3 had markedly reduced ability to bind the large soluble ligands fibrinogen and PAC-1 when activated with monoclonal antibody (mAb) PT25-2 but not when activated by Mn(2+) or by coexpressing the clamped alphaIIb with a beta3 subunit containing the activating mutation N339S. The clamp had little effect on the binding of the snake venom kistrin (M(r) 7,500) or alphaIIbbeta3-mediated adhesion to immobilized fibrinogen, but it did diminish the enhanced binding of mAb AP5 in the presence of kistrin. Collectively, our studies support a role for alphaIIb extension about the genu in the binding of ligands of 340,000 and 900,000 M(r) with mAb-induced activation but indicate that it is not an absolute requirement. Our data are consistent with alphaIIb extension resulting in increased access to the ligand-binding site and/or facilitating the conformational change(s) in beta3 that affect the intrinsic affinity of the binding pocket for ligand.
Blue
Robert
R
Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, New York 10021, USA.
Li
Jihong
J
Steinberger
Jonathan
J
Murcia
Marta
M
Filizola
Marta
M
Coller
Barry S
BS
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D002448:Cell Adhesion; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D004220:Disulfides; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D040201:Platelet Membrane Glycoprotein IIb; D005340:Fibrinogen; D003545:Cysteine